INTERNATIONAL REVIEW OF CYTOLOGY V132

VOLUME 132

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander

1949-1 988 1949-1 984 19671984-

ADVISORY EDITORS Aimee Bakken Howard A. Bern Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham M. Nelly Golarz De Bourne Elizabeth D. Hay Mark Hogarth H. Ron Kaback Keith E. Mostov Audrey Muggleton-Harris

Andreas Oksche Muriel J. Ord Valdimir R. Pantic M. V. Parthasarathy Lionel I. Rebhun Jean-Paul Revel L. Evans Roth Jozef St. Schell Hiroh Shibaoka Wilfred Stein Ralph M. Steinman D. Lansing Taylor M. Tazawa Alexander L. Yudin

Edited by Kwang W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

Martin Friedlander Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME 132

Academic Press, Inc. Harcoufi Brace Jovanovich, Publishers San Diego New York Boston London

Sydney Tokyo Toronto

This book is printed on acid-free paper. @ Copyright 0 1992 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. San Diego, California 92101 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW1 7DX

Library of Congress Catalog Number: 52-5203 International Standard Book Number: 0-12-364532-8

PRINTED IN THE UNITED STATES OF AMERICA 92939495

9 8 7 6 5 4 3 2 1

Contributors ..............................................................................................................

ix

Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants Toshiyuki Nagata. Yasuyuki Nemoto. and Seiichiro Hasezawa I. II. Ill. IV . V. VI . VII . VIII . IX. X.

Introduction .............................................................. ........ ..... Origin of TBY-2 Cells .... .................................................. Growth and Cytological Characteristics ..................... ..... Studies of Subcellular Organelles .................................................. Synchronization of Cells ............................................ ..... Changes in Cytoskeletons during the Cell Cycle Progression ....................... Cytological Investigation .................... Gene Delivery into Cells ............................. Supplementary Remarks . ............................................................ .................... ........................................................... References ................... ..................................................

1 2 3 12 15 19 25 26 27 28 29

Cells in the Marginal Zone of the Spleen Georg Kraal I. I1. Ill . IV. V.

Introduction ................................. .................................................. Marginal Zone ........................................................... .................... ................................................................ Cells of the Marginal Zone Function of the Marginal Summary ..................... .................... References .................................................

31 32 34 52 67 68

V

vi

CONTENTS

Role of the Cytoskeleton in Genome Regulation and Cancer Theodore T. Puck and Alphonse Krystosek ...................................................... I. Introduction ......_ Reverse Transformation Reaction and Role of the Cytoskeleton ...... II. 111. A Theory of Mammalian Cell Genome Regulation Involving the Gytoskeleton .... ..... * .................................................. A.. Application to Cancer ........................ ............................. IV. V. Other Evidence for Genetic Regulatory ..................... VI. Applications to Molecular Biology ......................... .......................................................... VII. Evolutionary Considerations VIII. Additional Relevant Information Concerning the Nature of the Cytoskeleton and Previous Theories of Its Function ..................................... IX. Concluding Remarks ............ .............................................. References ...........................

75 76 82 88 92 94 95 96 103 106

Properties and Uses of Photoautotrophic Plant Cell Cultures Jack M. Widholm .......................................... I. autotrophic Cell Cultures II. .......................................................... Ill. Specific Uses ......... IV. Conclusions .......,..,...........,.......... .................... .......................................................... References ..........................

109 111 148 167 170

Zymogen Granules of the Pancreas and the Parotid Gland and Their Role in Cell Secretion Adrien R. Beaudoin and Gilles Grondin I. Introduction ...... ....... ...... .............. ....... ......................................................... 11. ZG Morphometry: Influence of Various Physiological Parameters on Granule Size ............................................................................................. Ill. Fate of the ZG Membrane after Exocytosis: A Recycling Process ................. IV. ZG Cytochemistry: Relationship between the Golgi Apparatus and ZGs .......

177 178 182 186

CONTENTS

V . Freeze-Fracture Observations: Evidence That the ZG Membrane Undergoes Some Major Topographical Alterations of Protein and Lipids .... VI . ZG lmmunocytochemistry and the Concept of Nonparallel Secretion .......... v11. Pancreas ZG Membrane Proteins ........................................ .............................. VIII . Parotid ZG Membrane Proteins .. IX . ZG Membrane Lipid Composition .................................... X . ZG Ion Transport in the Pancreas ................................................................. XI . Cytoskeleton and ZG Movement ..................................... .............................. XI1. Concluding Remarks ........................... References ............... .....................................

vii 191 196 199 206 208 209 211 215 217

Molecular Analysis of Plant Signaling Elements: Relevance of Eukaryotic Signal Transduction Models

Klaus Palrne ................................................ I . Introduction ................................ II. Advantages of Plants as Models .................................................................. 111 . Characterization of Plant Development and Its Control ................................ IV. Different Strategies for Organizing the Body Patterns in Plants and Animals ............................... ................................................ ..................................... ............ V . Development of Concepts VI . Molecular Elements and Mechanisms of Cellular Signaling ......................... VII . Elements of Eukaryotic Signal Response Pathways ....... VIII . Phytohormone Perception and Cellular Responses IX. Current Models and Perspectives for Phytohormone X . Functional, Molecular, and Structural Analyses of Plant-Specific Modules for the Recognition of Second Messengers ..................................... ......................................... XI . Concluding Remarks and Future Prosp ................................................

255 272 274

Index .........................................................................................................................

285

223 224 225 229 232 234 235 242 246

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Adrien R. Beaudoin (177), Departement de Biologie, faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada Gilles Grondin (177), Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, QuebecJ1K 2R1, Canada Seiichiro Hasezawa (1), College of General Education, SeJo Junior College, Tokyo 157, Japan Georg Kraal (31), Department of Cell Biology, Free University, 1085 BTAmsterdam, The Netherlands Alphonse Ktystosek (75), Eleanor Roosevelt lnstitute for Cancer Research, Denver, Colorado 80206 Toshiyuki Nagata (1), Department of Biology, faculty of Science, University of Tokyo, Tokyo 113,Japan Yasuyuki Nemoto ( l ) , Department of 6iotechnology, faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo 184,Japan Klaus Palme (223), M~-~lanck-/nstitut f i r fflanzenzuchtung, D-5000 KOln 30, Germany Theodore T. Puck (75), Eleanor Roosevelt lnstitute for Cancer Research, Departments of Medicine and of Biophysics, Biochemistfy, and Genetics, University of Colorado Health Sciences Center, and University of Colorado Cancer Center, Denver, Colorado 80206 Jack M. Widholm (109), Department of Agronomy, University of Illinois, Urbana, lllinois 61801 ix

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Tobacco BY-2 Cell Line as the “HeLa” Cell in the Cell Biology of Higher Plants Toshiyuki Nagata,’ Yasuyuki Nemoto,t and Seiichiro Hasezawas,

’

* Department of Biology, Faculty of Science, University of Tokyo, Tokyo

113,

Japan Department of Biotechnology, Faculty of Technology, Tokyo University of Agriculture and Technology, Tokyo 184, Japan College of General Education, Seijo Junior College, Tokyo 157, Japan

*

1. Introduction

Although it was not until the introduction of established cell lines such as HeLa that molecular studies of animal viruses rapidly progressed, such cell lines have played an important role in the basic understanding of the molecular and cellular biology of mammalian cells, and many such examples can be observed in previous monographs (Willmer, 1965). More recently, understanding of the immortality of cell lines such as HeLa and L cells originating from cancer tissues helped elucidate the mechanism of tumorigenesis in animal cells (Baserga, 1985). On the other hand, no such plant cell lines can be studied from various aspects of interest, although the establishment of cell lines from plant tissues is relatively easy, and innumerable cell lines have been obtained from various tissues and species of higher plants. There are several cell lines, such as tobacco XD (Filner, 1965), soybean (Gamborg, 1970), Acer pseudoplatanus (Simpkins ef al., 1970), and Catharanthus roseus (Vinca rosea) (Misawa and Samejima, 1978), whose literature is extensive. However, the tobacco BY-2 (TBY-2) cell line, the subject of this chapter, has shown unique characteristics, exceptionally higher growth rates, and high homogeneity. As there may be some who have never heard of the TBY-2 cell line or its characteristics, details are described here, but at the onset some essential features are presented which we think are important. Although most people would recognize that a highly synchronous cell population is necessary for the study of plant cells, thus far most cell lines stated to have been synchronized had a mitotic index (MI) of 10-20% at

’

Present address: Department of Biology, Faculty of Science, University of Tokyo, Tokyo 113, Japan. Inremotional Review, of Cvrologv. Vol. I32

1

Copyright Q 1992 by Academic Press. Inc. All nghts of reproduction in any form reserved.

2

TOSHlYUKl NAGATA ET AL.

most. The serious question is about the remaining 80-90% of cells in the population. Nevertheless, such systems were traditionally called synchronized populations. The synchrony of the TBY-2 cell line, which we established, using aphidicolin, was 70-80% in terms of MI (Nagata et af., 1982). Thus, the basic understanding of the molecular and cellular biology of plant cells will probably progress by the use of this cell line, and the previous description will therefore have to be rewritten. Anyone who researches the cellular events of higher plants may have to use this cell line, or the question will be raised as to how a phenomenon that has been observed in other sources looks in TBY-2 cells. This statement may seem a bit overwritten, but after 9 years of establishing this experimental system, not afew people have noticed this point. This is the reason that this chapter has such an unusual title. Thus, we describe what has been done on this cell line, established almost 20 years ago, and what is going on in our laboratory. Supplementally, papers from Dr. H. Shibaoka’s laboratory are cited, as his group has successfully used our method.

II. Origin of TBY-2 Cells

The TBY-2 cell line was established from the callus induced on a seedling of Nicotiana tabacum L. cv. Bright Yellow 2 in the Central Research Institute of the Japan Tobacco and Salt Public Corporation (now the Tobacco Science Research Laboratory, Japan Tobacco, Inc.) (Kato et al., 1972).It has been propagated in the medium of Linsmaier and Skoog (1965) supplemented with sucrose and 2,4-dichlorophenoxyaceticacid (2,4-D). According to Kato et al. (1972), the TBY-2 cell line was the most proliferative among the examined materials of 40 species of Nicotiuna and three species of Populus, which suggests that this cultivar of tobacco may have some specific characteristics. Because of the higher growth rate and the absence of nicotine in the TBY-2 cell line, the Japan Tobacco and Salt Public Corporation developed a project to produce these cells on an industrial level to examine the possibility of using them as a raw material for cigarettes. After the stepwise increase of the size of culture vessel, first to 369 liters and subsequently to 1500 liters, and many technological improvements (Kato et al., 19761, the corporation built a pilot plant of a 20-kl culture tank, in which milder agitation was used to avoid mechanical damage to the cells, and the volumetric oxygen transfer coefficient was set at the rather lower value of 40-60 per hour in comparison to microorganisms. The possible contamination was prevented by using conventional polyvinyl alcohol filters in combination with membrane filters. During the successful operation of contin-

TOBACCO BY-2 CELL LINE

3

uous culture for 66 days, a specific growth rate ( P ) of ~ 0.044 per hour (average generation time, 15.8 hr) was obtained. There were practically no problems in culturing the TBY-2 cells by long-term continuous culture on an industrial level. However, because of cost performance of the operation and some problems with the quality of the products (e.g., proteinous smell), the trial was stopped (Kato, 1982).These trials showed that there is no limitation for mass culture. Subsequently, the production of ubiquinone 10 (coenzyme Qlo),a drug for heart diseases, from cloned cells of the TBY-2 cell line was tried successfully, producing 10-fold more ubiquinone 10 than did tobacco leaves as a natural source (Ikeda er al., 1978). However, as the production of this drug by Pseudomonas and Rhodotorula is much higher than that of plant cells, the plant cells were replaced with the microorganisms (Misawa and Samejima, 1978). Further bioengineering aspects of TBY-2 cells, which are outside the scope of this review and are not described here, were studied intensively at the Japan Tobacco and Salt Public Corporation. Detailed results have been discussed by Kato et al. (1976). The TBY-2 cell line is available from our laboratory for scientific use under the assumption that investigators will underwrite the agreement required by Japan Tobacco, Inc., where this cell line was initiated. However, this does not necessarily mean that any TBY-2 cell line shows the characteristics described in this chapter. We must stress that one of the authors (T.N.) received this cell line in 1980 and established a method of high synchrony in 1982 by the use of aphidicolin, but such a high synchrony can be attained only when this cell line is properly propagated. This is probably because, during maintenance in our laboratory, some selection of actively dividing cells could have been added.

111. Growth and Cytological Characteristics TBY-2 cells are propagated in the modified medium of Linsmaier and Skoog (1965), in which KH2P04and thiamine HCl are increased to 370 and 1 mglliter, respectively, and sucrose and 2,4-D are supplemented to 3% and 0.2 mg/liter, respectively (Nagata et al., 1981). Every week 1-1.25 ml of stationary phase cells are transferred to 95 ml of the fresh medium and cultured on a rotary shaker at 130 rpm at 27°C in the dark. Under this condition cells grow as shown in Fig. I , and after 1 week initial cells p =

1 dx

- -,

x dt

where x represents cell density and t represents time (in hours).

4

TOSHlYUKl NAGATA ET AL.

0

1

2

3

L

5

6

7

0

Time after transfer (days) Growth curve and mitotic indices of TBY-2 cells. Growth was monitored by counting the cell number per 1 ml at approximately 1-day intervals, and mitotic indices were determined at 12-hr intervals.

FIG. 1

multiply 80- to 100-fold. Such a high growth rate of plant cells has not been reported elsewhere, so far as we are aware. MIS of 5 4 % were observed 1-4 days after transfer. Such a high growth rate is partly dependent on the phosphate content of the medium, the optimum value being 510 mghiter, which is 3-fold that of the original Murashige and Skoog (1962) medium. However, this phosphate in the culture medium was consumed by the third day of culture, while other major constituents, such as sucrose, nitrate, and sulfate, were still at 37%, 54%, and 57% of the initial concentration, respectively (Kato et al., 1977). Thus, the consumption of phosphate by this cell line is distinctive. Furthermore, a preliminary study of 31P nuclear magnetic resonance (31P-NMR)disclosed even more interesting features. According to the results with 31P-NMR,the cytoplasmic phosphate was discriminated from the vacuolar one because of the pH difference of both compartments; the cytoplasmic and vacuolar pHs of TBY-2 cells were 7.5 and 5.7, respectively (T. Nagata, unpublished observations). In fact, "P-NMR showed

TOBACCO BY-2 CELL LINE

5

that when the stationary phase cells were transferred to the fresh medium, phosphate was rapidly accumulated in the cytoplasm and then was taken up into the vacuole, once the cytoplasmic phosphate pool was saturated. In the late log stage of culture, the accumulated phosphate in the vacuole was reutilized in the cytoplasm, as had been demonstrated in A. pseudoplatanus cultured cells by Rebeille er al. (1983), although the case of TBY-2 cells was more drastic. Apparently, the phosphate pool of TBY-2 cells was almost vacant at the stationary phase. During culture the vacuolated rod-shaped cells in the stationary phase became round cells enriched with cytoplasm, accompanied by an increase in specific gravity, when the cells are transferred to the fresh medium. The morphological changes in the organelles of plastids and mitochondria were most conspicuous when they were followed under an epifluorescence microscope after staining with the DNA-specific dye 4’6-diamidino-2phenylindole (DAPI). As shown in Fig. 2, a rapid increase in the fluorescence intensity of plastid nucleoids (DNA-protein complex) was observed soon after the stationary phase cells were transferred to the fresh medium, but after 2 days it decreased gradually. When the DNA content of plastid nucleoids was quantified with a supersensitive microspectrophotometer based on photon counting (Photonic Microscope System, Hamamatsu Photonics Co., Hamamatsu, Japan), it increased sharply after a lag of 6 hr and reached a maximum 28 hr after transfer, when it was approximately 13-fold the initial value (Fig. 3). To determine when plastid DNA synthesis occurred, autoradiography of the cells was performed after feeding [3H]thymidine. According to the labeling pattern of autoradiograms, cells were classified into four types. As shown in Fig. 4, in cells of the first and second types either the plastids (P+N-; Fig. 4A) or the nucleus (P-N+; Fig. 4C) was labeled, respectively. In the cells of the third type, both the plastids and the nucleus were labeled (P+N+;Fig. 4B), and in the last type neither the nucleus nor the plastids were labeled (P-N-; Fig. 4D).The proportions of these four labeling types of TBY-2 cells were followed during culture. The results (Table I) show that, during the first 24 hr of incubation, plastids were labeled in one-third of the cells (P+N-), while the nucleus alone was labeled (P-N+) in only 10% of the cells. The percentage of cells with labeled plastids (P+N- and P+N+)reached 83% on the first day and decreased to 12% on the second day (Table I). Thereafter, there was no labeling in the plastids, whereas, in contrast, the percentage of cells with labeled nucleus (P+N+ and P-N+) was 60% on the first and second days and then decreased gradually. The results of [3H]thymidine incorporation confirmed the preferential synthesis of plastid DNA during the first day of culture. Thereafter, plastid division was observed predominantly from the first to the second day of

FIG.2 Change in organelles in TBY-2 cells during culture. Cells harvested at day 0 (A and B), day 1 (C and D), day 2 ( E and F), and day 4 (G and H)after transfer to fresh media were treated with cellulolytic enzymes to remove cell walls and examined under a fluorescence microscope after staining with DAPI according to Kuroiwa ef a / . (1981). Left and right rows show fluorescence and phase-contrast micrographs, respectively, of the same fields. N , Nucleus; single arrowhead, plastid; double arrowheads, mitochondrion. Bar = 10 pm.

8

TOSHlYUKl NAGATA ET AL.

2

3

4

5

6

7

k x affer transfer bays) FIG. 3 Change in DNA content per plastid (pt) nucleoid (see Fig. 2) during culture of TBY-2 cells. After staining with DAPI, DNA content of pt nucleoids was determined, using the Photonic Microscope System (Hamamatsu Photonics Co., Hamamatsu, Japan) with T4 phage as a standard. The content is expressed as multiples of T4 phage, using an arbitrary unit, T. At each time point DNA contents of at least 50 pt nucleoids were counted. Vertical bars represent the standard error.

culture, and the peak of the number of plastids per cell reached maximum at the second day of culture (Fig. 5 ) . The total amount of plastid DNA per cell can be calculated from the number of nucleoids per plastid, the DNA content per plastid nucleoid (Fig. 3), and the number of plastids per cell (Fig. 5). This DNA content, expressed as multiples of TCphage DNA ( T ) , can be converted to copy number of plastid DNA as a factor of 1.18T (Yasuda et al., 1988). The results (Table 11) show that the copy number of plastid DNA per cell rose from 1000to 11,000by the first day, representing an 11-fold increase within 24 hr. From the second day this number decreased gradually, to 1000 copies per cell on the seventh day. Thus, plastid DNA was synthesized almost exclusively during the first day of culture, and this DNA was distributed to daughter cells through successive cell divisions.

FIG. 4 (A-D) Autoradiograms of four types of TBY-2 cells with different labeling patterns. Cells were fed [3H]thymidine at I-day intervals and labeled for 24 hr. The locations of the nuclei were determined under a fluorescence microscope after staining with DAPI. Cells were classified on autoradiograms into four types, according to their labeling pattern [note that labeled plastids showed clusters of silver grains (A and B)]: (A) only the plastids labeled (P+N-); (B) both the plastids and the nucleus labeled (P+N+);(C) only the nucleus labeled (P-N+); (D) neither the plastids nor the nucleus labeled (P-N-). Bar = 10 pm.

10

TOSHlYUKl NAGATA ET AL.

TABLE I Changes in the Proportion of Cells with Four Types of Labeling Patterns during Culture of TBY-2 Cells'

Incubation period (%) Labeling pattern P+NP+N+ P-N+ P-N-

0-1 days

1-2 days

2-3 days

3-4 days

4-5 days

5-6 days

33.6 49.9 10.8 5.7

5.2 7.0 52.9 34.9

0 0.6 28.3 71.1

0 0 14.5 85.5

0 0 8.4 91.6

0 0 0.7 99.3

6-7 days

0 0 0 100

Cells were incubated with [3H]thymidine for 24 hr at daily intervals, and autoradiograms were prepared. Cells on the autoradiograms were classified into four types (shown in Fig. 4). At least 500 cells were counted for each period.

Cannon et al. (1985) reported that the copy number of plastid DNA per photoautotrophic tobacco cell was 3-fold that of heterotrophic cells, but no such difference was found between similar cells of soybean (Cannon et al., 1986). This apparent discrepancy might have resulted from the incorrect assumption that plastid DNA per cell does not change much, but as Yasuda et al. (1988) showed, this is not the case. The question may be

100

.L 1

2

3

4

5

6

7

qme after transfer (days) FIG. 5 Change in the number of plastids per cell during culture of TBY-2 cells. Plastid numbers were determined under a fluorescence microscope equipped with phase-contrast optics, after staining with DAPI. Dumbbell-shaped plastids, suggesting plastid division, were frequently observed at 1-2 days of culture. For each point at least 200 cells were counted. Vertical bars represent the standard error.

11

TOBACCO BY-2 CELL LINE TABLE II Change in the Copy Number of Plastid DNA per Cell during Culture of TBY-2 Cellsa

Day of culture

T value per plastid nucleoid PIastid nucleoids per plastid Plastids per cell T value of plastid DNA per cell Plastid DNA copies per cell

0

I

2

3

4

5

6

2.8 6.6 46 850 lo00

39.7 3.9 60 9290 10,960

17.6 4.2 92.5 6840 8070

14.9 3.8 56 3170 3740

6.1 3.7 44 993 1170

3.1 5.1 53 838 989

3.2 5. I 54 881 1040

a The T values of plastid DNA per cell were calculated from the observed numbers of plastid nucleoids per plastid, the Tvalues of plastid nucleoids (Fig. 3), and the number of plastids per cell (Fig. 5 ) . These T values were converted to copy numbers of plastid DNA according to the equation copy number = 1.18T.

raised, then, as to whether TBY-2 cells are a peculiar example. A recent report by Takio and Nagata (1990) showed that, essentially according to the same method described above, plastid DNA was preferentially synthesized in the photomixotrophic cell culture of a moss, Barbula unguiculata, which had a higher growth rate, showed active photosynthesis under illumination, and retained well-developed chloroplasts (Takio and Nagata, 1990). From these results it can be said that the preferential synthesis of plastid DNA in TBY-2 cells has reflected faithfully the dynamics of the plastid DNA synthesis of plant cells. It is interesting that, even in the intact seedlings of wheat, the synthesis of plastid DNA is preceding that of chromosomal DNA (Miyamura et al., 1990). Mitochondria1 DNA of TBY-2 cells has been analyzed by Sato et al. (1991). Quantitative fluorescence microscopy of mitochondrial nucleoids stained with DAPI revealed that each mitochondria retained mitochondrial DNA in the size range of 120-200 kbp, while the deduction from the restriction enzyme analyses shows that the size is approximately 270 kbp, as shown in other papers (Sparks and Dale, 1980). This discrepancy suggests that each mitochondrion might not contain the whole sequence of mitochondrial DNA. On the other hand, the copy number dynamics of mitochondrial DNA do not keep pace with chromosomal DNA, and the synthesis of mitochondrial DNA localized between days 1 and 3 after transfer to the fresh medium (Sato et a / . , 1991). This information elucidates the replication dynamics of plant mitochondria. Thus, as a model material TBY-2 cells are suitable for studying the biochemistry and molecular biology of plant organelles.

12

TOSHlYUKl NAGATA ET AL.

IV. Studies of Subcellular Organelles To study the biochemistry and molecular biology of organelles, the isolation of such subcellular organelles is necessary, in intact condition if possible. It is not easy to isolate organelles from plant cells, as these cells are covered with thick cell walls. The preparation of protoplasts from TBY-2 cells, from which the isolation of organelles is easy, has been established, and a detailed procedure has been published (Nagata, 1987a,b), so only essential points are described here. Treatment of 3-dayold TBY-2 cells with 0.1% Pectolyase Y-23 and 1% Cellulase YC (both from Seishin Pharmaceutical Co., Nihonbashi-Koamicho, Tokyo) at 30°C for 50-60 min liberated protoplasts efficiently. The disruption of protoplasts suspended in lysis buffer [17% sucrose, 20 mM Tris-HC1 (pH 7.6), 0.5 mM EDTA, 1.2 mM spermidine, 7 mM mercaptoethanol, and 0.4 mM phenylmethylsulfonyl fluoride] can be easily accomplished by passage of the cells through 20-pm mesh placed in the apparatus, which was constructed to give a constant pressure of 0.5 kglcm’ to the filter to treat a large amount of material (approximately 1 liter) (Fig. 6). After removing the nuclei the plastid fraction was further purified on the stepwise sucrose density gradient. The plastids recovered from the interface of 40-60% sucrose retained intact plastid nucleoids under an epifluorescence microscope after staining with DAPI (Fig. 7A-C), and an electron-microscopic view showed the presence of matrix retaining higher electron opacity (Fig. 7D,E). When Nonidet P-40 was added to the plastid suspension (final concentration of 1%)and the suspension was stirred for 10 min, plastids were disrupted to release plastid nucleoids (Fig. 8).

N

FIG. 6 Schematic presentation of an apparatus to disrupt protoplasts suspended in a large volume. Intact organelles are recovered from the suspension of broken protoplasts (BPI. Released organelles can be purified by sucrose gradient centrifugation. AD, Auto-dispenser; FH, filter holder; IB, ice bath; IL, inlet tube; NM, nylon mesh; OL, outlet tube; OV, one-way valve; PG, pressure gauge; PP, suspension of protoplasts.

TOBACCO BY-2 CELL LINE

13

FIG. 7 Isolated proplastids. The same optical field of proplastids was observed under fluorescence and phase-contrast microscopes after staining with DAPI. (A) Fluorescence microscopy. (B) Fluorescence and phase-contrast microscopy. (C) Phase-contrast microscopy. (D and E) Isolated proplastids were observed under the electron microscope. Arrows show plastid nucleoids. Bars = 5 wm.

Biochemical study revealed that plastid nucleoid is the complex of plastid DNA and four species of proteins, whose molecular masses are 69, 31, 30, and 14 kDa, respectively (Nemoto et al., 1988). As these four proteins were liberated from the plastid nucleoids at different NaCl concentrations, their binding affinities should be different. It is also interesting that, when plastid DNA mixed with these four proteins was dialyzed, plastid nucleoids were reformed and looked very much like those freshly isolated from plastids under fluorescence microscopy. Since the released proteins from plastid nucleoids were different from those of chloroplast nucleoids, the true function of these proteins remains to be elucidated. Since the cytological study described in the previous section revealed that plastid DNA was synthesized preferentially, Takeda et al. (1992)tried to elucidate its replication mechanism. Copies of plastid DNA are multiple in plant cells and replicate autonomously, but their genomes are preserved

14

TOSHlYUKl NAGATA ET AL.

FIG. 8 (A) Isolated plastid nucleoids were observed under the fluorescence microscope after staining with DAPI. The same optical field was observed under (B)fluorescence and phasecontrast microscopes and (C) the phase-contrast microscope. (Dand E) The same specimens were observed under the electron microscope. Bars = 5 pm.

and succeed to descendant generations. It is intriguing to study the regulation mechanism of copy number dynamics of plastid DNA, as it is not known in higher plants. The first step to elucidate this mechanism should be to determine the replication origins of plastid DNA. There were reports by Kolodner and Tewari (1975) describing two replication origins located in the opposite strands, using the pea according to examination of the plastid DNA with electron microscopy. However, the D-loop strand observed by electron microscopy is not decisive, as it is very difficult to discriminate true replication origin from other possible artifacts (e.g., the denaturation loop, the R loop by transcription, and the displacement loop by recombination or replication). Other technical difficulties, such as length out of standard deviation of the expected value and binding of proteins to DNA, might accompany such artifacts. Thus, the replication origin of higher plant cells is an open question. On the other hand, the use of synchronously replicating plastid DNA was a key to determining the replication origin in Chlamydomonas reinhardtii (Waddel et al., 1984; Wang et al., 1984) and Euglena gracilis

TOBACCO BY-2 CELL LINE

15

(Koller and Delius, 1982; Ravel-Chapuis et al., 1982). Thus, the application of preferential replication of plastid DNA in TBY-2 cells after transfer to the fresh medium should be a candidate to determine the replication origin of plastid DNA. When protoplasts prepared from the stationary phase were transferred to the fresh medium, the preferential incorporation of [3H]thymidine into plastids was observed. As the replication origin could be recovered in the replication fork fraction, such fraction was concentrated by benzoylated naphthoylated diethylaminoethyl (DEAE) cellulose column chromatography after treatment with restriction endonucleases (Hay and DePamphilis, 1982).After the single-strand gaps and tails were filled with a Klenow fragment of Escherichia coli DNA polymerase I, newly synthesized DNA was labeled by the addition of [cx-~’P]~CTP according to the primer extension. When the plastid DNA was digested with several restriction endonucleases and hybridized with the labeled probes, the labels were observed only in specific sites, located in the 23 S rRNA region specified in the inverted repeats of the sequence data of tobacco plastid DNA (Shinozaki et al., 1986).On the other hand, the observation of plastid DNA by electron microscopy revealed D loops in this specific region, which coincided with the fraction labeled specifically in the replication fork fraction. Therefore, Takeda et NI. (1992) concluded that they could determine the replication origin of tobacco plastid DNA, the first such discovery in higher plants. It is worth mentioning that the replication origin of the plastid DNA of higher plants was determined by use of the TBY-2 cell line.

V. Synchronization of Cells The reproduction of cells is composed of the replication of genetic materials and the successive distribution of genetic materials as well as cell components to two daughter cells, and this recurrent progression of the cell cycle is a fundamental subject in cell biology. Although the first recognition of the cell cycle that comprises G I , S, G2, and M was proposed in the study of plant cells (Howard and Pelc, 1951), understanding of molecular events of the cell cycle in plant cells is far behind that of animal cells and that of Saccharnmytes cereuisiue (Baserga, 1985).Although the essential mechanism of the cell cycle should be common among eukaryotic cells, identification of the cdc-2 gene and the protein p34cd‘-2has recently been described in plant material (John et al., 1989; Feiler and Jacobs, 1990). This is due mainly to the lack of a suitable cell synchronization system in plant cells. The study of the cell cycle of plant cells is also intriguing, however, as

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there are several specific features of plant cells. Some specific features are the presence of the preprophase band (PPB), cortical microtubules (MTs), and phragmoplasts (Lloyd, 1987). The relationship between cortical MTs and the orientation of cellulose microfibrils is also intriguing. In considering these events we tried to establish high cell synchrony in plants, and finally TBY-2 cells were synchronized using aphidicolin and an MI of approximately 70-80% was recorded (Nagata et al., 1982). Aphidicolin is a drug purified from culture filtrates of a fungus, Cephalosporium aphidicolia, and is reported to be a specific inhibitor for DNA polymerase a (Ikegami et al., 1978). Before this work the highest cell synchrony was found in Haplopappus gracilis cell cultures, using hydroxyurea as described by Eriksson (1969, in which a 35% MI was recorded. Although the chromosome abberation was frequently observed by hydroxyurea treatment, such anomaly was rare in aphidicolin treatment. This is probably because hydroxyurea was used at the millimolar level and aphidicolin was used at the micromolar level. In this section we describe the basic protocol and some specific details on the synchronization method of TBY-2 cells. Although, for a review article, such a methodological description may be unusual, it is necessary, as one of us (T.N.) has been told rather often by other researchers that our protocol is not easy to reproduce. An important point is that the growth of TBY-2 cells should be fast; cells must multiply 70- to 100-fold in 1 week, as described in Section 111, which implies that this protocol could be applied to other plant cells, if they grow fast. Under this condition high synchrony should be attained easily. In fact, several laboratories, including that of H. Shibaoka of Osaka University, have successfully introduced this synchro-

;

o

o

l 75

Time after release [houid FIG.9 Change in the mitotic index ofTBY-2 cells after release from aphidicolin treatment. S. Gz, M , and G , represent the middle of these phases of the cell cycle.

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nization protocol to their subjects. Ten milliliters of stationary phase cells is transferred to 95 ml of the fresh medium that contains aphidicolin (5 mg/liter), which was dissolved in pure dimethyl sulfoxide at a concentration of 5 mg/ml and kept refrigerated until use. After a 24-hr incubation on the rotary shaker at 130 rpm at 27"C, the drug was removed from the culture medium by washing with 1 liter of the fresh medium. Upon washing the cell suspension was poured into a cylindrical funnel with a fused-in fritted glass filter (17 G2 Hario glass, Tokyo), in which the flow rate of the washing medium was controlled with a Hoffman clamp placed on a silicon tube connected to the bottom of the funnel. The whole system could be easily sterilized by autoclaving. A total washing time of approximately 15 min was suitable for obtaining higher synchrony, and the cells were subsequently resuspended in the same volume of the fresh medium. When we followed the change in MI, which was determined under a microscope after staining with lactopropionic orcein according to Eriksson (1965) or with DAPI according to Yasuda et al. (1988), the first and second peaks were located 10 and 23 hr, respectively, after the release from aphidicolin, which implies that one generation time was estimated to be approximately 13 hr. The duration of cells in the M phase was calculated to be 2 hr from the first peak (Fig. 9). The length of the S phase was determined by autoradiography and by microspectrophotometry, using propidium iodide. Figure 10 shows that the percentage of tritium-labeled nuclei increased immediately after the release from aphidicolin, and essen-

"6

3

6

9

!

firm after release lhows) FIG. 10 [3H]Thymidine incorporation into TBY-2 cells after release from aphidicolin treatment. Cells were incubated for 20 min with [3H]thymidine at the indicated times, and the percentage of cells with labeled nuclei was determined by autoradiography. The locations of the nuclei were determined by fluorescence microscopy after staining with DAPI.

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TOSHlYUKl NAGATA ET AL.

4°C %

E

c

5u

i3 2°C

0

3

6

9

12

Time after release (hours) FIG. 11 Change in the DNA content of TBY-2 cells after release from aphidicolin treatment. DNA content was determined by quantitative fluorescence microscopy after staining with propidium iodide. The 2°C and 4°C levels were calculated as the average of the values of 500 cells.

tially all cells incorporated [3H]thymidine into their nuclei during the subsequent 5-hr incubation. Fluorescence microspectroscopy after staining with propidium iodide also showed that the DNA content per cell doubled during the first 5-hr incubation (Fig. 11). Thus, the length of the S phase was estimated to be 5 hr. As seen in Fig. 9, G2 and G I are estimated to be 4 and 2.5 hr, respectively. This generation time of 13 hr is the shortest reported for plant cell suspension cultures (Gould, 1984), but this value could become much shorter, as the growth speed of TBY-2 cells is still gradually increasing. Although the cell synchrony of TBY-2 cells by aphidicolin treatment is exceptionally high, it is still insufficient, especially for the transition from the M phase to the G, phase of the cell cycle, as the extent of synchrony is gradually decreasing from the moment of release from aphidicolin treatment. To get much higher synchrony, the synchronized TBY-2 cells obtained after aphidicolin treatment were further treated with propyzamide, an antitubulin drug (Akashi et a / . , 1988), to induce mitotic arrest (Kakimot0 and Shibaoka, 1988). Propyzamide (1.6 mg/liter) was added to TBY-2 cells 6 hr after the release from aphidicolin treatment. When propyzamide was removed from the medium after 4 hr of treatment, mitotic arrest was released, the progression of the cell cycle restarted, and nearly 80-90% MI was observed soon afterward. As the effect of this drug was almost reversible, which is in sharp contrast to colchicine, the cell cycle progression was immediately recovered.

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VI. Changes in Cytoskeletons during the Cell Cycle Progression By using this highly synchronized cell popoulation, Hasezawa er al. (I991) and Hasezawa and Nagata (1991) followed characteristic features, along with the progression of the cell cycle of plant cells. Among other aspects they focused on the change in the cytoskeletons, as the orientation of cellulose microfibrils, which are main components of cell walls, are believed to be determined by MTs (Newcomb, 1969).Furthermore, as shown in stomata1 guard mother cell differentiation, the unequal cell division by septum formation initiates the direction of differentiation. As the septum formation is processed by a subcellular structure of phragmoplast, which is a complex of MTs, microfilaments (MFs), and some other components, it may be said that cytoskeletons determine the direction of differentiation of plant cells. Although previous works have clarified to some extent the role of cytoskeletons during the cell cycle (Lloyd, 1987), various important problems remain to be resolved, especially relating to the transitions between different cell cycle stages. Meanwhile, the substantial progress of the staining methods of cytoskeletons, especially of MFs, allowed observation of the detailed changes in the cytoskeletons. Although MFs were very labile to conventional fixative treatment (e.g., glutaraldehyde) and could not be seen under a fluorescence microscope, this difficulty was circumvented with the pretreatment of specimens with a buffer containing tropomyosin and rn-maleimidobenzoyl N-hydroxysuccinimide ester, a protein-stabilizing agent (Kakimoto and Shibaoka, 1987; Sonobe and Shibaoka, 1989). Thus, the triple staining with indirect fluorescent antibody to MTs, rhodamine-labeled phalloidin to MFs, and DAPI to chromosomes revealed the interrerlationship among these structures. Soon after the release from aphidicolin treatment, when the S phase began, the cytoplasmic MTs extending from perinuclear regions that had not been observed at G I reached cell membranes and were clearly observed, and the MTs were mostly overlapped with MFs (Fig. 12A and B). Accompanying this cytoskeletal change, the nucleus moved nearly to the center of the cells (Fig. 12C) and its position was supported by cytoplasmic strands consisting of MTs and MFs. At the G2 phase the formation of the PPB was most conspicuous. The PPB was connected to perinuclear MTs and partly overlapped with MFs, whose location was broader than that of the MTs (Fig. 12D-F). When a compact girdle-shaped PPB was observed, the structures, in which spindles were apparently organizing, were observed at the two pole positions. The spindle seemed to be produced at the expense of the PPB. By the time

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of mitosis, the cortical MTs disappeared completely and the location of MTs coincided with a spindle, while MFs seemed to surround the spindle and were not observed inside it (Fig. 12G-I). Toward the end of mitosis, a phragmoplast is formed, a specific structure in plant cells which is responsible for cell plate formation. MFs as well as MTs were major components of the phragmoplast (Fig. 12J-L). During the transition between cytokinesis and the G I phase, characterized as the presence of disintegrating phragmoplasts, short MTs were transiently observed on the surface of a nucleus (Fig. 12M, N, and Q), and then the MTs developed toward the direction of cell plates and elongated along the inner surface of the cell membrane to the distal end from the location of the forming cell plate. As a consequence a gradient of the distribution of MTs was observed transiently. In the G I phase no MTs were observed in the perinuclear region (Fig. 12P), and most of MTs were observed as more or less ordered cortical MTs (Hasezawa and Nagata, 1991). Thus, Hasezawa et al. (1991) and Hasezawa and Nagata (1991) followed the sequence of change in the cytoskeletons during TBY-2 cell cycle progression. This search revealed several novel features which had not been previously observed. First, the decisive moment was observed at which cortical MTs originate from the perinuclear surface. However, the passage through this moment was extremely short, and the origin of the MTs at the perinuclear surface was observed as a rare event, even after synchrony by aphidicolin treatment. In fact, as the landmark of the transition of the border of M / G I , they observed the coexistence of disintegrating phragmoplast and short perinuclear MTs (Fig. 12M) at a frequency of 0.08% (?0.03%) in the total population. To avoid certain ambiguities, the sequential synchronization method of aphidicolin and propyzamide was introduced to induce higher synchrony during progression from the M phase to the G I phase (described in Section V). Three hours after the release from propyzamide treatment, the frequency of the coexistence of disintegrating phragmoplast and short perinuclear MTs increased to 1.2% (?0.2%), while at this stage 52.2% (? 1.12%) of cells were located at the border of M/GI. If the total passage through

FIG. 12 The changes in microtubules (MTs) and microfilaments (MFs) during progression of the synchronized TBY-2 cell cycle with aphidicolin. The changes in MTs, MFs, and chromosomes were followed by a triple staining with fluorescent antibodies against MTs, rhodamine phalloidin, and DAPI, respectively. (A-C) S phase; (D-F) G2 phase; (G-I) M phase; (J-L) late M phase; (M-0) M E I ; (P-R) G I . (A, D, G, J, M, and P) Fluorescent antibodies against MTs; (B, E, H , K , N , and Q ) rhodamine phalloidin staining; (C, F, I, L, 0, and R) DAPI staining. A preprophase band is clearly seen in (D). A phragmoplast is clearly seen in (J). An arrow in (M)shows a disintegrating phragmoplast. while short MTs are observed in the perinuclear region of two daughter cells. Bars = 20 pm.

FIG. 12 (continued)

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M/GI is assumed to be approximately 1 hr, the passage through the right moment of the coexistence of disintegrating phragmoplast and short perinuclear MTs is only 2-3 min. It is important that even such a rare event could be interpreted according to the sequential synchronization method. Thus far, there have been two suppositions on the origin of cortical MTs. Earlier, Gunning’s (1980) school proposed, from electron-microscopic surveys, that the nucleating sites for MT arrays are located along cell edges. More recently, Clayton et (11. (1985) proposed, after staining with an anticentrosomal immune serum 5051 from human cells, that the nuclear envelope serves as the initiation site for the earliest MTs and subsequently ordered cortical arrays are formed. However, there was also an argument against the latter statement that the autoimmune antibody did not show any specificity (Wick, 1989). Thus, the significance of the observation by Hasezawa and Nagata (1991) is that they showed directly that short MTs initiate at the nuclear envelope and develop somewhat later to cortical MTs, but the short MTs in the perinuclear region were transiently observed and were not observed in the later stage. Since MTs have been shown to develop from the perinuclear region during the formation of PPB, a spindle, and a phragmoplast (Lloyd, 1987; Hasezawa et al., 1991; Katsuta et al., 1990), it can be generalized that there are MT organization centers on the perinuclear regions. However, this does not exclude the possibility that MTs are organized elsewhere than in the perinuclear region. Second, the phragmoplast has been characterized from the synchronized TBY-2 cells by the sequential synchronization method. Although the phragmoplast is from previous morphological studies (Lloyd, 1987), considered a plant-specific structure, its function remains to be elucidated. To study its function, it is desirable that the phragmoplast be isolated from the cells and be analyzed biochemically as well as structurally. However, as the phragmoplast forms only briefly (approximately 20-30 min) during each cell cycle, it was impossible to separate this structure according to the traditional synchronization methods. Only the highly synchronized cell system could realize this requirement. When the synchronized cells, with the sequential treatment with aphidiColin and propyzamide, were treated appropriately with cellulolytic enzymes (as described in Section IV), the protoplasts at the highest MI were obtained (Kakimoto and Shibaoka, 1988). When the enzyme treatment is started 30 min before termination of the propyzamide treatment, protoplasts are prepared 30 min after the release from this treatment. As these protoplasts formed phragmoplast synchronously, the simple disruption of the protoplasts gave phragmoplast-rich fractions, which could now serve for biochemical analysis. In fact, this fraction showed a highly intact phragmoplast under electron microscopy. The incubation of this fraction

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with [3H] UDP-glucose resulted in incorporation of the radioactivity into the central region of phragmoplasts, while [3H] UDP-xylose was incorporated into Golgi vesicles during electron-microscopic autoradiography (H. Shibaoka, personal communication). On the other hand, Asada et al. (1991) treated the protoplasts, which had been synchronized by the sequential synchronization procedure, with glycerin. Although the glycerinated model cells lost cell cycle progression, the phragmoplast could translocate MTs toward the minus ends concomitantly, with tubulin polymerization at the plus ends located in the equatorial region. The translocation could be visualized by fluorescence microscopy, when the cells were treated with tubulins labeled with dichlorotriazonil aminofluorescein. The translocation was induced effectively by GTP, but less effectively by ATP. Thus, it has been shown that the equatorial region of phragmoplasts seems to be associated with a mechanochemical enzyme that generates the force for MT translocation by hydrolyzing GTP. Thus, the study of the isolated phragmoplasts could contribute to understanding the biochemical and molecular features of plant phragmoplasts. Another characteristic of the cell cycle events of the plant cell is the migration of the nucleus from the periphery to the center of the cell, while in the GI phase the cell nucleus is located in the periphery. This migration has been examined in details by Katsuta et al. (1990), using the TBY-2 synchronized system. Although the nucleus was tethered with cytoplasmic strands consisting of both MTs and MFs, this migration was insensitive to cytochalasin D, an anti-MF drug, but sensitive to propyzamide, an anti-MT drug (Akashi et al., 1988), which suggests that the role of MTs is more important than that of MFs in this migration. Furthermore, the disruption of MTs by propyzamide prevented the formation of PPB-like MFs as well as PPB, while the disruption of MFs by cytochalasin D did not prevent the formation of PPB. Thus, the migration of the nucleus in the premitotic stage is primarily responsible for MTs, which could play a role such as a scaffold, and along with this structure the network of MFs is formed. On the other hand, in later stages, the MFs support the mitotic spindle as well as phragmoplasts, when the cytoplasmic MTs disappeared completely. Similarly, Katsuta and Shibaoka (1988) observed that when protoplasts from TBY-2 cells were cultured in the elongation medium as described by Hasezawa and Syono (1983), the nucleus became located in the central position of the cell and was supported only by MFs. In these cells the disruption of MFs by cytochalasin D brought about the translocation of the nucleus to the cell periphery. As the cells in this culture condition can be interpreted as G I phase in the cell cycle, the migration of the nucleus in GI phase should be controlled by MFs.

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Although thus far we have seen drastic structural changes in cytoskeletons during the cell cycle progression of TBY-2 cells, a subsequent intriguing question should be what is the control mechanism of such drastic change in the cytoskeletons. According to the accumulated information from studies using animal cells as well as yeast, phosphorylation plays an important role during cell cycle progression. The level of phosphorylation should be controlled by protein kinases as well as protein phosphases. Although such information is poor in plant cells, some drugs (e.g., okadaic acid and calyculin A) could be useful for such analysis; both of these drugs have been shown to be specific inhibitors for protein phosphatases I and IIa (Haystead et al., 1989; Kipreros and Wang, 1990). In fact, the addition of these drugs to each cell cycle stage of the synchronized TBY-2 cells blocked the progression of each cell cycle stage to the next. Two noticeable changes were observed: (1) the treatment of G 2cells with these drugs did not form a PPB at all, and (2) upon the addition of one of these drugs at G2/M, the progression of mitosis was accelerated significantly. Furthermore, progression of the cell cycle was severely blocked at the border of MIGl (S. Hasezawa, S. Kawasaki, and T. Nagata, unpublished observations). Although this observation is still indirect, phosphorylation seems to play an important role during the cell cycle progression of plant cells as well, and the drastic spatial changes of MTs during the cell cycle as a plant-specific phenomenon have been regulated by phosphorylation.

VII. Cytological Investigation of Cell Elongation Although elongation growth is a plant-specific phenomenon and there is a mass of literature at the tissue and organ levels (Wareing and Phillips, 1981), there has been a paucity of information on the cellular level. When protoplasts prepared from TBY-2 cells were cultured in the FMS medium (Hasezawa and Syono, 1983)supplemented with I-naphthalene acetic acid (0.1 mglliter) and 6-benzyl aminopurine (1 mglliter), conspicuous elongation of TBY-2 cells was observed, while in the medium supplemented with 0.2 mglliter of 2,4-D, active division was observed. In TBY-2 cells elongation growth can be separated from division growth by changing combinations of plant hormones. Thus, the regulation of the cell elongation by a combination of auxin and cytokinin is intriguing for analyzing the elongation mechanism of plant cells under this condition. Elongation of cultured protoplasts started from 2 days of culture, and after 1 week reached nearly 400 pm. When the orientation and development of MTs and MFs were

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followed in cultured protoplasts, MTs and MFs were oriented in random directions just after the culture, then became perpendicular to the long axis after 2 days of culture. Thus, the direction of orientation of MTs and MFs went parallel with the elongation of cells (Hasezawa et al., 1988,19891,but the causal relationship between cell elongation and the orientation of MTs remain to be determined.

VIII. Gene Delivery into Cells

Delivery of genes into the protoplasts from TBY-2 cells mediated by liposomes has been introduced only briefly and has been described previously in detail by one of us (Nagata, 1987a,b). Liposomes of phosphatidylserine and cholesterol encapsulating tobacco mosaic virus (TMV) RNA were prepared essentially according to the reversed-phase evaporation vesicle method (Szoka and Papahadjopoulos, 1978)and were mixed with TBY-2 protoplasts. Subsequently, 12.5% polyethylene glycol (PEG) 1540 or 10% polyvinyl alcohol (PVA) was added to the mixture of liposomes and protoplasts. After an incubation of 10 min (PEG) or 15 min (PVA) at room temperature, the PEG or PVA was removed by washing with a solution of 0.05 M glycine buffer (pH 10.5), 0.05 M CaC12,and 0.4 M mannitol. The protoplasts were then cultured for 24 hr in the modified Linsmaier and Skoog (1965) medium (described in Section 111), supplemented with 0.4 M mannitol and 1% sucrose. In this system the delivery of intact and functional TMV RNA could be assessed by staining with a fluorescent antibody raised against TMV. Under optimal conditions the functional RNA was introduced to 80% of the protoplasts. It should be noted that liposomes were taken up endocytotically by protoplasts by electron microscopy, although delivery has been performed during treatment, using a combination of PEG and PVA with high pH-high calcium buffer. The treatment of protoplasts in this way was originally developed for the fusion of protoplasts (Nagata, 1987a,b). Electroporation of genes into protoplasts from TBY-2 cells has been successfully performed, but, since details have been described by one of us (Nagata, 1989) in a previous volume in this series, only essential points are described here. After protoplasts suspended in a medium consisting of 5 mM 2-(N-morpholino)ethanesulfonicacid buffer (pH 5 . 8 ) , 70 mM KCl, and 0.4 M mannitol were mixed with DNA or RNA, an electric impulse was released from a condensor (100 FF), to which electricity had been stored from a 300 V power supply. Upon a simple impulse genes were introduced to inore than 90% of the protoplasts. Delivery into protoplasts of four cell cycle stages from highly synchronized TBY-2 cells was also

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feasible (Nagata et al., 1987). Stable transformants were obtained according to the procedure described above, when neomycin phosphotransferase I1 was used as a selectable marker (Okada e f al., 1986). The gene delivery into TBY-2 cells could be performed by aid of Agrobacterium tumefaciens at high frequency. An (1985) showed that when the tobacco NT-I cell line, a sibling of TBY-2, was cocultivated simply with A . tumefuciens, harboring a binary vector of pGA473 and pTi Bo542, kanamycin-resistant clones were obtained maximally at a frequency of 50%. pGA473 retains a kanamycin-resistant neomycin phosphotransferase I1 gene as a selectable marker in plants. This frequency was dependent on the culture stage and was highest after 3-4 days of subculture. Concurrently, the induction of VirD, VirB, and VirE genes in the Ti plasmid were observed maximally 1, 2, and 2-3 days after transfer, respectively. According to recent understanding of the transformation mechanism of plant cells by the Ti plasmid, first Vir genes are induced by the plant exudates; subsequently, by the action of Vir genes, the T strand will be excised from the Ti plasmid. The T strand, combined with DNA-binding proteins, is transferred to the plant cells and integrated into plant genomes by recombination (Zambryski et al., 1989). This story is one of the most attractive in the recent development of plant molecular biology. During the elucidation of this mechanism, tobacco NT-I (TBY-2) played an important role. As cited by An (1985), Vir genes were induced by cocultivation with tobacco cells. At the time of cocultivation Stachel et al. (1986) noticed that a low-molecular-weight heat-stable plant metabolite exudated from plant cells into the culture medium and induced the expression of Vir genes. This metabolite was soon identified as acetosyringone and related phenolics (Stachel et al., 1985; Bolton et al., 1986).

IX. Supplementary Remarks As we have described, TBY-2 cells grow fast and can be synchronized to a high degree, and thus are suitable for studying the cellular and molecular biologies of plant cells. Such a cell line is highly necessary, as information on plant cells at the cellular level is still much less than that on other eukaryotes of animal cells and yeast. Another important point is that molecular and subcellular studies are easily feasible, as mass culture of TBY-2 cells is readily available. However, there is a laborious point, as this material must be continuously cultured in a proper condition to obtain a high growth rate and high synchrony. This is due to the fact that practical freeze preservation method has not been developed for plant cells. Conse-

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quently, this material could accumulate possible point mutations during long-term culture, and so it may not be appropriate for study of the genome structure of plant cells. Thus far, the generation of whole plants from this material has not been found and although one of us (T.N.) is still trying to develop such a method, such efforts seem to be in vain. This problem could be overcome by fusion with other cells retaining regeneration ability, as Maliga et al. (1977) have shown. Even if we consider such drawbacks of this cell line, there is no doubt that this material is most suitable for basic study of the cellular and molecular biologies of plant cells. This material could probably be combined with Arabidopsis rhaliana, which has been shown to be suitable for genetic study, as this material has small genome (Meyerowitz and Pruitt, 1985). Finally, we can supply this material to anyone who would like to use it to increase understanding of the basic characteristics of plant cells.

X. Summary

TBY-2 derived from the seedlings of N . tabacum L. cv. Bright Yellow 2 grows fast and multiplies 80- to 100-fold in 1 week. After the stationary phase cells of TBY-2 were transferred to a medium containing aphidicolin for 24 hr and then released from treatment, high synchrony was obtained starting from the S phase. The subsequent arrest of cells at metaphase with propizamide and the release from this treatment offered higher synchrony, starting from the M phase. Using this synchrony system, the change in the cell cycle progression of TBY-2 cells successfully followed the change in cytoskeletons. Another important point is that one could do biochemical and molecular biological studies on this material, since mass culture of this material is readily feasible, although, so far, cell cycle events have been studies of only the morphological aspects. Such an example is the biochemical study of a phragmoplast, a plant-specific structure, which has not been separated by conventional methods. Acknowledgments We would like to express our deepest thanks to Takashi Matsumoto, Japan Tabacco, Inc., who ailowed one of us (T.N.) to use this cell line initially, and Nobutaka Takahashi, University of Tokyo, who made great efforts to help us with the purchase of this cell line. Thanks are also due to Hiroh Shibaoka of Osaka University, who allowed us to see several unpublished articles by his group and gave us helpful comments in preparing this manuscript. This research was supported in part by grants from both the Ministry of Education, Science and Culture of Japan and the Ministry of Agriculture, Forestry and Fisheries of Japan (to T . N . ) .

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References Akashi. T., Izumi, K., Nagano. E . , Enomoto, M., Mizuno, K., and Shibaoka, H. (1988). Plant Cell Physiol. 29, 1053-1062. An, G. (1985). Plant Physiol. 79,568-570. Asada, T., Sonobe, S . . and Shibaoka. H. (1991). Nature (London)350, 238-241. Baserga, R. (1985). "The Biology of Cell Reproduction." Harvard Univ. Press, Cambridge, Massachusetts. Bolton, G. W., Nester. E. W., and Gordon, M. P. (1986). Science 232,983-985. Cannon, G . , Heinhorst, G., Siedlecki, J., and Weissbach, A. (1985).Plant CellRep. 4,41-45. Cannon, G . , Heinhorst, S. , and Weissbach, A. (1986). Plant Physiol. 80,601-603. Clayton, L., Black, C. M., and Lloyd. C . W. (1985). J. Cell Biol. 101, 319-324. Eriksson, T. (1985). Physiol. Plant. 18,976-993. Feiler, H. S., and Jacobs, T. W. (1990). Proc. Natl. Acad. Sci. U . S . A . 87,5397-5401. Filner, P. (1965). Exp. Cell Res. 39,33-39. Gamborg, 0. L. (1970). Plant Physiol. 45, 372-375. Gould, A. R. (1984). CRC Crit. Reu. Plant Sci. 4, 315-344. Gunning, B. E. S. (1980). Eur. J . CellBiol. 23, 53-65. Hasezawa, S . , and Nagata. T. (1991). Bot. Aria 104, 206-211. Hasezawa. S . , and Syono, K. (1983). Plant Cell Physiol. 24, 127-132. Hasezawa, S . , Hogetsu, T., and Syono. K. (1988). J . Plant Physiol. 133,46-51. Hasezawa. S.. Hogetsu. T.. and Syono, K. (1989).J. Plant Physiol. 134, 115-119. Hasezawa, S . . Marc, J.. and Palevitz, B. A. (1991). Cell Motil. Cytoskeleton 18,94-106. Hay, R. T., and DePamphilis, L. (1982). Cell28,767-779. Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989). Nature (London)337,78-81. Howard, A., and Pelc. S. R. (1951). Exp. CellRes. 2, 178-187. Ikeda, T., Matsumoto, T.. and Noguchi, M. (1978). Agric. B i d . Chem. 41, 1197-1201. Ikegami, S . , Taguchi, T., Ohashi, M., Oguro. M., Nagano, H., and Mano, Y. (1978). Nature (London)275,458-460. John, P. C. L., Sek, F. J., and Lee, M. G. (1989). Plant Cell2, 1185-1193. Kakimoto, T.. and Shibaoka. H. (1987). Protoplasm 140, 151-156. Kakimoto. T., and Shibaoka, H. (1988). Protoplasma, Suppl. 2,95-103. Kato, A. (1982). J . Ferment. Technol. 60, 108-118. Kato, A., Kawazoe, S., Iijima, M., and Shimizu, Y.(1976). J . Ferment. Techno/.54,82-87. Kato, A., Fukasawa, A., Shimizu. Y . , Soh, Y.. and Nagai, S. (1977).J. Ferment. Technol. 55,207-212. Kato, K., Matsumoto, T., Koiwai, A., Mizusaki, S. , Nishida, K., Noguchi, M., and Tamaki, E. (1972). In "Fermentation Technology Today" (G. Terui, ed.), pp. 689-695. SOC. Ferment. Technol., Osaka. Japan. Katsuta, J., and Shibaoka, H. (1988). Plant Cell Physiol. 29,403-413. Katsuta, J., Hashiguchi, Y., and Shibaoka, H. (1990). J . Cell Sci. 95,413-422. Kipreros. E. T., and Wang, J . Y. J. (1990).Science 248, 217-220. Koller, B., and Delius, H. (1982). EMBO J . 1,995-998. Kolodner, R. D., and Tewari, K. K. (1975). J. Biol. Chem. 250,8840-8847. Kuroiwa, T., Suzuki, T., Ogawa, K., and Kawano, S. (1981). Plant CellPhysiol. 22,381-396. Linsmaier, E. M., and Skoog, F. (1965). Physiol. Planr. 18, 100-127. Lloyd, C. W. (1987). Annu. Rev. Plant Physiol. 38, 119-139. Maliga, P., Lazar, G., Joo, F., and Menczel. L. (1977). Mol. Gen. Genet. 157,291-296. Meyerowitz, E. M., and Pruitt, R. E. (1985). Science 229, 1214-1218. Misawa, M., and Samejima, H. (1978). In "Frontiers of Plant Tissue Culture 1978" (T. A. Thorpe, ed.), pp. 353-362. Univ. of Calgary, Calgary, Alberta, Canada.

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Miyamura. S., Kuroiwa, T.. and Nagata. T. (1990). Plant Cell Physiol. 31,597-602. Murashige, T., and Skoog, F. (1962). Physiol. Plant. 15,473-497. Nagata, T. (1987a).In “Methods in Enzymology” (L. Packer and R. Douce. eds.), Vol. 148, pp. 34-39. Academic Press, Orlando, Florida. Nagata, T. (1987b). I n “Methods in Enzymology” (R. Green and K. J. Widder, eds.), Vol. 149, pp. 176-184. Academic Press, Orlando, Florida. Nagata, T. (1989). I n / . Rev. Cytol. 116,229-255. Nagata, T., Okada, K., Takebe, I., and Matsui, C. (1981). Mol. Gen. Genet. 184, 161-165. Nagata, T., Okada, K., and Takebe, I. (1982). Plant Cell Rep. 1,250-252. Nagata. T., Okada, K., Kawadu. T., and Takebe, I . (1987). Mol. Gen. Genef.207,242-244. Nemoto, Y., Kawano, S., Nakamura, S. , Mita, T., Nagata, T., and Kuroiwa, T. (1988). Plant Cell Physiol. 29, 167-177. Newcomb, E. H. (1969). Annu. Reu. Plant Physiol. 20,253-288. Okada, K., Takebe, I., and Nagata, T. (1986). Mol. Gen. Genet. 205, 398-403. Ravel-Chapuis. P., Heizrnann, P., and Nigon, V. (1982). Nature (London)300, 78-79. Rebeille, F . , Bligny, R., Martin, J.-B., and Douce, R. (1983). Arch. Biuchern. Biophys. 225, 143- 148. Sato, M., Nemoto, Y., Kawano, S . , Nagata, T., and Kuriowa, T. (1991). Planta (in press). Shinozaki, K., Ohme, M., Tanaka. M.. Wakasugi. T., Hayashida, N.. Matsubayashi, T.. Zaita. N., Chunwongse, J.. Obokata. J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa. K., Meng, B. Y.,Sugita, M., Deno, H., Kamogashira, T., Yamada, K., Kusuda, J . , Takaiwa, F., Kato, A., Tohdoh, Shimada, H., and Suguira, M. (1986). EMBO J . 5, 2043-2049. Simpkins, I., Collin, H. A., and Street, H. E. (1970). Physiol. Plant. 23, 385-396. Sonobe, S., and Shibaoka, H . (1989). Protoplasma 148,SO-86. Sparks, R . B., Jr., and Dale, R. M. K. (1980). Mol. Gen. Genet. 180,351-355. Stachel, S . E., Messens, E., Van Montagu, M., and Zambryski, P. (1985). Nature (London) 318,624-629. Stachel, S . , Nester, E. W., and Zambryski. P. C. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 379-383. Szoka, F., Jr., and Papahadjopoulos, S . (1978).Proc. Natl. Acad. Sci. U.S.A.75,4194-4198. Takeda, Y., Hirokawa, H., and Nagata, T. (1992). Submitted. Takio, S., and Nagata, T. (1990). J . Plant Physiul. 137, 147-151. Waddel, J . , Wang, X. M., and Wu, M. (1984). Nucleic Acids Res. 12, 3843-3856. Wang, X. M., Chang, C. H., Waddel, J.. and Wu, M. (1984). Nucleic Acids Res. It, 3857-3865. Wareing, P. F., and Phillips, I. D. J. (1981). “The Control of Growth and Differentiation in Plants,” 2nd ed. Pergarnon, London. Wick, S. M. (1989). Cell Biol. In?. Rep. 9, 357-371. Willmer, E. N. (1965). “Cells and Tissues in Culture.” Academic Press, New York. Yasuda, T., Kuroiwa, T., and Nagata, T. (1988). Planta 174,235-241. Zambryski, P., Tempe, J., and Schell, J. (1989). Cell 56, 193-201.

Cells in the Marginal Zone of the Spleen Georg Kraal Department of Cell Biology, Free University, 1085 BT Amsterdam, The Netherlands

1. Introduction The spleen functions as an elaborate filter of the blood stream, through which particles such as bacteria and dead blood cells can selectively be removed based on the complex interplay between the blood stream and phagocytizing cells. At the macroscopic level the spleen can be divided into a red pulp compartment, distinguishable by the abundance of erythrocytes, and a white pulp compartment, consisting of large nodules of organized lymphoid tissue. It is in the red pulp that blood filtration takes place, whereas the white pulp is involved in the specific immunological defense against bloodborne antigens. The complexity of the structure of the spleen is directly related to the complexity of the vascularization of this organ. The splenic artery, which initially enters via the hilus, branches into smaller arteries which pass along the trabeculae of the spleen. After continuous branching small arterial vessels leave the trabeculae as so-called central arteries. They then become encompassed by a sheath of lymphoid tissue, the periarteriolar lymphocyte sheath (PALS). From the central artery small branches form the capillary meshwork of this white pulp area. Other branches traverse the white pulp into the red pulp, where they open as terminal arterioles. Here, the blood follows preferred channels which are lined not by endothelium, but by cytoplasmic processes of reticular cells, and is eventually collected in venous sinuses. Due to this open circulation, the blood comes into contact with the numerous macrophages of the red pulp and can effectively be filtered (Snook, 1950; MacPherson er al., 1973; Weiss, 1977). In addition, a closed circulation has been described with direct connections to venous sinuses. This closed circulation has been debated over the years, starting with the opposing findings by Knisely (1936) and by Mackenzie et al. (1941). More recently, using scanning electron microscopy combined with refined casting procedures, evidence for direct arInternariond Reuien, r!f'Cytolog,'. Vol. 132

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Copyright 0 1992 hy Academic Press, Inc. All nghts of reproduction in any form reserved.

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teriovenous connections has been presented (Kashimura and Fujita, 1987; Schmidt et al., 1988). Another part of the terminal arterioles directly ends in the marginal zone, either in open sinuses, also consisting of reticular cells, or directly between the reticulum cells of this area. There is increasing evidence that the spleen plays a rather unique role in the immunological defense particularly against bacterial antigens. Coincidentally, the spleen contains an unmatched area, not seen in any other lymphoid organ, right at the border of the PALS and the red pulp. In this marginal zone part of the arterial blood stream opens into sinuses and it is here that antigens come into contact with a set of conspicuous cell types with peculiar features. Here, an overview is given of the various nonlymphoid and lymphoid cell types localized in the marginal zone and the way they are arranged in the basic structure of this compartment. Based on the many data that exist on the function of these cells, the role of the marginal zone is discussed with special attention to the intriguing ways these cell types interact with each other in the defense against invading pathogens.

II. Marginal Zone

The marginal zone is generally described as a layer surrounding the PALS and B cell follicles, predominantly composed of intermediate-sized lymphocytes. Its presence was described as early as 1929 by MacNeal, who recognized its importance in phagocytosis. Gradually, the importance of this area as a functional entity in the spleen became appreciated, but not always fast enough as Milliken (1969) complained, “Although the perifollicular envelope has been described many times in both man and animals it is still not generally well enough known to be included in most histology textbooks.” In addition to the perifollicular envelope, the marginal zone has been described under a variety of names, such as “border zone” and “white pulp halo” (MacPherson et al., 1973). The most conspicuous anatomical feature of the marginal zone is the presence of a sinus in which part of the arterial blood stream opens. The marginal sinus consists of a series of anastomosing vascular spaces lying between the white pulp and the actual marginal zone. It is not a vessel, but a cleftlike space in which many capillaries terminate. The endothelial cells of the capillaries are continuous with the cells that line the sinuses. Between the endothelial cells and the subjacent white pulp is a basement membrane which is interrupted or fenestrated, thus providing a passage for cells from the marginal zone into the white pulp and vice versa.

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This general picture can be applied to most species investigated, but there are substantial differences among species. In the rat, in which the marginal sinus has been described for the first time (Andrew, 1946; Altschul and Hummason, 1947; Snook, 19501, they appear as small discontinuous spaces interconnected by short capillaries (Schmidt et al., 1985a). Larger spaces that have merged over a broader range of the periphery of the white pulp are found in the mouse (Schmidt et al., 1985b). In addition marginal sinuses have been reported in rabbits (Burke and Simon, 1970), dogs (Schmidt et al., 1983),and cats (Blue and Weiss, 1981). In the spleen of the cat, the spaces of the marginal sinus are quite inconspicuous, as is the whole marginal zone in this species (Blue and Weiss, 1981). The situation in humans has been a bit controversial. Although the presence of a marginal zone, as such, has been described in the human spleen (Takasaki, 1979; Saitoh et al., 1982; Brozman, 19851, the presence of a distinct marginal sinus has been debated. It was reported by Barnhart and Lusher (1979), but in an extensive study on a large number of methylmethacrylate-embedded human spleens, Van Krieken et af. (1985) concluded that no evidence for the existence of a marginal sinus could be found. Recently, however, Schmidt et ai. (1988)clearly demonstrated the presence of a marginal sinus, using scanning electron microscopy. These authors, with their elaborate experience with spleen morphology in various species, described the human marginal sinus as a flattened, almost continuous, system of anastomosing spaces, with fewer follicular capillaries terminating in the inner aspects of the sinus as compared with other species. The discrepancy between these findings and previous negative findings by other groups (Van Krieken et al., 1985; Nunes and Esperanca-Pina, 1985)may stem partly from the use of pathological spleens as well as from the use of different techniques to evaluate the results. Taken together, we must conclude that this important part of the marginal zone is present in all species examined, although it cannot be denied that there is a substantial interspecies difference. The basic structure of the marginal zone consists of a meshwork of reticular cells, which are concentrically layered around the PALS and the follicular areas, with a more dispersed three-dimensional appearance toward the red pulp (Veerman and Van Ewijk, 1975).The opening of a part of the arterial blood supply into the wider spaces of the marginal sinus, right at the border of the marginal zone and the white pulp, results in a blood flow with a lower resistance. This gives bloodborne cells an opportunity to reside in the marginal zone or to migrate into the white pulp and gives cells already in the marginal zone the opportunity to react with the incoming cells and antigens (Fig. 1).

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FIG. 1 The marginal zone of the mouse. The central arteriole (CA) branches into small capillaries that either pass through the marginal zone and end in the red pulp (RP) or open into the marginal sinus (MS). The marginal zone is composed of reticular cells (RC). Within this reticular framework the large marginal zone macrophages (MZM) and marginal zone B lymphocytes (MZ B Ly) are localized. At the inner border of the marginal sinus and the white pulp (WP), the marginal metallophilic macrophages (MMM) are situated.

111. Cells of the Marginal Zone A. Marginal Zone B Lymphocytes

As a result of the structure of the marginal zone, a number of bloodborne lymphocytes are continuously present in this area on their way to either the white or red pulp. As a population these passaging cells constitute only a minor fraction of the cells in the marginal zone. Most of the lymphocytes found here are intermediate-sized B lymphocytes. That the marginal zone is a major B cell area was already suggested by Keuning et a/. ( 1963) and was later confirmed by specific staining with anti-immunoglobulin (Ig) antibodies in mice (Goldschneider and McGregor, 1973; Weissman, 1975) (Fig. 2) and by the demonstration of Fc and C3 receptors in humans (Jaffe et al., 1975) and rats (Kumararatne et al., 1981). Additional proof that the lymphocytes in the marginal zone are predominantly B cells came from the findings that the marginal zone is not reduced in animals that are congenitally athymic, but that animals treated with anti-p antibodies from birth showed an almost complete depletion of the marginal zone (Kumararatne et af., 1981).

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FIG. 2 The B lymphocytes in the spleen are organized in follicles and in the marginal zone surrounding the white pulp. Note that the B lymphocytes in the marginal zone stain more intensely with the anti-IgM antibody. f, Follicle; rp, red pulp; p. penarteriolar lymphocyte sheath. Bar = 200 pm.

1. Do Marginal Zone B Cells Form a Separate Lineage? The marginal zone in the spleen is the only B lymphocyte-dependent area in the body where B cells are not organized in follicular structures. B cells in follicles are, unless the follicles become stimulated, small resting lymphocytes. Is there evidence that the B lymphocytes of the marginal zone, with their intermediate size, form a distinct population? In comparison with small B lymphocytes, marginal zone B cells show a strongly reduced expression of IgD on their surfaces (Stein et al., 1980; Gray et al., 1982; Hsu, 1985). When B cells become activated, they lose surface IgD (Kraal et al., 1986a), and the expression of the interleukin 2 (IL-2) Tac receptor on marginal zone B cells (Hsu, 1983, together with their size, is indicative of a state of activation. However, additional evidence suggests that the marginal zone B cells are not merely a population of p+F-activated B cells. It was demonstrated that marginal zone lymphocytes are extremely sensitive to treatment with cyclophosphamide, leading to a selective depletion of this area (Kumararatne et al., 1980). By using this depletion model, repopulation studies were performed to study

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the precursor identity of the marginal zone B cells (Kumararatne and MacLennan, 1981). Fetal liver cells or bone marrow cells from animals that had been depleted of recirculating cells by local irradiation of their spleens showed faster recovery of the marginal zone upon transfer into animals from which the marginal zone had selectively been depleted by cyclophosphamide. Conversely, normal bone marrow cells, as well as thoracic duct lymphocytes, were able to repopulate the depleted area. This led the authors to conclude that the immediate precursors of the marginal zone cells are recirculating cells and that the B lymphocytes in this area are mature and not “virgin,” as was suggested previously (Strober and Dilley, 1973). Albeit that the precursors of these cells can be found in the recirculating pool, the marginal zone B lymphocytes themselves are certainly not within that pool. Depletion of the recirculating lymphocytes by thoracic duct cannulation did not lead to appreciable changes in the marginal zone population, and local irradiation of one-half of the spleen resulted in significant depletion of the PALS and the follicles of adjacent nonirradiated areas, but again, not of the marginal zone (Kumararatne et al., 1981). Additional experiments from this group, with more precise phenotyping of the cells involved, confirmed these results. It was found that, after selective depletion of recirculating cells by local irradiation of the spleen with 32Pstrips, the majority of pf8+ B lymphocytes were eliminated from the spleen and the lymph nodes, but the p’8- marginal zone cells of the nonirradiated half of the spleen were spared (Gray et al., 1982). Together, these results clearly indicate that the marginal zone B cells are a unique population of B cells with low recirculatory capacity (MacLennan et al., 1982). Further evidence that the marginal zone B cells are unique comes from experiments by Bazin et al. (1982), in which rats are injected from birth with anti-IgD antibody. In these animals a complete suppression was found of the development of follicle-associated B cells, but not of the p f 8 marginal zone B cells. Although in the follicles of anti-IgD-treated rats B cells would develop occasionally, these cells were pf8- and also lacked the HIS24 marker (Kroese et al., 1987),which is also absent from marginal zone cells in the spleen (MacLennan and Gray, 1986). These results point to a development of the marginal zone $8- B lymphocytes independent from the follicular small p+8+ lymphocytes. This is somewhat contradictory to the finding that the precursors of this population can be found within the recirculating pool of predominantly follicular B lymphocytes (MacLennan and Gray, 1986). Attempts to identify B lymphocytes with the phenotype of marginal zone cells at other sites have not been conclusive, although indications for a small population of p+8- cells in lymph node follicles has been presented

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in rats (Bazin et al., 1982) and humans (Van den Oord et al., 1986; Van Krieken et al., 1989). Whether these cells are related to the marginal zone B cells is not clear. Are they the small population of precursor cells which are also putatively identified in the thoracic duct lymphocytes of the rat (MacLennan and Gray, 1986), or are they activated follicular or extrafollicular B cells that have lost IgD, as described for germinal center B cells (Stein et al., 1980; Jacobson et al., 1981)? Further phenotypic differences between follicular B cells and marginal zone B cells are the enhanced radiosensitivity of the latter (Bazin et al., 1986; Riggs et al., 1988). This radiosensitivity, as well as the enhanced sensitivity for cyclophosphamide, may be related to the fact that marginal zone B cells are more activated. The expression of CRl and CR2 receptors on marginal zone B cells is another unusual feature of these cells compared to other follicular B cells (Gray et al., 1984a). In humans recently, two monoclonal antibodies have been developed that can discriminate between marginal zone B cells and follicular B cells (Smith-Ravin et d., 1990). The UCL4D12 antibody recognizes marginal zone B cells and a small subpopulation in the follicles. These cells do not express surface IgD. The UCL3D3 antibody does not recognize marginal zone B cells, but reacts with follicular B cells. The two antigens stain reciprocal populations, although some overlap was reported. In Table I a comparison is made of the characteristics of marginal zone and follicular B cells. It is obvious that, with the help of antibodies such as UCL4D12 and UCL3D3, more insight into the role of the marginal zone B cells and especially their relationship with the follicular B cells can be obtained.

2. Relationship between Marginal Zone B Cells and CD5 B Cells The differential expression of IgM and IgD, as observed by flow cytometry, has led to the classification of murine B cells in three populations (Hardy et al., 1983, 1984). Population I expresses high IgD and intermediate levels of IgM and is most numerous in all mouse strains (except in the xidCBA/N mouse). Population 11, with high levels of both IgM and IgD, is found in all mouse strains in comparable frequencies, whereas population I11 constitutes a small population which is only found in the spleen, but not in the lymph nodes. It is characterized by the expression of low levels of IgD and high levels of IgM. Within this small population of type 111 cells the CD5 B cells are also found. CD5 B cells are unique in many respects. In addition to the expression of Ly-1 (in mice) or Leu-1 (in humans), they show distinctive levels of several other B cell markers and are associated with several special functions (see Herzenberg et al., 1986; Kipps, 1989). In the mouse they are found in high frequencies in the peritoneal cavity. In

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TABLE I Characteristics of Marginal Zone B Cells Compared to Follicular B Cells

B cells Characteristic ~~~

Marginal zone ~

Size Recirculation Cyclophosphamide sensitivity Radiation sensitivity Surface IgM Surface IgD IL-2 receptor (Tac) CRI/CR2 HIS24 (rat) CD23 KiB3 UCL4D12 (human) UCL3D3 (human)

Follicular

Reference

~

Intermediate -

Small

+

++

''

+t

t

tt

t

-

++

t

-

+ -

+ -

MacLennan et a / . (1982) Kumararatne e t a / . (1981) Bazin et a / . (1986) Gray et a / . ( 1982) Stein et al. (1980) Hsu (1985) Gray et d . (1984a) MacLennan and Gray ( 1986) Ling e l a / . (1987) Ling et a / . (1987) Smith-Ravin et a / . (1990) Smith-Ravin e l al. (1990)

the spleen they are rare, and in the lymph nodes, undetectable (Hayakawa et al., 1983, 1986; Hardy et al., 1984). In humans such an anatomical distribution is less distinctive. Substantial percentages, up to 30%, can be found in the peripheral blood and the lymph nodes (Plater-Zyberk et al., 1985; Hardy and Hayakawa, 1986; Kipps and Vaughan, 1987), whereas in the human spleen the frequency is somewhat lower (Freedman et al., 1987).

Interestingly, as has been found for the marginal zone B cells, CD5 B lymphocytes form a distinct lineage (Hayakawa et al., 1983). During ontogeny they are among the earliest detectable cells in the spleen, but their relative numbers decline rapidly when conventional B cell populations develop. At 6 weeks of age, they represent less than 2% of the total B cells in the spleen of a normal mouse. In transfer studies using allogeneic mice, evidence has been presented that the progenitors for CD5 B cells are mainly found in the peritoneal cavity, whereas precursors of the conventional B cell lineage are predominantly derived from the bone marrow (Hayakawa et al., 1985). CD5 B cells are implicated in the pathogenesis of autoimmune diseases. Mouse strains that develop autoimmune disorders, such as NZB mice and mice homozygous for the motheaten gene, have elevated numbers of

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39

splenic and peritoneal CD5 B cells (Hayakawa et al., 1983; Shultz and Green, 1976; Sidman et al., 1986). Moreover, it was demonstrated by in uitro sorting that the NZB CD5 B cells were the major producers of the autoantibodies found in this mouse strain (Hayakawa et af., 1983, 1985). On the other hand, CBA/N mice, which are immunodeficient in several respects (Amsbaugh er al., 1972; Scher et al., 1975), are devoid of any detectable CD5 B cells in the spleen and the peritoneum (Hardy et al., 1983; Hayakawa et al., 1986). In humans a similar relationship with elevated levels of CD5 B cells and autoimmune disorders has been implicated in rheumatoid arthritis, primary Sjogren’s syndrome, and systemic sclerosis (Plater-Zyberk et al., 1985; Maini er al., 1987; Dauphinee et al., 1988; Hardy er al., 1987). When the characteristics of the CD5 B cell and the marginal zone B cell are compared, many similarities can be found. Both cell types constitute a separate cell line with precursors that are distinct from the precursors of the follicular B cells. They develop early in life and, according to their IgD and IgM profiles, they both have the population 111 phenotype, with bright expression of IgM and low to negative expression of IgD. However, marginal zone B cells are defined primarily by their anatomical localization, whereas CD5 B cells are characterized by function and membrane phenotype in uitro. CD5 cells are numerous in the mouse peritoneal cavity. In a study in which allotypically different peritoneal cells and bone marrow cells were transferred into lethally irradiated mice (Kroese et al., 1989), it was shown that all conventional B cells in these mice express the bone marrow allotype and all CD5 cells and especially a large proportion of plasma cells in these animals express the peritoneal allotype. However, very few cells in the marginal zone were of the CD5 peritoneal allotype. Therefore, identity between the two populations is very unlikely, because the number of CD5 B lymphocytes in the murine spleen (less than 2% of total B cells) can never account for the large amount of marginal zone B cells seen in sections in comparison with the number of follicular B cells. Furthermore, in CBA/N mice, in which the CD5 B lymphocyte is absent, a marginal zone can be seen that, although smaller in size than in normal CBA mice, is populated by the typical marginal zone B cells (Kraal et al., 1988a). It cannot be excluded that in normal animals the CD5 B cells may form a minor portion of the marginal zone B cell population, but also in the red pulp many bright IgM-positive cells can be seen. The low expression of the CD5 marker on these B cells, which can be detected only by extremely sensitive flow cytometry, is of limited use in immunohistochemistry . Therefore, precise localization of the CD5 B cell population and the possible relationship between the two types of B cells must await the development of a positive way to discriminate CD5 B cells by immunohistochemistry.

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6. Macrophages When foreign substances such as India ink are injected into the blood stream of an experimental animal, they are initially localized in the marginal zone and only later are found in the red pulp. This is based on the structure of the blood supply, but also points to a well-developed phagocytic capacity of this area. When we look at the marginal zone in more detail, several types of macrophages can be distinguished, each with a defined location and set of characteristics. 1. Marginal Metallophilic Macrophages

The first description of the macrophages that are now called marginal metallophilic macrophages was given by Snook (1964). He described a rim of cells situated at the margin of the white pulp along the inner border of the marginal sinus. Strictly speaking, they are therefore situated in the white pulp, but right at the border with the marginal zone. However, generally, they are included in the populations of cells that inhabit the marginal zone. These cells showed affinity for silver and hence were called metallophilic cells. In spite of their acid phosphatase activity, they did not phagocytize well, although iron compounds could be taken up. The orientation of these cells, with slender cell processes into the white pulp, is very characteristic, especially in the spleens of small rodents. Also, in the cat macrophages have been described which are thought to be comparable to the marginal metallophilic macrophages (Blue and Weiss, 1981). These cells were found on the circumferential reticulum of the marginal sinus and could not take up thorotrast. In other species, including humans, the situation is not so clear, which may be related to the function of these cells. An important feature of the marginal metallophilic macrophages in the mouse is their strong nonspecific esterase activity, which is very sensitive to the organophosphatase inhibitor E600 (Eikelenboom, 1978). It has been demonstrated in in uitro studies that detoxification of endotoxin was mediated by organophosphatase-sensitive esterases (Keene, 1962; Smith et al., 1963).This type of esterase was found especially in the sinus-lining cells of the red pulp in rabbits, but not in macrophages (Ballantyne, 1968; Snodgrass, 1968, 1971; Snodgrass and Snook, 1971).This could imply that the detoxification process takes place in different cell types in different species. In mice, with their poorly developed red pulp sinuses, the marginal metallophilic cells may therefore have a function comparable to that of the sinus-lining cells of other species. In the spleen of the chicken at the outer border of the Schweigger-Seidel sheath, a single line of nonlymphoid cells with acid phosphatase activity but low phagocytic capacity was demonstrated (Eikelenboom et al., 1983). Based on the orientation of

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these cells and their function, the authors concluded that these cells were comparable to the marginal metallophilic macrophages of rats and mice. A monoclonal antibody was developed in our laboratory which specifically reacts with the marginal metallophilic macrophages in the mouse spleen (Kraal and Janse, 1986).This antibody, MOMA-1, shows complete correlation with nonspecific esterase activity and has a restricted expression on cells of the mononuclear phagocyte system. In the spleen it is restricted to the marginal metallophilic cells (Fig. 3). Its major expression outside the spleen is on medullary and subcapsular macrophages in the lymph nodes. MOMA-1 does not recognize the enzyme nonspecific esterase as such, because around 7 days after birth, MOMA-1-positive cells can be identified in a ring around white pulp areas before nonspecific esterase activity can be demonstrated. With the use of this antibody, the function of these cells in the marginal zone was studied. A series of experiments was started in which newborn mice were injected from birth with high doses of MOMA-I. In these experiments we focused on a role of the marginal metallophilic macrophages in the handling of antigens, influenced by their strategic position at the border of the marginal sinus (Kraal et al., 1988b). Multiple injections of the antibody led to drastic reduction of the number of cells, but not to a complete elimination of the macrophages. When these animals were chal-

FIG. 3 The marginal metallophilic macrophages are organized as compact rings of cells at the inner border of the marginal zone and the white pulp. Here, they are specifically recognized by the MOMA-I monoclonal antibody. Bar = 150 pm.

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lenged with various antigens, it was found that the responses against T cell-dependent and -independent type 2 responses were impaired, but not the response against a T cell-independent antigen. These findings were somewhat in contrast to earlier findings in which complete elimination of the macrophages from the marginal zone by liposome treatment did not lead to altered patterns in the immune response against these types of antigens (see Section IV,B,3). An explanation for this may be that, in newborn animals injected with MOMA-1, the distribution of the MOMA-1 cells is not so restricted in the spleen as it is in adults. Macrophage-like MOMA-1 cells can be observed in the red pulp in young animals, which may be an indication that during ontogeny different macrophages can express this marker and may be modulated by the antibody. Therefore, the treatment during the first 4 weeks as performed in our experiments could have resulted in effects on more macrophage populations than only the marginal metallophilic cells. Recently, a monoclonal antibody has been described which in the spleen selectively reacts with marginal metallophilic macrophages (Crocker and Gordon, 1989). This antibody, SER-4, was raised against the mouse macrophage-restricted hemagglutinin sheep erythrocyte receptor (SER). This receptor, which binds unopsonized sheep red blood cells through recognition of sialylated glycoconjugates, was originally described on resident bone marrow macrophages. Here, it was thought to play a role in the adhesive interactions with hematopoietic cells (Crocker and Gordon, 1986). This specificity for the marginal zone is in accordance with earlier reports in which the reaction of sheep erythrocytes with cells in the marginal zone has been demonstrated (Stejska and Fitch, 1970; Dukor et al., 1970). Stejska and Fitch (1970) described how sheep red blood cells adhere to the marginal zone and the marginal sinus of spleen sections from nonimmunized rats, and it was suggested that this reaction was caused by the action of “natural” antibodies (Stejska and Fitch, 1970; Thunold, 1973).When these interactions were studied in more detail, a variety of red blood cells from different species and also certain lymphocytes showed this agglutination phenomenon (Radaszkiewicz et al., 1979). Furthermore, by treating the sections or adhering cells with different agents, including enzymes, it became clear that the interaction was receptor mediated and directly correlated to the amount of neuraminic acid in the membrane of the adhering cells. The relationship between the SER, as described by Crocker and Gordon (1986), and the red blood cell receptor on marginal zone cells has been emphasized by Damoiseaux et al. (19911, who described a blocking function of the ED3 anti-rat macrophage antibody in the adherence of red blood cells on spleen tissue sections. ED3-positive macrophages in the spleen are restricted to the marginal zone, and it was found, as for SER, that the

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interaction between the ED3 cells and the adhering red blood cells was not dependent on divalent cations, was trypsin sensitive, and was mediated by sialic acid. In the rat distribution of ED3 is not restricted to the marginal metallophilic macrophages (Fig. 4), but this antibody also recognizes the adjacent marginal zone macrophages (Dijkstra er al., 1985a; see Section III,B,3). It is therefore conceivable that the receptor is expressed on both types of macrophages. In the mouse the situation is more restricted. The monoclonal antibody SER-4 specifically reacts with the marginal metallophilic macrophages but not with the marginal zone macrophages and is indistinguishable from MOMA-1 staining (Crocker and Gordon, 1989). Anti-SER antibody blocks the binding of erythrocytes to the mouse spleen marginal zone area, but MOMA-I is ineffective. A more physiological role for this receptor may be inferred from recent findings in our laboratory, where it was found that SER-4 as well as ED3 is involved in the binding of activated T and B cells to the marginal zone and the subcapsular sinus of the lymph nodes. This binding was studied, using a frozen section assay as developed for lymph node high endothelial venules by Stamper and Woodruff (1976)and by Butcher et al. (1979), and showed specific binding of mitogen-stimulated T and B cells, but not small lymphocytes in the marginal zone. Again, as found for the binding with sheep red blood cells, this interaction was mediated by macrophages, was dependent on sialic acid, and could be blocked completely by ED3 in the rat or SER-4 in the mouse. No effect was seen using antibodies against the a and /3 chains of LFA-1, VLA-4, or MOMA-1 (Van Den Berg et al., 1992). The localization of blast T and B cells at these sites may be related to a retention mechanism to hold memory cells or, in the case of B cells, plasmablasts. The medullary cords and the inner part of the marginal zone/outer PALS are the precise locations of plasma cells, ensuring an efficient deposition of antibodies into the passing blood or lymph. Taken together, the data show that the marginal metallophilic macrophage is a conspicuous cell type, strategically positioned along the borders of the marginal sinus, having high activity for nonspecific esterase and low phagocytic activity and expressing receptors for sialic acid-containing glycoconjugates. The first characteristic is indicative for a role in the degradation of incoming material (e.g., endotoxin); the latter is more suggestive of a role in the interaction with lymphocytes, perhaps in determining the migration into the white pulp area. Treatment of mice with a high dose of lipopolysaccharide (LPS) results in a decrease in the number of MOMA-1 macrophages in the marginal zone, together with a decrease in B cells and marginal zone macrophages in this area (Groeneveld et al., 1986). The disappearance of B cells after LPS and the concomitant appearance of B cells with similar phenotype in

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FIG. 4 Distribution of ED3-positive cells in the rat spleen. In the rat the ED3 antibody recognizes both marginal metallophilic and marginal zone macrophages. wp, White pulp; rp, red pulp. (A) Bar = 200 Fm; (B) bar = 50 pm.

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the splenic follicles is suggestive of a direct migration of the marginal zone B cells, related to the formation of memory cells under normal conditions (Gray et al., 1984b; Groeneveld et al., 1985). LPS treatment also results in a migration of MOMA-1-positive cells in the follicles. In the center of the follicles, the staining is found on tingible body macrophages and indicates that the MOMA-1 cells in the marginal zone may be related to this cell type (Groeneveld et al., 1986).

2. Marginal Zone Macrophages a . Disrribution and Characteristics This illustrious population of macrophages forms a ring of two or three layers dispersed throughout the marginal zone. They are big cells with long cell processes and appear to have close contacts with the surrounding marginal zone B lymphocytes. They are positioned throughout the marginal zone, from the marginal sinus to the border area with the red pulp. They are highly phagocytic, as can be seen from the presence of lytic enzymes such as acid phosphatase and esterase, and often contain, when examined at the ultrastructural level, phagolysosomes with necrotic erythrocytes and cellular debris (Dijkstra ef al., 1985b).They do not express major histocompatibility complex (MHC) class I1 antigens (Humphrey, 1981), but can be discriminated by specific monoclonal antibodies (see below). The marginal zone macrophages were first described as such by Humphrey (1981; Humphrey and Grennan, 1981) in a study of the tolerogenic and immunogenic effects of hapten-conjugated polysaccharides in the mouse. A marked variation of the tissue distribution was noticed when the localization of these conjugates was examined. Acidic polysaccharides localized mainly in the red pulp of the spleen, but in the case of neutral polysaccharides, an exclusive localization was detected in macrophages of the marginal zone. These neutral polysaccharides included various haptenated antigens such as dinitrophenol-Ficoll, hydroxyethyl starch (HES), and dextran, which are all considered to be thymus-independent type 2 (TI-2) antigens (Mosier et al., 1977). The subdivision of T cell-independent antigens into two types is based on the response these antigens elicit in CBAlN mice, which have an X-linked immunodeficiency that leads to changes in their B cell differentiation. These mice make near-normal immune responses against bacterial LPSs (TI-l), but are unable to respond to the group of TI-2 polysaccharides (Scher et al., 1975; Scher, 1982). Such an exclusive localization of TI-2 antigens in the marginal zone macrophages has prompted much interest in the role of the marginal zone in the immune response against this type of antigen. The unique combination of a distinct

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type of B cells with low IgD expression and the marginal zone macrophages is not found in any other lymphoid organ and is suggestive of a specific microenvironment.

b. Detection by Monoclonal Antibodies The marginal zone macrophages have been studied extensively in small rodents and in both rats and mice they can be recognized by monoclonal antibodies. In the rat the marginal zone macrophages express the ED3 antigen, but this antigen is also found on marginal metallophilic cells and red pulp macrophages, especially in the area close to the marginal zone (Dijkstra et al., 1985a). Outside the spleen the antigen shows a very restricted expression; it is found on macrophages in the medullary cords of the lymph nodes, which, interestingly, can also specifically take up the neutral polysaccharide TI-2 antigens (e.g., fluoresceinated Ficoll). In addition the ED3 antigen can be induced on cultured bone marrow macrophage precursors by factors from concanavalin Astimulated T cell supernatants, but is down-regulated by y-interferon (Damoiseaux et af., 1989). Also, at sites of inflammation during autoimmune disorders, the ED3 antigen can be found on some inflammatory macrophages, as demonstrated in experimental allergic encephalitis (Polman er al., 1986) and arthritis (Dijkstra et al., 1987). The ED3 antigen is associated with the SER, as demonstrated in suspensions and on sections (see also Section III,B,l), and as such must be considered a molecule with a function in cellular interactions involving sialic acid. Using several neoglycoproteins, Harms et al. (1990) were able to show that the ED3 macrophages in the marginal zone expressed glycosyl receptors specific for polysaccharides. It is not clear whether such glycosyl receptors must be considered receptors for TI-2 antigens. It has also been suggested that these types of receptors (e.g., the mannose receptor) play a role during inflammation by regulating the level of extracellular lysosomal hydrolases (Stahl et al., 1984). Whereas in the rat the ED3 antibody does not discriminate between marginal zone macrophages and marginal metallophilic macrophages, the situation in the mouse is more clear. Here, the antibody ER-TR9 specifically recognizes the marginal zone macrophages and, as shown in Fig. 5 , using double immunofluorescence, a complete correlation between the staining pattern with this antibody and the uptake of fluoresceinated Ficoll was demonstrated (Dijkstra er af., 1985b; Van Vliet et al., 1985). The ER-TR9-positive cells are the macrophages that take up the TI-2 antigens and, based on these characteristics, can clearly be separated from the MOMA-1-positive marginal metallophilic cells (Dijkstra er al., 1985b; Kraal and Janse, 1986). In the mouse they are localized as adiscrete rim of cells lying in the middle of the marginal zone at the periphery of the marginal metallophilic cells and the marginal sinus. Similar to ED3 expres-

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FIG. 5 (A) Marginal zone macrophages in the mouse are recognized by the monoclonal antibody ER-TR9. wp, White pulp. Bar = 50 pm. (B) Immunofluorescence of marginal zone macrophages after specific uptake of fluorescein isothiocyanate-labeled Ficoll.

sion in the rat, the ER-TR9 antigen is also expressed on medullary cord macrophages in the lymph nodes, but its expression in the spleen is extremely specific for only the marginal zone macrophages. This is further emphasized by the finding that the ER-TR9 antibody can effectively block the uptake of TI-2 antigens by the marginal zone macrophages (Kraal et af., 1989b). When ER-TR9 antibody was injected intravenously shortly before the injection of fluoresceinated sugars such as fluorescein isothiocyanate (F1TC)-Ficoll or FITC-HES, the uptake of these TI-2 antigens by the marginal zone macrophages was completely inhibited. It was intriguing that the antibody did not prevent the uptake of carbon or latex particles. These blocking studies clearly suggest that the ER-TR9 antigen is a receptor for polysaccharides, although it cannot be ruled out that the antibody attachment leads to changes in the cell membrane, after which the uptake or adherence of the sugars is inhibited indirectly. Indications for the latter effect come from experiments in which we injected polysaccharides prior to injection of the antibody. This protocol did not prevent the antibody from attaching to the cells, suggesting that the sugar receptor and the ER-TR9 epitope are not the same.

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In studies of the uptake of TI-2 antigens by the marginal zone macrophages in the rat, Chao and MacPherson (1990) made use of a tissue slice technique in which thin slices of spleen were incubated in uitro with FITC-HES. Under these conditions the cells were still fully capable of taking up the polysaccharide, apparently without the need for an efficient blood supply. The thin slice technique opened the way to direct investigation of the influence of various compounds on polysaccharide uptake. These authors showed that the uptake was abrogated by pronase and dispase, suggestive of a surface protein receptor, and that collagenase treatment could block the uptake, but not the surface binding, of the sugar antigen. Interestingly, the yeast cell wall derivatives mannan and zymosan were capable of blocking the binding and uptake, but, as in the case of our mouse ER-TR9 studies, neither interfered with the phagocytosis of carbon. Taken together, our data and those of Chao and MacPherson (1990) clearly indicate that the specific uptake of TI-2 polysaccharides is mediated via a receptor and that it is not so much the privileged location of the cells in the marginal zone but the specificity of the receptor that determines the uniqueness of these cells. Many bacterial capsule polysaccharides are contained within the group of TI-2 antigens, and with the development of specific antibodies such as ER-TR9 and related techniques, the role of these cells in the immune response against TI-2 antigens can be studied. 3. Turnover Characteristics of Macrophages in the Marginal Zone Using liposomes in which dichloromethylene diphosphonate (C12MDP) was encapsulated, Van Rooijen and Van Nieuwmegen (1984) found that a single intravenous injection of such liposomes effectively killed all macrophages in the red pulp and the marginal zone of the spleen. These macrophages are residing in the blood stream, phagocytize the liposomes, and die after accumulation of the C12MDP.Macrophages of the white pulp that are not directly reached by the blood stream are protected from this fate. That the macrophages were physically removed from the spleen was confirmed at the ultrastructural level (Van Rooijen et al., 1985). After elimination the various subsets-red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages-showed a striking difference in their reappearance kinetics. The red pulp macrophages were back in normal numbers after 1 week, the marginal metallophilic macrophages took 2 weeks to regain fully their position at the border of the marginal zone and the PALS, but the marginal zone macrophages reappeared very slowly and only after 1 month did the first cells start to localize in the marginal zone (Van Rooijen et af., 1989).The latter finding is

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in agreement with a low replacement rate under steady-state conditions of approximately 25% per month, as described by Humphrey and Sundaram (1985). Precursors of the macrophages, deriving from the bone marrow, are the nonphagocytizing monocytes which enter the spleen via the blood. Few data are available concerning the origin and kinetics of the macrophages of the spleen, and furthermore, in these reports the macrophage population in the spleen is considered as a whole (Van Furth and Diesselhof-Den Dulk, 1984). Calculations of the influx of monocytes into the spleen and of the local production of macrophages by DNA-synthesizing mononuclear phagocytes showed that under steady-state conditions 55% of the splenic macrophages are supplied by monocyte influx, and 45% by local production, indicating a dual origin of the macrophage population of the spleen. Furthermore, a mean turnover time of 6 days was found under normal steady-state conditions (Van Furth and Diesselhof-Den Dulk, 1984; Van Furth et a / . , 1985). The comparatively rapid start and short duration of the repopulation of the red pulp macrophages is in agreement with this short turnover time. It may depend on a relatively high proportion of their precursors among the cells entering. The late start and the long duration, especially in the case of the marginal macrophages, could be explained by a relatively low proportion of specific precursors. However, it could also point to differences in the microenvironment or a dependence on precursors from outside the organ. When under normal conditions the turnover of a population is predominantly dependent on local proliferation, the massive depletion of a population, as seen in the liposome experiments, may severely hamper the return. An interesting finding that came from the liposome depletion experiments was that the presence and retention of the marginal zone B cells did not seem to depend on the presence of the marginal zone macrophages. A specific interaction between the two cells was suggested by Humphrey (1981; Humphrey and Grennan, 1981), who found strong adhesion interactions between the marginal zone macrophages and the surrounding B cells. Initially, when the liposomes are administered, the marginal zone B cells tend to disappear. No other effects of the liposomes on lymphocyte populations were found. However, the marginal zone B cells were found to reappear in normal numbers in the marginal zone 12-16 days after treatment, long before any marginal zone macrophage can be observed.

C. Other Cell Types The marginal zone B cells, as well as the two types of macrophages described above, are specific populations of the marginal zone with a fixed

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location. In addition many other cells can be found in the marginal zone which must be regarded as cells passing through. For example, a substantial number of T lymphocytes can be found in this area, on their way to either the white pulp or the red pulp. Because of these decisive steps, they may reside in the marginal zone longer than expected, although very little is known about their kinetics in this region. Natural killer cells, as defined by the antibody NKH, have been identified in the human marginal zone (Witte et af., 1990) with a phenotype identical to that found in the blood. The same is true for small recirculating B cells, granulocytes, and monocytes (Buckley et al., 1987),which are distributed here by the blood. Also, the reports in the literature of the presence of progenitor cells in the marginal zone may come from a more accidental and temporary sojourn of bloodborne stem cells (Foon et al., 1984; Beschorner et al., 1985). 1. Dendritic Cells

The dendritic cell, which can be found in the blood, may fall into this category of passaging cells, but some recent findings merit attention in this respect. Dendritic cells are critical accessory cells in T cell-dependent primary immune responses (Steinman and Nussenzweig, 1980; Austyn, 1987). Although originally described as in uitro isolated cells, there is now an almost unanimous consensus that the dendritic cells can be seen as a larger population in which the Langerhans cells of the skin, the veiled cells in lymph, and the interdigitating cells of T cell-dependent areas in lymphoid organs are contained (Kraal et al., 1986b; Breel et al., 1987; Kraal, 1989). In the spleen dendritic cells are predominantly found in the PALS, where they can be recognized by their high constitutive expression of MHC class I1 molecules (Dijkstra, 1982). They are being increasingly regarded as a mobile population that can move from one site of the body to another. This can occur via the lymphatics, as has been demonstrated for Langerhans cells, or via the blood stream. It has been suggested that the major route for dendritic cell traffic is from peripheral nonlymphoid tissues to draining lymphoid organs. It is therefore not surprising that dendritic cells can be observed in the marginal zone (Witmer and Steinman, 1984) and the red pulp of the spleen, as any bloodborne cell eventually passes through these regions. However, recent observations point to the fact that the presence of dendritic cells in the marginal zone is more than an accidental passage. In a series of elegant experiments, Austyn and co-workers (KupiecWeglinski et al., 1988; Austyn et al., 1988a) looked at the migration of dendritic cells, using highly purified populations. In transfer experiments with radiolabeled cells, they were able to show that dendritic cells can migrate efficiently from the blood into the spleen, but that this migration is

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dependent on the presence of T cells. Their most intriguing finding was that, when purified dendritic cells were incubated on frozen sections of the spleen, a preferential adherence of these cells to the marginal zone area was found. Such specific adhesion was never found with purified macrophages or lymphocytes. The authors suggested that T cells in the PALS can modify the vascular endothelium to attract dendritic cells. This would indicate that marginal sinus-lining cells, or perhaps macrophages in this area, express adhesive molecules which can be selectively recognized by dendritic cells. In line with these findings is the selective expression of a dendritic cell-specific epitope of the CDl lc molecule, as recognized by the B418 monoclonal antibody. This antibody was developed in a series of antibodies against mouse integrins and was found to label only dendritic cells (Metlay ef al., 1990). Using immunohistochemistry with this antibody, the presence of dendritic cells in the PALS area of the white pulp was demonstrated, in agreement with observations using anti-MHC class I1 antibodies. In addition Metlay et al. described clusters of presumptive dendritic cells at the rim of the marginal zone at the border with the white pulp in areas where the marginal metallophilic macrophages were less abundant. This strategic position may be important in the process of antigen presentation to T cells that move from the marginal zone into the white pulp area.

2. Reticular Cells

The reticular cells of the marginal zone form the basic framework in which all other cells can move and interact. However, it is not clear how inert these cells are. As we consider the sinus-lining cells as reticular cells, it is obvious that these cells are different, as can be seen from their expression of the MECA-367 antigen (see Section IV,A,2). Evidence for further specialization within the stromal reticular cells has come from the studies by Weiss (1990). Based on studies of the course of malaria infections in mice, Weiss (1990) demonstrated the existence of a special stromal cell type in various compartments in the spleen: the barrier cell. This cell type is a fibroblastlike cell which can be activated to form complexes of contractile, branched, and even syncytial sheets. Activation may result from malaria infection and is probably elicited by IL-lu production by activated macrophages. In the marginal zone barrier cells may form vascular channels, leading to shunts between arterial and venous vessels, thereby bypassing the filtration beds of the red pulp. This results in the typical “asplenic” spleen seen in malaria infection, an important condition for the recovery of the infection. Whether barrier cells have a function in the normal spleen or

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only manifest themselves under conditions of severe infections is not clear. IV. Function of the Marginal Zone A, Recirculation of Lymphocytes

As a consequence of the function of the spleen’s being a filter in the blood stream, an enormous amount of blood passes through the spleen each day. It has been estimated that 3% of the cardiac output flows through the spleen, and with it an impressive number of bloodborne cells. Using a perfusion system in the pig, in which venous and arterial blood samples could be taken, it was estimated that 30 times the total pool of the animals’ recirculating lymphocytes passes through the spleen each day (Pabst and Trepel, 1975). Similar numbers have been found in the rat (Ford, 1969a). This way a continuous exchange of cells from the blood to the spleen takes place. Calculations in the rat, using radiolabeled cells, made it clear that the exchange rate of small lymphocytes from the blood into the spleen is on the order of 8 X lo7 lymphocytes per hour (Ford, 1969b).It is obvious from the distribution of the blood flow that the marginal zone plays an important role in this exchange process. Ten to 25% of the total lymphocyte flux passes through the marginal zone, and on average cells stay in this area for about 50 min. In contrast, it was found that 90% of the lymphocytes pass rapidly through the red pulp, with an average transit time of only 5 min, whereas the white pulp received 10% of the cells, with a transit time of 45 hr (Hammond, 1975; Ford, 1968, 1969a,b). The relatively long dwelling time in the marginal zone may be the direct result of the reduced blood flow in this area, but may also be indicative of selective interactions which result in an active migration of part of the lymphocytes into their respective compartments in the white pulp. Are there indications that the marginal zone plays an active role in this process? Studies in which the migration of lymphocytes was determined using autoradiography have made it clear that both T and B cells accumulate in the marginal zone (Nieuwenhuis and Ford, 1976). Lymphocytes that enter the white pulp first move into the outer PALS, a rapid process which starts within minutes after entry into the marginal zone. Here, a clear distinction is seen between T and B lymphocytes. T cells segregate from B cells by penetrating further into the central PALS, whereas B cells continue toward the base of the follicles along the outer PALS. From here they enter the corona of the follicles. T cells were never found to enter the corona (Nieuwenhuis and Ford, 1976; Van Ewijk and Nieuwenhuis, 1985). Recently, Pellas and Weiss (1990)claimed that this route from the marginal

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zone into the white pulp is probably less important than the entrance of lymphocytes into the white pulp via distal parts of the PALS. These smaller distal parts are directly surrounded by red pulp. From here both T and B lymphocytes move toward the larger PALS and segregate into their respective areas. Lymphocyte migration in the lymph nodes and mucosa-associated tissues is governed by the direct interaction of the lymphocyte with specialized venules in these organs (Butcher, 1986; Kraal er al., 1987; Yednock and Rosen, 1989).These high endothelial venules express adhesion molecules: vascular addressins (Streeter et al., 1988a,b). Differential expression of these addressins in different organs plays an important role in directing lymphocytes into particular lymphoid organs. Lymphocytes express a variety of homing receptors that may interact with these vascular addressins and other ligands (Gallatin et al., 1986; Jalkanen er al., 1986; Berg et al., 1989; Duijvestijn and Hamann, 1989). As more information on the structure of the various adhesion molecules on both endothelium and lymphocytes becomes available, a complex picture of cellular interactions is emerging (Springer, 1990). In the spleen, however, there are no histologically discernible high endothelial venules. In addition it has been found that the migration patterns in the spleen are different from those in other lymphoid organs. For example, treatment with trypsin or phospholipases severely affected localization in the lymph nodes, but had no effect on the homing of lymphocytes into the spleen (Woodruff and Gesner, 1968; Ford er al., 1976; Freitas and De Sousa, 1976). On the other hand it has become clear that in the spleen a specific selection takes place of the lymphocytes that enter the organ. This has been demonstrated in the mouse for T and B lymphocytes as well as for T cell subpopulations. B cells have a preference over T cells to home in the spleen, whereas within T cells a preference of CD8 cells has been found (Stevens et al., 1982; Kraal et al., 1983). The B cell preference is comparable to the situation observed at the level of the high endothelial venules in Peyer’s patches; the T cell subset migration is comparable to that in the peripheral lymph nodes. Together, these findings clearly indicate that the migration and localization in the spleen are certainly not random, but that selective forces interfere with the entrance of lymphocytes, especially from the marginal zone into the white pulp. 1. A Role for Macrophages in Lymphocyte Homing?

Cell types in the marginal zone that could be responsible for such selection are either macrophages or sinus-lining cells at the border of the marginal

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zone and the white pulp. Several authors have claimed a role for macrophages in directing the traffic of lymphocytes in the spleen (Ford, 1975; Freitas and De Sousa, 1976, Brelinska and Pilgrim, 1983). By careful examination of semithin and ultrathin sections in combination with autoradiography, Brelinska and Pilgrim (1983, 1984) showed that labeled migrating lymphocytes accumulated within some regions of the marginal zone in close proximity to marginal zone macrophages before migrating into the PALS. However, these observations could merely lead to “guilt by association. ” Using the method of macrophage elimination with ClzMDP liposomes, as described in Section 111,B,3, we tried to study more directly the role of macrophages in the retention of lymphocytes in the marginal zone. By using various time intervals after depletion of the macrophages, spleens could be studied that were (1) completely devoid of their macrophages, (2) had only red pulp macrophages, but no macrophages in the marginal zone, or (3) had red pulp macrophages and marginal metallophilic macrophages, but no marginal zone macrophages (Kraal et a/., 1989a). In these experiments Ly-5 congenic mice were used to enable an exact localization of individual cells in spleen sections in combination with a computer-aided quantification system. This way small differences between transferred cells in various regions of the spleen could be measured. Complete elimination of the macrophages in the spleen resulted in a reduction in the number of lymphocytes that would enter the marginal zone and migrate into the white pulp PALS. Repopulation of the red pulp macrophages did not alter this phenomenon, indicating that it was not the result of altered blood flow. When the experiments were performed using animals with spleens in which the marginal metallophilic macrophages had repopulated as well, again a clear difference in localization of the transferred lymphocytes was observed. This clearly suggested that the strategically positioned marginal metallophilic macrophages had no function in directing lymphocyte traffic. Only when the marginal zone macrophages had also reappeared in normal numbers in these animals was a distribution of transferred cells comparable to the control situation observed. Similar experiments using isolated T versus B lymphocytes showed that the elimination of the macrophages had a profound effect on the localization of B cells, whereas it hardly affected T cells (Kraal et ul., 1989a). Although these experiments clearly show that the presence of the marginal zone macrophages is important for optimal retention and localization of B lymphocytes in particular, the results cannot rule out that the effects are accomplished by nonspecific factors. The spleen has an extremely complicated blood flow in the region of the marginal zone. Therefore, it could be that the absence of marginal zone macrophages only, when the majority of other macrophage subpopulations is in its original position, leads to small

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changes in microcirculation. Lymphocytes may then have fewer opportunities to be retained in the marginal zone. That T and B cells show such differences in these experiments may result from the greater intrinsic migratory activity of T cells. Therefore, unless more direct approaches can be found to measure the interaction between unstimulated recirculating lymphocytes and macrophages in the marginal zone, it remains uncertain whether the macrophages play any pertinent role in the migration and retention of lymphocytes in this area.

2. Can Sinus-Lining Cells Direct Lymphocyte Traffic? Another cell type which may be involved in lymphocyte traffic in the spleen is the sinus-lining cell of the marginal sinus. Interestingly, it has been found recently that sinus-lining cells of the spleen react with the MECA-367 antibody. The MECA-367 antibody reacts with the vascular addressin which is present on high endothelial venules in mouse Peyer’s patches and mesenteric lymph nodes (Streeter et al., 1988a). In uiuo injection of the antibody completely abrogates the entry of lymphocytes from the blood into these organs and blocks the in uitro interaction of lymphocytes with high endothelial venules. Examination of spleen sections stained with MECA-367 revealed a distinctive staining pattern in the marginal zone of the spleen, at the border of the white pulp, where the marginal metallophilic macrophages are localized (Fig. 6). Using double immunohistochemistry with MECA-367 and MOMA- 1 antibodies, it was hard to discriminate whether different cell types were involved. To determine whether MECA-367 stained the marginal metallophilic macrophages, staining was performed in animals that had been depleted of their macrophages using the ClzMDP liposomes. In these spleens no staining could be observed of the MOMA- I marginal metallophilic macrophages 24 hr after liposome administration, but the distinctive pattern with MECA-367 was still present. It was therefore concluded that the MECA-367 staining was present not on macrophages in the marginal zone, but on a more stromal element of this area. Electron microscopy finally made it clear that the determinants detected by the MECA-367 antibody were indeed present on sinus-lining cells, long slender cells with few cytoplasmic organelles that form the inner border of the marginal sinus with the PALS (Kraal et al., 1992). The strategic position of these cells and the positive staining with the mucosal vascular addressin prompted us to study the possible role of the addressin in lymphocyte homing into the white pulp. Therefore, high doses of the antibody were injected intravenously, followed by transfer of syngeneic cells which had been labeled with a fluorescent dye. Preliminary

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FIG. 6 (A) The sinus-lining cells of the marginal sinus are recognized by the vascular addressin MECA-367. Arrows indicate the open spaces of the marginal sinus. rp. Red pulp; wp, white pulp. Bar = 100 pm, (B) Fifteen minutes after transfer migrating fluorescein isothiocyanate-labeled cells are visualized by staining with anti-FITC antibody. From the marginal zone (mz) cells migrate into the outer periarteriolar lymphocyte sheath.

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results so far have given no conclusive answers as to the role of the MECA-367 determinant. Several membrane molecules have been characterized on the lymphocytes’ cell surface which are involved in adhesion to endothelium and high endothelial venules in particular (Springer, 1990). None of these adhesion molecules or homing receptors has been found to interfere with the homing or localization of lymphocytes into the spleen, insofar as they have been tested for such a function, with the exception of the anti-guinea pig antibody CT4 (Kraal et al., 1986~). This certainly points to differences in the localization and distribution mechanism in the spleen compared to other lymphoid organs. However, due to the complexity of the spleen, specific effects of certain molecular interactions may also be masked. For example, due to the enormous participation of the red pulp in the distribution of bloodborne lymphocytes, any possible effects of blocking antibodies or certain treatments on migration into the white pulp may not be noticed when determining the total number of cells that have entered the spleen. Therefore, a more precise look at the different compartments of the spleen (e.g., by immunohistochemistry in combination with quantification methods) may yield a better approach to studying such effects.

6. Antigen Processing 1. Response against TI-2 Antigens and Presence of the Spleen The spleen plays a major role in the protection against bacterial infections. This can be attributed to both its enormous phagocytic capacity and the production of specific antibody. The spleen is especially important in the protection against virulent bacteria with polysaccharide capsules such as Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus inJuenzae, as can be seen from the often overwhelming infections with these organisms in postsplenectomy patients. The production of specific antibodies against capsular antigens is an important step in the immune response against these types of bacteria. Antibody-coated opsonized bacteria will be cleared from the blood by the liver as well as by the spleen. Polysaccharide antigens are a peculiar type of antigens and the antibody responses against these antigens differ markedly from responses against protein antigens. The response is largely T cell independent; hence, they are called TI (thymus-independent) antigens (Sela et al., 1972). Within the group of TI antigens, two types can be distinguished: TI-1 and TI-2. TI-I antigens (e.g., lipopolysaccharides) are completely T cell independent, whereas TI-2 antigens need accessory cells to elicit immune responses.

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The TI-2 group of antigens encompasses bacterial polysaccharides, bacterial polyribophosphates, blood group substances, but also synthetic polysaccharides such as dextran and Ficoll. TI-2 antigens are usually defined as those antigens that do not give rise to an antibody response in CBA/N mice (Scher, 1982). There is compelling evidence that the presence of the spleen is an absolute prerequisite for dealing with TI-2 antigens. Furthermore, a role of the marginal zone in particular has been suggested. What are the data that have led to these assumptions, and what is the precise role of the marginal zone? Splenectomized patients are at special risk for overwhelming infections with capsulated bacteria (Singer, 19731, but controversy existed over the ability of such patients to mount immune responses against bacterial polysaccharides. Often, antibody responses could be found, suggesting that lymphoid tissues at other sites were able to compensate for the absent spleen (Amman et al., 1977; Siber et al., 1978). However, previous exposure to these antigens when the spleen was still present could not always be ruled out, and specific antibodies are often found before operation. In an elegant study Amlot and Hayes (1985) were able to demonstrate the importance of the spleen in these types of infections. In a large group of splenectomized patients, they investigated the effects of immunization with a synthetic TI-2 antigen, DNP-Ficoll, that the patients could not have met before. Antibody responses against this antigen were much lower in the splenectomized group than in controls. Interestingly, when patients had been primed with the antigen before splenectomy, they responded normally against the DNP-Ficoll upon reimmunization. Amlot and Hayes concluded that the spleen is of major importance during the first exposure against these types of antigens, but once the antigen has been encountered, antibody responses may occur at other sites. These findings explain why splenectomy patients, because of previous exposure, often make normal antibody responses against various bacterial polysaccharides. However, such patients are at special risk for infections with bacteria not met before. Further evidence for the splenic dependence of antibody responses against TI-2 antigens has come from experiments in mice. Splenectomy in these animals revealed that antibody responses against T cell-dependent antigens such as trinitrophenol-keyhole limpet hemocyanin (TNP-KLH) were largely unaffected, but that the response against various TI-2 antigens was greatly impaired (Amlot et al., 1985). In uiuo and in uitro experiments in which the lymph nodes and the spleen were compared showed, in fact, that lymph node cells could not support the response against TI-2 antigens unless some accessory cell from the spleen was provided (Goud et al., 1988). All of these data point to an important role of the splenic

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microenvironment and the accessory cells for antigen presentation and B cell activation during the response against TI-2 antigens. During ontogeny the ability to mount immune responses against polysaccharide antigens appears quite late. Vaccination in humans with, for example, pneumococcal polysaccharides does not lead to protective antibodies in children under approximately 18 months of age (Cowan et al., 1978; Rosen et al., 1983; Douglas and Miles, 1984), illustrated by the susceptibility for infections with capsulated bacteria in neonates (Pabst and Kreth, 1980; Klein, 1981). This coincides with an immaturity of the spleen marginal zone in this period. The main characteristics of the infant marginal zone are the lack of CD21 expression and the high expression of IgM and IgD on B lymphocytes in this area (Timens et al., 1990). This phenotype is considered to be not yet a mature stage of B cell development, and cells in this stage do not react optimally to antigenic stimuli (Tedder et al., 1984; Hokland et al., 1985). Many authors have tried to link the special function of the spleen in TI-2 antigens to a special type of B cell. In humans this has not been successful so far. In a comparative study on the in vitro response of human B cells to pneumococcal polysaccharides, it was found that the predominant B cell responding in adult B cell populations expressed the FMC7 antigen. However, in neonates, which are unresponsive to pneumococcal polysaccharides, FMC7 is normally expressed on B cells (Rijkers et al., 1987). In the mouse more evidence for separate subsets has been found with the CBA/N strain. Based on studies of T cell-independent antigens and B cell differentiation antigens, B cells could be divided into immature and mature subsets. Expression of the antigens Lyb3, -5, and -7 was correlated with the more mature phenotype, which is lacking in the immunodeficient CBA/N mouse (Huber et al., 1977; Ahmed et al., 1977; Subbarao et al., 1979). It is this mature type of B cell which is responsible for the immune response against TI-2 antigens. However, based on Lyb5 expression and the differential expression of IgM and IgD, the majority of B cells in the lymph nodes fall within this mature B cell subset, indicating that additional factors are required for optimal TI-2 responses (Huber, 1982; Hardy et al., 1983). Experiments in which the response against the TI-2 antigen TNP-Ficoll was analyzed, using B cells which were carefully depleted of accessory cells and T cells, made it clear that, for optimal TI-2 immune responses, the presence of lymphokines was required (Endres et af., 1983; Pike and Nossal, 1984; Dekruyff et al., 1985). Using purified lymph node cells and spleen cells, Goud et al. (1988) were able to induce an immune response against TNP-Ficoll with lymph node B cells when IL-1 was added to the cultures. Addition of IL-2 or IL-4 was not sufficient for the induction of

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this response. In recent experiments these authors have described a regional differentiation of B cells to respond to TI-2 antigens. Mesenteric lymph node B cells, but not peripheral lymph node B cells, could be stimulated in virro by TNP-Ficoll, and the response of peripheral lymph node B cells could be induced by the addition of IL-5 or IL-6 (Goud et al., 1991). Such a stimulating effect of IL-5 on B cells in the response against TNP-Ficoll could not be found using peritoneal B cells (Wetzel, 1990). In this respect an interesting observation on the expression of the IL-5 receptor on splenic B cells has recently been published (Rolink et al., 1990). Using a monoclonal antibody against the IL-5 receptor, R52.120, it was found that 2-4% of splenic B cells expressed high levels of the receptor. These cells were IgM positive, but Ly-1 negative. Analysis of the variable-region heavy-chain gene family utilization revealed that these cells did not differ from the vast majority of the rest of the splenic B cell pool. Up-regulation of the IL-5 receptor on B cells that do not express detectable levels of the receptor by various activation protocols, including LPS, anti-IgM antibodies, interleukins, and combinations, has been unsuccessful so far. This led Rolink et al. to conclude that B cells with high levels of IL-5 receptor may represent a different lineage. Are they the cells involved in the TI-2 responses? They are undetectable in the lymph nodes, but their position in the spleen seems to be random (A. C. Rolink, personal communication). Attempts to modulate the TI-2 response by injection of anti-IL-5 receptor antibody have not yet given conclusive answers.

2. Deficiencies in TI-2Immune Responses In addition to the X-linked immunodeficiency of the CBA/N mouse, other deficient responses against polysaccharides and relationships with the spleen have been described. In the C57BR/cdj mouse hormonal factors have been implicated in the poor responses against polysaccharides (Cohn, 1986; Cohn and Schiffman, 1988). C57BR/cdj mice are capable of producing low antibody levels against SXIV polysaccharide in the absence of the spleen. Although in the CBAlN mouse smaller marginal zones were found (Kraal et al., 1988a), in the C57BR/cdj mouse no anatomical differences could be demonstrated in the presence and distribution of B lymphocytes and macrophage subpopulations in the marginal zone of the spleen (G. Kraal and D. A. Cohn, unpublished observations). Allotype-linked genes have also been implicated in the response against polysaccharides, but the restrictions in these systems are often not as stringent as in the CBA/N mouse. In humans the response against the bacterial polysaccharide SXIV is correlated with the G2m(n) immunoglobulin allotype, but not the response against the SIII polysaccharide

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(Ambrosio et al., 1985). A comparable situation has been described in the mouse (Makela et al., 1980; Pansanen and Makela, 1984).

3. Accessory Cells and the Response against TI-2 Antigens The above findings indicate that the responses against TI-2 antigens are probably under multiple control, not in the least by the presence and structure of the spleen. Several findings seem to run parallel: ( I ) immune responses against polysaccharide antigens appear late in ontogeny, coinciding with a relative immaturity of the marginal zone of the spleen, (2) they are dependent on the presence of the spleen, and (3) the B cells responsible for the response are within a mature subset that also appears rather late in ontogeny. The majority of marginal zone B cells as such, with their intermediate size and low surface IgD expression, do not fall within the category of cells involved in TI-2 responses. It is therefore more likely that the importance of the spleen and the marginal zone in the immune responses against polysaccharides lies in the presence of an accessory cell. Of course, the extremely intriguing finding that marginal zone macrophages selectively take up TI-2 antigens and are positioned strategically in the marginal zone has led to many assumptions on the role of these macrophages as accessory cells in TI-2 immune responses. How important are the marginal zone macrophages? As outlined in Section 111,B,2, the marginal zone macrophages specifically take up neutral polysaccharides and can be distinguished by monoclonal antibodies such as ER-TR9 in the mouse and ED3 in the rat. The uptake of polysaccharides is probably mediated by a protein receptor and can be inhibited by mannan and zymosan (Chao and MacPherson, 1990) or, in the case of the mouse, by the ER-TR9 antibody. This polysaccharide inhibition does not prevent the cells from phagocytizing carbon. Claassen et al. (1986a,b) assumed that when the marginal zone macrophages are directly involved in the processing and presentation of TI-2 antigens, a direct association must be visible between these cells and the specific antibody-forming cells. However, using a method to visualize B cells containing specific antibody, no relationship could be found between the localization of anti-TNP antibody-forming cells and marginal zone macrophages in the spleen when rats were injected with the T celldependent antigen TNP-KLH or TNP-Ficoll. Only 2 days after injection of the antigens, the first antibody-forming cells could be detected in tissue sections. They were predominantly located in the outer PALS and in the coaxial sheaths of lymphoid tissue surrounding the terminal arterioles, but no antibody-forming cells were found in the marginal zone. Furthermore, the localization of these cells was indistinguishable after TNP-KLH or TNP-Ficoll immunization, suggesting that a specific role of the marginal

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zone macrophages in responses against TNP-Ficoll is not likely, although not impossible. In mice the ER-TR9 antibody reacts specifically with the marginal zone macrophages and is furthermore capable of blocking the uptake of neutral polysaccharides such as Ficoll. Therefore, experiments were begun to determine whether responses against TI-2 antigens could be modulated by this antibody. Injections of the antibody prior to injection of the antigen led to a complete blockade of antigen uptake by these macrophages, but no reduction in the antibody response was observed as determined by plaqueforming cell assay (Kraal et al., 1989b). We then used the antibody to eliminate marginal zone macrophages, by coupling purified antibody to gelonin. Gelonin is a toxin that can be obtained by extraction from the seeds of the umbellifer Gelonium mult$orum. The gelonin is a 30-kDa molecule that interferes with protein synthesis by ribosome inactivation (Gasperi-Campani et al., 1980; Stirpe et al., 1980).It has the same intracellular target as ricin, but it is not toxic for intact cells; therefore, it is relatively safer to use than ricin. Using ER-TR9 coupled to gelonin, we were able to specifically eliminate the marginal zone macrophages in the spleen. However, even in these animals a normal response against TNPFicoll could be demonstrated. These results made it unlikely that the marginal zone macrophages were directly involved in antigen handling in TI-2 antigen responses, although it cannot be ruled out that their function can be taken over by other antigen-presenting cells. Our results were quite in contrast to earlier experiments in which the method for macrophage elimination (Van Rooijen, 1989), as outlined in Section III,B,3, was used to study the role of the marginal zone macrophages (Claassen et al., 1986b).Intravenous injection of C12MDPencapsulated in liposomes led to a complete elimination of red pulp macrophages as well as the macrophage populations in the marginal zone of the spleen. After immunization with a T cell-dependent antigen (TNP-KLH) or a T cell-independent antigen (TNP-LPS), no differences were found in antibody responses between treated and control mice. However, in these mice an injection with TNP-Ficoll as a TI-2 antigen led to a strong decrease of the antibody response in the macrophage-depleted animals. These results are certainly suggestive for a role of the marginal zone macrophages in the immune response against TI-2 antigens. However, in later experiments these authors were not able to reproduce these results. This may have been the result of the liposome preparations used in the earlier experiments, in which an effect was also seen on the B cells in the marginal zone of the depleted spleen, an effect not observed in later ClzMDP-liposome preparations. In fact, there are now several reports, using different approaches, that clearly indicate that the marginal zone

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macrophages have a limited role, if any, in the response against TI-2 antigens. An interesting observation concerning the limited role of the marginal zone macrophages was made using autotransplants (Claassen et al., 1989). In mice splenic tissue was autotransplanted, leading to restoration of splenic tissue after initial necrosis. In these autotransplants the different compartments of the spleen develop again, and during the course of this development the capacity of the tissue fragments to mount an immune response against TNP-Ficoll was studied. Four weeks after surgery a normal marginal zone was seen with its typical relatively large B cells. At the border of the marginal zone and the PALS, the marginal metallophilic MOMA-I macrophages were present, but at this time no marginal zone macrophages could be seen. In fact, after only 10 weeks restoration of the marginal zone macrophages was found, as determined by immunohistochemistry for ER-TR9. However, only 4 weeks after transplantation a full restoration of time course and peak of anti-TNP titer against TNP-Ficoll was observed, and normal numbers of antibody-forming cells were observed in the splenic tissues, despite the absence of marginal zone macrophages. These findings clearly indicate that it is not so much the marginal zone macrophages that are important for the initiation of the immune response, but rather the structure of the marginal zone itself. What, then, is the function of the marginal zone macrophage? The large phagocytic capacity of the marginal zone macrophages and their strategic position point to an important role in antigen processing. However, experiments in which macrophages from the spleen were eliminated by liposome treatment clearly showed that these cells were dispensable in the response against protein as well as polysaccharide antigens. The reason for this may lie in the fact that the nature of these antigens does not require elaborate processing and that they can be presented efficiently by less phagocytizing cells, such as dendritic cells or B cells themselves. This would argue that the response against particulate antigens is more dependent on the presence of the marginal zone macrophages. This was investigated by Delemarre ef al. (1990a), who depleted mice of their splenic macrophages, as well as the macrophages in popliteal lymph nodes, and looked for the responses against haptenated sheep red blood cells as particulate antigens. In contrast with the findings for soluble antigens, a strong decrease in the response was found after macrophage depletion in the spleen. Surprisingly, no effect was seen of the depletion of lymph node macrophages on the response against particulate antigens. This is in agreement with earlier observations by this group that in the lymph node depletion of macrophages by subcutaneous liposome administration did not result in changes of the immune response against soluble

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antigens (Delemarre et al., 1990b). The differences in response between macrophage-depleted spleen and lymph nodes and the haptenated sheep red blood cells could be the result of administration of the antigen. Intravenously injected erythrocytes directly enter the spleen and are phagocytized by the marginal zone macrophages. This handling is obviously necessary before the antigens can be presented and recognized. In the case of subcutaneous administration of the erythrocytes for lymph node sensitization, it can be envisaged that transport via the tissue fluids and afferent lymph may have damaged the red blood cells sufficiently, leading to antigenic fragments that can be presented directly without further processing. The important role of the marginal zone macrophage in the processing of the haptenated red blood cells was demonstrated in a time course experiment after depletion of splenic macrophages (Delemarre et af., 1991). As outlined in Section III,B,3, the various macrophage populations in the spleen show large differences in their repopulation kinetics after elimination by liposome treatment. Red pulp macrophages return relatively fast, but especially the marginal zone macrophages take several weeks for complete reconstitution. This way a splenic architecture can be accomplished which selectively lacks the marginal zone macrophages. Using such animals, Delemarre el af. were able to show that only normal responses against the sheep red blood cells could be found after full restoration of the marginal zone macrophages. Marginal zone macrophages must therefore be considered potent phagocytizing cells positioned strategically in the marginal zone. The selective uptake of bacterial polysaccharides must be seen as an additional advantageous characteristic by which bloodborne bacteria stick to the macrophage cell membrane without the need for prior opsonization.

4. Immune Response and Role of the Marginal Zone a. Primary Immune Responses The importance of the marginal zone lies in the reduced arterial blood flow that opens in this area, enabling phagocytizing cells to clear the blood of possible pathogens. For an immunological reaction to occur, it is necessary that antigens are presented to T and B cells with the relevant antigenic specificity. It has been demonstrated that for primary T cell-dependent immune responses, dendritic cells are necessary (Steinman and Nussenzweig, 1980; Austyn, 1987). Dendritic cells are mainly present in the T cell-dependent PALS areas of the spleen. In the PALS, especially in the outer PALS just inside the marginal zone, many activated lymphocytes can be found after antigenic stimulation. Here, the classical plasmacellular reaction takes place (Langevoort, 1963). It is also a site of extensive cell traffic: Both T and B cells enter the outer PALS after leaving the marginal zone, and it is here that cell contacts can be made.

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How can antigen that enters the spleen via the marginal zone reach the outer PALS to be presented to T and B cells? The situation in the spleen may well be analogous to that in the lymph node. Langerhans cells in the skin pick up antigenic fragments and transport the antigen on their surface to draining lymph nodes via the afferent lymphatics. As veiled cells, they move to the T cell-dependent paracortical area of the lymph node, where they can be found as dendritic interdigitating cells. Here, as in the outer PALS of the spleen, extensive lymphocyte traffic takes place by means of the many high endothelial venules present in this area. This way a continuous renewing of the potential of antigen specificities in terms of B and T cell antigen receptors is ensured, and optimal interaction among T, B, and dendritic cells can take place. A similar situation may occur in the spleen, where it has been demonstrated that dendritic cells can enter from nonlymphoid sites via the blood stream (Austyn et al., 1988a;Larsen et al., 1990). From the marginal zone, where they initially localize, migration takes place into the outer PALS, where interaction with lymphocytes occurs. The antigen they transport may have been derived from their original sites, but antigenic fragments produced by the macrophages in the marginal zone may also be taken up by the dendritic cells during their sojourn in the marginal zone and transported to the outer PALS. This way cooperation between marginal zone macrophages and dendritic cells ensures an optimal presentation of antigen in the (compared to the marginal zone) less turbulent environment of the outer PALS. As demonstrated, in vitro T cells and dendritic cells rapidly cluster independently of antigen and MHC (Inaba and Steinman, 1986).In these clusters a selection takes place of antigen-specific T cells; T cells that do not possess the appropriate T cell receptors leave the clusters (Austyn et al., 1988b).Translated to the situation in uiuo, this means that in the outer PALS continuous enrichment takes place of antigen-specific T cells, which, in turn, can activate passing B cells. Eventually, this leads to antibody-forming cells that can be found in large numbers in the outer PALS, but never in the marginal zone (Fig. 7). b. Secondary Immune Responses Some of the activated antigen-specific B lymphocytes do not turn into antibody-secreting cells, but develop into memory B cells. This is thought to occur in the follicles under the influence of the follicular dendritic cells, in combination with formed immune complexes (Klaus et al., 1980; Van Rooijen, 1990a). Memory cell formation involves somatic mutations of the immunoglobulin V regions and class switching (Kocks and Rajewsky, 1989; Kraal et al., 1982), leading to circulating memory cells that are long lived. Interestingly, indications have been given that memory B cells are located in the marginal zone. When rats were immunized with T cell-dependent hapten-protein conjugates,

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FIG. 7 Localization of antibody-forming cells. Specific anti-trinitrophenol (TNPbforming cells in a section of mouse spleen 5 days after injection of TNP-KLH (keyhole limpet hemocyanin). The antibody-forming cells are localized in the outer periarteriolar lymphocyte sheath and around a terminal arteriole (ta), but not in the marginal zone. rp, Red pulp; wp, white pulp; ca, central arteriole. Bar = 150 prn.

substantial numbers of hapten-binding cells could be found in the marginal zone (Liu et al., 1988).These cells had been in the cell cycle shortly before arriving in the marginal zone and showed the characteristic phenotype of marginal zone B cells, expressing IgM, but not IgD. Following reimmunization with the hapten conjugate, these cells disappeared from the marginal zone, and hapten-binding cells were found in the PALS areas and follicles. In these compartments the cells were found to be in the active cycle, giving rise to both antibody-forming cells and, again, marginal zone hapten-binding cells. These authors concluded that these cells are memory cells because they have obviously undergone antigen-specific proliferation and can be induced to reenter the cell cycle after renewed antigen exposure. Hapten-binding cells could not be found after immunization with TI-2 polysaccharide antigens, in concordance with the poor memory formation found with such antigens. However, coimmunization with LPS could lead to hapten-binding marginal zone cells specific for polysaccharide antigen and subsequent secondary responses (Zhang et al., 1988). The presence of memory cells in the marginal zone is in line with our recent findings that marginal metallophilic macrophages in the mouse and ED3

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macrophages in the rat express a receptor which can interact with activated T and B lymphocytes and which may be involved in the retention of activated memory cells at these sites (see also Section III,B,I). Therefore, in the case of a secondary response, B cells themselves can transport antigen to the PALS area and present it to T lymphocytes. The antigen, as in the case of the primary antigen presentation by dendritic cells, could have been processed by macrophages in the marginal zone (Van Rooijen, 1990b). Such ideal localization of memory B cells with intimate contacts with marginal zone macrophages in an area where all bloodborne antigens pass creates an optimal line of defense.

V. Summary The marginal zone of the spleen forms an intriguing area in which a variety of cell types are combined. Several of these cell types seem to have a fixed position in the marginal zone, such as the marginal zone macrophages, the marginal metallophilic macrophages at the inner border, and, to a lesser extent, the marginal zone B cells. For other cell types-T lymphocytes, small B cells, and dendritic cells-the marginal zone is only a temporary residence. It is this combination of relatively sessile cell populations and the continuous influx and passing of bloodborne immunocompetent cells that turn the marginal zone into a dynamic area, particularly apt for antigen processing and recognition. In no other lymphoid organ can such a unique combination of cells and functions be found. The opening of the arterial blood stream in the marginal sinuses results in a reduction of the velocity of the blood stream, and antigens are initially screened in the marginal zone. To this, extremely potent phagocytic cells, the marginal zone macrophages, are present which can take up and phagocytize large foreign particles, such as bacteria and effete red blood cells. Further filtration of the blood takes place in the filtration beds of the red pulp. The marginal zone macrophages express membrane receptors for bacterial polysaccharides which lead to efficient phagocytosis, probably even in the absence of prior opsonization. Antigenic fragments produced this way can be taken up by dendritic cells that enter the spleen by the blood as part of a mobile surveillance immune system. Dendritic cells present antigen to T cells in the outer area of the T cell-dependent PALS, leading to clustering and enrichment of antigen-specific T cells. Antigens in the marginal zone can also directly associate with memory B cells thought to reside here for longer times, having intimate contact with the

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marginal zone macrophages. B memory cells then migrate into the PALS and present antigen to T cells. The marginal zone therefore functions not only as an area of initial filtration and phagocytosis of antigens from the blood, but also as a site of lymphocyte emigration. Some of the incoming T and B lymphocytes in the recirculating pool enter the white pulp from the marginal zone. The underlying force and selective molecular mechanisms that guide this migration are unknown. Both B and T lymphocytes recirculate through the outer PALS area on their way to the follicles and the inner PALS, respectively. This way the outer PALS forms an extremely important area in which there is a continuously changing repertoire of antigen receptor specificities in the presence of antigen-presenting cells. This explains the efficiency of the spleen in the induction of specific immune responses. The importance of the spleen in terms of specific immunity lies predominantly in its capacity to deal with TI-2 antigens. The marginal zone macrophages, with their receptors for TI-2 antigens, however, are found not to be an absolute requirement for these type of responses. It seems more likely that the efficacy to deal with TI-2 antigens is determined by a combination of factors, such as the local production of interleukins, the susceptibility of certain B cell subsets to these interleukins, and the presence of marginal zone macrophages. Acknowledgments I am indebted to all the members of the Department of Cell Biology for their support over the years. Special thanks are due to Christine Dijkstra and Nico Van Rooijen for their helpful suggestions and discussions during the preparation of this review and for the pleasant way in which our collaboration has always taken place.

References Ahmed, A., Scher, J. I . , Sharrow, S. O., Smith, A. H., Paul, W. E., and Sachs, D. H. (1977). J . Exp. Med. 145, 101-1 10. Altschul, R., and Hummason, F. A. (1947). Anat. Rec. 97,259-264. Ambrosio, D. M . , Schiffman, G., Gotschlich, E., Schur. P. H., Rosenberg, G. A., De Lange, G. G., Van Loghem, E., and Siber, G. R. (1985). J . CIin. fnuest. 75, 1935-1942. Amlot, P. L., and Hayes, A. E. (1985). Lancet 1, 1008-1011. Amlot, P. L., Grennan, D., and Humphrey, J . H. (1985). Eur. J . fmmunol. 15,508-512. Amman, A. J., Addiego, J., Wara, D. W., Lubin, B., Smith, W. B., and Mentzer, W. L. (1977). N . Engl. J . Med. 287,897-900. Amsbaugh, D. F., Hansen, C. T., Prescott, P., Stashak, P. W., Barthold, D. R., and Baker, P. J. (1972). J . Exp. M e d . 136,931-949. Andrew, W. (1946). Am. J. Anat. 79, 1-73.

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Role of the Cytoskeleton in Genome Regulation and Cancer Theodore T. Puck*,+,*and Alphonse Krystosek' * Eleanor Roosevelt Institute for Cancer Research, Denver, Colorado 80206

' Departments of Medicine, and Biophysics, Biochemistry, and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262 * University of Colorado Cancer Center, Denver, Colorado 80262

1. Introduction Perhaps the most important conceptual problem in mammalian genetics involves understanding cellular genome regulation. The only comprehensive model for genome regulation available so far has been that which functions in cells such as Escherichia coli. This simple system involves a single chromosome in which regulated genes are, for the most part at least, controlled by the operon mechanism. Thus, groups of one, two, or three contiguous genes are simultaneously turned on or off in accordance with the reproductive needs of the organism in a particular medium. By and large, therefore, E. coli cells with the same gene structure behave in identical fashion. The situation is very different in the mammalian cell. The genome contains approximately 1000 times as much DNA as E. coli, and it is distributed among scores of chromosomes. All the cells of the body have the same chromosomes and genes. However, the cells of tissues such as brain, liver, and pancreas each display a different spectrum of gene activities. Moreover, the cells of these various tissues also display different and characteiistic morphologies which can be identified by microscopic examination. In this chapter we present evidence for the existence of two levels of genome regulation in the mammalian cell and show that the cytoskeleton and perhaps other fiber systems are involved in this regulation. Another of the unsolved problems of mammalian cell biology involves the molecular nature of cancer. While the existence and biochemical activities of a large number of different oncogenes have been demonstrated, and many metabolic differences between the behavior of a variety of cancer cells and their normal counterparts have been elucidated, a Intemarional Rruirw of C?toluyy, Vol. 132

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comprehensive picture of the fundamental difference in gene regulation between normal and cancer cells is still not available. In this review we apply the two-tiered theory of regulation of the mammalian genome (Puck et al., 1990) to the problem of cancer. We present evidence that, in an impressive number of cancers, the first stage of the regulatory system in which the cell cytoskeleton is involved is defective. Experimental evidence and theoretical conclusions are presented. In addition, speculative extensions of these conclusions are considered. Space does not permit review of the voluminous literature on the structure and properties of the mammalian cell cytoskeleton. A small and fairly arbitrary selection of previously demonstrated facts is presented in Section VIII.

II. Reverse Transformation Reaction and Role of the Cytoskeleton A. Rationale of the Approach

Cancer represents a distortion of normal metabolic regulation. Cancer cells exhibit a loss of differentiation properties and a concomitant loss of growth control. While occasional cancers retain some of their differentiation characteristics, as in the ability to take up iodine by thyroid cancer cells, a marked change occurs in at least some cellular differentiation characteristics. Finally, the morphology of cancer cells is almost invariably changed to so large an extent that cancer cells can be identified morphologically under the microscope. While this latter fact is generally taken as evidence of deep-seated change in the cytoskeleton of most cancer cells, specific instances of fairly gross cytoskeletal changes in malignancy have been described (Porter er al., 1974). Carcinogenesis involves three distinct steps. The first is initiation, which can be brought about by genetic mutation. The second step is promotion, a molecularly undefined change that must occur after the initiation step. Finally, the process called progression occurs over a fairly prolonged period, during which the cancerous cells become steadily more malignant, presumably through evolutionary selection. A model of the cancer process should also account for the promotion process. The phenomenon of reverse transformation (discussed below) offers a unique opportunity to study the molecular nature of cancer. Studies comparing cancer and normal cells isolated from the same tissue and grown in tissue culture always involve some uncertainty about the origin of the cell lines compared. It is possible that these may contain differences not

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directly related to the malignancy process under study. In reverse transformation the fact that the same cells can be transferred back and forth between the normal and malignant condition insures validity of the comparisons.

6. Phenomenon of Reverse Transformation Significant illumination of the molecular basis of malignancy has come from study of the reverse transformation reaction. This phenomenon, in which cancer cells revert to the normal phenotype in the presence of cell-specific agonists and return to their original malignant behavior on its removal, was first described for the cancerous Chinese hamster ovary (CHO) cell (Hsie and Puck, 1971) and the rat sarcoma cell (Johnson et al., 1971). CHO possesses all of the malignant cell stigmata, including the ability to kill injected nude mice. CHO and its subclone, CHO-K1 has been used as a model cancer cell for studies in our laboratory and others for many years. More recently, phenomena closely resembling reverse transformation have been described in a variety of other cells in different laboratories, and other names, such as redifferentiation, inducible differentiation, or reversion of malignancy, have been applied by other workers to describe what appears to be essentially the same or a very similar process. While cAMP appears to be the most universal of the reversetransforming agents identified so far, a number of other molecular species have also been described with similar effects in particular malignancies (Bloch, 1984; Freshney, 1985).

1. Components of the Reaction Upon treatment of CHO-K1 cells with cAMP derivatives, a series of changes takes place, some of which begin within a matter of minutes and others of which are not established for approximately 48-72 hr. Reverse transformation in CHO-Kl cells can be demonstrated by the addition of a variety of agents which increase the cAMP concentration within the cell (i.e., dibutyryl CAMP, 8-Bromo CAMP, isobutyl methyl xanthine, or forskolin), and the effect is intensified by the simultaneous addition of agents such as testosterone, testololactone, or prostaglandin (Porter et at., 1974; Puck et al., 1972; Puck and Wenger, 1973). The sequence of events which occurs upon raising the level of cAMP inside the CHO-Kl cells is as follows: (1) New patterns of protein synthesis and phosphorylation are established (Gabrielson et at., 1982; Chan et al., 1989; Miranti and Puck, 1990). (2) A deep-seated reorganization of the cytoskeleton occurs in which the fibrous elements change from a sparse

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and relatively disorganized system to a dense pattern of largely parallel elements like that typical of normal fibroblasts (Hsie and Puck, 1971; Hsie et al., 1971; Puck and Jones, 1973). (3) The cell morphology changes so that the compact pleomorphic form of malignant form rearranges to assume the morphology of a typical fibroblast. (4) A variety of changes in the cell membrane occur, including the disappearance of a set of oscillating knobs or blebs, the deposition of fibronectin around the cell membrane, changes in active transport, and the capping response to particular lectins (Nielson and Puck, 1980; Storrie, 1973, 1974, 1975). (5) Density control of growth and elimination of growth in suspension are initiated (Puck, 1979, 1987). (6) A marked increase in the sensitivity of a specific fraction of the DNA to hydrolysis by DNase I occurs. We have named the latter reaction genome exposure (Schonberg et al., 1983; Ashall et al., 1988; Puck, 1983; Ashall and Puck, 1984). Weintraub and Groudine (1976) first demonstrated that in chick red blood cells, fibroblasts, brain cells, and red blood cell precursors, different DNAs are hypersensitive to hydrolysis when isolated nuclei are treated with DNase I, and they proposed, therefore, that active genes are preferentially digested by this enzyme. Garel et al. (1977) showed large tissue-specific sensitivities of particular genes in different tissues and proposed that this sensitivity is a measure of the transcriptional potential of a given cell type. Our work has applied these considerations to cancer. We have shown that in a variety of malignant cells, the amount of the hypersensitive DNA is greatly reduced over that found in the corresponding normal cells, and we proposed that conversion of genes from the relatively resistant to the hydrolysis-sensitive state (which we call gene exposure) is the necessary first step for gene expression. We also have demonstrated the necessary role of the cytoskeleton in this reaction (Ashall and Puck, 1984; Krystosek and Puck, 1990; Miranti and Puck, 1990; Ashall et al., 1988; Puck, 1987). The genome exposure reaction is fairly massive, affecting approximately one-tenth to one-third of the genome (Ashall and Puck, 1984).DNA with high hydrolytic sensitivity produced by reverse transformation is called exposed DNA, as opposed to the remainder, which we call sequestered DNA. Data indicating the approximate time course of some of these reactions have been presented (Ashall et al., 1988).

2. Cancer as a Phenomenon Involving a Decrease Rather Than an Increase in Active Genes The fact that all of the cancer cells tested so far demonstrate a significantly reduced level of gene exposure implies that fewer active or potentially active genes are present. While at first this appears to be at variance with

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the traditional view of cancer as a hyperactive cell, the apparent contradiction is readily resolved: In the malignant cells specific differentiation genes are sequestered and therefore cannot function. These include the genes responsible for the tissue-specific control of reproduction. Hence, the cancer cell lacking these restraints continues to reproduce without limit. Thus, the cancer cell is more active reproductively because it lacks the functions of the differentiated growth control genes.

3. Demonstration That in the Cancerous State CHO-K1 Cells Lose the Rim of Exposed Genes around the Nuclear Periphey Hutchison and Weintraub ( 1985) first demonstrated a peripheral distribution of DNase I-sensitive DNA around the nuclear periphery of the immortalized, but noncancerous, L cell. Nick translation experiments demonstrated that the region of distribution of exposed DNA which sets apart the normal from the malignant form of the CHO-Kl cell is confined to a shell encompassing the periphery of the nucleus (Krystosek and Puck, 1990). We have demonstrated that both ovary-derived normal fibroblasts and the CAMP reverse-transformed cells exhibit a clear region of DNase I-sensitive DNA around the nuclear periphery, but this exposed region of DNA is absent in the malignant form. Other cancer cells behave similarly (Krystosek and Puck, 1989)(Table I and Fig. 1). All of the noncancer cells tested by us so far display the peripheral nuclear pattern of exposed DNA. It appears valid to conclude, then, that at least an important class of cancers represents a defect in genome exposure. This defect is of a surprisingly large magnitude, rendering cancer cells hardly labeled in our standard nick translation procedure, which intensely marks the nuclear shell of normal cells (Krystosek and Puck, 1990). 4. Requirement of an Intact Cytoskeleton for Restoration of the Region of Exposed DNA in the Reverse Transformation of Malignant CHO-K1 Cells Of particular importance is the fact that cytoskeleton-disorganizingagents such as colcemid or cytochalasin B prevent the basic features of reverse transformation. This applies not only to effects such as the changes in cell morphology, but perhaps more significantly to the reappearance of the shell of exposed DNA in the CHO-Kl cancer cell treated with reverse transformation agents (Ashall and Puck, 1984; Krystosek and Puck, 1990). These findings indicate a direct relationship between the cytoskeleton and the shell of exposed DNA around the nuclear periphery, which constitutes

TABLE I Demonstration of Genome Exposure Defect in All of the Cancer Cells Tested, but None of Their Normal Cell Counterpartsa

Cancer cell

Presence of a visible region of genome exposure in the nuclear periphery

CHO-K I Chinese hamster ovary

0

CHO-KI Chinese hamster ovary

0

ASV-transformed vole fibroblast IT

0

Rat glioma C6

0

LAN-I human neuroblastoma LAN-2 human neuroblastoma LAN-5 human neuroblastoma PC12 rat pheochromocytoma

0

NG108-IS neural mouse-rat hybrid, tumor Colo 679 human melanoma RPMI7932 human melanoma HeLa human cervical carcinoma U937 human lymphoma

0

0

0 0

0 0

0 0

Noncancer comparison cell cAMP reversetransformed CHOKI Normal Chinese hamster ovary fibroblast cAMP reversetransformed ASVtransformed vole fibroblast cAMP or hydrocortisone reverse-transformed C6 Retinoic acid reversetransformed LAN- I Retinoic acid reversetransformed LAN-2 Retinoic acid reversetransformed LAN-5 Nerve growth factor reverse-transformed PC12 cAMP reversetransformed NG108IS Normal human melanocyte Normal human melanocyte 8-CI cAMP reversetransformed HeLa Retinoic acid reversetransformed U937

Presence of a visible region of genome exposure in the nuclear periphery

+

+ + +

+ + +

+ + +

+

+ +

Genome exposure was measured by in siru nick translation. A typical example is shown in Fig. I . Normal fibroblasts, lymphocytes, and melanocytes, as well as the 10 reversetransformed malignant cells, demonstrated typical peripheral nuclear labeling of DNase I-sensitive nuclear chromatin, as in Fig. 1B. All 12 cases of cancer cells tested showed little or none of this exposed DNA pattern (Krystosek and Puck, 1990; Krystosek er uf., 1992) (Fig. IA). ASV, avian sarcoma virus. 80

FIG.1 Demonstration of the difference in DNA exposure pattern revealed by nuclei of (A) a typical cancer cell and ( B ) the corresponding normal or reverse-transformed cell. (A)The cell nuclei are malignant CHO-KI cells; (B) the same cell after reverse transformation with CAMP derivatives. Both types of cells were subjected to the nick translation procedure, which permits visualization of the exposed DNA region (1.e.. DNase I-sensitive region) as a shell around the nucleus (Krystosek and Puck, 1990). Each of 12 different cancer cell lines tested behaved as in (A), while their normal or reverse-transformed counterparts resembled that in (B) (Krystosek et al.. 1992). 81

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the differentiating genetic feature between the malignant and reversetransformed states of cells such as CHO-Kl. It should be noted that de Graaf et al. (1990) studied the threedimensional distribution of DNase I-sensitive chromatin regions in interphase nuclei of embryonal carcinoma cells and of a presumably differentiated variant of these, but found the same patchy distribution of DNase I-sensitive chromatin regions preferentially, but not exclusively, located at the nuclear periphery in both cells. Further study of the change in DNase I sensitivity of nuclear DNA at different stages of the transition between normal and malignant cells is required. 111. A Theory of Mammalian Cell Genome Regulation Involving the Cytoskeleton

We have proposed a two-tiered scheme for the regulation of the mammalian genome which has an extra step beyond that recognized in E . coli (Puck et al., 1990). This scheme accounts for the mode of action of genes that are differentiation specific in the mammalian organism. The first step in the activation of such tissue-specific genes is their conversion from the sequestered to the exposed state. This involves transfer of the appropriate DNA from the interior of the nucleus to the region of the nuclear periphery and the necessary conformational changes and specific protein interactions that render such DNA susceptible to hydrolysis by DNase I. We propose that this conversion of these genes to sensivity to DNase I hydrolysis also renders them susceptible to reaction with inducers, repressors, and other transcriptional factors. The second step, then, which resembles the corresponding process in bacteria, is activation or inactivation of the exposed genes as a result of interaction with appropriate effector molecules in the surrounding medium. Thus, it is necessary, but not sufficient, for differentiation-specific genes to be exposed before they can be activated. This first step of exposure may occur early in lineage determination during normal development, and it may be separable in time from actual transcriptional activation. Similarly, the restoration of cytoskeletal control of nuclear function in reverse transformation allows increased genome exposure and access of DNA to specific transcriptional regulators. A. Role of the Cytoskeleton

Study in many laboratories of the action of a large number of growth and differentiation agonists and antagonists in the mammalian cells has revealed a critical role played by membrane receptors and their associated structures, by phosphorylation and dephosphorylation phenomena, by a

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variety of oncogenes, by calcium and other cations, as well as our own demonstration that an organized cytoskeleton is necessary for the genome exposure reaction. Our theory of genome regulation has provided a unifying conceptual picture for the roles of all these elements. The most unusual feature of this particular formulation may be its ascribing a genome regulatory role to the cytoskeleton, a structure whose regulatory role was not previously generally recognized (Willingham et nl., 1977; Alberts et al., 1989). The picture that we have developed proposes a joint action of the mammalian cell fiber systems, presumably including the extracellular matrix, the three components of the cellular cytoskeleton, and the nuclear matrix. It has been proposed for some time that the nuclear matrix plays an important role in genome regulation (see, e.g., Pienta et a/., 1989).Demonstration that the cytoskeleton is directly involved in the process of genome exposure led us to a more general picture involving an interaction of the fiber systems as a whole. Our model of the role of the fiber system in controlling genome exposure has been suggested by the known effect of the cytoskeleton during mitosis (Puck, 1977).In this phase of the life cycle, attachments are established, linking the microtubules by way of the kinetochore to the centromere of each chromosome. Concomitant with the establishment of these connections, chromosomal condensation around the region of attachment occurs so that the overall length of the chromosome decreases by a factor of 20,000 as compared to naked DNA; the thickness increases appropriately, and gene expression ceases. We have taken this chromosome condensation process as a model of the sequestration process, which includes higher-order packing and protein interaction, the exact physicochemical nature of which remains to be elucidated in specific situations. The two essential processes are hypothesized to be the changes in specific regions of chromosomal sequestration or exposure, and the changes in attachment of specific fibers to appropriate points along the genome. The connection between these two processes remains to be elucidated.

B. Role of Repetitive DNA Sequences

Until the advent of the nick translation technique, it was not possible to visualize directly the location of exposed DNA. Data have been presented to show different degrees of DNA exposure and sequestration within the cell (Krystosek and Puck, 1990). The chemical relationships, if any, between DNA of various degrees of sequestration and the diverse heterochromatin patterns revealed by conventional staining procedures are so far unknown.

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We further proposed that during interphase specific regions of the chromosomal DNA are sequestered and others are exposed to match the differentiation needs of the particular tissue cell. This is accompanied by differential attachment of particular elements of the fiber system to specific points along each chromosome through the agency of specific attachment proteins. As a result certain chromosomal regions are transported to the nuclear periphery and are maintained largely in the exposed condition. Others are moved to the nuclear interior, where they are mostly in the sequestered state. By this formulation the interphase nucleus appears as a highly dynamic structure, in which specific regions of each chromosome are regulated with respect to geometric location and degree of exposure and sequestration in reactions controlled by the cell fiber system plus the auxiliary participating proteins. Sequestration is viewed as a result of higher-order packing and specific protein attachment, which occurs as a result of the attachment of specific fibers to particular points along the chromosome in a fashion resembling the condensation reaction of mitosis. The interphase events differ from those of mitosis in the following characteristics: When the attachment between the fibers and the chromosome occurs at the centromere, the entire chromosome condenses, whereas in interphase the specific attachment sites are distributed along each chromosome and produce limited regions of sequestration. In the case of mitosis, it is the microtubules which attach to the kinetochore protein, which, in turn, is linked to the centromere DNA. We cannot yet specify which fiber structures and which protein structures participate in the attachments, which presumably occur during interphase. It is possible that nuclear matrix fibers take part in these reactions. Also not yet understood are the specific chemical dynamics that induce sequestration or exposure as a result of different kinds of fiber interaction with different sites along each chromosome. However, the picture that emerges proposes that each differerntiation state is defined by the specific pattern of DNA sequestration and exposure, which occurs throughout the genome as a result of the specific fiber network established and its interactions with the chromosomes through the agency of specific participating proteins. The analogy with the events of mitosis can be extended. During mitosis attachment of the microtubules occurs only at the centromere region of each chromosome, which contains no coding sequences, but rather a high density of repetitive DNA stretches. We therefore postulate that herein lies the function of other families of repetitive sequences distributed throughout the entire mammalian genome. We suppose that these, together with their associated proteins, serve as the fiber binding sites and therefore focal points for specific and limited DNA conden-

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sation which is required for each step of differentiation. By this picture the distribution of a large fraction of the total DNA in these repetitive sequences becomes readily understandable, as is the fact that these repetitive sequences are highly conserved, indicating the existence of an important genetic function, rather than their constituting “junk DNA.” Experiments demonstrated that all three elements of the cell cytoskeleton are involved in the reverse transformation reaction (Ashall and Puck, 1984; Chan et al., 1989). Thus, disorganization of either the microtubules alone by colcemid or the microfilaments by cytochalasin B prevents reverse transformation and its associated genome exposure. Moreover, when either of these two fibrous elements is disorganized by appropriate chemical treatment, the intermediate filaments also collapse or greatly condense, as revealed by immunofluorescence experiments with appropriate antibodies (Lazarides, 1980). We propose as a working hypothesis that different proteins attach to different repetitive sequences under different metabolic conditions. These, in turn, may serve as connecting links to particular types of terminal sequences of the fiber system network, which eventually end up at specific chromosomal loci. Thus, we visualize that whether a given chromosomal region remains expanded and exposed, or condensed and sequestered, is, in large part, determined by the nature of the protein attached to its key repetitive sequences and the integrity of the fiber network of the cell. We conceive that various families of repetitive sequences can become associated with more than one possible protein, depending on the states of differentiation and metabolic activity in which the cell finds itself. Cell type-specific nuclear proteins have been described (Fey and Penman, 1988). The biosynthesis of specific nuclear proteins and the metabolic fluxes controlling patterns of cytoskeletal assembly are lineageand environment-controlled processes. The net result of these regulatory actions is that genomic configurations are set both by the history of the cell and its current contact with soluble and solid-state regulatory molecules. The ability to access the uniquely exposed genes allows appropriate gene responses to cell stimuli, completing an interlocking feedback loop. This scheme has a particular implication for cancer. It explains the behavior we described in a study of specific protein attachments to a representative human repetitive sequence that occurs 10,000 times in the genome. In a series of 10 human cancer cells, a particular protein of 66 kDa was found to attach specifically to this repetitive sequence. In 10 normal human cell lines this protein was not found (Law et al., 1989). These characteristics suggest a candidate protein for involvement in the inappropriate DNA sequestration process observed in aberrant cells.

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C. Existence of an Information Transfer System Involving the Cytoskeleton and Extending from the Cell Membrane t o Multiple Specific Sites on Each Chromosome

The picture that emerges involves a transfer of information from the cell surface via the cytoskeleton to the nucleus, resulting in exposure of a particular set of genes at the nuclear periphery. The cell membrane is deeply involved in these changes, since, upon the addition of CAMP to the CHO-Kl cell, the violently oscillating membrane knobs or blebs disappear, fibronectin becomes deposited at the cell membrane, changes arise in cell surface antigen activity and concanavalin A binding to these, cell adhesivity to glass and plastic surfaces changes, and specific transport activities are altered. It has been demonstrated (Burridge et al., 1988) that transmembrane junctions link the extracellular matrix and the microfilaments. Associations between membranes and microtubules have also been described (Murray, 1984). It seems quite possible, then, that similar junctions occur between the cytoskeletal fibers and those of the nuclear matrix. Thus, the cytoskeletal system of the mammalian cell forms an integral component of an information transfer mechanism, operating between the external environment and the DNA of the nucleus, bringing about a specific chromatin pattern of gene exposure and sequestration that is specific for each state of differentiation. By this visualization the cell cytoskeleton and the cell nucleus are not static structures, as they appear under ordinary microscopic examination of the cell. Instead, these structures are highly dynamic, rearranging in accordance with changes in cell differentiation so that a different specific set of genes finds itself in the exposed condition, arranged in the nuclear periphery. A rotational motion of chromatin has been described by DeBoni (1988), but its relationship to the placement of DNase-accessible chromatin is not known. We have also proposed that the housekeeping genes are clustered around the nucleoli and are exposed in both normal and malignant cells (Puck et af.,in press). Presumably the nucleoli act in the same way as does the nuclear surface, in furnishing access to newly synthesized gene products to egress from the nucleus.

D. Role of Phosphorylation Processes and Ca2+ Interactions

This conceptualization also furnishes a possible role for the different phosphorylation and dephosphorylation reactions that have been shown to occur as a result of cell treatment with a variety of growth and differentia-

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tion agonists and antagonists. We have proposed that at least one function of the bewildering array of phosphorylation changes that go on in response to many kinds of chemical stimulation is to change the geometry of the fiber network within the cell so as to cause a different array of chromosomal sites to be targeted, and hence a different pattern of chromosome exposure and sequestration to be achieved. It is conceivable that it also changes the specific protein attachment pattern to the critical DNA sequences involved in the genome exposure reaction. This concept also affords a possible explanation for the puzzling role of Ca2+fluxes in the cell activation which occurs in proliferation and differentiation (Bunn et al., 1990). Phosphorylation of protein molecules, which occurs in reverse transformation as well as in normal differentiation, creates a point of intense electronegativity at a particular site. We postulated that simple calcium binding to and neutralizing of these sites, as well as the formation of ion bridges, would change the configuration of the network and allow it ultimately to attach to new configurations along the chromosomes. There are probably multiple roles for an increase and decrease in intracellular calcium concentration following cell membrane stimulation. A rise in free Ca2+activates the actin filament-severing and -capping protein gelsolin, which could be a prelude to rearrangements in the polymer network. Calcium is also important in many metabolic systems, including the activation of kinases that phosphorylate cytoskeletal and nuclear proteins. Transmembrane signaling, which initiates Ca2+ fluxes, thus sets into play a complex set of covalent and noncovalent binding phenomena, among which change in the fiber system interactions with chromatin may be especially important. The model presented here visualizes the cytoskeleton as a connecting link transferring information from specific receptors and other important macromolecules on the cell surface and including connectors with the extracellular matrix, forming a continuous fibrous network that ultimately ends up on specific sites on each chromosome so as to cause specific condensation of the DNA chain and consequent sequestration of specific genes in the neighborhood of these attachment sites. Transcription proteins, which, by and large, are subject to regulation by Ca2+fluxes and phosphorylation, may thus be involved in these molecular actions. Indeed, Workman and Roeder (1987) have shown that the TATA box-binding factor (TFIID) modulates nucleosome assembly, allowing ready access to RNA polymerase. The description of sequence homology between a region of vimentin and the protein dimerization domain of the c-fos protein is intriguing in this regard, suggesting that cytoskeletal filaments may impinge on defined motifs at transcriptional loci (Capetanaki et al., 1990). The lamins are distributed in the same peripheral nuclear location as exposed chromatin, although they may not be physically coincident with

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the farthest reaches of nuclear DNA (Paddy et al., 1990). The protein machinery necessary for the exposure reaction now requires definition. In pursuit of such a program, we are currently studying the spatial localization of transcription factors, nuclear protooncogenes, and other nuclear proteins in transformed and reverse-transformed cells. The repetitive DNA sequences-which we visualize as serving to attach to particular proteins, which, in turn, attach to end groups of the fiber network to bring about a genome exposure pattern specific to various differentiation states-should thus constitute a new code different from the triplet DNA code that specifies the amino acid chain coded for by each gene. These considerations offer a relatively simple model explaining how cells of the different tissues with identical genomes display different metabolic patterns and also possess different and characteristic morphologies. It has long been recognized that cell morphology depends in a critical way on cytoskeletal structure (Byers and Porter, 1964). Thus, changes in the cytoskeletal network designed to bring about different patterns of genome exposure would concomitantly produce a different cell morphology in each tissue. Specialized polarities (e.g., the adhesion belts of epithelial cell sheets and the axons of neurons) contain cytoskeletal assemblies that are spatially segregated or built with isoforms of the major components. Thus, the different cytoskeletal conformations are visualized as establishing different chromosomal connecting patterns, as well as determining cellular morphology and participating in functions such as locomotor mechanisms. It is conceivable that different subsets of cytoskeletal complexes may be specialized for different kinds of functions within the cell. While we as yet have no evidence that the nuclear matrix fibers form part of this system, it remains a distinct possibility for future experiments to resolve. Preliminary electron micrographs (W. D. Meek and T. T. Puck, unpublished observations) showed that intermediate filaments of the cytoskeleton appear to attach to the perinuclear region of the nucleus, but the molecular identity of the targets is still obscure.

IV. Application t o Cancer A. Theoretical Considerations

The phenomenon of reverse transformation in which it was shown that malignant cells could be caused to assume a normal phenotype in the presence of a specific metabolite such as CAMP(Hsie and Puck, 1971) led

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to an experimental program in our laboratory that produced recognition of the role of the cytoskeleton in the process of genome exposure. The CHO-K1 cell which was reverse transformed by CAMPderivatives underwent deep-seated change in all three components of the cytoskeletal structure when the process of genome exposure was restored in this cell, demonstrating a relationship between the cytoskeleton and the cancerous condition. The malignant CHO-Kl cell was shown to have lost a large part of its exposed genome as compared with normal cells from the same tissue, or with the same CHO-Kl cell after its reverse transformation. We have since undertaken measurements of genome exposure in a variety of cancers. As of the time of this writing, in each of 12 cancers examined, the genome exposure process has been found to be greatly reduced in the cancer cell as compared with its normal counterpart or with itself after reverse transformation by appropriate agents. These results indicate that, at least in a very substantial class of cancers, the genome exposure step is defective. This finding, at first, seemed surprising, since we had previously entertained the idea that the cancer cell with greater growth activity would be more active genetically than normal cells. Reflection about the process, however, reveals that the behavior observed is to be expected. In the cancer cell a smaller number of specific differentiation genes must be active as compared with the normal cell, since it has lost differentiation characteristics. Its decreased genome exposure indicates a decreased number of specific differentiation genes with the potentiality for activation. These genes include the growth control genes specific for each tissue. Thus, elimination of growth control can make the cancer cell more active metabolically, but fewer genes are active or potentially active. It is important to note that the conceptual picture presented here views cancer as a disease due to a defect in genome exposure which causes loss of growth regulation, as well as other specific differentiation properties. Thus, it becomes clear why both of these features are associated in cancer. It is common to emphasize the unrestricted multiplication of malignant cells as the principal cause of pathology. The distortion of the normal differentiation properties and the abnormal protein synthetic and processing patterns may well be equally important in the pathological process. The defect in the cell cytoskeleton that characterizes the malignant cells studied here also explains why cancer cells are identifiable by direct microscopic examination. The abnormal cytoskeleton produces changes in cell morphology that can be recognized under the microscope. Our proposal accounts for the concomitant association of deficits in cytoskeletal organization with the changed state of the chromatin of the nucleus (Krystosek and Puck, 1990).

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6. New Practical Possibilities These considerations open up the possibility of new approaches to cancer diagnosis, cancer staging, and the monitoring of therapy. Examination of the genome exposure process with respect to the total amount of DNA exposed and specific identity of the exposed genes in different types of cancer as opposed to normal tissue cells may make possible more precise definition of the presence, extent, and degree of cell pathology in the malignant process. Such studies are in progress. These considerations also offer the possibility of a new approach to chemotherapy. Current practice is almost exclusively based on the principle of administering a cytotoxic agent in the hope that it may kill cancer cells more readily than normal cells. However, almost invariably, these agents are also highly lethal for normal cells, as has been demonstrated by the single-cell survival curve procedure (Puck and Marcus, 1956). It is this circumstance that limits effective treatment so strongly to situations in which early diagnosis is possible. When the size of the malignancy is still small, the necessary amount of toxic agent is reduced, as shown by the survival curve, so that the body can afford to lose the relatively small number of normal cells which would be killed during the required therapeutic regimen. For cancers of larger size, more intensive treatment is required. The loss in normal cells resulting from such therapy cannot be tolerated so easily. The reverse transformation reaction offers the possibility that, for different types of cancer, specific reverse transformation reagents can be found which restore normal phenotype to the cancer cells. Since, in every case demonstrated so far, the reverse transformation agent is a normal metabolite, rather than a toxic agent, there is reason to hope that its administration is far less destructive to the normal cells of the body. A variety of different reverse transformation inducers have already been described in different cell systems, including CAMP, 12-0-tetradecanoyl phorbol-13-acetate (TPA), nerve growth factor, and retinoic acid. Further clarification of cytoskeletal dynamics in genetic regulation may ultimately lead to a rational design of agents capable of restoring normal fiber organization in cancer cells, and concomitantly normal nuclear regulation. If reverse transformation therapy should prove feasible, in many cases it might not have to be continued indefinitely. Addition of CAMPderivative or other appropriate reverse transformation agents restores to appropriate cancer cells the ability to differentiate and control their otherwise unregulated multiplication. If a terminal nonmultiplying differentiation state exists, then presumably, when the last of the genetically altered cancer cells has been able to reach this terminal state under the influence of reverse

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transformation, such therapy might be capable of being discontinued. Obviously, interesting studies lie ahead. The genome exposure phenomenon offers a possibility for understanding the still mysterious process known as cancer promotion. In situations which have so far been carefully studied, it has been demonstrated that, while the first or initiation process in carcinogenesis constitutes a mutational step, no malignancy can result unless a second step involving administration of a promoter such as TPA occurs (Berenblum, 1954). Our theoretical formulation opens up the possibility that this second step may be a change in genome exposure. Thus, a cell exposed to a randomly acting mutagen presumably suffers mutations in both exposed and sequestered parts of the genome. Since the latter represents the majority of the genes in most differentiated tissues, most of the mutations occur in the sequestered genes that are not transcribed. Due to the inactivity of such genes, any malignant change is not expressed, and hence, the cancer remains a potentiality. Treatment of such cells with a promoter may cause exposure of sequestered genes, which could permit expression of the altered genetic information. The affected cell could then continue its path toward establishment of the cancerous process. The ability of the classic promoter, TPA, to affect cell contacts and adhesions, cytoskeletal functions, and protein kinase systems clearly places it in the same metabolic paths that we have proposed as converging on nuclear structure. These speculations also raise the question as to whether exposed and sequestered genes may have the same or different susceptibilities to mutation by chemical agents on the one hand, and ionizing radiations on the other. We have tentatively visualized the DNA sequestration process as consisting of a combination of higher-order supercoiling plus protein interaction. These two processes could conceivably act in opposing fashion in determining the intensity of staining by DNA dyes. This may be the reason that the defect in DNA exposure in cancer failed to be observed earlier by conventional chromatin-staining procedures. Finally, these considerations offer a possible general explanation of what constitutes a protooncogene. By definition such structures are genes whose mutation can cause cancer. By the formulation presented here it would appear that protooncogenes would include all genes whose normal function is essential for the genome exposure process. We have demonstrated the necessary role of the cytoskeleton in this genome exposure process. We have also developed the conception that an information transmission system extending from the extracellular space through the membrane and its contained receptors, continuing through the cytoplasm through the agency of all three components of the cytoskeleton, and ending up in the elements controlling genome exposure is necessary for the

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production of the specific information transmission arrangement required by each state of normal differentiation. A large number of genes must control the normal functioning of this informational delivery system. Mutation in any of the genes necessary for the integrity of this process could damage the exposure process. Genes whose mutation produces a defect in genome exposure large enough (i.e., so as to lose regulatory access to genes critical for growth control and differentiation) to produce cancer would then be protooncogenes. Thus, for example, the I9-kDa protein encoded within the adenovirus E l B gene specifically associates with and disrupts nuclear lamina and intermediate filament assemblies of cells into which its coding sequence can be transfected (White and Cipriani, 1989, 1990). The presence of the phenomenon that we now call genome exposure was first demonstrated by the laboratories of both Axel (Garel e f al., 1977)and Weintraub (Weintraub and Groudine, 1976),who showed a selective sensitivity to DNase I of approximately 20% of the genome of different cell types. These genes represented the active or potentially active genes. Our work has demonstrated the change in genome exposure in cancer cells, the restoration of this action when cancer cells are treated with an effective reverse transformation agent, and the role of the cytoskeleton in this reaction; our work has also developed a general theory of cytoskeletal control of genome exposure as the regulatory step responsible for the pathology of at least a large class of different cancers (Puck et al., 1990). Other workers (Ben-Ze’ev, 1986; Capco et al., 1982; Pienta et al., 1989) have discussed various aspects of extracellular matrix, nuclear matrix, and cytoskeletal organization which seem entirely consistent with the present proposals.

V. Other Evidence for Genetic Regulatory Actions of the Cytoskeleton

The use of cells cultured in uitro has provided refined opportunities to test cellular responses to drugs and other actions that affect specific extracellular and intracellular fibers. A number of other genetic modulating actions of the cytoskeleton have been described which require study to determine whether genome exposure or other mechanisms are involved. Rumsby and Puck (1982) showed that colcemid suppressed the induction of the growthassociated enzyme, ornithine decarboxylase, when fresh medium is added to cultured normal fibroblasts. This suggested the involvement of the organized array of microtubules in the signal transmission pathway. This requirement was not present, however, in malignant HeLa cells or in the

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spontaneously transformed CHO-K I cells, suggesting that such cancer cells have lost this necessity for an organized cytoskeleton. 3T3 variants capable of adipocyte differentiation have been a favored model for studying the sequence of events in phenotypic change. Spiegelman and Farmer (1982) showed an early decrease in the biosynthetic rates for vimentin, a- and 0-tubulin, and p- and A-actin during differentiation of 3T3-F442A preadipocytes. This decrease in replenishment of cytoskeletal components was due to altered levels of the corresponding mRNA molecules. These authors suggested that a remodeling of the cytoskeleton is necessary to achieve the adipocyte morphology and the expression of lipogenic enzymes. A follow-up study (Spiegelman and Ginty, 1983) indicated that cell attachment to fibronectin matrices could prevent these later changes in lipogenic gene expression. This effect could be overcome by exposing cells to cytochalasin D or by keeping cells in a rounded configuration. The interpretation given by these authors was that fibronectin produced an inappropriate transmembrane stabilization of the cytoskeleton which interfered with the normal program of cytoskeletal dynamics necessary for changing the pattern of gene expression. There is a large literature on the modulation of cellular growth and differentiation and gene expression by extracellular matrix components which appears to be consistent with the general theoretical conceptions presented here. For example, the study by Blum and Wicha (1988) of mammary gene expression demonstrated that culture of mammary epithelial cells with laminin induces the synthesis of specific milk proteins. Inclusion of cytochalasin D or colchicine in the medium for 24 hr inhibited the accumulation of a-casein, transferrin, and a-lactalbumin. This effect was shown to be at the level of accumulation of mRNAs for these proteins. The laminin-inductive effect on gene expression thus depends on the integrity of the actin and tubulin polymers. Ben-Ze’ev (1986) has reviewed the literature on the modulation of cell shape and growth control by the degree of spreading on the tissue culture substrata. Variation in these cell culture parameters produced changes in the biosynthesis and organization of the cytoskeleton, especially the intermediate filaments. Other changes can be produced by alterations in cell contact and spreading. Our model would predict concomitant ramifications for changes in nuclear chromatin conformation and specific gene expression in differentiation. As noted by Ben-Ze’ev (1986), cytoskeletal domains can also influence adherens junctions producing further complexities in the types of signal transmissions stemming from modulating the cellular fiber system. Beyond participating in activating appropriate gene expression, the cellular fiber systems may guide the individual events of transcription, nuclear export, and cytoplasmic translation. Transcriptional loci for active

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genes have been shown to be associated with the nuclear matrix (Ciejek et al., 1983). Nascent Epstein-Barr virus transcripts in lymphoma cells are resistant to the Triton X-100 extraction used in nuclear matrix/ cytoskeleton preparations, suggesting a physical association of the transcripts themselves with the matrix (Xing and Lawrence, 1989). It has been known since 1977 (Lenk et al., 1977) that the bulk of the polyribosomes are bound to the cytoskeleton; this anchoring may be mediated by mRNA poly (A) tails (Taneja et al., 1989). Such anchoring was shown for both total cellular mRNA (Taneja et al., 1989) and actin mRNA (Sundell et al., 1989). However, Lawrence and Singer (1986) have also shown, by in situ hybridization, that, while actin and tubulin mRNA have a peripheral cytoplasmic translation site, vimentin mRNA is more highly concentrated closer to the nucleus. There may well be anchoring signals that confer a degree of specificity of mRNA association with different domains of the cytoskeleton. It is not known whether these are encoded in messenger transcripts, by cytoskeletal associated proteins, or both. Since the effects of the cytoskeleton are so diverse, specific mechanisms would be expected to regulate the cellular levels of their component molecules. Evidence has been presented for both tubulin (Ben-Ze’ev et al., 1979; Cleveland et al., 1981) and actin (Leavitt et al., 1987) that their mRNA levels are regulated by the assembly state of their respective proteins. According to this autoregulation scheme, tubulin mRNA fails to accumulate when the pool of tubulin monomers increases, a condition under which further tubulin synthesis would not be necessary. The effect on tubulin mRNA stability appears to be at the level of both inhibition of transcription and decay of existing mRNA (Cleveland et al., 1981). The fact that much mRNA is cytoskeleton bound (Taneja et al., 1989) raises questions about the fate of other mRNAs when the polymerization state of the cellular fiber systems is altered.

VI. Applications t o Molecular Biology

The general picture developed here proposing a two-step regulation of specific differentiation genes in mammalian cells, one of which is exposure of the appropriate genes and the other being the turning on and off of exposed genes in accordance with the needs of the cells of each specific tissue, opens up large regions for study in the molecular biology of mammalian cells. It becomes necessary to reexamine the data on transcriptional control in mammalian cells to determine whether the primary exposure step, the subsequent regulation of exposed genes, or both are

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involved in individual cases and along consecutive pathways of development. It is also necessary to identify the exposed and sequestered genes in normal differentiated tissues and to map genes that are exposed simultaneously, since these may define functional mapping of chromosomal domains important in specific differentiation processes. Our theory leads to the expectation that the DNA interval between consecutive repetitive sequences of the appropriate kind may constitute such a domain. The molecular structures of the chromosomes and their various interacting proteins involved in the exposure and sequestration processes must be defined. In the latter group the lamins-which have been demonstrated to possess a nuclear peripheral distribution paralleling that of exposed DNA, the topoisomerases, zinc fingers, isoprenylated proteins, cytoskeletal proteins, G proteins, and various histone proteins, among others-must be examined for their possible participation at various levels in the exposure reaction. The genes controlling formation of these proteins and other aspects of the exposure reaction must be identified and mapped. Ionic effects involved in genome exposure must be investigated. The role of phosphorylation and dephosphorylation of the various phases of genome exposure and sequestration must be defined. The effects of life cycle stage and cell-cell interaction require study. The role of DNA methylation is important, as is that of the various non-amino acid coding sequences in the genome. A search must be made to see whether problems such as aging and particular genetic diseases such as Marfan’s syndrome, which has been shown to involve a disturbed fiber system (Godfrey et al., 1990a,b), involve the genome exposure process. Understanding of the differentiation mechanism in the immune system, which appears to include unique complexities, appears to present no particular difficulties to the genome exposure theory. Antibody-producing cells presumably have their own unique genome exposure pattern, like cells of any other tissue. In addition, however, it is necessary to postulate that the exposed gene set undergoes a series of more or less random breakages and recombinations under the influence of a set of enzymes specific to the immune system differentiation process. As a result the capacity to encode for a huge set of different antigen recognition sites is conferred on the set of cells of this tissue.

VII. Evolutionary Considerations

The theoretical developments presented here would lead to the expectation that different classes of single-gene mutations can exist in the mammalian cell, depending on whether the mutation occurs in an exposed gene, a

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sequestered gene, or genes which control the exposure process. We have already considered the case of exposed versus sequestered genes in Section IV,B. Different situations arise in considering mutation occurring in the genes controlling the exposure mechanism itself, as opposed to mutation in the target genes controlled by the exposure mechanism. In the former situation, since exposure can change the state of a considerable fraction of the genome in one operation, mutation in one of the genes controlling the exposure process itself may well affect expression of a great number of genes simultaneously. On the other hand, mutation in a specific locus that forms one of the final target sites of the exposure process would produce a much more limited change. In the former case one might well find a large variation in the developmental process of the organism affected. It is conceivable that these two different kinds of mutation might be connected with different modes of evolutionary development. Evolutionary changes seem to fall into at least two categories. On the one hand, changes have been observed that appear to be gradual and affect variation within a given species. On the other hand, there seems to be evidence for large quantum jumps in evolutionary development. It is conceivable that the latter might be due to mutation in genes controlling the exposure process. It is clear that a new era of fundamental understanding and power over human health is at hand. The promise of important advances in human health and well-being which will result from these further studies is for all mankind. This promise can only be achieved in a world at peace.

VIII. Additional Relevant Information Concerning the Nature of the Cytoskeleton and Previous Theories of I t s Function The scheme of gene regulation presented above makes certain demands on the components of the cellular fiber systems. This summary is intended to indicate that the components indeed show the needed degree of complexity in their diversity, developmental regulation, coordinated interactions with other cell components, and modulation of function by the ion flux and protein kinase systems expected for their participation in initiating phenotypic change. The huge literature on the roles of the cytoskeleton in functions such as cell polarity, secretion, and intracellular transport is not covered here.

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A. Developmental Considerations The cytoskeletal components function in the earliest stages of fertilization and development. Actin participates in the acrosomal reaction of sperm at egg contact, and microtubule assembly is implicated in pronuclear migration to achieve syngeny and the acquisition of a nuclear lamina coating for the paternal genetic information (Schatten and Schatten, 1987). Vincent et a f . (1987) have studied the role of microtubule assembly in fertilized amphibian eggs undergoing the first rounds of DNA replication. A rotation of the subcortical cytoplasm relative to surface markers was found to set up the orientation of the embryonic axis. Evidence was provided that microtubules mediate both the rotation and the response to rotation (i.e., dorsalization or axis polarization), and a “positive feedback model” was invoked in which the direction of tubulin assembly dictates the orientation of rotation, whereas rotation itself favors a continued tubulin assembly in the biased direction. Evidence has been presented for a functional domain in the actin cytoskeleton of Ascidia eggs that may partition morphogenetic determinants in muscle cell precursors (Jeffery and Meier, 1983; Jeffery, 1984). Contraction of the actin network in this domain may be the motive force for ooplasmic segregation. The association of mRNAs with the cytoskeletal framework could be part of the mechanism of differential partitioning of transcripts during ooplasmic segregation. In other cases drug inhibition studies have implicated microtubules as participating in moving morphogenetic information (e.g., the teleplasm domain in the fertilized egg of the leech, Helobdella triserialis) (Astrow et al., 1989). Studies with cytoskeletal inhibitors, cell fractionation, and in siru hybridization have shown that microtubules are involved in the gross translocation of messenger molecules to the vegetal hemisphere of Xenopus oocytes (Yisraeli er al., 1990), whereas microfilaments are important at the stage when mRNA is discretely anchored at the cortex. Regulated release from anchoring allows specific messages (e.g., the RNA for V g l , a member of the transforming growth factor 0 family) to become distributed as a broad band at the vegetal pole, where it remains and is translated during early embryogenesis. Fibroblast growth factors and transforming growth factors participate in mesodermal induction. The cytoskeleton thus participates (even before fertilization) in the placement of genetic transcripts for inductive proteins in a key location at the vegetal pole, where the protein product will function only after several rounds of cell division. By and large, however, the role ascribed to the cytoskeleton before 1975 did not include any role in direct genome regulation.

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6. Extracellular Matrix The extracellular environment contains a diverse assemblage of glycoproteins and polysaccharides that mediate cellular form and communication. Both in tissues and in culture, cells are in contact with their own and other cells’ secretory products. Collagen and elastin are major fibrous proteins and are embedded in a hydrated gel of polysaccharides. These latter components vary according to tissue type. Chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, and keratin sulfate have molecular weights ranging from 5000 to 50,000 and vary in their repeating disaccharide units and degree of sulfation. All are linked to core proteins, which also vary as members of a gene family. Hyaluronic acid exhibits molecular weights ranging from 4000 to several million and is unlinked to protein. About 20 distinct a-chain collagen genes have been described, yielding protein products of different carbohydrate, hydroxylysine, and hydroxyproline contents. These are assembled into various triple-helical collagen molecules that can organize into fibrils showing distinct tissue distribution. Among the cellular adhesion proteins, vitronectin and fibronectin are concentrated at the focal adhesions. Many minor cellular secretory products become localized in the pericellular space. These include proteases and their inhibitors and protein growth factors that bind to heparin. By such a localization scheme binding to a receptor (e.g., the transmembrane growth factor receptors) and the consequent cellular signaling appear as parts of an interconnected solid-state communication process. Protease retention in the extracellular matrix could be important, since specific cleavage of molecules such as fibronectin can yield new forms of activated signaling agents. C. Focal Adhesion Sites

The plasma membrane contains surface proteases, ectoprotein kinases, glycosidases, and receptors for diverse signaling molecules. Elements of the cytoskeleton attach to protein complexes at the membrane. All of these sites provide opportunities for modification of the cell fiber system and cell signaling, as well as changes in cell morphology. For example, cell-cell and cell-substratum contacts are both mediated by transmembrane glycoproteins: the cell adhesion molecules and the integrins, respectively. The first integrin described was the fibronectin receptor consisting of large a and /3 chains. The receptor for laminin is also an integrin assembled from different genetic a- and P-chain isotypes. Members of this receptor family

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share a common structure, including a binding domain recognizing the Arg-Gly-Asp sequence of adhesion proteins and an intracellular domain from which cytoskeletal fiber systems are organized. Cell-substratum adhesions occur focally at the basal surface of cultured cells and represent laterally immobile concentrations of integrins and matrix proteins. At the cytoplasmic face of these complexes, actin polymers are anchored through the agency of an interacting collection of proteins, including a-actinin, fibrin, vinculin, and talin (Burridge et al., 1988). Cellular response to stimuli such as epidermal growth factor results in disassembly of the rigid adhesions, allowing new arrangements of actin (Schlessinger and Geiger, 19811, a feature that we postulate occurs in analogous fashion between the fiber system and the genome during signal transmittal. Notably, the inner faces offocal adhesion sites are also the loci of accumulations of viral src protein (Rohrschneider, 1980), possibly implicating disruption of anchoring sites as one mechanism of loss of fiber integrity in transformation.

D. Microtubules Microtubules are heteropolymers composed of CY and p chains. Vertebrates have at least six genetic isotypes of each subunit (Sullivan, 1988). These proteins are subject to further posttranslational modification. There are, for example, 12 isoforms of p-tubulin resolved by electrophoretic methods (Field et al., 1984). a-Tubulin chains are subject to cycles of modification on the C terminus by addition of tyrosine in a ligase reaction (Gundersen and Bulinski, 1986) and also acylation of lysine residues (Sullivan, 1988).These reactions occur differentially on tubulin dimers and polymers, and the status of the C terminus may regulate elongation during assembly. P-Tubulin has been reported to be phosphorylated during the differentiation of mouse neuroblastoma cells (Gard and Kirschner, 1985). The C-terminal domains of both chains are functionally specialized (Maccioni e?al., 1985) for assembly and binding to the microtubule-associated proteins (MAPS). Ideas about tubulin functions have been guided by the “multitubulin hypothesis,” which stated that variations in the tubulin chains could lead to assembled microtubules designed for different functions (Fulton and Simpson, 1976). Although widely believed, experimental proof has been difficult to gain. For example, Saccharornyces cereuisiae carries out all of its life functions with a coding diversity of only one p- and two a-tubulin genes. In several approaches, using immunological reagents for the identification of distinct P-chain isotypes and intraspecific transfection of cloned

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genes, Cowan’s laboratory (Cowan er al., 1987) demonstrated free intermingling of P-isotypic forms, as opposed to discrete segregation of the pools. Microtubule heterogeneity is further increased by the variable association of the assembled polymers with different subspecies of the MAPs (Olmsted, 1986). MAP2 may be multifunctional, mediating interactions with microfilaments, calmodulin, and protein kinase type 11. Several MAPs are subject to phosphorylations that can modify their function. Kinase systems can thus impinge on the microtubule system, even though a-tubulin itself has not been demonstrated to be a substrate. The dynamic instability model of Kirschner and Mitchison (1986) proposes that individual microtubules can exhibit either long-term stability o r rapid cycling between growing and shrinking forms. Such changes might well be involved in the dynamics that we postulate to occur in the genomic response to cell stimuli. E. Microfilament

There are, in mammals, six actin proteins, most of which are distributed in specific muscle types. P and y isotypes are, however, general to most cell types. The microfilament polymers are assembled by subunit interactions between individual actin subunits, and assembly can be inhibited by sequestration of free monomers by the protein profilin. As polymers, microfilaments have properties not possessed by the individual monomer (e.g., deformability , cross-linking to polymers of tubulin, and simultaneous contact with the plasma membrane and the nucleus). The microfilament system is distinguished by its diversity of cellular localizations. Polymerized actin is most recognizable as the light bands of myofibrils and the stress fibers coursing through fibroblasts and terminating at the focal contacts. High concentrations of actin polymers figure prominently in specialized structures such as the microvilli of intestinal epithelial cells, the adhesion belt of epithelial cell sheets, the acrosome of sperm, the lamellipodia of motile cells, and the vigorous microspikes of the elongating neuronal growth cone. Similar to muscle, in which the troponins mediate interaction with Ca2+, myosin, and actin, interactions in nonmuscle cells are regulated by other proteins. Filamin dimers cross-link actin filaments, modulating its deformability characteristics. The calcium-binding protein gelsolin allows severing and rearrangements of actin polymers in response to Ca2+ flux.

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F. Intermediate Filaments

The intermediate filament (IF) proteins are genetically more diverse and their function is less understood than that of the other intracellular fiber systems. The IFs of epithelial cells are extraordinarily varied, containing approximately 20 distinct forms, whereas cells of mesenchymal origin express only the 53-kDa vimentin polypeptide. Muscle and glial cells express specific isotypes, called desmin and glial fibrillary acid protein, respectively. Neurons, on the other hand, contain a triplet of neurofilament proteins of 60, 100, and 130 kDa. Unlike tubulin and actin, IF proteins are elongated molecules with common central a-helical regions that participate in the interactions of self-assembly. The various IF proteins differ at the N- and C-terminal portions. The distribution of different IF types suggests their involvement in differentiated cell functions, but their absence in some eukaryotes may rule out major roles in genetic regulation. On the other hand, their penetrance into the nuclear space and homology with the lamina proteins suggest possible involvement in nuclear function. Vimentin is subject to phosphorylation, a process that has been suggested to regulate its assembly state at mitosis (Chou et al., 1989). Vimentin phosphorylation occurs early in the reverse transformation reaction of CHO-K1 cells, which restores malignant cells to their normal phenotype (Chan et al., 1989). At the end of this reaction, the IFs have changed from the highly condensed state of the transformed cell to the fibrous array characteristic of the normal fibroblast. Phosphorylation, in this case, facilitates assembly, showing that modification effects can be complex. The structural integration of IFs with microtubules and microfilaments is known from the action of drugs such as colcemid, which collapses both microtubules and IFs (Lazarides, 1980). The detailed molecular basis and importance of these polymer interactions are not yet understood. Methods for studying the dynamics of IF transitions are now becoming available, and new members of this diverse gene family have recently been discovered (Steinert and Liem, 1990).

G. Nuclear Matrix The detergent-insoluble residue of the nucleus is sometimes termed the “karyoskeleton” or, depending on the exact methods of extraction, the nuclear matrix or nucleoids. This structure includes the remnants of nuclear pore complexes and the nuclear lamina, which underlies the nuclear envelope of higher cells. The major proteins of the lamina are called lamins

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and consist of three major forms in mammals. Lamin A is a larger form of lamin C, whereas lamin B is encoded separately, but is fairly homologous. Lamin sequences suggest that they are a separate group of the IF gene family and perhaps even the most ancestral of the members. The lamins are elongated molecules similar to the IFs and capable of selfassembly. The significance of generation of lamins from precursor forms, the existence of minor lamins, and posttranslational modification by isoprenoid incorporation (Beck et a / . , 1988) are not presently understood. Hyperphosphorylation of the lamins mediates the disassembly of the nucleus at mitosis. Lamin B subunits, however, remain associated with nuclear envelope vesicles, presumably serving as nucleation sites for lamina reformation (Gerace and Blobel, 1980). Lamin B also appears to serve as the IF attachment site at the nuclear envelope (Georgatos and Blobel, 1987). A large number of nuclear matrix proteins exist. Their spatial relationship to chromatin and lamin has not, however, been clarified. Large loops of supercoiled DNA are known to be anchored at nuclear matrix attachment sites. By and large, however, the intimate details of chromatin association with the lamina and the more internal components of the nucleus are not understood. Recent advances from several laboratories further support our original concept of a genome regulatory role for the cytoskeleton (Puck, 1977). Nathan and Sporn (1991) have reviewed cytokine action and emphasized the extracellular matrix, cell surface receptors, and cytoskeletal changes as key features of initiation of a cellular response by these regulatory molecules. Lux et al. (1990) have described a repeated amino acid sequence motif in erythrocyte ankyrin which may mediate organization of the cytoskeleton at the plasma membrane. This motif is also discerned in DNA coding sequences of various morphogenetic regulators such as the Notch gene of Drosophifa mefanogaster, suggesting membrane/ cytoskeletal interactions as part of the mechanism of action of its gene product. This same ankyrin repeated motif has now been described in the cloned sequence of the pleotypic transcription factor NFKB (Kieran e t a / . , 1990). This factor, in the inactive state, had been known to be excluded from the nucleus; it now seems that it could actually be cytoskeletal bound. Interaction of oncogene and proto-oncogene products with the cytoskeleton is a burgeoning area which will help define roles in signal transduction. The c-mos protein has been shown to be a kinase which associates with and phosphorylates tubulin (Zhou et al., 1991). The kinase which phosphorylates vimentin at mitosis has been identified as the cell cycle control kinase, p34cdc2,which also phosphylates histone HI (Chou et al., 1990). This result emphasizes the coordinated organization of key events in cell cycle dynamics and blurs the distinction between nuclear and

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cytoskeletal compartments as separate entities. Collard and Raymond (1991) have investigated cell shape and signal transduction and concluded that an organized intermediate filament network is required to transmit a signal invoked by TPA in HEp-2 carcinoma cells through the nuclear membrane, where the conformation of lamin A tail domains is altered. Finally, the involvement of kinesinlike protein in various forms of chromosome segregation (Zhang et al., 1991) suggests that “motor” proteins which utilize ATP should be investigated with regard to changes in chromatin sites associated with the genome exposure reaction.

IX. Concluding Remarks We have tried to show that mammalian cells, unlike E . coli, operate with at least two different levels of genome regulation. The first governs the transition from sequestered to exposed genes. The second regulates the transition of exposed genes between active and inactive states. It is proposed that specific differentiation states involve exposure of different sets of genes. Approximately 30% of the genome in mammalian cells is converted from the sequestered to the exposed stage in a single action governed by effectors such as CAMP, retinoic acid, and nerve growth factor, acting on transformed cells. The extent of such transitions within normal cells during development remains a challenge for future investigation. We have shown that the cell cytoskeleton is a necessary structure in producing the specific genome exposure pattern for each cell type. Exposed genes characteristic of a specific state of differentiation are located in a shell distributed around the nuclear periphery. It is postulated that the cytoskeleton and probably other cellular fiber systems (e.g., the nuclear matrix) control sequestration or exposure by attachment to specific sites along the chromosomes in interphase. Chromosomal condensation in mitosis, which accompanies microtubular attachment to the centromere via the kinetochore, is taken as a model for the corresponding exposure and sequestration transitions that occur during interphase. Thus, the nucleus is viewed as a highly dynamic and integrated structure containing machinery for extrusion into the peripheral shell of chromosomal regions whose exposure is required in each specific state of differentiation. It is believed that the repetitive sequences distributed throughout the mammalian genome serve as chromosomal attachment sites (with the aid of specific proteins) during interphase, as occurs with the centromere-kinetochore complex during mitosis. The difference between mitosis and interphase, in

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this respect, would lie in the fact that sequestration occurs only over limited chromosomal domains in interphase, instead of over the entire chromosome, as in mitosis. Attachment of specific proteins to particular families of repetitive sequences may produce the necessary binding to specific fiber proteins and to the protein assembly of the nuclear membrane region, so as to maintain the specific exposure domains of each chromosome attached at the nuclear periphery during the cell’s sojourn in its particular differentiation state. We have postulated that specific phosphorylation and dephosphorylation actions, together with calcium binding at specific phosphorylation points, participate in reorganizing the cytoskeletal network, which results in ultimate targeting of different points on the genome. This action produces different specific patterns of genome exposure and sequestration characteristic of each state of cell differentiation. By this picture the mammalian cell cytoskeleton becomes part of an information transmission system extending from the cell membrane and its specific receptor sites, through the cytoplasm, and terminating in specific points on each chromosome, so that specific domains of exposure and sequestration result. Genome exposure is necessary, but not sufficient, for gene activation. These studies have led to recognition of a new genome regulatory function of the cytoskeleton and its many different associated proteins. While aspects of the differential sensitivity of genetic loci in the mammalian cell to hydrolysis by DNase I had been reported by other workers, the involvement of this phenomenon in cancer was a most surprising development. Each of the first 12 cancer cells we examined have demonstrated grossly defective genome exposure as compared with the corresponding normal or reverse-transformed cell. We have proposed that at least a large class of cancers represents pathology due to defective genome exposure. The abnormal morphology and behavior of cancer cells are explained as a defect in the exposure system, which includes the cell cytoskeleton. These considerations offer the possibility of a new definition for a protooncogene as any gene whose mutation can destroy the information transmission system that brings about exposure of the specific gene regions required in the state of differentiation of the particular cell considered. By this formulation cancer becomes a pathological state in which specific differentiation genes are no longer accessible to the cell. Since these include the genes that control cell reproduction, the cancer cell is free to multiply without limit. This conception provides a new and simplified view of the nature of cancer which promises new approaches to therapy and prevention. Indeed, agents such as retinoic acid, which we have shown to be restorative

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of genome exposure in specific malignancy, are now being investigated for efficacy in clinical trials. The principle of the use of reverse transformation, with its attendant restoration of normal genome exposure, may well be much more hopeful for cancer treatment than the current widespread use of cytotoxic agents. These various considerations have been applied to molecular biological, developmental, and evolutionary aspects of mammalian cell behavior. These considerations also suggest that studies of mutagenesis and its consequences in mammalian cells must give separate consideration to three different types of genes: exposed genes, sequestered genes, and genes controlling the exposure process. We have proposed a metabolic scheme for integrating extracellular stimuli, ion fluxes, kinase action, cytoskeletal organization, and chromatin structure. In situ hybridization with nucleic acid probes will be particularly important in learning whether repetitive and single-copy DNA loci change position in the nucleus during the process of exposure. Methods must be developed for studying how the termini of cytoskeletal fibers interact with nuclear substructure. The present period in biomedical science is witnessing tremendous accomplishments in the mapping of the human genome. In addition to the specification of the precise linear order of the location of the different genes on their chromosomes, it is now also becoming necessary to determine exposure domains of the chromosomes (i.e., to map the genes that become exposed and sequestered together in different states of differentiation and to define the domains so formed). This may be called a functional mapping of the genome. In addition, however, it is also becoming necessary to understand the detailed molecular mechanisms by which different gene regions and genes are exposed and sequestered and the nature of the gene activation processes operating on exposed genes. Finally, this hinges on a detailed molecular understanding of signal transduction pathways, including the sequential ordering of the events contained therein, their branchings, their redundancies, and their metabolic concomitants. The cytoskeleton appears to serve as one extended modifiable structure for passing metabolic signals to the nucleus. The resulting changes in gene expression then bring about new informational transmission from the nucleus to the rest of the cell and its environment. The roles of structures such as the Golgi apparatus and the endoplasmic reticulum also need delineation at the molecular level. The tools for such molecular understanding seem now to be largely available. The goal that appears to be achievable should constitute a new detailed and powerful understanding of cell function in health and disease.

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Acknowledgments This work was aided by grants from the Lucille P. Markey Charitable Trust, the American Cancer Society, the Raphael Levy Memorial Foundation, and the Walter Orr Roberts Memorial Fund. T.T.P. is an American Cancer Society Professor.

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Properties and Uses of Photoautotrophic Plant Cell Cultures Jack M.Widholm Department of Agronomy, University of Illinois, Urbana, Illinois 61801

I. Introduction

Plant tissue cultures have provided easily manipulated systems for cytological, physiological, biochemical, and genetic studies. This goal is also being attained in the case of photosynthesis with the many quality PA' cultures now being produced in ever-increasing numbers. PA cultures utilize photosynthesis to provide energy and carbon for growth, so no sugar or other energy-producing compounds are added to the culture medium. A number of excellent studies have been conducted with PM cultures in which some sugar is provided, but these are generally not included in these discussions, since the added sugar must have an effect on the metabolism of the cells and thus complicates the general results. Several PA cultures have been grown as callus on agar-solidified medium. Such systems have a number of problems, including most significantly the apparent presence in the solidifying agent of a substance(s) that can provide carbon and energy for cell growth. McHale (1985) showed that

' Abbreviations: B5, Gamborg et af. (1968) medium; BA, 6-benzyladenine; Cj, photosynthesis by which COz is fixed initially by RuBPcase into a C, compound; C,, photosynthesis by which C 0 2 is fixed initially by PEPcase into a C4 compound: CA, carbonic anhydrase (carbonate dehydratase); CAM, crassulacean acid metabolism; Chl, chlorophyll; 2.4-D, 2.4-dichlorophenoxyacetic acid; DCMU, diuron. 3-(3,4-dichlorophenyI)-11 I-dimethylurea; DMSO, dimethyl sulfoxide: H , heterotrophic; HEPES, N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid; IAA. indole-3-acetic acid; IJ0, 50% inhibitory concentration; IRGA, infrared gas analyzer; LS, Linsmaier and Skoog (1965) medium: MS, Murashige and Skoog (1962) medium; NAA, naphthaleneacetic acid; NMR, nuclear magnetic resonance; NR, nitrate reductase; PA, photoautotrophic; PCV, packed cell volume: PEPcase, phosphoenolpyruvate carboxylase; PGA, 3-phosphoglyceric acid; PM, photomixotrophic; PMSF, phenylmethylsulfonyl fluoride; PSI, photosystem I; RuBPcase, ribulose-bisphosphate carboxylase: UV, ultraviolet. lnrernarional Review

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agar- or agarose-solidified medium without sugar would support the growth of chlorophyll-containing Nicotiana tabacum cells when incubated in the light in C02-free air (15-93% dry weight increase in 21 days). Rey et al. (1989) showed a 54% dry weight increase with Nicotiana plumbaginifolia cells grown in darkness on an agar-solidified medium lacking sugar for 21 days. Callus cells also grow as multicellular masses that would not allow light or gas exposure to the internal layers, and as such the exterior and interior would most likely be quite different physiologically. The most uniform systems should be suspension cultures. Since higher plants are usually photosynthetic organisms, one would think that it would be quite easy to initiate and maintain PA cell cultures, but generally the culture initiation is a slow difficult process that appears to involve selection o r enrichment which may or may not involve selection for some mutant cell type. No definitive studies have been performed to study PA cultures genetically, however. That the suspension cultures are truly PA should be proved for each cell line by showing that growth is dependent on photosynthesis and can continue for a long period (i.e., several culture cycles). Thus, the medium should have no sugar or high levels of any other compound capable of producing energy. The growth should be dependent on light and COz and be inhibited by low levels of photosynthetic herbicides such as atrazine or DCMU. PA plant cell cultures have been reviewed several times (Yamada et al., 1978; Horn and Widholm, 1984; Dalton and Peel, 1983; Hiisemann, 1985; Horn and Dalton, 1984; Neumann and Bender, 1987; Widholm, 1989), but continued progress, especially in the quality of the cultures and their experimental utilization, warrants updating and analysis of the topic area. I have attempted to present the data in similar units, so that comparisons can be made easily between the different experimental results. In many cases accurate conversions cannot be made, since insufficient data are presented. For example, some results are given on the basis of dry weight, and I include a conversion to fresh weight, assuming that the dry weight was about 7-10% of the fresh weight. Most data are presented on a per milligram of Chl or per gram of fresh weight basis, but sometimes results are given per milligram of protein or per cell without enough additional information to allow conversion. Another problem involves light intensity measurements, sometimes given in lux or foot-candles, which are measured on the human eye response spectrum, or in micro-Einstein units, which measure photons in the 400- to 700-nm wavelength region (photosynthetically active radiation), so they cannot be accurately converted. However, since most of the measurements are with fluorescent bulbs, which should have defined light spectra, some rough approximations can be made to convert the illuminance measurements (lux and foot-candle) to

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irradiance units (pEm-'sec-'). I have chosen the numbers given by Sato et al. (1987), with 1000 lux = 15 pEm-'sec-' (1 foot-candle = 10.76 lux, and 1 p E = 1 pmol of photons). This review first presents descriptions of the initiation of PA cultures of various species of higher plants, listed chronologically in order of the first publication. Summaries of the general growth and photosynthetic characteristics are also included. Following this summaries of the overall culture initiation methods, medium requirements, and growth and photosynthetic characteristics are given. Then, several sections describing the many specific uses of PA cultures are presented, followed by conclusions.

II. Initiation and Characterization of Photoautotrophic Cell Cultures

A. Cultured Cells 1. Nicotiana tabacum and Nicotiana plumbaginifolia

Since N . tabacum (tobacco) has been one of the most commonly cultured species, it is perhaps inevitable that the first, as well as the most, studies have utilized this species. The first report of a PA plant cell culture was that by Bergmann (1967a,b), who was able to grow N . tabacum cv. Samsun suspension cultures in MS basal medium with no growth regulators or sugar. The MS basal medium contained minerals, 4.1 pM nicotinic acid, 0.3 pM thiamine HCI, 0.5 pM pyridoxine, and 0.55 m M inositol. The cultures were illuminated with fluorescent light (5000 lux, about 75 pErn-'sec-') and were aerated by bubbling 1% COZin air through the liquid medium in bubble tubes. The cells had a doubling time of about 5 days. Chandler et al. (1972) were able to grow certain cloned cell strains obtained from N . tabacum pith in suspension culture for up to three transfers, with 0.5 pM 2,4-D and 0.2 p M kinetin as growth regulators with no sugar. One-third of the clones did not form Chl and died, while some of the others grew slowly and exhibited high rates of photosynthesis, Hill reaction, and respiration. Berlyn and Zelitch (1975) grew N . tabacum callus on both agar-solidified medium and in shallow layers of liquid medium consisting of LS salts, 1.5 pM NAA, 1.5 p M isopentenyladenine, 0.55 mM inositol, and 0.3 p M thiamine with continuous illumination (120-235 pEm-'sec-'). The cultures in liquid medium grew faster in the presence of higher COz concentrations, with about a 2-fold increase in dry weight being attained in

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21 days even after eight subcultures. The highest Chl level measured was 43 pg/g of fresh weight. The PA N . tabacum callus grown on agar-solidified medium was studied further by Berlyn et al. (1978). The callus showed about a2-fold increase in dry weight during each 21-day passage and contained about 40 p g of Chl per gram of fresh weight. The maximal C 0 2 fixation rate, 125 pmol/mg of Chl per hour, was attained with about 0.9% COz, dark fixation being about 5% of this. Increasing the O2 concentration to 60% with air C02 levels completely inhibited growth during the second subculture period. To eliminate the complication of the partial feeding due to the presence of agar shown by McHale (1985), McHale et al. (1987) grew the tobacco callus on polyurethane pads soaked with liquid LS medium with 1.6 p M NAA. The callus growth was stimulated by elevated C02 levels, 1% C02 giving about a 2-fold increase in dry weight in 21 days (doubling time, about 13 days). Yamada and Sat0 (1978) grew pith-derived N . tabacum cv. Samsun NN callus under 2000 lux (about 30 pEm-2sec-1) continuous light on LS basal medium with 10 pit4 NAA, 1 pit4 kinetin, and 3% sucrose with double the original vitamin concentration. After several years' growth this culture contained 70 p g of Chl per gram of fresh weight, and studies with different light and sucrose levels showed that illumination of 6000 lux (about 90 pEm-'sec-') o r higher and sucrose levels of 0.5% or lower were required for maximal Chl accumulation. PA growth was attained in agarsolidified medium with illumination (4000 and 8000 lux, about 60 and 120 pEm-2sec-1) and a 1% C 0 2 atmosphere, which was sustained for about 1 year with 10 subcultures. The Chl levels were as high as 90 pg/g of fresh weight, and the fastest doubling times were about 9 days. The cultures would not grow continuously with air levels of C02. PA and PM N . tabacum cv. Samsun NN callus cultures which contained 48 pg of Chl per gram of fresh weight were able to fix C 0 2 in the light and dark at rates of 132 and 4.7 pmol/mg of Chl per hour, respectively (Nishida et al., 1980). Fixation product analysis showed that 31% of the PA culture fixation was through C4compounds and 43% in the PM culture. When the levels of the C02 fixation enzymes were measured in these cultures, the RuBPcase activity was 39 pmol of C 0 2 per milligram of Chl per hour, and the PEPcase activity was 177 pmol of C02 per milligram of Chl per hour in the PA cultures (Sato et al., 1980). The PM cultures had similar respective levels, 36 and 130 pmol of C 0 2per milligram of Chl per hour. In view of the very low RuBPcase :PEPcase activity ratio in the PA culture, it is surprising that the amount of Cq labeling is not higher than that observed by Nishida et al. (1980). Yamada et al. (1981) attempted to grow a PA N . tabacum cv. Samsun NN cell suspension in a 5-liter jar fermenter, but found that, even with

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aeration with 1% C02, the O2 level had to be lowered to 14% to obtain growth (less than one doubling in fresh weight in 17 days). The cultures were illuminated continuously (8000 lux, about 120 pEm-'sec-') at 26°C. Impeller type and speed, as well as aeration rate, were optimized in these experiments. The PA N . tabacum cv. Samsun N N culture can be grown in liquid medium in two-tiered flasks with 1-2% C02 under continuous illumination (120 pEm-'sec-') in LS basal medium with 1.11 m M inositol, 2.4 p M thiamine HCl, 1 0 p M NAA, and I p M kinetin (Sato et al., 1987,1989).The Chl levels were about 170 pglg of fresh weight, and the doubling time was about 14 days. The Hill reaction and PSI activities of isolated chloroplasts were 70-80 and 140-160 pmol of O2per milligram of Chl per hour, respectively. A PA suspension culture was initiated from a H suspension culture of the N . tabacum x Nicotiana glutinosa fusion hybrid, T3glC (Horn et al., 1983a), which had the nuclear genomes from both species and the plastids and the mitochondria from N . glutinosa. When the H suspension in MS basal medium with 1.81 p M 2,4-D was placed under continuous illumination (30 pEm-'sec-') with longer times between subcultures, yellowing or greening occurred (Xu et a/., 1988). PM cultures with 30-100 p g of Chl per gram of fresh weight formed from the H cells in MS basal medium with 3% soluble potato starch, 7 p M kinetin, and 0.5 p M picloram. Subsequently, the starch level was reduced to 1%. PA cultures were initiated in a medium containing MS salts, complex vitamins (Horn et al., 1983b), 0.3 p M picloram, 5.4 p M NAA, 0.93 p M kinetin, 5 m M HEPES, and no starch or sugar, with 5% C 0 2 blown into the flasks through an inlet syringe needle stuck through the rubber stopper. Another needle served as the outlet, and both needles had small Millipore (Bedford, MA) filters attached to prevent contamination. The PA culture, which was denoted NTG-P, grew only slightly in ambient COz levels and not at all in the dark. The maximal Chl level was 237 pglg of fresh weight, and this did not increase after more than 2 years when the cells were grown in a minimal medium, with the growth regulators as the only organic compounds (Goldstein and Widholm, 1990). The RuBPcase activities also did not change much, being about 40-60 (initial) and 214-266 (total) pmol of C 0 2 per milligram of Chl per hour. The in uiuo activation levels were 30% or less at both times. The PEPcase activities were high at both times, being about 80 pmol per milligram of Chl per hour originally (Xu et al., 1988) and 146 pmol when last examined (Goldstein and Widholm, 1990). The doubling time was about 12 days. Carriere et al. (1989) initiated a PA N . tabacum suspension culture from a PM culture grown in MS basal medium with 10.7 p M NAA, 0.89 p M BA, and 1.5% sucrose upon transfer to MS basal medium with no growth

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regulators or sucrose in a bubble column aerated with 2% C02 under 18-hr illumination (100 pErnw2sec-') daily at 25°C. The PA cultures were maintained for several months by transferring one volume of medium into three volumes every 2 weeks. The Chl content was 70 pglg of fresh weight. A PA suspension culture of N . tabacum was initiated from a PM suspension grown in Mitchell and Gildow (1975) medium with 1.07 pM NAA, 0.89 pM BA, and 1% sucrose by inoculating cells into MS medium with 4.5 pM 2,4-D and 2.3 p M kinetin. A two-tiered flask system was used with 2% C 0 2 , continuous illumination (100 pErn-*sec-'), and shaking (Ikemeyer and Barz, 1989). The Chl level of the PA cells was about 100 pg/g of fresh weight, and the doubling time was about 9 days. A rapid method for establishing N . p l ~ m & u g ~ PA ~ ~callus o ~ ~cultures, u using mesophyll protoplasts from 3-month-old plants grown in a growth chamber, has been described by Rey et af. (1989). Protoplasts were plated in a modified Bourgin et al. (1979) liquid medium with 4.5 p M 2,4-D, 1.8 p M BA, 0.45 M mannitol, and 0.25% glucose, and this was diluted after 5 days with an equal volume of the same medium without glucose and 2,4-D, but with 0.57 pM IAA, 0.1% casein hydrolysate, and 0.3 M mannitol. The unsealed plates were incubated in a Plexiglas chamber flushed with air (0.05% C02) with continuous illumination (120 pEm-'sec-'). The glucose was exhausted from the medium by day 12. The cultures were diluted 2-fold with the same fresh medium again on days 12 and 20, and on day 28 the suspensions were plated on the second medium, containing 0.8% agar. After 28 days in a 2% COZatmosphere or 35-42 days in air, 1- to 3-mm-diameter calli had formed, which were transferred to the same medium for continued growth. The suspensions formed by day 28 contained 143 pg of Chl per gram of fresh weight, had an IRGA C 0 2 fixation rate of 107 pmol per milligram of Chl per hour and a dark respiration rate of 76 pmol of 0 2 per milligram of Chi per hour. After growth for 42 days with air C 0 2 levels, calli pieces (60 mg each) were then transferred to fresh medium and kept under air or 2% C02 for 21 days. The respective fresh weights for the air versus high C02 were 210 and 370 mg, the Chl levels were 156 and 165 pg/g of fresh weight, the net photosynthesis rates were 1 1.5 and 0 pmol of C02 per milligram of Chl per hour with 0.034% COZand 109 and 45 with 1% COZ,and the dark respiration rates were 6.5 and 20 pmol of 0 2 per milligram of Chl per hour. Thus, both C02 conditions allow growth, but the high C02-grown cells have higher respiration and a lower capacity to fix C02 in the light. The possible reasons for the lower photosynthetic capacity of the 2% COZ-grown N . plumbaginifoh calli were increased dark respiration, decreased RuBPcase activity, and feedback inhibition of photosynthesis (Rey et ul., I990a). Since it has been shown by McHale (1985) that agar can support cell

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growth, the N . plurnbaginifolia calli, which were grown on agar-solidified medium in these experiments, were grown in the light and in the dark with air or 2% COz levels for 21 days (Rey et al., 1989). Dry weight increases of 54% were observed in the dark, while the net light-induced increases (minus that in the dark) were 200% and 600%, respectively. Thus, light contributes about 80% and 90%, respectively, to the growth. Estimates of the respective doubling times from the growth results presented above would be about 21 and 8 days. When the N . plurnbaginifolia cells are placed in liquid medium without sugar, plant regeneration begins and a homogeneous cell suspension cannot be obtained (P. Rey, personal communication). Thus, there are several examples of PA growth of cultures of tobacco and other Nicotiana species. While the cultures can grow well, none of the growing cultures contains more than about 200 pg/g of fresh weight of Chl, while, as shown later in this chapter, many other well-established cultures of other species have much higher levels. The low Chl levels are not characteristic of N . tabacum leaves, since Takeda el ul. (1989) reported leaf Chl levels of 2500 pg/g of fresh weight. 2. Ruta graveolens In the only report of P A R . graveolens cultures, Corduan (1970) described callus cultures which were grown for about 2 years on 0.65% agarsolidified medium with no other organic compounds in a 1% COz atmosphere and 2000 lux (about 30 pErn-'sec-') fluorescent light. Since the medium contained agar, there may be a question about the true PA nature of the culture, as discussed above. However, in 14C02fixation experiments performed in the light, incorporation of label into sugars, sugar phosphates, organic acids, and amino acids was observed. The dark fixation was much lower and was predominantly into amino acids, as measured by thin-layer chromatography.

3. Chenopodium rubrum Hiisemann and Barz (1977) described the initiation of a PA suspension culture of C. rubrurn from a green PM culture initiated from hypocotyl callus growing on MS salts and vitamins, 0.1 pM 2,4-D, and 2% sucrose. The suspension was initially placed in a sucrose-free medium in a sealed two-tiered flask, in which the C 0 2 concentration was maintained at 0.5% by the proper proportions of K2C03and KHC03 ( 2 M concentration). The cultures were grown under continuous illumination ( 19 Wm-', about 87 pEm-2sec-') at 28 k 1°C. Growth was slow initially, but the rate increased when the COz level was raised to 1 .O% and the 2,4-D level was lowered to 0.01 pM. The cell-doubling time was about 12 days, and the Chl

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content was 84 p g / g of fresh weight. These cells fixed C02 in the light in 1% C02 at a rate of 185 pmol/mg of Chl per hour, and the corresponding dark rate was 2.6. At a later time the PA C. rubrum culture contained 200 pg of Chl per gram of fresh weight, fixed C 0 2in the light at a rate of 190 pmol/mg of Chl per hour, and showed a doubling time of about 5 days when grown continuously in a 2% C02 atmosphere with 0.1 pM2,4-D (Hiisemann et al., 1979). In comparison PM cells grown on 2% sucrose grew more rapidly, contained less Chl (50 pg/g of fresh weight), and had similar photosynthetic CO2 fixation rates. The dark C 0 2fixation rate of the PA cells was about 3% of that in the light, while the PM dark rate was 25% of the rate in the light. The 14C02photosynthetic fixation products after a 30-min labeling period with either exponential or stationary phase PA cells were about 67% in the typical C) fixation compounds, with most of the remainder in C4 fixation compounds such as malate. The PM cells showed a reversal in this ratio, with the Cq fixation products predominating. Short-term labeling showed that C. rubrum leaves and PA and PM cultures all fixed COz predominantly via RuBPcase into PGA and sugar phosphates. Measurement of the C02 fixation enzymes showed that leaf extracts had a RuBPcase : PEPcase activity ratio of 4.7, while the ratios for exponential and stationary phase PA and for PM cells were 0.5, 1.2, and 0.18, respectively. The respective activities in the exponential and stationary phase PA cells were about 107 and 96 pmol of C02 per milligram of Chl per hour for RuBPcase and 214 and 78 for PEPcase. These results for C02 fixation products and enzyme activities are not completely consistent, since the enzyme levels indicate a higher predominance of C4 fixation than that shown by the fixation products. Hiisemann (1981) was able to obtain a C. rubrum PA suspension culture which would grow in a mineral medium without any growth regulators, vitamins, or other organic compounds in two-tiered flasks with 2% C02. The culture was produced by selectively transferring the darkest green viable cells. Studies with these cells showed that RuBPcase and PEPcase activities were almost equal during exponential growth, while RuBPcase predominated in the stationary phase. Short-term I4Co2labeling patterns mirrored these enzyme changes. The C02 fixation rate was about 8090 pmol/mg of Chl per hour in the light and 2-3.7% of this in the dark. The Chl levels were 20-30 pg per lo6 cells (about 200-300 p g / g of fresh weight). Husemann et a / . (1984) characterized the C. rubrum PA culture microscopically and physiologically during the entire batch culture cycle to show that the cells have different metabolism during the log and stationary growth phases. During the log phase the cytosolic and mitochondria1 activities are higher than that of the chloroplasts, as shown by enzyme

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activity, carbon flow, starch, and Chl changes. During the log phase there is preferential synthesis of cellular components such as lipids and proteins, while in the stationary phase general cellular component synthesis decreases and the photosynthate is shunted into starch. These results would also indicate that the stored starch is used to partially support the log phase growth. Thus, batch culture growth is not steady state, but consists of changing metabolic patterns. Generally, the PA C. rubrum cell Chl levels are reported to be near 0.2 mg/g of fresh weight, but Ashton and Ziegler (1987) reported that their strain, which they obtained from Hiisemann in 1980, contained about 1 mg of Chl per gram of fresh weight. These cells were maintained with a 16-hr light (75 pEm-2sec-') and an 8-hr dark cycle, while the cells are usually grown under continuous light of a similar intensity (19 WmP2, about 87 pEm-'sec-') (Hiisemann er al., 1984). The C . rubrum PA suspension culture was also grown as a batch culture in a 1.5-liter specially constructed airlift fermenter in the MS salts medium without any organic components (Hiisemann, 1982). The 2% C02 atmosphere was circulated through the medium, which was maintained at 25°C with a light intensity of 8000 lux (about 120 pEm-2sec-1). Under these conditions the cells grew somewhat slower than in the 30-ml batch culture and also had lower protein, Chl, RuBPcase, PEPcase, and COZ fixation levels. Hiisemann (1983) also constructed a continuous culture system using the 1.5-liter airlift fermenter and used conditions described previously (Hiisemann, 1982), except that the light intensity was 10,000 lux (about 150 pEm-2sec-1), and a pumping system was used to simultaneously add fresh medium and withdraw the cell suspension. By using a dilution rate of 0.16 per day, a steady-state cell level was reached within 8 days, giving a cell-doubling time of about 100 hr. Under these conditions a cell density of about 1.1 x 106/mlwas maintained with about 1 1 pug of Chl and 230 pg of protein per milliliter. The C 0 2assimilation rate was about 100 pmol of COZ per milligram of Chl per hour, with dark fixation levels equal to 3% of the light levels. The cells also contained starch, glucose, and sucrose, as well as nitrate and nitrite reductases, isocitrate dehydrogenase, malate dehydrogenase, and pyruvate kinase activities. The RuBPcase : PEPcase activity ratio was about 0.9, with activities of about 100 pmol/mg of Chl per hour. In further metabolic studies of the C. rubrum cell line, Herzbeck and Hiisemann (1985) showed that exogenously supplied 14C-labeledmalate was metabolized largely to organic acids (citrate, succinate, and fumarate) and to amino acids, which confirms the citric acid cycle involvement in the utilization of the C4fixation products. These results fit with the hypothesis, put forth by several authors, that the elevated levels of PEPcase during

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exponential growth feeds C4 compounds through the citric acid cycle to produce the amino acids needed for protein synthesis during the rapid growth phase. 4. Cytisus scoparius

Cytisus scoparius (Scotch broom) callus cultures were started from seed by Yamada and Sato (1978) on LS basal medium with 10 p M NAA, 1 p M BA, and 3% sucrose in light. The callus was subcultured every 21 days by selectively transferring the greenest tissue. After about 2 years the callus was cultured on the same agar-solidified medium without sucrose in a 1% COz atmosphere with continuous 4000- or 8000-lux illumination (about 60 or 120 pErn-'sec-'). Sustained growth was obtained over three subcultures, but the medium did contain agar. Chl levels up to 230 pg/g of fresh weight were attained. Sat0 et al. (1979) reported that PA C. scoparius callus grew from 50 mg of dry weight to a mean of 97 mg over four successive 4-week subcultures and contained a mean level of 133 pg of Chl per gram of fresh weight. This paper also describes some characteristics of a PM C. scoparius culture. The PA and PM C . scoparius callus I4CO2 fixation rates were near 63 pmol per milligram of Chl per hour in the light and were 2.6 and 7.0, respectively, in the dark (Sato et al., 1980). The label in C4 compounds such as malate was 41-44% of the total after 5 min in the light and about 100% in the dark. This high C4 labeling pattern fits with the fixation enzyme ratio of 0.47 for RuBPcase : PEPcase activities for both cell lines, the RuBPcase activity being 37 prnol of COZ per milligram of Chl per hour. Sato et al. (1981) reported that increasing the PQP3 content and lowering the NAA concentration to 1 p M with 1 p M BA, optimized C. scoparius PA growth. The doubling time was about 14 days, and the callus Chl content was 90-100 pg/g of fresh weight. All of the C. scoparius cultures described above were grown on agarsolidified medium, but recently a new culture was established which will grow PA in liquid medium (F. Sato, personal communication).

5 . Peganum harmala Suspension cultures of P . harmala were grown PA in two-tiered flasks with 2% COz in MS basal medium with 0.1 p M 2,4-D o r NAA with continuous light (8000 lux, about 120 pEm-2sec-') (Barz et al., 1980). The culture consisted of single cells and 10-30 cell aggregates with photosynthetic activity of 157 pmol of C 0 2 per milligram of Chl per hour. The lipid and harman alkaloid compositions were studied, as presented later, but no further details about the culture characteristics were presented.

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6 . Spinacia oleracea

Callus cultures were initiated by Dalton and Street (1976) from S. oleracea (spinach) seedlings on agar-solidified MS basal medium with 0.55 m M inositol, 20 p M biotin, 1.4 m M glutamine, 0.93 p M kinetin, 0.54 p M NAA, and 2% sucrose. Suspension cultures were initiated from callus in liquid medium of the same composition, except that the NAA was removed. The green S. oleracea suspension culture was grown in continuous culture in a 1.7-liter lab fermenter with 1% fructose and continuous light (Dalton, 1980). When the fructose content was lowered to 0.5%, the cell Chl level increased %fold, and the ratio of photosynthesis to respiration increased 22 times. When all fructose and other organic compounds were omitted and 1% COz was bubbled through the fermenter with increased light (120 pEm-*sec-'), PA growth was attained. Under three different dilution rates and light intensities the Chl levels were 1050-1390 pglg of dry weight (about one-tenth of this on a fresh weight basis), and the rate of photosynthesis measured as O2evolution was 406-546 pmol/mg of Chl per hour. The highest dilution rate maintained a cell-doubling time of about 5 days. This culture is ideal, since it grows rapidly on a medium with no organic compounds added. 7 . Datura stramonium and Datura innoxia

Cultures were initiated from leaf segments of aseptically grown D. stramoniirrn plants placed on agar-solidified LS basal medium with 5 p M NAA and 0.05 p M BA in Petri dishes in a 1% C 0 2 atmosphere with 3000-5000 lux (about 45-75 pEm-2sec-') continuous illumination at 2529°C by Yasuda et al. (1980). Some green callus formed, which was subcultured seven times at 21-day intervals. The callus increased by 55% in fresh weight during a 21-day period, contained 130 pg of Chl per gram of fresh weight, and did evolve 0 2 in the light (26 pmol/mg of Chl per hour). Since these cultures grew only slowly for an extended period on a medium without added sugar, the presence of agar in the medium raises a question about their true PA nature. Callus of D. innoxia, which was originally initiated by Ranch and Giles (1980), was grown on 0.8% agar-solidified MS basal medium with 3% soluble potato starch, 0.5 p M picloram, and 7.0 p M kinetin (Xu et al., 1988). Green callus which formed on this medium was used to initiate a PM suspension culture in the same medium without agar and with 1% starch. The cultures were incubated in 300 pEm-*secC' continuous light at 30°C on a gyratory shaker. A PA culture (denoted DAT-P) was initiated from

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this culture in liquid medium with MS salts, complex vitamins (Horn et al., 1983b), 0.3 pM picloram, 5.4 pM NAA, 0.93 p M kinetin, 5 mM HEPES (pH 7.0), and no starch or sugar. CO;? (5%) was blown into the flask through a 16- or 17-gauge needle stuck through the rubber stopper on the Erlenmeyer flask, with another needle as an exit. Both needles had a small Millipore filter unit glued onto them to maintain sterility. The flasks were partially covered initially with aluminum foil to lower the light intensity, and the foil was gradually removed during the initial 4-to 10-week period. The culture was transferred when dark green growing tissue was visible and then was subcultured every 6 weeks. After 18 months of PA culture, the DAT-P cells contained 101-226 pg of Chl per gram of fresh weight and grew with a doubling time of about 14 days (Xu et al., 1988). No growth occurred in the dark, and only one doubling occurred in the light in ambient CO;?levels (600-700 pl of C02 per liter in the culture room) in a 28-day growth period. The cells grew in small clumps, with cell diameters of about 50pm with numerous chloroplasts. The IRGA demonstrated that C 0 2 was fixed at the rate of about 26 pmoll mg of Chl per hour in 2% 02, while dark evolution was very high (about 65 pmol of C02 per milligram of Chl per hour. The 14CO;?fixation rates in the light and in the dark in 2% 0 2 were near 38 and 3.5 pmol/mg of Chl per hour, respectively. The initial RuBPcase activity levels varied widely (6.2-25.2 pmol of CO;?per milligram of Chl per hour) and were about 17-27% of the total (activated) activities. PEPcase activities were near 40 pmol of C02 per milligram of Chl per hour. The DAT-P culture was further characterized during about a 2-year period on a more minimal medium in which all vitamins and the HEPES buffer were removed (Goldstein and Widholm, 1990). During this period the Chl content increased gradually from 146 to 758 pg/g of fresh weight, with a cell-doubling time of about 12 days. The initial RuBPcase activity increased to 60 pmol of C02 per milligram of Chl per hour, with a total activity of 171, so the activation level in the cells was about 35%. Addition of the protease inhibitors antipain, leupeptin, and PMSF did not affect the initial or total activity levels of RuBPcase. The PEPcase activity also increased to 20 pmol of COz per milligram of Chl per hour. 8. Hyoscyamus niger Yasuda et al. (1980) placed leaf pieces of aseptically grown H. niger plants on agar-solidified LS basal medium, as described above with D . stramonium, and cultures were obtained which could be maintained on the medium without sugar for five 3-week passages. The culture showed a 198% increase in fresh weight during a 3-week period and contained 31 p g of Chl per gram of fresh weight. The rate of 0 2 evolution in the light was 138 pmol per milligram of Chl per hour.

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9. Arachis hypogea A callus culture was initiated from A. hypogea (peanut) cotyledons by Kumar (1974) on MS basal medium without inositol, but with 4% sucrose, 1.86 pM kinetin, and 0.6% agar. The culture was described to be PA by Bender et al. (1980) and has been maintained on agar-solidified sugar-free medium for about 7 years with normal air C 0 2 levels in the light (Bender et al., 1985). The green callus was then grown in continuous light (100 pEm-2sec-') with air C 0 2 levels at 21°C in a Niter laboratory fermenter (3 liters of medium). Starting with 7-8 g of fresh weight of cells, the daily fresh weight increment for the 6-week period was 250 mg, which would give a doubling time of almost 6 weeks. Short-term CO2-labeling studies showed greater initial fixation into C4 compounds, rather than C3 fixation products. The A . hypogea cells were grown continuously in a liquid medium containing only inorganic compounds with ambient C02 levels, with a doubling time of over 42 days (Neumann et al., 1989). Addition of sucrose to the culture medium decreased I4CO2 fixation into compounds formed via the C3 pathway (RuBPcase), but not that Of C4 (PEPcase). This change in fixation pattern is correlated with the levels of RuBPcase and PEPcase found in the cells grown with the different sucrose levels. The results presented thus far indicate that the A. hypogea tissue culture can grow slowly in ambient C 0 2 levels in minimal medium and therefore may be a good system for further study. 10. Asparagus ofjcinalis

PA suspension cultures of A . ofjcinalis (asparagus) were grown as batch cultures with 14-day subculture periods for 5 years in algal cultivation tubes and were then studied extensively when grown in a steady-state turbidostat for 500 hr (Peel, 1982). The medium was MS basal and organics with 1.37 m M glutamine, 5.4 p M NAA, and 2.3 M kinetin. Two percent C02 was bubbled through the culture tube, which was maintained at 25"C, with 16 hr of 120 pEm-2sec-' light and 8 hr of darkness. The cultures were maintained at either a high (about 5 mg of dry weight per milliliter) or low density (about 2 mg of dry weight per milliliter). The Chl levels were steady at 1 .O and 4.4 mglg of dry weight for the high- and low-density cultures, respectively, which would be about one-tenth of these values if calculated on a grams of fresh weight basis. The respective rates of O2 evolution of samples removed from the turbidostat were 162 and 230 pmol/mg of Chl per hour, while the dark O2 utilization rates were 58% and 66% of the light O2 evolution rates. The cells grew rapidly, with high- and low-density doubling times of 5.8 and 1.9 days, respectively. The lower photosynthesis and longer doubling time for the high-density culture indicate that light may be limiting the performance of the high-density cultures.

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PA A . oficinalis cultures were also initiated by Chaumont and Gudin (1985) by fir;st selecting green friable callus obtained from leaves on MS macroelements, Nitsch (1968) microelements with modified Nitsch and Nitsch (1965)organics, 2% lactose, 1.37 mM glutamine, 5.4p M NAA, and 0.44 p M BA. PM suspension cultures were initiated from this callus and were grown with continuous light (130 pEm-*sec-'). PA cultures were then obtained by gradually reducing the lactose content by dilution, in the presence of a 1% C 0 2 atmosphere. PA growth was obtained in both a batch-fed airlift culture tube system or a continuous or semicontinuous stirred system (Street et al., 1971).Net positive O2evolution was obtained within 2-8 weeks. After 4 months of growth under PA conditions, the doubling time in the airlift and stirred culture systems were 21 and 7 days, respectively. The two reports of PA A . ofjcinalis cultures demonstrate that fastgrowing cultures can be obtained (Peel, 1982;Chaumont and Gudin, 1985), but no further information has been reported concerning these cultures.

11. Glycine max Callus was initiated from cotyledons of G. rnax (soybean) cv. Corsoy on MS salts, complex vitamins (Horn et al., 1983b), 5.4 p M NAA, 0.93 p M kinetin, 0.6% agar, and 1% sucrose under continuous illumination (Horn et al., 1983b). Following selective transfer of the greenest callus for many subcultures, a PM suspension was initiated in the same medium without agar under continuous illumination (200-300 pEm-*sec- I ) on a gyratory shaker at about 28°C. PA cultures were initiated from the PM cells under the same conditions in the same medium without sucrose, with 5 mM HEPES (pH 7.0) added. The atmosphere in the flask contained 5% COZ, which was blown in through a syringe needle. Growth was variable and slow initially, but by the 17th biweekly transfer the cells had a Chl level near 400 pglg of fresh weight and a doubling time of about 5 days. Growth was completely inhibited by 0.5 p M DCMU and required light and elevated C02. The maximal I4CO2fixation rate measured late in the culture period was about 90 pmol/mg of Chl per hour when dark fixation was low. This PA culture was named SB-P. The SB-P cells were much more highly vacuolated and contained fewer chloroplasts and less starch than did soybean leaf cells (Figs. 1 and 2). The SB-P culture was characterized by Rogers et nl. (1987) after several years of continuous PA growth since its initiation in 1982 by Horn et al. (1983b). The culture medium had been simplified by removal of HEPES Electron micrograph of SB-P (Glycine max) cells, taken in 1985, showing the large vacuole and low chloroplast density (D. R . Duncan, unpublished observations). Bar = 3 pm.

FIG. 1

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and all of the vitamins, except 0.3 pM thiamine. The cells grew at a linear rate, with a doubling time somewhat less than 7 days and contained 1500-2000 p g of Chl per gram of fresh weight. The I4CO2 fixation rate in the light was about 30-65 pmol/mg of Chl per hour, while the dark rate was 8% or less of that in the light. Light-induced O2 evolution rates averaged about 80 pmol/mg of Chl per hour, while the dark respiration rates were usually 10-20 pmol of O2 per milligram of Chl per hour, but a peak of 40 was observed 4 days after transfer. RuBPcase activity was relatively stable during the culture period, with levels of about 40 (initial) and 6090 pmol of COz per milligram of Chl per hour (total), while the PEPcase peaked at about 85 pmol early in the culture period but was usually near 30 pmol for most of the period. Light micrographs of the SB-P cells show that the cells in 1985 (Fig. 3) had a much lower number of chloroplasts than do the cells today (Fig. 4). the Chl level of the cells in 1985 was about 400 pg/g of fresh weight, while the level today is near 2000 pg/g of fresh weight. A second PA soybean suspension culture, which was denoted SBl-P, was initiated by Rogers and Widholm (1988) from stems of another genotype, PI 437833, on L2-based medium (Phillips and Collins, 1981) with 1.81 pM 2,4-D, 25 pM NAA, 10 p M kinetin, 3% sucrose, and 0.8% agar with 250 pEm-'sec-' continuous illumination. Selected green callus was grown on MS salts, 0.3 p M thiamine, 5.4 p M NAA, 0.93 pM kinetin, 1% sucrose, and 0.8% agar. Selected green callus was then placed into the same medium without agar and incubated on a gyratory shaker with continuous illumination, and a selection process for green viable cell clumps occurred. After 15 transfers at 2-week intervals, the PM culture Chl content had increased from the initial level of 52 to 700 pg of Chl per gram of fresh weight. The PA culture was initiated from the PM cells in the same liquid medium, but without sucrose, under a 5% C 0 2 atmosphere blown into the flask. About 90% of the cells bleached and died and green viable cell clumps were selected for, and after 35 weeks with 15 transfers 90% of the cells were green and viable and the culture Chl content was about 1500 pg/g of fresh weight. The culture was composed of aggregates of 30-50 cells with from 50-90 x lo6 cells per gram of fresh weight. The SBl-P cells were similar to SB-P cells in most respects, including a doubling time of 5 days, an O2 evolution rate of 75 pmol/mg of Chl per hour, I4CO2 incorporation rate of about 50 pmol/mg of Chl per hour, a low dark fixation rate of about 7.5% of that in the light, high 0 2 inhibition of C 0 2 FIG. 2

Electron micrograph of soybean (Glycine max) leaf mesophyll cells showing the small vacuole and high chloroplast density (D. R . Duncan, unpublished observations). Bar = 3 Frn.

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FIG. 3 Phase-contrast micrograph of PA SB-P (Glycine max) cells taken in 1982 when the

chloroplast number and the Chl content were relatively low (M. E. Horn. unpublished observations). Bar = 25 pm.

fixation, a COZcompensation concentration of about 230 pl per liter, initial and total RuBPcase activities of about 40 and 65 pmol of COZper milligram of Chl per hour, respectively, and a PEPcase activity of 40-1 10 pmol of C02 per milligram of Chl per hour. This study shows that when a second soybean PA culture (SBl-P) was initiated from a different genotype, the resulting culture had characteristics very similar to that of the other independently initiated line (SB-P). The SB-P and SBI-P cultures were studied further by Roeske et al. (1989). The respective l4COZfixation rates for the strains were 60-90 and 130-170 pmol/mg of Chl per hour, and the respective total RuBPcase activities were 156 and 262 pmol of C 0 2per milligram of Chl per hour. The cultures were light activated for 5 min with 350 pEm-*sec-' white light before enzyme extraction, so the initial RuBPcase activity of SBI-P was

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FIG. 4 Phase-contrast micrograph of PA SB-P (G/ycinr m a x ) cells taken in 1991 when the chloroplast number and the Chl level had increased greatly (C. Goldstein, unpublished observations). Bar = 25 Fm.

93% of the total, but the SB-P was not activated by this treatment and was only 55% of the total. The PEPcase activities were 19 pmol of C02 per milligram of Chl per hour. Dark respiration rates were 36 and 13 pmol of 0 2 per milligram of Chl per hour for SB-P and SBl-P, respectively. The labeling patterns found in I4CO2 pulse-chase experiments showed that less than 5% of the label was incorporated into malate, and most was through the C3 pathway, which fits with the relatively high RuBPcase : PEPcase ratio. Goldstein and Widholm (1990) reported that the SBl-P cells, but not the SB-P cells, could grow continuously in a medium devoid of thiamine, with only MS salts and growth regulators present. The SBI-P cells maintained a Chl level of about 1700 pgig of fresh weight, with a doubling time of 8-9 days. The RuBPcase initial and total activities were 67 and 150 pmol of C 0 2per milligram of Chl per hour, and the corresponding PEPcase activity

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was 24. The SB 1-P cells were able to grow for 11 subculture cycles of 3 weeks under ambient C02 levels (600-700 pl of C02 per liter), while the SB-P cells did not adapt well to these conditions. These studies show that the two soybean PA cultures which have been grown continuously for many years under PA conditions have gradually improved their photosynthetic characteristics, so are now similar to leaves in many respects. 12. Solanum tuberosum

Callus was initiated from S. tuberosum (potato) tubers on MS basal medium with 14 p M 2,4-D, 0.93 p M kinetin, 0.11 p M folic acid, 0.11 p M biotin, 0.1% casein hydrolysate, 2.5% sucrose, and 0.9% agar (LaRosa et al., 1984). Green friable callus (20-50 pg of Chl per gram of fresh weight) was placed into the same medium lacking agar and incubated on a shaker with 90-1 10 pErnp2sec-' fluorescent light (15-hr light period). PM cultures were initiated from the H culture with at least 50 pg of Chl per gram of fresh weight in media with 0.5% sucrose and then with 0.25% sucrose. After several passages the PM cells with about 100 pg of Chl per gram of fresh weight were inoculated into a medium lacking sucrose and were incubated in a chamber containing 2% COz, in which PA growth was obtained. The cultures were subcultured every 30-60 days over a 3-year period. The PA culture contained 100-200 pg of Chl per gram of fresh weight, fixed C 0 2at the rate of about 30-70 pmol per milligram of Chl per hour, and had a doubling time of about 8 days. The PA S. tuberosum cells have recently been used to select an atrazineresistant mutant (Smeda er al., 1990).The PA cells are still growing after 9 years under about double the original light intensity (16 hr of light at 29°C and 8 hr of dark at 26 "C) (P. C. LaRosa, personal communication). The Chl level is about 450 pg per gram of fresh weight, and the O2evolution in the light is 150-175 pmol per milligram of Chl per hour. W. Digitalis purpurea

Callus was induced from D. purpurea seedlings on M S salts with 3 p M thiamine HCI, 17 p M IAA, 3% sucrose, and 0.8% agar (Hagimori et al., 1982). After 30 days the callus was transferred to the same liquid medium with 5.7 p M IAA and was incubated on a reciprocal shaker under continuous illumination at 28°C. These cultures were grown under PA conditions, with 1% C 0 2in a two-tiered flask with no sucrose in the medium (Hagimori et al., 1984). The fresh weight increased 2-fold in 3 weeks and 3-fold in 6 weeks, with no growth in the absence of added C02. The Chl level was 220 pg/g of fresh weight, the cell photosynthetic 0 2 evolution rate was

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87 pmol per milligram of Chl per hour, the Hill reaction rate with isolated chloroplasts was 26.2 pmol of 0 2 per milligram of Chl per hour, and the RuBPcase activity was 94.9 pmol of C 0 2 per milligram of Chl per hour. The D . purpurea culture appears to have potential, but in the work reported so far the batch culture was grown for only one passage under PA conditions and continued growth was not described. 14. Morinda lucida

Callus was initiated from green twigs of M. lucidu plants on B5 basal medium with 9.0 p M 2,4-D, 2.7 p M NAA, 2.9 p M IAA, 0.93 p M kinetin, 2% sucrose, 0.3% casein hydrolysate, and agar under continuous white light (3000-4000 lux, about 45-60 pEmP2sec-') (Igbavboa er al., 1985). A suspension was initiated from the callus in the same medium without agar under continuous light (6000 lux, about 90 p Em-2sec-'), with periodic filtering to remove large clumps. After several passages most of the cells were green and PA cultures were formed when sucrose was gradually omitted from the medium. The cultures were grown with 2% COZ in two-tiered flasks or in an airlift fermenter. The PA cells grew slowly, with a doubling time of over 14 days, with Chl levels of about 130-276 p g / g of fresh weight. The PA culture was maintained in the two-tiered flask system with 2% C 0 2 and was used further in studies of anthraquinone synthesis and cytological changes (Yamamoto et al., 1987), so the culture can grow continuously. The culture medium does, however, contain 0.3%casein hydrolysate, which would make some contribution to support the cell growth. However, the cells can grow without this additive (W. Husemann, personal communication).

15. Daucus carota Bender er al. (1985) described a D. carota (carrot) root explant system in which photosynthetic cultures are formed during incubation in liquid medium (Neumann, 1966) with 2% sucrose, 0.28 m M inositol, 1 I .4 p M IAA, and 0.46 p M kinetin under continuous light (about 100 pEm-2sec-'). The ultrastructural and photosynthetic development of this system was described by Kumar er al. (1983). The explants began to turn green after 4-5 days, and if placed in sugar-free liquid medium in a lab fermenter after 10 days, the cultures grew with a 932-mg daily fresh weight increment (with an initial fresh weight of 7-8 g), which would indicate a doubling time of about 3 weeks. These results were obtained with ambient C 0 2 levels and continuous illumination. If these cultures continue to grow upon further transfer in liquid medium, then the system would be one of the few to grow well with ambient COz levels.

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16. Catharanthus roseus PM suspension cultures of C. roseus (periwinkle) cv. Little Delicata were maintained in B5 medium with 4.5 p M 2,4-D and 3% sucrose under continuous illumination (40 pEm-*sec-') at 26°C (Tyler et al., 1986). PA cultures were initiated from the PM culture in liquid B5 medium with 5.4 pM NAA, 4.6 p M kinetin, and no sucrose, with a 2% C02 atmosphere flushed through the flask under 100 pEm-'sec-' illumination. The culture was maintained for about 1 year with 4- to 6-week subculture intervals. The cells contained about 50-105 p g of Chl per gram of fresh weight, had maximal 0 2 evolution rates of about 90 pmol per milligram of Chl per hour, contained high starch levels, and doubled in number every 19 days. The chloroplasts had a less extensive thylakoid system than did leaf mesophyll cells.

17. Euphorbia characias Callus was initiated from E. charucias stems on M S basal medium with 41 n M biotin, 6.3 pM cysteine, 1.37 m M glutamine, 5.4 p M NAA, 4.4 p M BA, 0.8% agar, and 3% sucrose (Hardy et al., 1987a) with 90110 pEm-2secC' illumination for 12 hr at 25°C and 12 hr of dark at 20°C. Green friable callus was selectively transferred and was subsequently suspended in the same liquid medium and incubated on a gyratory shaker at 25°C with 18 hr of 90-100 pEm-*sec-' illumination daily. When the sugar of the medium was exhausted, cell growth ceased and Chl was at its highest level (32 days); PA cultures could then be initiated by transfer to fresh medium in three different reactors aerated with 2% C 0 2 . When the transition from PM to PA of the Euphorbia cells was followed in two reactors, a similar pattern was noted; Chl and photosynthetic capacity both increased after the sugar in the medium was exhausted (Hardy et al., 1987b). Another Euphorbia PM suspension culture was initiated by Chagvardieff et al. (1988) in MS basal medium with 5.4 p M NAA, 4.4 p M BA, and 1.5% sucrose under 18 hr of daily illumination (90-100 pEm-2sec-') at 25°C on a gyratory shaker. PA cultures were produced from these PM cultures in bubble columns in MS salts, Morel and Wetmore (1951) vitamins, l .37 p M glutamine, and 0.55 p M inositol, with no growth regulators or sugars (Rebeille et al., 1988). The strain growing with 2% COz had been subcultured every 2 weeks for 2 years by transferring one volume into three volumes of fresh medium for a 7-day doubling time. A strain was also grown for several months in airlift conical flasks on an orbital shaker with bubbled air. These cells were subcultured every month and grew with a doubling time of 20 days. Gross photosynthesis of these strains was 138192 pmol of C 0 2 per milligram of Chl per hour, and the Chl levels were as

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high as 10 mgig of dry weight (about 700 pgig of fresh weight) in cells in the stationary phase (which was the time when these values were maximal) (Carrier er al., 1989). Thus, this recently initiated Euphorbia PA culture appears to grow well both in air and on 2% C02 and has a high Chl level; they should be ideal for many studies. 18. Amaranthus cruentus and Amaranthus powellii

Callus was initiated from seedlings of the C4 photosynthesis species A . cruentus and A . powellii on MS basal medium with 1.81 p M 2,4-D, and suspension cultures were initiated from the callus after 28 days using the same medium (Xu et al., 1988). When the suspensions were grown under 300 pEm-2sec-1 continuous illumination with a reduced transfer frequency, the cells became yellow or green and then when transferred to MS basal medium with 3% soluble potato starch, 7 p M kinetin, and 0.5 p M picloram, the Chl levels climbed to 30-100 pg of Chl per gram of fresh weight. The starch was then reduced to 1%, and PA cultures were initiated from these PM cultures in liquid medium with MS salts, complex vitamins (Horn et al., 1983b), 0.3 p M picloram, 5.4 p M NAA, 0.93 p M kinetin, 5 mM HEPES (pH 7.0), and no starch or sugar with a 5% C 0 2 atmosphere. The flasks were partially shaded initially, and when dark green growing tissue was visible, the cultures were transferred to fresh medium, with subsequent transfers every 6 weeks for at least 18 months. The respective A . cruentus and A . powellii PA cultures, which were denoted ACR-P and APO-P, doubled in fresh weight every 8-14 days and contained 114-294 pg of Chl per gram of fresh weight. The APO-P cells required high C 0 2 and light for growth (the ACR-P cells were not tested). The I4CO2 fixation rates for the suspensions were 9-39 pmol of C 0 2 per mjlligram of Chl per hour, with dark rates of 10-20% of those in the light. The ACR-P and APO-P PEPcase levels were about 21 and I3 pmol of C 0 2 per milligram of Chl per hour, respectively. The respective RuBPcase varied widely, with maximal initial activities of 33-50 and total activities of 79-99 pmol of C02 per milligram of Chl per hour, which shows initial activation levels of 42-51%. The ACR-P line was lost, but the APO-P culture, when studied about 2 years later after being grown in a medium containing only minerals and the growth regulators, had increased Chl levels near 1700 pg/g of fresh weight (Goldstein and Widholm, 1990). The doubling time was about 12 days. The RuBPcase activity had not increased, being 39 (initial) and 85 pmol of COZ per milligram of Chl per hour (total), for a 46% activation level. The PEPcase levels had, however, doubled to 26 pmol of C02 per milligram of Chl per hour.

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19. Gossypium hirsutum Callus cultures were initiated from aseptic cotyledons and hypocotyls of 2-day germinated cotton seedlings, G. hirsutum cv. Stoneville 825, by Blair et al. (1988). Suspension cultures, initiated from the callus in MS basal medium with 1.81 pM 2,4-D, were maintained under low light for 9 months, with 10-day subculture intervals. A green suspension was produced when placed under continuous light (200 p Em-2sec-') during the mid-log phase and was then subcultured at the stationary phase into MS salts, complex vitamins (Horn et al., 1983b), 1% sucrose, 5.4 pM NAA, and 0.93 p M kinetin. A variegated mixture of green and nongreen cells formed, which contained about 29 p g of Chl per gram of fresh weight. When these cultures were incubated in the same medium without sucrose, but with 5 mM HEPES (pH 7.0)added in a 5% C02 atmosphere, about 90% of the cells died and the remaining cells grew slowly as 5-mm-diameter hard clumps. After four or five subcultures in 6 months, the doubling time was about 14 days, and the Chl content was 300 pg/g of fresh weight. Growth of these cultures required light and 5% C02 and was 64%inhibited by 1 p M DCMU. After 19 months the cell aggregate size had decreased to less than 0.5 mm in diameter, and the Chl content was 500-600 pg/g of fresh weight. The doubling time was about 4 days during the early log phase and 8 days later on. The light-stimulated C 0 2 fixation (measured by IRGA) by the PA G. hirsutum culture, denoted COT-P, was stimulated as the 0 2 concentration was decreased, indicating that photorespiration was occurring. Dark respiration ranged from 30 to 44 pmol of C 0 2 per milligram of Chl per hour. The I4CO2 fixation rate in the light was about 24 pmol/mg of Chl per hour, the dark rate being much lower. The initial and total RuBPcase activities were 46 and 63 pmol of C02 per milligram of Chl per hour, respectively, while the PEPcase level was near 76 pmol. When the COT-P cell homogenates were mixed with spinach leaf or SB-P cell extracts and RuBPcase activity was measured, no decrease in the total activities were seen. This indicates that the COT-P cells do not contain gossypol or other secondary compounds often found in cotton leaves, which are known to inhibit enzyme activity. The COT-P cells were adapted to grow for periods of up to 24 months under ambient C 0 2levels (600-700 pl of C 0 2per liter in the culture room) (Xu et al., 1988), with subculture every 3 weeks. This strain was denoted COT-PA. The cells contained 218-607 p g of Chl per gram of fresh weight and had a doubling time of about 14 days, a light I4CO2 fixation rate near 18 pmol/mg of Chl per hour (the dark rate being about 25% of this value), initial and total RuBPcase activities of about 33 and 53 pmol of C02 per milligram of Chl per hour, respectively, and a PEPcase activity of about 13.

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More recently, Roeske et al. (1989) reported that the COT-P and COTPA cells are similar in appearance and cell size (35-60 p m in diameter), being highly vacuolate, with chloroplasts studding the periphery. Both strains fixed C 0 2 at the rate of 100-130 pmol/mg of Chl per hour, and similar O2 inhibition of COZfixation was observed. Illumination of cells in 350 pEm-’sec-’ light for 5 min before RuBPcase extraction produced about 94% activation, in comparison with the total activity found after in uitro activation. The total RuBPcase activities for COT-P and COT-PA were 117 and 162 pmol of CO2 per milligram of Chl per hour, respectively, and the PEPcase activity was about 20. 14C02labeling showed incorporation to be largely by C3 fixation, which fits with the high RuBPcase : PEPcase activity ratio. These cells were grown in a simplified medium with MS salts, 0.3 p M thiamine, 5.4 pM NAA, 0.93 p M kinetin, and 0.3 pM picloram. Goldstein and Widholm (1990) have grown the COT-P cells in a liquid medium without any vitamins for about 30 months and now find the Chl level to be about 900 pg/g of fresh weight, the doubling time to be 7 days, and the initial and total RuBPcase activities to be 42 and 151 pmol of CO2 per milligram of Chl per hour. Thus, the RuBPcase is only 27% activated in uiuo. The PEPcase activity was 16 pmol of C02 per milligram of Chl per hour. The cotton cultures have changed during a few-year period, to have high Chl and RuBPcase levels and low PEPcase activity. The C02 fixation products are also similar to those of leaves, so these cultures, growing in either ambient or elevated C02 levels, form an ideal system for PA cell studies. 20. Dianthus caryophyllus A PA suspension culture of D . caryophyllus (carnation) was grown in MS basal medium with 10.8 p M NAA and 0.39 p M BA, stirred at 150 rpm with 100-120 pEm-’sec-’ continuous illumination at 25°C with 1% COZ (Rebeille, 1988). The culture grew with a doubling time of about 12 days and contained about 200 p g of Chl per gram of fresh weight. Photosynthesis measured as O2 evolution in the light varied greatly, with a maximum of about 17.5 pmol of 0 2 per milligram of Chl per hour, with a steady dark respiration rate near 10 pmol of 0 2 per milligram of Chl per hour. Cellular sucrose and glucose levels were lowest during exponential growth, when photosynthetic activity was highest, while starch levels were low at all times. Since the C o t compensation concentration was found to be below the air C 0 2 level, the carnation culture was incubated with atmospheric COZ levels, and growth did occur for 1 year, with a doubling time of about 24 days. The Chl levels and photosynthesis rates were similar to those of the

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1% C02-grown PA cultures, but the respiration rate and the sugar content were lower. The air-grown culture had lower fumarase levels, indicating a lower mitochondria content, which would correlate with the lower respiration. The PA D. caryophyllus suspension culture was also being grown in flasks on an orbital shaker and in bubble columns with air bubbled through both systems under continuous fluorescent illumination ( 1 50- 180 p Emp2 sec-l) (Avelange et al., 1990). The suspensions grew as 100-150 cell aggregates, with a doubling time of 15-20 days. Chl levels were 100150 pg/g of fresh weight, and typical net photosynthesis and respiration rates were about 110-170 and 40-1 10 pmol of O2 per milligram of Chl per hour, respectively. The D. caryophyllus suspension cultures grow well at both air and elevated C 0 2 levels, so they are good cultures for further studies.

B. Culture Initiation and Medium Requirements

While there have been many methods used to initiate PA cultures, most cultures have been produced by using a relatively long procedure whereby H callus cultures are initiated from some plant tissue, green portions are selectively transferred, PM suspensions are initiated and maintained for a period, and finally the cells are placed in a medium lacking sugar, in which they begin to grow as PA cultures. Some cultures, however, were initiated more rapidly, as discussed below. Since Chl levels are related to photosynthetic capacity, conditions which favor Chl accumulation are used in the culture initiation procedure. Light is, first of all, necessary, since Chl biosynthesis requires light. The use of a cytokinin is almost universal, at least initially, since these compounds appear to stimulate chloroplast development and do induce the synthesis of mRNA-encoding chloroplast proteins in leaves (Flores and Tobin, 1986). Kumar et al. (1984) showed that kinetin increased the Chl levels, the number of chloroplasts per cell, and the degree of thylakoid development in cultured carrot root explants. In studies with PM N . tabacum cultures, Bergmann (1967a,b)found that the growth regulator 2,4-D depressed Chl levels and, in turn, photosynthesis, while NAA did not have this effect. The addition of 25 or 50 p M 2,4-D depressed photosynthesis rates within 1 hr, so this auxin seems to have a potent detrimental effect on PA cultures. Others, including Hiisemann and Barz (1977) and Yamada et al. (1978), have also bound 2,4-D to be detrimental to greening. As a result most PA cultures are grown with NAA as the auxin. However, several cultures are grown with 2,4-D [tobacco (Chandler et al., 1972; Ikemeyer and Barz, 1989), P. harmala (Barz et al.,

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1980), and potato (LaRosa et al., 1984)]. While IAA was used in several cases, it would appear that, due to light sensitivity, this auxin would be degraded rapidly. There is some indication that PA cultures have fewer requirements for exogenous organic compounds (vitamins and growth regulators), since some of the cultures [C.rubrum (Husemann, 1981) and S. oleracea (Dalton, 1980)] grow without any additions, even though most H cultures require growth regulators and thiamine. That light may be important in vitamin needs is shown by studies with S . oleracea cultures, which require organic factors when grown in the dark (Dalton and Peel, 1983), while the PA culture required none (Dalton, 1980). Likewise, tobacco cultures required thiamine when grown in the dark, but not when grown in the light (Bergmann and Bergmann, 1968). Goldstein and Widholm (1990) found that the PA cultures SBl-P, COT-P, DAT-P, NTG-P, and APO-P grew well without thiamine, but SB-P did not. Why one soybean strain required thiamine and one did not is not known. However, Ohira et al. (1976) found that soybean, tobacco, and rice suspension cultures required thiamine, while R . graueolens and peanut did not. Thiamine levels decreased to near zero in cultures unable to grow and remained steady in those that did grow. Light had no effect on the growth or thiamine levels in these studies. Thus, we can conclude that not all H cultures require thiamine and that light may or may not always affect this requirement. A desirable goal with PA cultures is to eliminate all organic constituents from the medium in order to have absolute PA growth. The carbohydrate source and amount also affect Chl levels, as demonstrated by Edelman and Hanson (1971), who showed that 3% sucrose inhibited Chl accumulation in two carrot cultures, while other sugars may or may not, depending on the carrot strain. Neumann and Raafat (1973) also showed that sucrose decreased the Chl content of carrot cultures. Likewise, studies by Dalton and Street (1977), with a PM spinach suspension culture growing in a medium with no organics except sugar, showed that the readily utilizable sugars, sucrose, glucose, fructose, and maltose, when present above a certain level in the medium (0.25% for sucrose), inhibited Chl accumulation, while the poorly utilized sugars, inulin or raffinose, supported only slow culture growth, but stimulated Chl accumulation. This concept was taken one step further when starch was used as a sugar source for growing cultures (Blair et al., 1988; Xu et al., 1988). Apparently, sugar is released slowly, so is limiting to growth and thus enhances Chl accumulation. Most of the PA cultures are grown with MS and LS minerals. Sat0 et al. (1981) found that changing the N, K + , Cu2+,Mn2+,or Fe3+concentrations of the LS basal medium did not affect PA growth, while increasing the

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PO:- concentration 4-fold did increase the growth rate and the Chl content. We found that changing the levels of Mg2+, PO:-, Ca2+, or the combined microelements, or altering the NH;; ratio of the MS or LS formulation did not enhance the growth of the SB-P cells appreciably (M. E. Horn and J . M. Widholm, unpublished observations). Continuous illumination from fluorescent lamps (100-300 p Em-'sec-' ) is used with most PA cultures. However, the Euphorbia PA culture (Rebeille et al., 1988) was grown with an 18-hr photoperiod, and the asparagus PA culture (Peel, 1982) was grown with a 16-hr photoperiod. Continuous light should supply more energy to the cultures and possibly decrease the dark respiration energy loss, but a dark period might allow normal metabolic processes that occur in plants at night (e.g., starch utilization) to be performed. A number of culture vessels have been used to grow PA cultures, including Petri dishes, two-tiered flasks, bubble tubes, flasks with a controlled atmosphere blown through, and several kinds of fermenters. The two-tiered flask system is closed to the atmosphere and maintains elevated COz levels (usually 1-2%) by having a K2C03 KHC03 solution of the proper proportions in the lower flask. This closed system should allow the build-up of gases such as ethylene, which was shown by Dalton and Street (1976) to inhibit Chl accumulation in spinach suspension cultures. Since a number of cultures have grown quite well in the two-tiered flask system, as listed above, gaseous build-up is apparently not a problem, at least with certain cultures. The C.rubrum cells did not grow any better in an open system, and several new cultures (e.g., S . oleracea, C . roseus, Lycopersicon peruvianum, and Digitalis lanata) have also been grown PA in the closed two-tiered flask system (W. Husemann, personal communication). Most PA cultures are grown with 1% or higher levels of COz. However, there are several PA cultures capable of growth with ambient air COz levels. It is important to realize that, while the normal air COz level is about 0.035%, the levels found in culture rooms may be much higher, as our culture room is usually between 0.06%and 0.07%. The cultures which are apparently capable of continuous growth in air with reasonable doubling times are the carnation (Rebeille, 1988; Avelange et al., 1990) and Euphorbia (Rebeille et al., 1988; Carrier et al., 1989) suspensions. Husemann et al. (1990) recently reported that the C.rubrum suspension can also grow in air. These cells have been growing for about 2 years, with 2- to 3-week subculture intervals, with a 150-230% increase in cell number and fresh weight each time (W. Husemann, personal communication). We have reported that several cultures grow in ambient COz levels (e.g., COT-PA, SB1-P, and NTG-P); however, these cultures do not grow indefinitely and die after 8-24 months (Goldstein and Widholm, 1990). The other cases of growth with air COZlevels are with agar-solidified medium

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[peanut (Bender et al., 1985)and N . plumbaginifolia (Rey et al., 1989)l. In the latter example it was shown that 20% of the growth obtained in the light could be obtained in the dark, so the growth is actually PM. The peanut as well as carrot cells were also grown with air C02 levels in liquid medium in a fermenter, in which slow growth occurred, but the experiment lasted only 42 days, so continuous growth was not clearly demonstrated (Bender et al., 1985). The only PA cultures which have been initiated from Cq species are the Amaranthus lines APO-P and ACR-P (Xu et al., 1988). The plants themselves would normally fix C 0 2 initially via the C4 pathway through PEPcase, but the cultures have higher levels of RuBPcase, giving a RuBPcase :PEPcase activity ratio of 3-5 in both lines (Xuet a!., 1988; Goldstein and Widholm, 1990). This outcome is perhaps not surprising, since PEPcase and RuBPcase are localized in two different cell types in Cq plants which are arranged in a specific fashion to form bundle sheath and mesophyll tissues. Tissue cultures are not generally organized spatially into specific tissue types. It is possible that manipulation of these cultures in certain ways could cause differentiation into the mesophyll cell type with high PEPcase, but this has not been done. That photosynthetic differentiation may be inducible in tissue cultures has been demonstrated with callus of a CAM plant, Kalanchoe blossfeldiana, which was grown PM with 3% sucrose (Mricha et al., 1990). When grown under 16-hr daily illumination, the cultures perform C3 photosynthesis, and when switched to 9-hr daily illumination, there is a switch toward CAM, as shown by increased levels of PEPcase and malic acid. The response is controlled by phytochrome, since 30-min interruptions of the long night with red light prevents the change to CAM. The activity of phytochrome was also demonstrated by the red-light stimulation of swelling of K . blossfeldiana callus protoplasts, which was reversible by far-red light. So far, there are no reports of PA cultures from the cereals. This is perhaps, in part, due to a general lack of uniform greening of cereal cells in culture. We have seen greening with Zea mays cultures only in localized regions of callus where shoots are forming. One can ask the question, What proportion of the serious attempts to initiate PA cultures is successful? While most unsuccessful attempts are probably not reported, Dalton and Peel (1983) described work over a several-year period with Psoralea bituminosa cultures in which greening was enhanced, but PA was not attained. These workers have previously reported success with spinach (Dalton, 1980) and asparagus (Peel, 1982). Yamada and Sat0 (1978) initiated PA cultures of C. scoparius and N . tabacum, but were unsuccessful with Phellodendron amurense at the same time. Likewise, Yasuda et al. (1980) were successful with D.stramonium and H . niger, but were not with Atropa belladonna. In our own

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work success has been attained with two soybean genotypes, cotton, D . innoxia, a Nicotiana fusion hybrid, and two Amaranthus species (Horn et al., 1983b; Rogers et al., 1987; Xu et af., 1988), but we were unsuccessful with Amaranthus hybridus (both wild type and triazine resistant), A . powellii (triazine resistant), and Amaranthus retrojexus (Xu et al., 1988). Most attempts to initiate PA cultures probably begin with cultures that already contain high Chl levels, so the success rate would probably be lower if one began with a random sample of culture lines. Some systems may routinely produce PA cultures quickly, including the initiation from D. stramonium and H . niger leaf pieces placed directly under PA conditions (Yasuda et al., 1980), from N . plumbuginifolia protoplasts (Rey et d., 1989), and from carrot root pieces, which grew after sucrose was exhausted from the H medium (Bender et al., 1985). The first three systems were on agar-containing media, while the latter one was grown for only 42 days in a fermenter. The N . plumbaginifolia cells do not form a homogeneous cell suspension when placed in liquid medium without sugar, but begin to regenerate plants (P. Rey, personal communication). Thus, further documentation is needed to confirm that a large proportion of normal cells can quickly adapt to grow PA continuously. Since most cultures have been initiated by a long-term gradual selection process, one can also wonder whether the final PA culture results from adaptation or selection of either epigenetic variant or mutant cells. The proportion of cells that survive is usually low, but is probably greater than lop6, which is the usual frequency of spontaneous mutations. That the PA cells are different from wild-type cells is indicated by the ability of the PA C. rubrum and SB-P cells to rapidly go from H to PA by changing the growth conditions (Ziegler and Schiebe, 1989; Erdos et al., 1986). The original production of the two PA cultures involved long selection periods (Hiisemann and Barz, 1977; Horn et af., 1983b).The cultures also continue to change during long-term PA growth, usually by increasing Chl and RuBPcase levels (Goldstein and Widholm, 1990). C. Photosynthetic Characteristics

One way to describe the photosynthetic characteristics of PA cultured cells is to compare them with leaves. This approach has relevance, since one goal of the tissue culturist is to produce an in vitro system identical to that of leaves for the study of photosynthesis. As far as Chl levels are concerned, the PA cultures contain about 30-2000 pg/g of fresh weight, most levels being below 200. Leaves, on the other hand, usually contain 2000 pg/g of fresh weight or more, even when grown under the relatively low light intensities used for PA cultures in most laboratories (about

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one-tenth that of sunlight). Thus, only a few lines (e.g., SB-P, SBl-P, and APO-P) which have about 2000 p g i g of fresh weight are near the leaf levels. Euphorbia PA cultures and leaves also have similar Chl levels (about 10 mgig of dry weight, which would be near 1000 p g i g of fresh weight), but these levels are about one-half those of normal leaves. These cultures with high Chl levels, however, do not grow more rapidly or have other photosynthetic properties that distinguish them from other PA cultures. One reason for the low Chl level in the N . tabacum cultures is that the number of chloroplasts per PA cell is near 90, while the leaf cells contain over 200 (Takeda et al., 1989). The leaf cells are also smaller, so the amount of Chl per gram of fresh weight is much higher (2500 pg versus 180 pg in PA cultures). The relatively low abundance of chloroplasts in Nicotiana PA cultures is illustrated in Fig. 5, which shows NTG-P cells which routinely contain only about 200 pg of Chl per gram of fresh weight. In contrast the APO-P cells shown in Fig. 6 have a much higher density of chloroplasts, which correlates with the high Chl level of these cells (about 1700 p g i g of fresh weight). The chloroplast ultrastructure in PA cultures is similar to that of leaf chloroplasts, however, for N . tabacirm (Brangeon and Nato, 1981), C. rubrum (Hiisemann et al., 1984), and soybean (Horn and Widholm, 1984; Erdos et al., 1987). Electron micrographs of SB-P cell and leaf mesophyll cell chloroplasts are shown in Figs. 7 and 8, which show a similar thylakoid structure. Since the PA cultures contain chloroplasts, certain lipids characteristic of photosynthetic tissues have also been detected. Analysis of the PA C. rubrum cells detected high levels of mono- and digalactosyldiacylglycerides and also lower levels of sulfoquinovosyldiacylglycerides and diacylglycerophosphoglycerides (Hiisemann et a / ., 1980). H C . rubrum cells contained only traces of these compounds. The PA cells contained a higher proportion of linolenic acid and lower linoleic acid than the H cells, which is also characteristic of leaves. Likewise, as in leaves, the linolenic acid was predominantly esterified in monogalactosyldiacylglyceridesand digalactosyldiacylglycerides. Barz et al. (1980) compared the lipid contents of H, PM, and PA P . harmala cultures and found that the PA, and to a lesser extent the PM, cultures contained higher levels of mono- and digalactosyldiacylglycerides. There were also clear differences in the fatty acid compositions of individual lipid classes and the different cell strains. Martin et al. (1984) compared the lipid content of soybean PA and H cultures and leaves and found that the PA cells and leaves both contained sulfoquinovos yldiacylglycerol and mono- and digalactosyldiacylglycerols. The H cells contained none of the first compound, but did contain the latter

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FIG. 6 Phase-contrast micrograph of PA APO-P (Amaranthus powellii) cells, taken in 1991, showing a high abundance of chloroplasts, which correlates with the high Chl content (C. Goldstein, unpublished observations). Bat = 25 pm.

two. The chloroplasts contained most of the neutral lipids, diacylglycerol, and triacylglycerol, as well as diacylglycerol acyltransferase, the last step in triacylglycerol synthesis. The syntheses of fatty acids from acetate and of lysophosphatidic acid by glycerophosphate acyltransferase are also localized in the chloroplasts. Leaf tissue had five times as much total glycerolipids as H cells and two times as much as PA cells. The cultures had more than 10 times the neutral lipid content of leaves, and the level fluctuated, as if these lipids might serve as energy storage. Growth temperature did alter the fatty acid composition. Starch levels were much lower than leaves in PA cells during rapid growth, but did become about one-half the leaf level later in the culture period. Takeda et al. (1989)compared PSI and Hill reaction activities of isolated chloroplasts of N . tabacum leaves and PA and PM cultures and found a wide variation in leaves of different ages, the activities of mature leaves being about double that of the PA cells on a per milligrams of Chl basis. FIG. 5 Phase-contrast micrograph of PA NTG-P (Nicotiana tabacum x Nicotiana glutinosa fusion hybrid) cells taken in 1991 which correlates with a low Chl content, showing a low chloroplast abundance (C. Goldstein. unpublished observations). Bar = 25 pm.

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FIG. 7 Electron micrograph of a SB-P (Glycine max) cell chloroplast taken in 1985 (D. R . Duncan, unpublished observations). Bar = 1 pm.

The PA cell activities were constant, except for a large decline in the late stationary phase. There was about twice as much PSI as Hill reaction activity in all systems. To determine whether any components of the PA cell chloroplasts were deficient, thylakoid membrane polypeptides were isolated and separated by one-dimensional lithium dodecyl sulfatepolyacrylamide gel electrophoresis. The PA cultures did contain lower levels of several components, including the (Y and /3 subunits of the coupling factor CFl , which correlates with differences in the ratio of uncoupled to coupled electron transport in isolated thylakoid membranes. These studies must be pursued further to describe in more detail the possible effect of the altered chloroplast component levels on the photosynthetic performance of the PA cultures. Since most of the PA cultures were initiated from C3 species plants, which rely on RuBPcase for COz fixation, the level of this enzyme should be of prime importance. The RuBPcase total activity levels can vary from

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FIG. 8 Electron micrographof a soybean (Glycine m a r ) leaf mesophyll cell chloroplast (D. R. Duncan, unpublished observations). Bar = 1 p m .

very low levels up to over 200 pmol of COZper milligram of Chl per hour, as shown with SBl-P (Roeske et al., 1989), NTG-P (Goldstein and Widholm, 1990), and N . plurnbaginifolia (Rey er al., 1990a). These levels are still much lower than those reported for spinach and soybean leaves of about 500 pmol of COZper milligram of Chl per hour (Rogers et al., 1987; Blair et al., 1988). Most reports of RuBPcase activities are for activated levels, which do represent the enzyme capability, but not the actual in vivo activity level. We usually measure both the initial activity (that found in extracts of cells immediately after homogenization) and the total activity (that found in the cell extracts after activation with saturating Mg2+ and HCO;). In leaf extracts the initial activity is usually over 90% of the total, and similar values were found by Roeske el c11. (1989) with COT-P, COT-PA, and SBl-P cultures if the cells were activated with

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bright light (350 pEm-'sec-') for 5 min prior to homogenization. However, the initial RuBPcase activity of these same lines taken directly off the growth room shaker would only be about 30-50% of the total activity (Goldstein and Widholm, 1990).The RuBPcase activation levels in the other PA cultures we work with (e.g., SB-P, APO-P, DAT-P, and NTG-P) are also in this range. Rey ef al. (1990a) found RuBPcase activation levels to be below 40% in both the air- and 2% COz-grown cultures PA N. plumbaginifolia. These results indicate that the RuBPcase in PA cultures is usually not more than 50% active under normal conditions. The low RuBPcase activation levels are apparently not due to a lack of the enzyme rubisco activase, which is involved in the light-induced activation in plants (Salvucci et al., 1986). The enzyme appears to be present, since Roeske et al. (1989) were able to activate the RuBPcase in several PA cell lines with 5 min of high-intensity light, and Western blot assays with SB-P cell extracts, using a spinach rubisco activase antibody, indicated the presence of activase (J. M. Werneke, S. M. D. Rogers, and J. M. Widholm, unpublished observations). Roeske er al. (1989) found that bright light would not activate the RuBPase of the SB-P cells to over 56% of the total activity, which could indicate a deficiency in rubisco activase in these cells. It would appear that higher light intensities would be desirable for growth of the PA cultures, since plants grown under low light conditions have both lower initial and total RuBPcase activities (Boardman, 1977; Torisky and Servaites, 1984). Supporting this conclusion is the marked increase in activation of RuBPcase from several PA cultures, caused by short high-intensity light treatment (Roeske et al., 1989).Yamada and Sato (1978) and Nato et al. (1983) also noted increases in Chl levels with increasing light intensities. However, when we attempted to gradually increase the light intensities with the SB-P cells, the cells never survived in light above 400 pErn-*sec-* (S. M. D. Rogers and J. M. Widholm, unpublished observations). The highest light intensity reported for growing PA cultures is about 300 pEm-*sec-'. Roeske et al. (1989) found that there was a decrease in the total RuBPcase activity in SBl-P cells when incubated for short times in the dark (37% inhibition in 35 rnin and 43% in 130 rnin). This dark-induced decrease in enzyme activity could indicate that the PA soybean cells, like soybean leaves, synthesize the dark inhibitor carboxyarabinitol-1-phosphate(Servaites et al., 1986). There is no evidence that this inhibitor is present in light-grown PA cultures, however. Many PA cultures have high levels of PEPcase in relation to RuBPcase, and this can vary during the growth phase in batch culture. The high PEPcase activity is characteristic of the C4 fixation pathway, where C4 compounds are formed in mesophyll cells and are rapidly transported to

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the bundle sheath cells, where C 0 2is released to be refixed by RuBPcase. This system concentrates CO2 to lessen photorespiration, which is initiated when RuBPcase uses 0 2 , rather than C02, in the fixation reaction (reviewed by Ogren, 1984). The rate of metabolism of the C4 compounds formed by PEPcase fixation in PA cultures is, however, slow (Nishida et al., 1980; Hiisemann et al., 1984, 1990; Herzbeck and Husemann, 1985; Roeske et al., 1989), and therefore it is assumed that the C4 compounds feed into the tricarboxylic acid cycle and glycolysis, so are used for amino acid and other related syntheses. Feeding studies with ['4C]malate give further evidence for this utilization route (Hiisemann et al., 1990). Several of the batch culture PA systems show characteristic patterns of higher PEPcase, respiration, and structural component synthesis and lower RuBPcase and Chl during the rapid growth phase, with a reversal of these trends during the stationary phase (Husemann, 1981; Husemann et al., 1984; Rogers et al., 1987; Chagvardieff et al., 1990). These changes also fit with the concept of anaplerotic C 0 2 fixation by PEPcase during rapid growth to feed carbon to the tricarboxylic acid cycle for amino acid and the other biosyntheses which would be needed at this time. Most of the PA cultures have higher levels of PEPcase and lower levels of RuBPcase activity than do leaves, in which the RuBPcase : PEPcase activity ratio is 4.7-7 for C. rubrum (Husemann et al., 19791, spinach (Blair et al., 19881, and soybean (Rogers et al., 1987). One possible reason for this difference is that PA cultures are actively growing, while the leaves used in the comparisons are mature and are no longer growing (Rogers et al., 1987). Developing leaves of C3 plants show a gradual change from a low to a high RuBPcase : PEPcase activity ratio, with a similar change in the C 0 2 fixation product pattern (Kisaki et al., 1973). However, several cultures have gradually changed during many years in culture and now have greatly increased RuBPcase : PEPcase activity ratios, due both to RuBPcase increases and PEPcase decreases. These cultures include COT-PA, COT-P, SB-P, SBI-P, and APO-P, which have activity ratios of 6-14 (Roeske et al., 1989; Goldstein and Widholm, 1990). That the PA cultures are capable of photorespiration was demonstrated in many studies, by showing that increased O2 levels inhibit photosynthesis, that increased C 0 2 can reverse this inhibition, and that I4CO2 is incorporated into glycine and serine (the studies include, among others, those by Berlyn et al., 1978; Sat0 et al., 1979; Nishida et al., 1980;McHale et al., 1987; Roeske et al., 1989; Rebeille, 1988; Rey et al., 1990a). Berlyn et al. (1978)also showed that high 0 2 inhibits the growth of PAN. tabacum callus. The accumulation of glycolate in light in the presence of the glycolate oxidase inhibitor a-hydroxy-2-pyridinemethanesulfonicacid and the metabolism of exogenous [14C]glycolateto C02, glycine, and serine (Berlyn et a / . , 1978) also indicate the presence of the photorespiratory

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pathway. Rey et al. (1990a) found that aminoacetonitrile, an inhibitor of glycine decarboxylase, an enzyme in the photorespiratory pathway, inhibited PA N. plurnbaginifolia callus growth only in low C02 (0.05%), but not with high C 0 2 (2%). Photorespiration would only be occurring with low C02, and the inhibitor would prevent recycling of the carbon back to the Calvin cycle. The need for elevated C02 levels for the growth of most PA cultures also supports the presence of photorespiration. The recent use of mass spectrometry to measure 1 6 0 2 / 1 8 0 2 exchange rates with PA Euphorbia and carnation cells has also shown that a portion of the O2uptake in the light is due to photorespiration (i.e., is inhibited by C02) (Rebeille, 1988; Carrier et al., 1989; Chagvardieff et al., 1990). However, increasing the C02 only decreased the carnation cell 0 2 uptake by 40-50%, while the decrease with detached leaves was 75-80% (Rebeille, 1988). The remainder of the O2 uptake in the light would be due to mitochondrial respiration and possibly the direct photoreduction of oxygen at the PSI accepter site [Mehler-type reaction (reviewed by Badger, 1985)l. The latter reaction was indicated since the 0 2 uptake in the light, with photorespiration suppressed (high C02), was greater than the dark respiration of PA Euphorbia cultures (Carrier et al., 1989; Chagvardieff et al., 1990) by as much as 45% in the early stationary phase. The direct photoreduction of O2at the PSI acceptor site (Mehler-type reaction) may be either a trap for energy overflow or a regulatory pathway for pseudocyclic electron flow to furnish ATP. The Mehler-type reaction has been seen in the leaves of plants (reviewed by Badger, 1985). Avelange et al. (1991) measured both C 0 2 and O2 gas exchange, using " 0 2 and I3CO2and mass spectrometry with the PA carnation suspensions. Under optimum photosynthetic conditions of high light and high C02, C02 influx equaled 75% of 0 2 evolution. Light decreased the rate of C 0 2evolution from that in the dark by 50%, but the O2 uptake was unchanged. These and other results indicate that mitochondrial respiration is somewhat inhibited in the light and that C02 recycling is not responsible for the decrease in C02 evolution. When the C02 and 0 2 gas exchange was also measured with the PA Euphorbia suspension culture, a similar decrease in C02 efflux (40%), but no change in 0 2 uptake, were found when the cells were placed in the light (Avelange and Rebeille, 1991). By following the kinetics and the effect of light flashes and electron transport inhibitors, the 0 2 uptake by mitochondria was indeed found to be inhibited somewhat in the light, but this was compensated for by increased chloroplastic 0 2 uptake, presumably by a Mehler-type reaction, to give no net change in 0 2 uptake. The C02 compensation concentrations (C02 concentration in which C02 fixation and C 0 2release are equal under light) of most PA cultures are higher than those observed with leaves (cultures, about 0.015-0.035%

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CO,; leaves, 0.005-0.008%) (Tsuzuki et al., 1981; McHale ef al., 1987; Rogers et a / . , 1987;Blair et a / . , 1988;Carrier ef al., 1989;Chagvardieff et al., 1990).In C3 plants the COz compensation concentration is thought to result from the balance between C 0 2fixation by RuBPcase and the release by photorespiration (Ogren, 1984). The PA cultures do have higher COZ release than do leaves, both in the dark and in the light, probably due to greater mitochondria1 respiration. This greater respiratory COz release in the light would seem, then, to cause the increased C02 compensation concentration usually observed. The inherent cause of the increased respiration in PA cultures may be growth, which requires energy utilization for cell structural component synthesis. Chagvardieff et al. (1990)found that respiration in PA Euphorbia cultures behaved like a derived function of growth which would be similar to the developing leaf, in which the high respiration in the developing leaves correlated with an increased C02 compensation concentration (Ticha and Catsky, 1981). In direct comparisons of leaves and PA cultures, the largest difference noted is usually the higher dark respiration rate found with PA cultures. With Euphoriba the amounts of Chl, the photosynthesis rates (02evolution), the photorespiration rates, and light-stimulated oxygen uptake rates were similar in PA cultures and leaves, but the PA culture dark respiration rate was at least four times higher (Carrier et al., 1989). This higher respiration rate was accompanied by a 2- to 5-fold higher C02 compensation concentration. The PA carnation cells also had a photosynthesis rate similar to that of leaves when calculated on a per milligrams of Chl basis, but the dark respiration rate was 700% that of leaves (Rebeille, 1988). Again, the C 0 2compensation concentration was increased by 2- to 3-fold. For some reason, low compensation concentrations have been found for PA N . tabucum cells grown on polyurethane pads wet with medium [0.0066%(McHale et al., 1987)] and SB-P cells spread on a Petri dish as a thin layer [0.0090% (Rogers et al., 1987)].The C 0 2compensation concentration was 0.0230% for the SB-P cells in liquid medium, which is near that found for several other PA cultures. It would appear that the liquid medium itself should not affect the measurements, since Servaites and Ogren (1977)found the values for isolated soybean leaf mesophyll cells in liquid medium to be similar to that of leaves. The 2- to 4-fold higher C 0 2 compensation concentration of the PA cultures would raise the C 0 2 concentration requirement somewhat, but should not raise it enough to cause the high COz requirement of most PA cultures. Studies with COT-PA cells show that there is no COZ concentrating mechanism, since the internal inorganic carbon concentration equaled that of the surrounding medium in short-term experiments done with quenching by centrifugation through silicone oil (Roeske et ul., 1989). This con-

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clusion is also supported by the observation that 0 2 has been shown to inhibit photosynthetic carbon fixation in many PA cultures. In contrast some algae can concentrate inorganic carbon through an inducible HCO; transport system (reviewed by Spalding et al., 1983). Two experiments have shown that C02, not HCOj, is the form taken up by PA cells from the culture medium. Rebeille et al. (1988) showed that C02 was taken from the culture medium by PA Euphorbia cells by massspectrometric measurements of the I3CO2 : "C02 ratio when HI3COjwas added in the presence or absence of CA, which would speed up the equilibrium. Roeske et al. (1989) measured I4C incorporation by COT-PA cells when Hl4CO; was added to the medium in the presence or absence of CA. In this case there was a lag for the first 30 sec in I4C incorporation, which was abolished when CA was added. Rebeille et al. (1988) also showed that C 0 2 was the species released by the cells during respiration. The question as to why most PA cultures require elevated C02 levels for growth is an important one, which is unanswered at present. One possibility raised by Tsuzuki et al. (1981) is that CA may be deficient in the cells, which would limit the C 0 2 and HCO; interconversions. These workers found that PA C. scoparius and N . tabacum callus cultures had CA levels lower than 9% of those of leaves of the same species. The C02 compensation concentrations of the cultures were more than three times those of cells isolated from leaves. However, results obtained with PA cotton and soybean cultures indicate that CA levels do not affect the ability to grow on ambient C02 levels (Roeske et al., 1989). Spinach, cotton, and soybean leaves had high CA levels, as did the SB1-P culture (76% as much as spinach leaves). The SB-P culture had about one-quarter of the SB 1-P level, while the COT-PA and COT-P cultures had no detectable CA, either immunologically or by enzyme assay. Of these strains COT-PA grows best with ambient C02 levels, yet it contains no CA. SBI-P cells grow with ambient C02, but SB-P, which had some CA activity, does not grow at all on low C02 (Goldstein and Widholm, 1990). Thus, we conclude that there is no correlation between CA activity and the ability to grow with low C02.

111. Specific Uses A. Light Regulation

Whole plants are known to have several photoreceptor systems, including: protochlorophyllide, which, upon absorbing red light, is converted to chlorophyllide catalytically, which then leads to Chl biosynthesis; phytochrome, which has two interconvertible forms-P,, which absorbs red

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light (660 nm), and Pf,, which absorbs far-red light (730 nm) (red light converts P, to Pf,, which is the active form); and blue and/or UV light receptors of more than one form, including blue UV-A and UV-B forms (Schafer et al., 1990). Chloroplast development is controlled by phytochrome and by the protochlorophyllide conversion needed for Chl biosynthesis (Tobin and Silverthorne, 1985). However, in studies with nonchlorophyllus Crepis capillaris (Hiisemann, 1970), N . tabacum (Gross and Richter, 1982), and C. rubrum (Richter et al., 1987)cultures, blue, but not red, light would induce and maintain Chl accumulation in a low sucrose medium. Phytochrome has been detected, however, in soybean, parsley, and carrot suspension cultures (Schafer et al., 1990) and has been shown to be active as a photoreceptor in the SB-P cells in the induction of the accumulation of Chl alb-binding protein mRNA, since red light induces and far-red light reverses the induction (Lam et al., 1989). This study also implicates calmodulin in the signal transduction process, since a calmodulin antagonist abolishes the mRNA induction. Phytochrome has also been implicated in the day length-induced changes of callus of the CAM plant, K. blossfeldiana, from C3 to CAM metabolism and in the stimulation of swelling of protoplasts isolated from this callus (Mricha et al., 1990). Phytochrome, blue UV-A, and the UV-B receptors are all involved in the UV-B-induced flavonoid synthesis system in parsley cells (Schafer et al., 1990). The effect was documented in this study by measuring cellular mRNA levels and transcription run-off mRNA levels for certain of the biosynthetic enzymes. However, the phytochrome contribution to the response could not be demonstrated with a newly initiated parsley suspension culture. Thus, some PA cultures (e.g., N. tabacum and C . rubrum ) do not seem to have the same array of photoreceptors as plants, while SB-P may. These characteristics may not necessarily be species specific, since two different parsley cultures also respond to light differently. These cultures can provide a wide variety of unique responses which could help simplify light response studies by the use of certain strains. More details on the lightinduced responses are presented in the next section.

8. Differentiation Many of the cell systems have been used to describe the transition from H to PM and then to PA growth stages. These transitions are similar to those seen in developing leaves. A differentiation system which would be unique to cultured cells, however, is the readily reversible H to PA to H (and so on) system, which has been described with both SB-P and C.rubrum cells. A readily reversible greening and bleaching system has been developed

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with the SB-P cells by Erdos et al. (1986, 1987) and by Gillott et al. (1991) in which Chl disappeared and carotenoids declined to very low levels within 25 days when the PA cells were grown in a medium with 3% sucrose in the dark. The cultures can be grown under H conditions for periods of at least 16 weeks (eight subcultures), and when placed in the light with 5% C02 7 days after subculturing (when the sucrose in the medium was exhausted), the cells grew and accumulated Chl, carotenoids, RuBPcase, and Chl alb-binding protein. The Chl increase could be seen within 2 hr and by 12 days had reached about 600 pg/g of fresh weight. The Chl db-binding protein began to accumulate within 6 hr, and the rate of increase was greater than the increase in mRNA, indicating that the synthesis is controlled at both the transcriptional and translational levels. The plastids appear to be normal mature chloroplasts with starch grains in cells grown in the light and are amyloplasts with starch grains in cells grown in the dark. The cultures formed during the transition to the light conditions continue to grow as normal-appearing PA cultures when transferred to fresh medium without sugar. It has also been possible to prepare protoplasts from SB-P cells, grow them back to cell colonies in a medium with sugar, and then reinitiate PA suspensions which grow just as well as the original culture (Chowdhury and Widholm, 1985). The PA C. rubrum suspension cultures, grown in the dark with 2% sucrose for over 8 months, lost all Chl (Ziegler and Schiebe, 1989). When reinoculated into a culture medium lacking sucrose in 16 hr of daily illumination (75 pEm-2sec-') with 2% C02in two-tiered flasks, the cells formed Chl and the COZ fixation enzymes, as well as other enzymes typical of photosynthesis, and began to grow, with a general delay of about 10 days. The culture was then apparently capable of continued PA growth. The PA SB-P and C. rubrum cultures can be reversibly bleached by growth in the dark with sucrose and then be converted back to PA by transfer back to PA conditions (i.e., light, no sugar, and elevated C o d . These systems should be ideal for studies of the transitions between the two growth states. This may be possible with many other PA cultures, but we have found that the COT-P cells turn brown and die when placed into a medium containing 0.1% or more sucrose (L. C. Blair, C. S. Goldstein, and J. M. Widholm, unpublished observations). The COT-P cells survive with starch as the carbon source, however.

C. Molecular Biology There have been a number of studies describing the responses of cultured cells to light. Gross and Richter (1982) demonstrated that Chl synthesis in

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N . tabacum suspension cultures was induced by blue, but not red, light. Labeling experiments showed that both the large and small subunits of RuBPcase were synthesized, and the small-subunit mRNA level increased upon blue light treatment (Hundrieser and Richter, 1982). The synthesis of Chl, other pigments, the psbA peptide, and large and small subunits of RuBPcase were shown to be induced by blue light, and the coordinate accumulation of mRNA for the latter three proteins was demonstrated by Richter and Wessel(1985). The parallel increase in mRNA levels indicates a coordination between nuclear and plastid transcription. The blue, but not the red, light dependence for chloroplast formation was also demonstrated with C. rubrum, in which the mRNA levels for six chloroplastic proteins were induced (Richter et al., 1987). A transcriptionally active chromosome system was developed whereby DNAprotein complexes were isolated which would transcribe plastid genes in uitro. The activity was higher with the transcriptionally active chromosomes from blue light-induced cells. Some cDNA clones from mRNA of genes which are induced rapidly by blue light in C. rubrum cells have been isolated by Kaldenhoff and Richter (1990), with the goal of finding genes which are involved in signal transduction or coordination of the expression of the light-induced genes. These genes include a glycine-rich protein, a 0-tubulin-like protein, and an acidic ribosomal-like protein. Further work is needed to determine the exact role of these gene products in the light responses. Comparisons of the methylation of both plastid and nuclear DNAs in independently derived long-term green and white Acer pseudoplatanus (sycamore) suspension cultures show that most genes producing products involved in photosynthesis were methylated in the white, but not the green, cells (Ngernprasirtsiri et al., 1988, 1989). This methylation comelated with the lack of transcriptional activity observed with in uitro RNA polymerase assays. These results indicate that DNA methylation may regulate the gene expression of both plastid and nuclear genes in these special cell lines. Plastid DNA amounts were measured in several N . tabucum suspension cultures and plants by hybridization of labeled plant DNA to plastid DNA bound to filters (Cannon et al., 1985). Dark-grown cultures contained about 3300-4800 copies per cell, while green PM or PA cultures contained 9500-12,000 copies, which is equal to 14.5-18.3% of the total cellular DNA. Values taken from the literature for N . tabacum leaves were 26005800 copies and for roots, 600 copies. The PA suspension used here was a N . tabacum strain cv. Samsun NN, obtained from F. Sat0 and Y. Yamada. In quantitative studies with soybeans, the amounts of plastid DNA were about 26% of the total cellular DNA in PA, PM, and H SB-P suspension cultures, while the levels were !2.3% in leaves, 18.9% in seeds, and

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6.1-8.9% in roots of soybean plants of the same genotype (Cannon et al., 1986a). Thus, analyses of chloroplast DNA levels in PA N. tabacum and soybean cultures show about a doubling of plastid DNA when compared with leaves of the same species. The N . tabacum, but not the soybean, culture had lower plastid DNA levels when grown in the dark. Yasuda et al. (1988), however, showed by in situ fluorescence measurements (4’,6-diamidino-2-phenylindolestained), that the plastid DNA copy number per cell can change by 10-fold during the growth cycle in batchcultured N. tabacum cells grown in the dark. The number of copies per cell increased from 1000 at day 0 to 11,000on day 1 and declined back to about 1000 by day 4, when the number stabilized. Cannon et al. (1985) reported 3300-4800 copies per N. tabacum cell grown in the dark, which might reflect, according to the results of Yasuda et al. (1988), the time during the culture cycle when the cells were analyzed. However, the chloroplast DNA levels of the N. tabucum (Cannon et al., 1985) and SB-P cells (Cannon et al., 1986a) did not vary appreciably during the culture cycle (G. C. Cannon, personal communication). Studies by Heinhorst et al. (1985) with H N. tabacum cultures have shown that nuclear and plastid DNA syntheses are not closely coupled, since inhibition of one, with specific inhibitors, does not affect the other. The reasons for the differences in the relative stability of the plastid DNA levels seen by Yasuda et al. (1988) and by Cannon et al. (I985,1986a, G. C. Cannon, personal communication) could be due to the different DNA quantitation methods or to the different cell lines used. The SB-P cells have been permeabilized by treatment with L-alysophosphatidylcholine, which allowed uptake and incorporation of labeled nucleotides into DNA (Cannon et al., 1986b). Diethyl ether or DMSO were less effective. The incorporation was not affected by an inhibitor of DNA polymerase a, aphidicolin, and incorporation was only into organellar DNA. Molecules as large as DNase I (molecular weight, 40,000) could enter the cells and release the newly incorporated labeled nucleotides. The permeabilized cells also incorporated labeled precursors into RNA and protein. To study DNA replication, the N. tabacum cv. Samsun cell strain, from F. Sat0 and Y. Yamada, grown under PM conditions, was labeled for 48 hr with [I4C]thymidine (Infante and Weissbach, 1990). The cells were rinsed and then incubated with [3H]thymidine and 5-bromodeoxyuridine (for density labeling) for 48 hr. DNA was then isolated from purified nuclei, chloroplasts, and mitochondria and was then centrifuged in CsC12 equilibrium density gradients. DNA from each of the three compartments had all increased in density, showing that all molecules were replicated in the

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48-hr labeling period. If the chloroplast DNA was heated to separate the DNA strands, the 14C- and 3H-labeled strands banded at different densities, demonstrating the presence of the old light strand and the new heavy strand. DNA helicase activity was partially purified from isolated chloroplasts from SB-P cells grown PM (Cannon and Heinhorst, 1990). The strand displacement activity required a nucleoside triphosphate and Mg2+ or Mn2+,but did not require a free unannealed single-stranded or replication forklike structure. DNA polymerases were also purified from isolated chloroplasts and mitochondria from SB-P cells (Heinhorst et at., 1990). There were two forms in chloroplasts and one from mitochondria, all of which have estimated molecular weights of 85,000-90,000. The activities require KCI and Mn2+ and are resistant to DNA polymerase a inhibitors. Takeda et al. (1990) compared polypeptides from PA N. tabucum cultured cells grown in liquid medium with those extracted from young healthy leaves on two-dimensional gels. The cultured cells had many additional polypeptides, four of which, upon microsequencing, showed NHz-terminal amino acid sequences similar to those of the stress proteins osmotin and chitinase. These stress proteins were also found in the N. tabacum cells grown under PM or H conditions, in a newly initiated H cell line, in regenerating shoots, in old leaves, and in roots and leaves infected with tobacco mosaic virus. These results indicate that all cultured cells are under stress and express a stress response when compared to young healthy leaves. Anderson (1988) found that the addition of 3-30 pM jasmonic acid to SB-P cells increased the levels of a 30-kDa polypeptide which crossreacted with a mouse antibody made against the soybean vegetative storage protein (Anderson et al., 1989). Treatment with abscisic acid, BA, or gibberellic acid did not increase the protein. Treatment of soybean leaves with jasmonic acid caused accumulation of both the 28- and 30-kDa crossreacting peptides. The vegetative storage proteins are thought to be a short-term storage form, since these proteins accumulate in soybean plants following removal of sink tissues through depodding. The SB-P cells accumulated only the 30-, but not the 28-kDa form, however. Averyhart-Fullard et al. (1988) purified two hydroxyproline-rich proteins from SB-P cell walls, one of which had an amino acid composition similar to that encoded by a soybean cDNA which hybridizes to both a moderately abundant mRNA from SB-P cells and germinating seedlings. The cDNA sequence predicts a decameric repeat of Pro-Pro-Val-TyrLys-Pro-Pro-Val-Glu-Lys, which would fit with the amino acid analysis of 20% proline, 20% hydroxyproline, 20% lysine, 16% valine, 10% ty-

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rosine, and 10% glutamate (Datta et al., 1989). This protein represents a new class of hydroxyproline-rich cell wall proteins which is similar to extensin, but has a different repeat structure, which includes serine. Several other molecular studies have been conducted with PA cultures, as described in Sections III,A, B, and H. D. Metabolic Regulation

As described earlier, a number of studies have described the suppressive effect of high concentrations of sugars on Chl levels in cultured cells, although no mechanism for this effect has been elucidated. Studies with PA and PM cultures have shown that photosynthesis is inhibited rapidly by sugar, so the long-term loss of Chl may be a secondary effect. When different amounts of sucrose (0.75-2.5%) were added to PM potato cultures, the Chl concentration per milliliter of medium was not altered appreciably over a 16-day period, although the concentration per gram of fresh weight was decreased at the higher sucrose levels, due to increased rates of growth (LaRosa et al., 1984). In contrast CO:! fixation was decreased by 35% within 1 hr by 0.25% or higher sucrose concentrations. Rebeille (1988) found that photosynthesis was highest during the log growth phase, when sucrose and fructose were lowest in the PA carnation cells. There was a rapid increase in sucrose, glucose, and other carbohydrates when 20 mM sucrose was added to the medium, and photosynthesis declined by 75% in 24 hr. When 20 mM glucose was added to the PA carnation cultures grown in air C02 levels, carbohydrates and phosphorylated compounds began accumulating immediately and respiration increased, while photosynthesis decreased markedly (Avelange et al., 1990). The rise in respiration in the light, which may be due to increased mitochondrial capacity, as shown by increases in two marker enzymes, was sufficient to account for the decreased photosynthesis. After 24 hr photosynthetic 0 2 evolution and C02 fixation began to decline, even though RuBPcase activity was stable. After 48 hr the maximal activity of lightinduced phosphoribulokinase had decreased by 70%. This decrease in the enzyme that produces RuBP might be responsible for the decrease in photosynthesis. Neumann et al. (1989) found that PA peanut cells incorporated less 14C02into C3 pathway compounds when sucrose was added to the culture medium, although Cq fixation was not changed. The fixation levels correlated well with the levels of the respective enzymes, RuBPcase and PEPcase. Hiisemann et al. (1989, 1990) showed that raising the pH of the incuba-

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tion medium from 4.5 to 6.0 or 7.0 during I4CO2fixation by C . rubrum cells increased the percentage of the label in malate from about 20% to 40%. This increase in the medium pH increased the vacuolar pH from about 5.2 to 6.4 and that of the cytoplasm from about 7.15 to 7.35, as determined by 31P-NMRspectrometry. Subcellular fractionation studies localized PEPcase, the enzyme which would produce malate in the short-term I4CO2 fixation experiments, in the cytosol, where the medium induced pH changes were small. However, in vitro PEPcase assays showed that the activity increased by about 100% with 0.2-unit pH increases in the range from 7.2 to 7.8, which could explain the increased malate synthesis. Comparisons of the 14C02fixation products after a l-hr pulse and 5-hr chase showed that C. rubrum cells incubated at pH 6.0 had greatly increased I4C in lipids, amino acids, protein, and cell walls. This pattern indicates that malate is metabolized through the tricarboxylic acid cycle and glycolytic pathways in the C . rubrum cells. Spilatro and Anderson (1988) showed that as PM SB-P cells use up the sugar in the medium and become PA, most of the glycolytic and sucrose metabolic enzymes measured did not change appreciably in activity, except for pyrophosphate : fructose-6-phosphate phosphotransferase, which was about 3-fold higher in the PM stage. The activator of this glycolytic regulatory enzyme, fructose 2,6-biphosphate, was always present in very high concentrations, so the authors concluded that changes in the enzyme levels, rather than in the activator levels, control the glycolytic pathway in the SB-P cells. Nitrogen metabolism has been studied extensively with the PA C . rubrum suspension cultures. In the initial report Campbell et al. (1984) found that the PA C. rubrum cells took up NH'J preferentially during the first week of batch culture, then took up both NH: and NO; for the next week until the NH'J was exhausted, and by the end of 38 days, one-third of the NO; remained in the medium. The levels of several nitrogenassimilatory enzymes were measured during the culture period. These PA cultures, which were grown on a 16-hr light 8-hr darkness regimen, showed diurnal fluctuations in NR activity, which was cycloheximide sensitive, above a basal level, which disappeared in the stationary growth phase as the NR activity decreased (Renner and Beck, 1988). The addition of NH'J increased NR activity by increasing cytoplasmic NO; levels due to the mobilization of vacuolar NO; and by stimulating NOS uptake (Beck and Renner, 1989). This study also demonstrated that nitrite reductase, glutamine synthetase, glutamate synthase, and NADP-glutamate dehydrogenase were chloroplast localized, while NR, NAD-glutamate dehydrogenase, and certain aminotransferases were cytoplasmic. Beck and Renner (1990) followed nitrogen fluxes and compartmentation in the PA C. rubrum cells during a batch culture cycle by following NH:

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and NOT uptake and incorporation, nitrogen-metabolizing enzyme activities, and compartmentation in purified chloroplasts and vacuoles. The enzyme activities were more than sufficient to handle the nitrogen flows, so the limiting factors would apparently be NH: and NO; uptake. A vacuolar NOT pool was formed during the division phase, and this persisted in the stationary phase, when net protein synthesis ceased and free amino acids also accumulated in the vacuole. Flow diagrams were constructed to describe the nitrogen fluxes during the predivision, cell division, transition to stationary, and stationary phases. The soybean suspension culture, SB-P, was grown under H, PM, and PA conditions, and the NR activity was studied (Nelson et al., 1984). When NO; was in the medium, NR activity with an apparent K , identical to that of the soybean leaf-inducible form was present in the cells, and the addition of glutamine did not repress the activity. There was no NR activity when H and PM cells were grown with glutamine as the sole nitrogen source. Nitrogenous gas evolution, which is a characteristic marker of the constitutive soybean leaf NR, was never observed with any age culture under any growth conditions. These studies show that the constitutive NR, which is always expressed in leaves, is not expressed in H, PM, or PA cultures, while the inducible form can be induced by NO;. When PA Euphorbia suspension cultured cells were y-irradiated (250 Gy), cell division ceased and sugar and dry matter accumulated (Chagvardieff et al., 1989). The Chl level, photosynthetic capacities, and O2 uptake rates in the light and darkness of the irradiated and control cells were similar between 4 and 9 days after irradiation, even though the sugar levels were elevated several-fold. Stationary phase nondividing cells usually had lower metabolic rates. Thus, the irradiation treatment makes it possible to stop cell division in any growth phase, allowing metabolic studies without the complication of growth stage. E. Membrane Properties

Electrical membrane potential and resistance studies using microelectrodes were conducted with the PA C. rubrum cells grown with MS salts and vitamins and 100 nM 2,4-D (Ohkawa et al., 1981). The results indicate that these cells have an electrogenic hyperpolarizing ion pump and a hexose-specific saturable electrogenic membrane channel. The cell membrane transport properties measured are similar to those of leaf mesophyll cells. Similar conclusions were reached in a study by Biichner et al. (1981) in which turgor pressure and water transport properties were measured with single PA C. rubrum cells or leaf mesophyll cells, using a miniaturized

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pressure probe. Both cell types had similar water exchange times, volumetric elastic moduli, and hydraulic conductivities. Proton pumping was studied, using a microsomal fraction from the PA C. rubrum cultures which was presumably of tonoplast origin (GogartenBoekels et al., 1985). The ATP-dependent proton accumulation was measured by the pH-dependent shifts in acridine orange absorption. The pumping is highly specific for ATP and insensitive to vanadate. The transmembrane pH collapses upon the addition of the protonophor carbonylcyanide-m-chlorophenylhydrazone. The pump operates at a constant rate which is independent of the ApH. These studies indicate that PA cultures have properties similar to those of leaves, so they should be good model systems for the study of membrane properties. F. Cell and Subcellular Biology

Since PA cultures usually consist of small clumps of cells, microscopic observation of the cells is simplified, which can expedite many studies. The suspension cultures grow as variable-sized clumps, with cells from about 30-70 p m in diameter, although the cells are usually not round (Rogers and Widholm, 1988; Xu et al., 1988; Roeske et al., 1989). Leaf cells are usually smaller; soybean cells are 13 X 66 p m (Cosio et al., 1983) and N. tabacum leaf cells are about lo7 per gram of fresh weight, while there were 2.2 x lo6 in PA cultures (Takeda et al., 1989). The PA cells are usually highly vacuolate, with the chloroplasts in the cytoplasm near the cell wall, and all cells contain chloroplasts, from our unpublished observations. PA N. tabacum cells contain less than half as many chloroplasts as leaf cells and are larger, so they contain only 7% as much Chl per gram of fresh weight (Takeda et af., 1989). Detailed comparisons of chloroplast numbers have not been made with other species. Rapidly dividing PA C . rubrum cells contained chloroplasts surrounded by numerous large mitochondria, while stationary phase cells had fewer mitochondria around the chloroplasts, which then contained large starch granules (Hiisemann er al., 1984). The chloroplasts in the PA C. rubrum cells appeared to be normal, with stromal lamellae, grana stacks, and osmiophilic globuli. The greening of dark-grown N. tabacum suspension cultures, when placed in the light in a medium with sucrose, involves the gradual change from the H highly vacuolate cells, with amyloplasts virtually devoid of lamellae, but filled with starch grains and osmiophilic globules, to less vacuolate cells with chloroplasts containing well-developed thylakoids and grana stacks, with no starch within a 14-day period (Brangeon and

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Nato, 1981). Peroxisomes were identified in the green cells. The PEPcase activity peaks during the rapid growth phase, when respiration is highest, and RuBPcase increases only during the stationary phase, at a time when 02 evolution also increased (Nato et al., 1981). Chloroplast development during the greening of SB-P cells involved a large proliferation of membranes within 12 hr in the starch-containing amyloplasts, which originally contained few internal membranes (Horn and Widholm, 1984; Erdos et al., 1987; Gillott et al., 1991). These plastids then elongate, the membrane cisternae proliferate, and by day 12, when the Chl and carotenoid levels become maximal, mature chloroplasts are formed, with thylakoids and grana stacks. Upon continuous growth under PA conditions, the grana stacks enlarge and more starch accumulates. These chloroplasts are similar in appearance to those found in soybean leaves. There were no obvious changes in other subcellular components in the SB-P cells during the greening transition, except that the PA cultures appear to have no peroxisomes (D. R. Duncan, unpublished observations). When the SB-P cells were placed in the dark with sucrose in the medium, the transition from PA to H involved a rapid decline in Chl (complete loss in 25 days) and loss of intergranal thylakoids, leaving isolated grana which dissociate, swell, and degenerate (Gillott et al., 1991). Amyloplasts are formed which contain a few lamellae, which may correlate with the low carotenoid content that persists in the H cells. Some of the dedifferentiating plastids appear to degenerate through a multilobed amoeboplast stage. Cell viability has been determined, using the red exclusion dye, phenosafranin, with several PA cultures (Blair et al., 1988; Roeske et al., 1989) and protoplasts (Chowdhury and Widholm, 1985). Another exclusion dye, Evans blue, was used with soybean mesophyll cells (Cosio et al., 1983), so it should also be effective with PA cells. Fluorescein diacetate, which is taken up by viable cells and is hydrolyzed to the fluorescent compound fluorescein (yellow) (Widholm, 1972), should also be useful for viability determination. Thiemann et al. (1989) measured viability of PA C . rubrum cells by their ability to generate reducing equivalents to reduce nitroblue tetrazolium chloride, which was easily quantitated in the ethylacetate phase by spectrophotometry . This method is similar to the triphenyltetrazolium chloride reduction assay, in which the reduced formazan dye can be extracted into ethanol to be measured by absorption (Towill and Mazur, 1975). There have been many subcellular fractionation studies with protopIasts prepared from PA cultures. The protoplasts are ruptured osmotically or by forcing them through a fine mesh, and the organelles are purified by differential centrifugation or density gradient centrifugation [basic method described by Willmitzer and Wagner (1981) for H cultures]. The studies

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include those by Beck and Renner (1990), who isolated vacuoles and chloroplasts from C. rubrum for the measurement of compounds and enzymes to follow the flux of nitrogen in growing cultures; Husemann et al. (19891, who isolated chloroplasts from C . rubrum to determine the cytosolic localization of PEPcase; Martin et al. (1984), who measured the lipid composition and activities of lipid biosynthetic enzymes in gradientfractionated SB-P; Cannon et al. (1985), who purified nuclei from N . tabacum for DNA measurement; Heinhorst et al. (1990), who purified chloroplasts and mitochondria from SB-P and purified and characterized DNA polymerases from each organelle; and Infante and Weissbach (1990), who purified chloroplasts, mitochondria, and nuclei from tobacco for DNA isolation. Characterization of Chl a fluorescence with SB-P, SBl-P, DAT-P, COT-P, and COT-PA suspension cultures has shown, first of all, that these systems are relatively free of much of the fluorescence spectra distortion caused by the severe reabsorption encountered with leaves (Xu et al., 1988, 1989). All of the cells contained PSII-CP43 and -CP47 and PSI antenna systems identical to that of chloroplasts from leaves. In more intensive studies with the SB-P and SBl-P cells, calculations from fluorescence induction curves indicated a ratio of 6 : 1 of mobile plastoquinone to bound plastoquinone (Xu et al., 1989). Double-flash fluorescence measurements indicated an equal quantity of bound plastoquinone QB and its reduced form QB, which is a higher proportion of QB than that found in dark-adapted isolated thylakoids. The half-times of QA decay were similar to that of spinach chloroplasts, but the ratio of the fast and slow components was somewhat different. Atrazine and DCMU inhibited electron flow, as if they could totally replace QB at its binding site. There were differences in herbicide sensitivities noted between SB-P and SB 1-P cells and spinach chloroplasts. These results indicate that Chl a and components of the electron transport systems are similar in PA cultures and leaves and that such studies can be performed with whole cells without the serious fluorescence spectra distortion problems seen with leaves. Flow cytometry can be used with separated cells or protoplasts prepared from PA cultures to measure the Chl content by autofluorescence of individual cells to study the Chl distribution and changes in the population during the culture period (K. Nanda and J. M. Widholm, unpublished observations). This could allow sorting of the high Chl-containing cells for further growth. Likewise, nuclei from cultures can be stained with DNAspecific fluorescent dyes (e.g., ethidium bromide and mithramycin), and the DNA contents can be determined to measure ploidy level, cell cycle parameters, and the effects of chemicals which can increase (colchicine) or decrease [isopropyl-N(3-~hlorophenyl)carbamate]DNA contents

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(K. Nanda and J . M. Widholm, unpublished observations). There are many other possible uses of flow cytometry. G. Genetic Engineering

At this time there have not been reports of the genetic engineering of PA cultures (e.g., transformation, selection, or protoplast fusion) other than selection for herbicide resistance, but the systems are available. Such systems should be especially useful for transformation with photosynthetic herbicide resistance genes or antibiotic resistance which can affect chloroplasts (e.g., streptomycin and spectinomycin), since stringent selection should be possible. Chloroplast transformation has recently been accomplished in higher plants with N . tabacum leaves, by particle bombardment using a plastid 16 S rDNA gene carrying spectinomycin and streptomycin resistance (Svab et al., 1990). Tissue culture was used to select the resistant cells, which remain green, and to regenerate plants from them. The use of cultured cells for transformation may be advantageous, since certain inhibitors can be applied to attempt to lower the plastid DNA copy number. A lower DNA copy number should enhance the transformation efficiency. Studies with H Solanum nigrum suspension cultures have demonstrated that the plastid DNA copy number can be reduced somewhat by nalidixic acid and novobiocin, both DNA gyrase inhibitors (Ye and Sayre, 1990). One disadvantage of using long-term PA cultures in genetic engineering work is that they are probably not capable of regenerating plants, due to the long period of time in culture; however, the chromosome numbers can remain normal, as shown for the SB-P and SB1-P cultures (Chowdhury and Widholm, 1985; Rogers and Widholm, 1988). Protoplasts were prepared from the SB-P cells and were cultured in a medium containing glucose and sucrose under continuous light (Chowdhury and Widholm, 1985). Up to 25% of the protoplasts divided to form cell colonies. The protoplasts were as sensitive as the SB-P cells to atrazine when viability and Chl levels were measured, while H soybean cells were not very sensitive. This indicates that the SB-P protoplasts rely on photosynthesis to a large extent for energy. The colonies that developed from the protoplasts contained high Chl levels, and when placed in liquid medium under PA conditions (no sugar with 5% COz), continuous PA growth was obtained. The growth rates and Chl levels were similar to those of the original SB-P cells. The preparation of protoplasts from PA cultures and the reformation of PA cultures from these protoplasts has not been described with other species, but this should be possible.

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Protoplast-derived callus, obtained from a Nicotiana sylvestris mutant which lacked the photorespiratory pathway enzyme serine : glyoxylate aminotransferase, like the whole plant, which had been selected by bleaching under air COz conditions, did not grow under air COz conditions (photorespiratory conditions), while the wild type did (McHale et al., 1989). Both mutant and wild-type cells grew under nonphotorespiratory conditions (1% Cot). Both cell types also grew at similar rates when 0.3% sucrose was added to the medium and fixed I4CO2at similar rates, which indicates that sucrose can help maintain Calvin cycle intermediate levels, which are limiting in the mutant under photorespiratory conditions. As a result of the block in the photorespiratory pathway, the mutant always had higher serine levels than did the wild type, and these levels were decreased by high C02, which would decrease flow into the pathway. A total of 170 revertant colonies, from a total of 678,000 mutant colonies plated in the photorespiratory conditions, remained green and formed shoots amid the background of necrotic cells. When the shoots were retested under air COZ conditions, 23 remained green and grew further. All of the revertants had regained some serine :glyoxylate aminotransferase activity, about half of them showing approximately 50% activity. Progeny obtained from one of these revertants segregated 3 : 1 for normal plants, indicating that there was reversion to activity for one gene in the diploid culture. H. Herbicide Effects and Selection for Resistance

PA cultures have been treated with photosynthetic herbicides initially to determine whether, indeed, the cultures were dependent on photosynthesis for growth. These studies would include those by Horn et al. (1983b), in which SB-P cell growth was inhibited completely by 0.5 pMDCMU, while the SB-M cells on 1% sucrose grew about 70% as well with DCMU as the control in the light without DCMU. SB-H cells grown on 1% sucrose were inhibited only 9% by the DCMU, and the growth of the SB-M cells on 1% sucrose with DCMU was the same as the SB-H cells on 1% sucrose. These results indicate that 0.5 p M DCMU inhibits all photosynthesis, but has little effect on sugar-supported growth. Ashton and Ziegler (1987) showed that atrazine or DCMU inhibited both the photosynthesis and growth of PA C . rubrum cells more effectively than those of PM cultures grown with 1% or 2% sucrose. These photosynthetic electron transport inhibitors were inhibitory to the PM cell growth after the sucrose was exhausted from the medium. Paraquat inhibited growth and induced Chl destruction in both PA and PM cells about equally. The effects of 12 different herbicides were determined with PA, PM, and H N . tabacum suspension cultures and with N . tabacum seedlings by Sato et al. (1987). The PA culture was generally more sensitive to each herbi-

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cide. This was especially true for the photosynthetic herbicides atrazine, DCMU, propanil, nitrofen, and paraquat, the ratio of the 150 values for the H cells to that of the PA cells varying from 60 to 10,000. The seedling response was generally similar to that of the PA cultures in all cases. The commonly used photosynthetic electron transport inhibitor, DCMU, showed 150 values for the PA culture, seedlings, and PM and H cultures of 0.02,0.03, 1.0, and 200 p M , respectively, which was the best example of a correlation of sensitivity with the contribution of photosynthesis to growth. The seven herbicides that should not affect photosynthesis directly, diphenamid, dinoseb, 2,4-D, glyphosate, sodium chlorate, 1,3dimethyl-4-(2,4-dichlorobenzoyl)-5-hydroxy-pyrazolate, and bialaphos, inhibited growth of each of the four systems in a narrow concentration range, as one might expect. Thiemann et al. (1989) grew PA C . rubrum cultures in 24-well microtiter plates on a shaker with 1.5 ml of cell suspension per well and added the herbicides metribuzin, diuron, propanil, or dinoseb or the Fusarium species phytotoxin, fusaric acid. The cells were monitored for growth (PCV and cell number), Chl content, O2 evolution, viability, and Chl fluorescence. Lethal concentrations were determined for each compound, indicating that the system could be used to test a large number of compounds rapidly in a small area. Several different measurements could be taken quite easily during the course of the assay with this system. A PM N . plumbaginifolia callus culture system was described by Cseplo and Medgyesy (1986)in which growth was stimulated several-fold by light when the medium sucrose or glucose concentrations were very low (0.2-0.3%). Photosynthetic electron transport inhibiting herbicides caused bleaching and inhibited the light-dependent growth, but had little effect on callus grown heterotrophically on 3% sucrose. Nonphotosynthetic herbicides inhibited the growth of both PM and H cultures about equally. The PM N . plumbaginifolia culture system described above was used with ethylnitrosourea-mutagenized N . plumbaginifolia leaf protoplasts by screening for green colonies which formed in the presence of 100 p M terbutryn, a photosynthetic electron transport inhibiting herbicide, in a medium containing 0.3% sucrose with 16 hr of light [I500 lux, about 22 pEm-’sec-’(Cseplo et al., 19SS)l. Electron transport in isolated chloroplasts from one of the selected resistant strains (TBR2) was more than 750 times more resistant to inhibition by both terbutryn and atrazine than was the wild type. There was little difference in sensitivity to DCMU, and the selected strain chloroplasts were more sensitive than wild type to dinoseb. This cross-resistance pattern is similar to that found with some triazine-resistant weeds. Regenerated plants were also resistant to terbutryn, and the resistance was inherited maternally. Sequencing of the TBR2 mutant plastid-encodedpsbA gene which codes

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for the quinone-binding protein, which is also the triazine herbicide binding site, showed two base changes (Pay et al., 1988). The base change at position 791, G to A, results in a serine-to-asparagine substitution. The C-to-T change at position 933 does not change the amino acid, so is silent. The base change at position 791 changes Ser-264, as has been found in all triazine-resistant plants sequenced thus far. However, the changes found in all of the other five selected weeds have been to glycine (summarized by Shigematsu et al., 1989). Rey et al. (1990b) were able to select atrazine- and DCMU-resistant colonies from ethylnitrosourea-mutagenized N . plumbaginifolia protoplasts grown under conditions which were nearly completely PA (Rey et al., 1989). Protoplast-derived colonies, 28 days after preparation, were plated in 1 pM atrazine or 0.2 pM DCMU, and green growing colonies formed at a frequency near lop5 in the mutagenized cells. Plants were regenerated from some colonies that showed resistance when sprayed with normally toxic concentrations of the respective herbicides. Atrazine-resistant N. tabacum cells were selected with 100 pM atrazine under PM conditions in liquid medium with 3% sucrose over a 10-month selection period, with transfers every 28 days (Sato et al., 1988). The surviving green clumps were recovered, and when tested under both PA and PM conditions, they showed about a 200-fold increase in atrazine resistance in comparison with the wild type. The electron transport by isolated chloroplasts from one strain showed a 560-fold increase in resistance to atrazine inhibition and a 40-fold increase in resistance to DCMU. Sequencing of the psbA chloroplast gene showed that there was a G-to-C change in the codon 264 (AGT to ACT), which changes the amino acid from serine to threonine. The PA potato suspension (LaRosa et al., 1984) was used to select an atrazine-resistant cell line which also showed an amino acid change at position 264, from serine to threonine (Smeda et al., 1990). Eight C. rubrum cell strains were selected under PA conditions resistant to the triazine herbicide metribuzin, and the strains also showed crossresistance to atrazine and DCMU (Thiemann and Barz, 1990). The strains are resistant due to decreased metribuzin binding to the quinone-binding protein, and this change in structure does not seem to affect electron transport. Analysis of the psbA gene sequence shows the changes to be in positions other than 264, including 219 (valine to isoleucine), and also at positions 220,229,251,266,270,272, or 273, sometimes in combinations of two or three (C. Schwenger, D. Nabor, and W. Barz, unpublished observations). Thus, all of the tissue culture-selected triazine-resistant mutants are different from the five resistant weeds which all have codon position 264 changes from serine to glycine (Shigematsu et al., 1989). The resistant

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N. plurnbuginifolia has a serine-to-asparagine change and N. tabacum and potato have serine-to-threonine changes, while the C. rubrum-resistant strains all have changes in positions other than 264. The C. rubrum strains also apparently do not have impaired photosynthetic electron transport, which has been shown in the triazine-resistant weeds to decrease biomass yield and competitive ability. These unique mutants obtained through tissue culture make one wonder whether the tissue culture selection provides some inherent environment which will always produce changes to amino acids other than glycine, or whether the results are only a result of chance. The answer to this question requires further study. Further studies of the atrazine-resistant N. tabacum cell strain selected by Sat0 et al. (1988) showed that electron transport in isolated thylakoids was very resistant to many triazines and moderately resistant to various substituted urea herbicides (Shigematsu et al., 1989). The resistance patterns were similar to those seen with triazine-resistant A. refroflexus with the triazines, but were greater with the ureas. Prediction of the secondary structure of the mutant psbA gene, with threonine in position 264, indicates that resistance is mainly due to a conformational change in the binding site. In contrast the usual mutation to a glycine residue provides resistance mainly to triazines, but not the urea herbicides, due to the lack of the serine hydroxyl group, and not due to a conformational change. Thus, studies of the triazine-resistant mutants obtained by in uitro selection show that unique changes in the psbA gene can be obtained. I. Secondary Compound Accumulation

Plant tissue cultures have been investigated extensively for the production of commercially valuable plant-derived compounds such as pharmaceuticals, flavorings, colorings, and fragrances. These compounds are usually secondary compounds so are not necessary for normal plant or cell growth. Despite a great deal of effort, most systems do not yield enough of the desired compound to compete with the plant itself as an economical source. One possible way to enhance the accumulation of certain secondary compounds which are found in leaves or other green portions of the plant would be to initiate green photosynthetic cultures. The reasons for the accumulation of certain compounds in leaves could be the localization of the biosynthetic enzymes in chloroplasts, which are not present in nongreen tissues, or the production of substrates via photosynthesis, which are not present in nonphotosynthetic tissues. I review here only the studies with PA cultures, even though there have been numerous studies with green PM cultures and on the effect of light on

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cultures in general (reviewed by Seibert and Kadkade, 1980; Dalton and Peel, 1983; Husemann et al., 1989). Barz et al. (1980) found that the harman alkaloids harmin, harmalin, harmol, and harmalol were present in H, but not PA, cultures of P. harmala, while PM cultures had only very low levels of harmin and harmalol. This fits the accumulation pattern of the plant, where these alkaloids are found mainly in the seeds, roots, and stems. However, quinazoline alkaloids, which are found in P. harmala leaves, were not detected in either PA or H cultures. Studies of the synthesis of glucose esters of p-coumaric and ferulic acids in C. rubrum cultures have shown accumulation in PM cells, since both light and sucrose (above 0.5%) induce the key biosynthetic enzyme phenylalanine ammonia lyase (Strack et al., 1984). As a result the esters are barely detectable in PA cultures, but if 0.5-1% sucrose is added, phenylalanine ammonia lyase and the phenolic esters accumulate. When digitoxin levels were measured in PM and PA D. purpurea cultures and in shoots forming from tissue cultures, both cultures had extremely low levels, while the shoots had more than 10 times these levels (Hagimori et al., 1984). Digitoxin normally accumulates in the leaves. The quantities of lipoquinones (e.g., phylloquinone, a-tocopherol, plastoquinone, and ubiquinone) in the PA M. lucida suspension culture were equal to those found in the leaves of greenhouse-grown plants (Igbavboa et al., 1985). In contrast anthraquinone glycosides, which are found in plant roots, were found only in traces in the PA culture, but their accumulation could be induced in both PA and PM cultures when placed in the dark with a supply of sucrose. When the anthraquinones begin to appear, the lipoquinones disappear. Thus, the PAM. lucida culture mimics the plant in which lipoquinones are chloroplast associated and anthraquinones are found in the roots. Yamamoto et al. (1987) confirmed the observation that anthraquinone accumulation was associated with heterotrophy in the PA and PM M. lucida cultures. They also showed that anthraquinone-accumulating cells had plastids which were distorted in shape and endoplasmic reticulum which was highly elongated. Further studies are needed to determine whether, indeed, these alterations are in some way associated with anthraquinone synthesis. PA C. roseus cultures were initiated to determine whether certain alkaloids, such as vindoline, vincristine, and vinblastine, which accumulate in periwinkle leaves, but not in H tissue cultures, might accumulate in chloroplast-containing cultures (Tyler et al., 1986). The culture did not contain detectable activity levels of two of these three enzymes in the vindoline biosynthetic pathway (De Luca et al., 1987). Since the results were negative, the presence of chloroplasts and active photosynthesis are

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not sufficient to cause accumulation of these alkaloids under these conditions in the cell line used. Husemann er a f . (1989) reported that PA C. roseus suspension cultures have very low levels of indole alkaloids and tryptamine and that, upon reversion to PM and then to H cultures, these constituents increase greatly, and this correlates with an induction of tryptophan decarboxylase, the first enzyme in the indole alkaloid biosynthetic pathway. Cotton leaves are known to contain gossypol and other secondary compounds known to cause enzyme inactivation. However, while actual chemical analyses were not performed, when COT-P cells were mixed and cohomogenized with spinach leaves, there was no loss of activity of RuBPcase in the extract (Blair et af., 1988). Similar results were also found with the labile NR from SB-P cells. These results indicate that COT-P cells, although photosynthetic, do not contain high concentrations of certain secondary compounds usually found in cotton leaves. Ikemeyer and Barz (1989) found that nicotine and chlorogenic acid levels were high in H, moderate in PM, and very low in PA N. tabacum suspension cultures, while trigonelline (N-methyl-nicotinic acid) was high in PA, low in PM, and very low in H cells. Nicotinic acid-N-glucoside was present in all cultures. Nicotine is known to be synthesized in tobacco roots. Treatment with a fungal elicitor from Phytophthora megasperma cell walls caused substantial accumulation of the phytoalexin capsidiol in the H and PM cells and only low levels in the PA cells. More than 70% of the capsidiol accumulation occurred in the culture medium. Elicitation did not affect the levels of nicotine, trigonelline, or nicotinic acid-N-glucoside. Since isoprenoid biosynthesis appears to be largely localized in the chloroplast, the biotransformation of the monoterpene alcohol geraniol to nerol, geranial, and neral was studied during a 24-hr period with PA, PM, and H cultures of four species (Carriere et af., 1989). The cultures used included E . characias (PA, PM, and H), N. tabacum (PA and PM), C. roseus (PM and H), and G . max (H). While there was variable biotransformation and geraniol disappearance, the species, rather than the growth state, determined the pattern. The paraffinic hydrocarbon compositions of PA (2% C 0 2 grown), PM, and H cultures and leaves of E . characias were studied by Carriere et a f . (1990). The PA cultured cells had a hydrocarbon composition very similar to that of leaves, being enriched in odd-carbon chain-length compounds, while the composition of the PM and H cultures were quite different. The total hydrocarbon levels of the PA and PM cultures were 63% and 5% of that of leaves on a dry weight basis, respectively. Thus, the sole dependence on photosynthesis for growth, as in the case of PA culture, markedly alters the hydrocarbon composition. An L . peruuianum suspension culture, grown PA for about 3 years in the

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two-tiered flask system, has been treated with an elicitor preparation from the tomato pathogen Fusarium oxysporum f. sp. lycopersici (A. Beimen and W. Barz, unpublished observations). This treatment causes a rapid incorporation of phenolic materials (p-coumaric acid, ferulic acid, pcoumaroyltyramine, feruloyltryamine, p-hydroxybenzaldehyde, and vanillin) into the cell wall, indicative of an antifungal defense reaction. These cell wall changes make the PA cells highly resistant to the enzyme usually used to prepare protoplasts. Thus, the use of PA cultures gives mixed responses as far as secondary compound accumulation is concerned, so it may or may not be a viable method for increasing the accumulation of any desired compound. Several of the studies have used H, PM, and PA cultures for comparisons and have included the reversion of PA to H by adding sucrose and growing in the dark. Such studies should allow a thorough and clear assessment of the effect of mode of growth on secondary compound accumulation.

IV. Conclusions

A large number of PA cultures have been described from about 24 different higher plant species. The ones that could be considered to be the most ideal, based on the length of time in continuous culture, Chl and photosynthetic enzyme levels, growth on minimal medium, and growth rate, would include lines of N. tabacum, C. rubrum, spinach, D.innoxia, asparagus, soybean, potato, Euphorbia, A. powellii, cotton, and carnation. Lines that can grow with ambient COz levels include Nicoriana species, C. rubrum, soybean, Euphorbia, cotton, and carnation. All PA cultures have COZ compensation concentrations higher than leaves, and this appears to be correlated with much higher dark respiration rates. Most cultures require a relatively long initiation period, except for the N. plumbaginifolia protoplast system (Rey et al., 1989), but these cells begin to regenerate plants and do not form a homogeneous cell suspension in liquid medium without sugar (P. Rey, personal communication). The PA suspension cultures have the usual advantages of suspension cultures: ease of transfer, direct contact of most cells with the medium, relatively homogeneous cell population, ease of microscopic observation for general viewing and viability determination, freedom from microorganisms, readily extractable tissue, easily changed medium, readily controlled conditions to give reproducible results, and growth in many different vessels, including large fermenters. However, the cultures are not identical to leaves in many respects, so studies with PA cultures can

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always leave doubt about their validity in comparison with what occurs at the whole-plant level. The PA suspensions grow in liquid medium, so they are obviously different from leaves, especially mature leaves, which are not growing or dividing. The importance of the liquid phase in gas exchange must also be considered. Avelange et al. (1991) considered the suspension cultures to have advantages over leaves for gas exchange studies, since there are no stomates or gas-water interphases to complicate the measurements. The lack of translocation of photosynthate from the PA cells in comparison with leaves likewise is a difference that would affect cellular metabolism. That all cultured cells appear to produce some stress proteins (Takeda et al., 1990) indicates that the culture environment is also stressful to the cells. While this review is limited to PA cultures from higher plants, bryophytes appear to be much more easily cultured. For example, cultures of the liverwort (Marchantia paleacea) contains Chl even when grown in the dark with glucose, and then when placed under PA conditions, the cells begin to grow without any lag period (Ngumi et at., 1990). The optimal growth conditions were illumination of at least 28 pEm-'sec-' with 2.5% COz. The PA cultures can be used to study some unique phenomena, such as the reversible change of chloroplasts to amyloplasts and then back to chloroplasts upon growth in the dark with sugar in the medium, followed by growth in the light without sugar, respectively. Both the C. rubrum (Ziegler and Schiebe, 1989) and SB-P (Erdos et al., 1986,1987) cultures are amenable to these manipulations. The selection for triazine herbicide resistance in uitro has produced mutants that have unique changes in the psbA gene, different from those seen previously in resistant weeds selected in the field. Each of the five weeds shows a serine-to-glycine change at position 264 (summarized by Shigematsu et al., 1989). The serine is changed to asparagine in the N. plumbaginifolia mutant (Pay et al., 1988) and to threonine in the N. tabacum (Sato et al., 1988) and potato (Smeda et al., 1990) mutants, and the C. rubrum mutants, which reportedly do not have impaired electron transport like the weeds do, have changes only in codons other than position 264 (C. Schwenger, D. Nabor, and W. Barz, unpublished observations). There have been very few genetic studies with PA cultures, but many experiments have shown that tissue culture can induce a high frequency of mutations in regenerated plants, which has been defined by Larkin and Scowcroft (1981) as somaclonal variation. In studies with PA cultures, chloroplast DNA was isolated from N. tabacum leaves and PA and PM cultures and was treated with 12 restriction endonucleases. This DNA was

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then separated by agarose gel electrophoresis, on which no differences were found in the fragment pattern (Sato et al., 1989; F. Sato, personal communication). Likewise, Southern hybridization with a RuBPcase large-subunit probe showed no differences, and the chloroplast psbA gene sequence from cultured cells was identical to that of the plant (Sato et al., 1988). Southern hybridization with a nuclear rDNA probe showed no differences in pattern after EcoRI and Hind111 digestion, although the amount of rDNA in the cultured cells was only one-quarter of that of the mesophyll cells (Sato et al., 1989). The chromosome number of the SB-P and SBI-P cells have remained normal (i.e., 40) when examined at two different times (Chowdhury and Widholm, 1985; Rogers and Widholm, 1988). The general lack of a phytochrome response for chloroplast development in favor of blue light control, which is contrary to the responses seen in plant leaves, shows that certain cultures may be very valuable for studies of certain photoreceptors, perhaps without the complication of interference by others. Roeske et al. (1989) found that the SB-P cells would not light-activate RuBPcase in bright light, which would indicate that rubisco activase may be deficient in this strain. Also, the constitutive NR of soybean leaves is not expressed in the PA, PM, or H cultures of the SB-P cells. The PA cultures could provide a unique system for the production of certain valuable secondary compounds, based on the concept that, if leaf cells produce the compounds, then so should the cultures. However, the results have been mixed. This possibly could be due to the growth of the cultures in contrast to mature leaves, which can accumulate the secondary compounds, but are not growing. This fits with the finding that many plant cell culture systems also accumulate secondary compounds only after growth has ceased (summarized by Sakuta and Komamine, 1987). Molecular biological and metabolic studies can be readily accomplished with PA cultures due to the ease in changing the medium and other conditions and in extracting the compounds central to the study. There are many aspects that require further study with PA cultures, including studies of the cellular hereditary material. Such studies would determine whether there are genetic changes during adaptation, whether all PA cultures have higher plastid DNA levels than leaves, whether all the chromosome numbers are normal, whether the plastid DNA undergoes especially high rates of somaclonal variation, which lead to the unique triazine-resistant amino acid changes, and whether other unique mutants can be selected. The PA culture system should naturally be able to select for any mutation or adaptation which increases the growth and survival rate. This would include decreased photorespiration, so long as low C 0 2 levels are used. The likelihood of such changes occurring should increase

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with time in culture. There is evidence showing a continuous change over a several-year period which can produce more leaflike cultures (higher Chl and RuBPcase and lower PEPcase). Further comparisons of the PA culture and leaf chloroplast component ratios are needed, since it appears that the RuBPcase : Chl ratios are lower in most PA cultures and certain activities (e.g., PSI and Hill reaction) and components (e.g., CFI) are also lower at least in PA N . tabacum cultures (Takeda er al., 1989). More attempts must be made to determine whether the light intensities can be increased to make the PA cultures more leaflike, that is, increase RuBPcase activation levels and total activity and perhaps enhance the photosynthetic electron transport capacity, as increased light levels can do in plants (Torisky and Servaites, 1984; Boardman, 1977). Higher light not only could increase the intrinsic photosynthetic capacity of the cultures, but would also make more energy available to support growth. The PA suspension cultures should be ideal for studies of various stresses (e.g., salt, osmotic, heat, and cold) on photosynthesis or chloroplast development, since the stress can be easily applied, and there are no structural components such as stomates to complicate the response. In the two decades since Bergmann (1967a,b) described the first PA higher plant culture, many additional cultures have been initiated and studied, so that many good systems are now available. Researchers should now utilize these cultures for physiological, biochemical, molecular biological, and genetic studies, which can be ideally carried out with such systems. Acknowledgments The unpublished research was supported by funds from the McKnight Interdisciplinary Photosynthesis Research Program, the SOH10 : U1 Center of Excellence in Crop Molecular Genetics and Genetic Engineering, and the Illinois Agricultural Experiment Station. I wish to thank the following for helpful comments and for additional information, including unpublished results: L. C. Blair, D. E. Buetow, G. C. Cannon, D. R. Duncan, C . S. Goldstein, M. E. Horn, W. Hiisemann, W. G. W. Kurz. P. C. LaRosa, K. H. Neumann, W. L. Ogren, P. Rey. F. Sato, and C. Xu. I also wish to thank R. Olson of the University of Illinois Center for Electron Microscopy and L. Rayblrrn for assistance with the microscopy. I also wish to thank Sharon Gocking for typing the manuscript.

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Widholm, J. M. (1989). In “Primary and Secondary Metabolism of Plant Cell Cultures” (W.G. W. Kurz. ed.), Vol. 11. pp. 3-13. Springer-Verlag. Heidelberg. Willrnitzer. L., and Wagner, K. G. (1981). Exp. Cell Res. 135,69-77. Xu, C., Blair, L. C., Rogers. S. M. D.. Govindjee. and Widholm, J . M. (1988). Plant Physiol. 88, 1297-1302. Xu, C.. Rogers, S . M. D.. Goldstein, C., Widholm. J . M., and Govindjee (1989).Photosynth. Res. 21,93-106. Yamada. Y., and Sato, F. (1978). Plant Cell Physiol. 19,691-699. Yamada, Y., Sato, F.. and Hagimori, M. (1978).I n “Frontiers of Plant Tissue Culture 1978” (T. A. Thorpe, eds), pp. 453-462. Univ. of Calgary, Calgary, Alberta, Canada. Yamada, Y., Imaizumi, K., Sato, F., and Yasuda, T. (1981). Plant CellPhysiol. 22,917-922. Yarnamoto, H., Tabata, M., and Leistner, E. (1987). Plant Cell Rep. 6, 187-190. Yasuda, T., Hashimoto, T.. Sato. F., and Yamada, Y. (1980). Plant Cell Physiol. 21, 929-932. Yasuda, T., Kuroiwa, T.. and Nagata, T. (1988). Planta 174, 235-241. Ye, J., and Sayre, R. T. (1990). Plant Physiol. 94, 1477-1483. Ziegler, P., and Schiebe, R. (1989). Plant. Cell Enuiron. 12, 725-735.

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Zymogen Granules of the Pancreas and the Parotid Gland and Their Role in Cell Secretion Adrien R. Beaudoin and Giiles Grondin Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Qukbec J l K 2R1, Canada

I. Introduction

The exocrine acinar cells from the pancreas and the parotid gland are adapted for the synthesis and secretion of digestive proteins. The secretory mechanisms of these acinar cells, particularly those of the pancreas, have been the subject of extensive studies. As early as 1898, Pavlov and associates (Pavlov, 1910) demonstrated the significance of the nervous mechanism of stimulation. The regulation of secretion was clarified to some extent by the discovery of peptide hormones such as secretin (Baylis and Starling, 1902) and pancreozymin (Harper and Raper, 1943). Microscopic studies had previously shown that cells of the exocrine glands, including those of the pancreas (Heidenhain, 1875), contain characteristic granules and that the number of these granules decreases when secretion is stimulated. By means of differential centrifugation of homogenate from dog pancreas, Hokin (1955) was able to isolate a fraction that contained chiefly zymogen granules (ZGs) and showed that this organelle had a higher concentration of proteolytic activity, amylase, and lipase than the whole homogenate. This observation was definitive in that it clarified the role of ZGs as storage sites of digestive enzymes in exocrine glands. With the advent of electron microscopy, autoradiography, and new cell fractionation, immunological, and biochemical techniques, these organelles could be studied in great detail. In this review we summarize some of the knowledge acquired on the pancreas and the parotid gland ZGs in the past two decades. Two other aspects, closely related to this subject, have been recently reviewed: One deals with the transport and secretion of digestive proteins (Beaudoin and Grondin, 1991 ;Grossman, 1988),whereas the other focuses on the composition of pancreatic juice (Beaudoin et al., 1989). Inremarional Review of Cvtology. Val. 132

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In Section I1 of this review, we consider the overall variations of the ZG compartment and the variations of the granule size under a variety of physiological conditions. In this task we were greatly assisted by the excellent review of Cope (1983). Then we examine the origin and fate of the ZGs as observed by cytochemical methods. In this section some of the concepts proposed by Farquhar and Palade (1981) have been outlined. In Section V we summarize the contribution of freeze-fracture techniques to the definition of the ZG membrane architecture. In Section VI the content of ZG is analyzed by immunocytochemical methods and the localization of secretory proteins is related to some current views on secretion. In the two following sections the electrophysiological properties of ZGs and the influence of the cytoskeleton on secretion are discussed. Finally, the observations presented in this review are integrated into a scheme of ZG maturation.

II. ZG Morphometry: Influence of Various Physiological Parameters on Granule Size

Like many other glandular tissues, the pancreas and the parotid gland process their macromolecular products by cisternal packaging and exocytosis (Cope, 1983; Case, 1978; Grossman, 1988; Beaudoin and Grondin, 1991). Their secretory products are segregated from the cytosol from the beginning and are transported through the cell and discharged, as a result of a series of fission-fusion reactions between adjacent endomembranous compartments (e.g., endoplasmic reticulum, Golgi complex, vesicles, secretion granules, and plasmalemma). As all of these compartments can be identified by the electron microscope, these tissues and the exocrine secretory process lend themselves to morphological quantification. Changes in compartments should reflect the passage of macromolecules through the cells. Thus, information concerning the formation, packaging, and discharge of secretory products can be obtained stereologically. Furthermore, analysis of the membrane envelopes themselves can provide information about membrane dynamics during the secretory cycle (Cope, 1983). Estimates of the size of parotid acinar cells vary between 550 and 1100 pm3, and those of the pancreas vary between 700 and 1700 pm3. Variations in cell size for a given species are probably attributable to differences in tissue-processing and analytical techniques, rather than to biological differences among strains. Although they look similar, significant morphological differences exist between pancreas and parotid exocrine acinar cells. These differences include the relative amounts of

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rough endoplasmic reticulum (RER) and the ratio of RER to secretion granules (Bolender, 1974; Amsterdam and Jamieson, 1974; Ferraz de Carvalho et al., 1978; Nevalainen, 1970; Bloom et ul., 1979). Indeed, pancreatic acinar cells have about twice the volume of the RER and about one-half the volume of secretory granules of parotid acinar cells. These values may reflect the greater synthetic capability of the pancreatic acinar cell and suggest that either its products are more highly condensed than those of the parotid gland or that the pancreas cells store less of their output in granular form than do parotid cells (Cope, 1983). In addition to secretory granules, one finds in the Golgi area various types of vesicles, including some minigranules (Beaudoin and Grondin, 1987). There are also small vesicles in transit between compartments; that is, vesicles shuttling back and forth between the RER and the Golgi apparatus, between the Golgi apparatus and the granules, and between the Golgi saccules and the plasmalemma. As the diameter of many of these vesicles is sometimes about the same order as section thickness, their numbers are difficult to quantify. One of the major factors that affect the number and size of secretory granules of these exocrine cells is their physiological state. In the rat pancreas the ZGs normally occupy 5-20% of the cell volume. Their number is generally higher after a short period of fasting. The mouse pancreas does not follow this pattern. Indeed, a 50% reduction in granule content after a 24-hr fast has been reported (Carlsoo et a / . , 1974). This is accompanied by a reduction in protein synthesis and a collapse of RER cisternae. Starvation causes crinophagy, or autolytic digestion of granules (Nevalainen and Janigan, 1974). In the rabbit values as high as 40% of the cell volume have been reported (Cope and Williams, 1973a; Bedi et af., 1974). In contrast, feeding causes a marked reduction (50%) in the granule compartment in rat pancreas as compared to fasted controls (Ermak and Rothman, 1981). Similar values were reported for the frog pancreas after 4 hr of feeding (Slot and Geuze, 1979). A comparable reduction in the granule compartment (40%) in rabbit parotid gland was observed within 1 hr of feeding (Carlsoo ef ul., 1974). The average size of the ZGs is also greatly influenced by stimulation (Fig. 2a-c). In the rat pancreas, in a resting state, the granule population shows approximately a gaussian size distribution with respect to diameter (0.7 and 1.0 pm) (Nadelhaft, 1973; Liebow and Rothman, 1973; Beaudoin et al., 1984; Aughsteen and Cope, 1987). Some studies reported a bimodal distribution of granule diameters with a minor peak in granule diameter around 0.5 and 0.6 pm and a major peak between 0.8 and 1.0 pm (Sato and Take, 1975). Yoshimura (1977) noticed that, among Wistar rats, the pancreata of some had two populations of granules and others had only one.

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Intriguingly, this bimodal distribution was also observed in stimulated pancreas, The average size of the small-granule population corresponds roughly to that of the minigranules (0.2-0.3 pm) described by Beaudoin and Grondin (1987). We have found that these minigranules are not present in all cells on a given section of pancreas. Secondly, their occurrence is variable from one species to another, being, for example, more frequent in pigs than in rats. In the rat azaserine-induced pancreatic tumor, some cells are filled with minigranules, whereas others contain a normal population of granules (Beaudoin et al., 1986b). A shift in the size-frequency distribution of granules has been reported in the rat pancreas after either feeding (Ermak and Rothman, 1981) or hormonal stimulation in vivo (Beaudoin et al., 1984; Aughsteen and Cope, 1987). Ermak and Rothman (1981) proposed that the number of granules discharged is not always in proportion to the amount of secretory material released by the gland. They suggested that granules shrink as their contents are released by diffusion-like processes. Beaudoin et al. (1984) proposed that this shift is, rather, due to a selective discharge of large granules and the concomitant formation of smaller new granules, especially after hyperstimulation. This view is shared by Aughsteen and Cope (1987). One must be cautious when interpreting data on granule size, since it could be influenced by many physiological and methodological factors. For example, cells in the vicinity of Langerhans islets are larger and contain more granules than are usually encountered in the rest of the tissue (Bendayan, 1984). After stimulation the so-called “periinsular cells” appear to retain more granules than cells more distant from the islets or the “teleinsular cells.” In addition some heterqgeneity has been observed among the teleinsular acini. The accuracy of granule size determinations is greatly reduced in stimulated glands, because the acini do not respond in synchrony to secretagogues. Indeed, differences in the number of granules was observed after feeding or following stimulation of the pancreas by pharmacological doses of secretagogues, some acini being more rapidly depleted of their granules than others (Phaneuf et al., 1985; Roberge et al., 198 1). Intriguingly, autoradiographic studies on the incorporation of [3H]leucineinto proteins of acini isolated form the pancreas of a fasting rat demonstrate great variations in labeling among acini (see Fig. 1). Another important parameter which may have been overlooked in the morphometrical studies mentioned above is circadian variation of both granule size and number (see Uchiyama and Saito, 1982; Uchiyama and Watanabe, 1984), which occur even under fasting conditions (A. R. Beaudoin, G. Grondin, and P. St-Jean, unpublished observations). The size distribution of the ZGs in the parotid gland cells shows some points of similarity with that observed in the pancreas. A unimodal size distribution of granules has been found in the starved rabbit (Cope and

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FIG. 1 Autoradiography illustrating the variations in the rate of protein synthesis among the acini isolated from the pancreas of a fasting rat. Proteins were pulse-labeled for 5 min with [3H]leucine and chased with cold leucine for 90 min in v i m . Note the differences between acini in both the density and localization of silver grains, indicating different rates of processing. Compare the density in l a (solid and open arrows) and localization in l b (arrowheads and arrow). Acini were prepared according to Roberge er ul. (1981). and autoradiography was carried out according to Kopriwa and Leblond (1962).

Williams, 1973b, 1981; Bedi et al., 1974), the mean granule size being close to 1 pm. As was reported for the pancreas, bimodal distribution in granule size has been observed in the rat parotid gland (Itoh, 1977). After feeding the cells of the rabbit parotid gland showed a greater reduction in the number of granules than in the total volume of granules per cell (Bedi ef al., 1974), indicating a preferential discharge of older granules that were smaller and more condensed than usual. Simson et al. (1974) observed the same phenomenon in the rat parotid gland following repeated stimulations with isoproterenol. Following stimulation in uiuo both the parotid gland and the pancreas demonstrate rapid granule depletion, whereas the pattern of regranulation is much slower (Amsterdam et al., 1969; Nevalainen, 1970; Geuze and Kramer, 1974; Cope and Williams, 1980, 1981). New granules usually begin to appear about 3-4 hr after the onset of secretion, then their volume rises sharply for the next 4-6 hr, during which time the rate of accumulation falls off, with complete restitution of granule stores after 12-16 hr (Cope and Williams, 1980, 1981). The size distribution of granules during regranulation has been analyzed for the rabbit parotid gland (Cope and Williams, 1981). Initially, the gland produces small granules relatively quickly, so that by 8 hr the cells con-

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tained nearly twice as many granules as the unstimulated controls. These, however, were only about two-thirds the volume of granules. Thereafter, the total volume of secretory material per cell continued to rise, but the number of granules per cell dropped to a number similar to that in the control group. A similar pattern of granule maturation was reported in the rat parotid gland, except that the transformation to larger granules, which occurs between 8 and 12 hr in the rabbit, was delayed in the rat (Itoh, 1977). Cope (1982) suggested that the gland first produces small granules with a mean diameter of about 0.5-0.6 pm, but as the gland fills up and the granules come into close contact, they fuse to form larger ones. The studies reported above show that, during fasting, there is generally an accumulation of large granules in both the pancreas and the parotid gland. A study of the pancreas suggests that the larger granules would concentrate in close vicinity to the apical plasma membrane. When the pancreas is stimulated either by feeding or by administered hormones, a reduction of mean granule diameter is generally observed, which reflects a selective exocytosis of large granules. If stimulation is maintained, newly formed granules are smaller, indicating that secretagogues exert an influence on the packaging process (see Fig. 2).

111. Fate of the ZG Membrane after Exocytosis: A Recycling Process

During exocytosis the ZG membrane fuses with the luminal plasmalemma, allowing the discharge of granule content. Occasionally, one could observe some membranous material in the gland lumen, suggesting that pieces of membrane are also expelled during secretion. In this respect Battistini et al. (1990) recently showed that, in response to stimulation, some particulate y-glutamyl transpeptidase is found in pancreatic juice. However, it cannot be excluded that this enzyme, which is believed to be present in ZG membrane of the rat pancreas, can also be released from ductal cells, as proposed by Yasuda et al. (1986). Despite this observation, which may involve minute amounts of membrane, it is believed that the granule membrane is almost entirely withdrawn into the cell by endocytosis (Kalina and Robinovitch, 1975; Herzog and Farquhar, 1977; Oliver and FIG. 2 Influence of stimulation on the ZG and the Golgi apparatus (Go) of the rat pancreas. (a) Pancreas after an overnight fast. (b) Pancreas after two injections of urecholine. (c) Pancreas after infusion of a cocktail of secretagogues. Note the swelling of the Golgi apparatus and the reduction in both number and size of the residual ZGs (arrowhead). Lu, Acinar lumen. Experimental conditions are as described by Beaudoin et al. (1984).

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Hand, 1978; Farquhar and Palade, 1981). Amsterdam et al. (1969) reported an increase in the number of smooth vesicles within the cytoplasm of the rat parotid gland during secretion. Geuze and Kramer (1974) made counts of vesicles following pilocarpine-stimulated secretion on the rat pancreas. They also reported increases in the number of coated vesicles per square micron of apical cytoplasm, which reached a peak 3 hr after the onset of secretion. However, it is unlikely that this peak marks the period of maximum membrane withdrawal. Indeed, exocytosis and endocytosis are usually considered to occur more or less simultaneously (Kalina and Robinovitch, 1975; Tamarin and Walker, 1976), with a peak in the number of vesicles about 2 hr after most of the granules have discharged their content. This peak in the numerical density of vesicles appears to coincide with the time that cytoplasmic volume is at its lowest level (Nevalainen, 1970). Cope (1982) suggested that this peak does not reflect a real increase in the number of vesicles per cell, but simply the concentration of vesicles into a smaller cytoplasmic volume. As reviewed by Farquhar and Palade (1981), results of early morphological studies with electron-dense tracers had established that, after exocytosis, membrane is recovered intact (i.e., exocytosis is coupled to endocytosis). However, because the tracers were found to be subsequently transported primarily or exclusively to lysosomes, it was concluded that the recovered surface membrane was degraded. The idea that the secretory granule membrane was recovered and degraded in lysosomes, rather than being reutilized or recycled, prevailed for some time until the studies by Holtzman et a / . (1977). In retrospect it is clear that these early studies were limited by the fact that the tracers used (usually horseradish peroxidase and native ferritin) were charged protein molecules that acted primarily as content markers. Hence, they were useful for following the fate of the vesicle contents, but not that of the vesicle membrane. Several groups (Farquhar, 1978; Herzog and Farquhar, 1977; Ottosen et al., 1980; Wilson et al., 1981; Herzog and Miller, 1979; Herzog and Reggio, 1980) later showed that, after exocytosis, the retrieved membrane is funneled through the Golgi complex. Evidence was obtained by using several tracers that had not been utilized before, that is, dextrans (uncharged relatively inert polysaccharide molecules) and cationized ferritin (which is known to bind electrostatically to membranes and, therefore, act as a membrane marker). As mentioned by Farquhar and Palade (1981), the most likely explanation for the bulk of this traffic in secretory cells is that it represents the recovery of granule membranes to be reutilized in the packaging of secretory granules; that is, it represents a recycling of granule membrane. Fate of the ZG membrane has also been studied during pancreas development. Indeed Carneiro and Sesso (1987) performed a morphometric

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evaluation of ZG membrane transfer to Golgi saccules following exocytosis in pancreatic acinar cells from newborn rats. More specifically, acinar cells from unfed newborn rats and suckling rats for 4,8, and 16 hr were examined morphometrically in semi- and ultrathin sections. In the cells of the unfed newborn rats numerical and volume densities of the ZG and the volume of the Golgi apparatus are, respectively, the highest and the lowest observed during peri- and postnatal life. Cisternae of the RER appear irregularly disposed among the ZG. Once feeding starts, cytoplasmic volume becomes progressively reduced until the 16th hour, owing to sustained exocytosis of ZG contents. The decline in the numerical density of ZGs between 0 and 4 hr revealed the minimum number of ZGs exocytosed in the first 240 min. The sum of the membrane surfaces measured in the various subcellular compartments [RER, condensing vacuoles (CVs), Golgi saccules, Golgi apparatus-associated microvesicles, “other structures,” apical and basolateral plasmalemmae, and mitochondria] did not vary significantly in the various groups of rats. After 4 and 8 hr the net amount of cellular ZGs is sufficient to account for the expressive increase in membrane surface occumng at these times in CVs, Golgi saccules, and microvesicles. The curves showing the membrane surface decrease in ZGs and the increase in the Golgi saccules appear to express a precursorproduct relationship. The results of topochemical reactions are consistent with the interpretation that part of the ZG membrane internalized after exocytosis, induced by alimentary stimulus, is reused to expand and/or form trans [thiamine pyrophosphatase (TPPase)-positive] and trans-most [acid phosphatase (AcPase)-positive] Golgi saccules. In the latter study there was no significant increase in apical plasma membrane in the first hours of suckling, despite a pronounced decrease in ZG content. The lack of increase in apical plasma membrane suggested an immediate retrieval of the ZG membrane (Carneiro and Sesso, 1987). However, some observations by Romagnoli (1988), on lobules of adult rats in uitro during a 10-min incubation period, showed a significant increase in apical surface upon stimulation. Indeed, the apical plasma membrane surface area and the number of ZGs less than 20 nm from the apical plasma membrane significantly increased and were directly correlated with increases in secretion. The diameter of ZGs decreased when lobules were stimulated by lop6M carbamylcholine, but increased at lo-’ and lop5 M concentrations as compared with controls (see Fig. 2c). In the parotid gland exocytosis and the ensuing membrane retrieval are restricted to a clearly delimited and easily recognizable portion of the plasmalemma and are not dispersed over the entire cell surface. Second, optimal stimulation of acinar cells with P-adrenergic agonists results not only in the rapid discharge of most secretion granules, but also in a clear

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enlargement of the acinar lumen (Amsterdam er al., 1969; Batzri et al., 1971). Such a situation, which is at variance with that observed in the exocrine pancreas, indicates that, at the luminal surface, membrane input (exocytosis) and output (retrieval) are not kept in balance during sustained stimulation. The obvious discrepancy between the large membrane surface disappearing from the granule pool and the considerable, though not massive, increase of the luminal plasmalemma suggests that the two processes are not completely dissociated (Koike and Meldolesi, 1981). In the experiments by Amsterdam et al. (1969), the acinar lumen remained clearly enlarged as long as 2 hr after isoprenaline injection, whereas in freeze-fracture studies membrane patches of the granule type were detected at the luminal surface until the fourth hour (Koike and Meldolesi, 1981). Moreover, a slow time course of membrane retrieval can be deduced from the careful stereological data in the rabbit parotid gland (Cope, 1983). Amsterdam et al. (1969) showed that the lumen perimeter increased markedly in the first 30 min of isoprenaline stimulation in the rat parotid gland. This increase corresponded well with the membrane added by exocytosis. By 2 hr the acinar lumen perimeter had already returned to normal values, with a massive increase in the number of apical vesicles. From the experiments described above it is clear that time after stimulation is a critical parameter when one wants to interpret the changes in acinar lumen in relation to ZG exocytosis. It appears that, at short term after stimulation in both the parotid gland and the pancreas, there is an enlargement of the lumen then the process of membrane retrieval is initiated and gradually catches up with the process of membrane addition by exocytosis. However, an equilibrium between the two processes is apparently reached more rapidly in pancreas than in parotid acinar cells. The intensity and duration of stimulation are major parameters that could shift this equilibrium one way or the other. The alterations of cell compartments associated with pancreas secretory activity are summarized in Table I.

IV. ZG Cytochemistry: Relationship between the Golgi Apparatus and ZGs

Cytochemistry has proved to be an invaluable tool to define the biochemical characteristics of the Golgi apparatus, and the relationship of this organelle to secretory granules, in a variety of cells. The central role played by the Golgi apparatus in the formation of secretory vesicles was recognized long ago by light microscopists. As mentioned by Farquhar and Palade (1981), early electron-microscopic studies (Sjostrand and Hanzon, 1954; Haguenau and Bernhard, 1955; Farquhar and Rinehart, 1954) noted

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ZYMOGEN GRANULES TABLE I Alterations of Cell Compartments Associated with Secretory Activity in the Pancreas"

Experimental conditions ~

Fasting

Cellular compartments cis-Golgi saccules trans-Golgi saccules and the trans-Golgi network trans-Golgi area microvesicles Condensing vacuoles ZGs Endocytic vesicles Acinar lumen

Normal Normal

Few Few (large) Many (large and dark) Few Small (few microvilli)

Stimulated Normal Increased (volume and membrane surface) Many Many (small) Few (small, pale) Many < I hr enlarged, with few microvilli 1-3 hr, small, with many microvilli >3 hr, normal

" Unless otherwise stated, the fasting condition corresponds to a deprivation of food for 16-36 hr. The stimulated condition corresponds to intense stimulation for about 3 hr, by either intravenous infusion of a secretagogue or repeated stimulation. As demonstrated by Phaneuf er a / . (1985). some acini appear to be insensitive to secretagogues following short-term stimulation.

the close association between secretory granules and Golgi elements. Shortly thereafter, several investigators (Farquhar and Wellings, 1957; Palay, 1958) published electron micrographs in which material resembling the contents of secretory granules was clearly recognized within Golgi elements. Subsequent morphological and autoradiographic studies (reviewed by Whaley, 1975; Beams and Kessel, 1968; Farquhar, 1971; Bainton et al., 1976) established that, in most cell types, the concentration and packaging of secretory products usually occur in the dilated rims of the trans-most cisternae. However, in a few cell types (e.g., the exocrine pancreas and the parotid gland), concentration takes place in specialized condensing vacuoles, which are separate from the stacked cisternae. In either case concentration results in the production of a storage granule with a condensed content and a membrane acquired in the Golgi complex. The fact that concentration commonly takes place in the dilated ends of the Golgi cisternae raised the intriguing question of how concentration is brought about in the dilated ends of a continuous compartment. As re-

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viewed by Farquhar and Palade (1981), the first information on this problem came from the experiments by Jamieson and Palade (1971), who showed that concentration in both condensing vacuoles and ZGs was maintained in situ in the absence of ATP synthesis. The findings led to the conclusion that concentration is not dependent on a continuous expenditure of energy. AcPase and TPPase cytochemistries have provided some insights into the details of the secretory pathway involving the CVs of the pancreas and the parotid gland (Novikoff et al., 1977; Hand and Oliver, 1984; Fujita and Okamoto, 1979; Beaudoin et al., 1983; Rambourg et al., 1988). These studies showed that CVs were formed from the trans-Golgi apparatus and the Golgi-associated endoplasmic reticulum-lysosome system (GERL). This region of the cell is included in what is now known as the trans-Golgi network. Novikoff et al. (1977) defined the GERL concept as a lysosome formation system. According to them, the GERL consisted of smooth endoplasrnic reticulum, multivesicular bodies, small vesicles, coated vesicles, and lysosomes. From their observations they proposed that in the pancreatic cell the nascent secretory vesicles or CVs are expanded cisternal portions of the GERL, a structure originating from the endoplasmic reticulum. The studies by Hand and Oliver (1977) confirmed the role of the GERL in granule formation in exocrine cells. Their observations suggested that the GERL might be derived from the inner Golgi saccule and indicated a close relationship between the GERL and the endoplasrnic reticulum. They also mentioned that endoplasmic reticulum-GERL continuities were encountered only rarely, and the marked differences in enzyme content suggested that the transfer of proteins is probably minimal. Later, Fujita and Okamoto (1979) reported that AcPase and TPPase were both present in the trans-Golgi saccule, in rigid lamellae, on CVs, and on coated vesicles in the trans area of the Golgi apparatus. They proposed to consider the GERL of Novikoff et al. (1977) as part of the Golgi apparatus. Beaudoin et al. (1983) reported cytochemical distributions of AcPase, TPPase, and ATP-diphosphohydrolase activities on thin sections of rat pancreas and on isolated ZG membranes. AcPase was found in the rigid lamellae separated from the Golgi stacked saccules, CVs, and the transGolgi saccules, but it was not detected in purified ZG membranes. TPPase was detected in trans-Golgi saccules, purified ZG membrane and occasionally ZG membrane in situ, and the plasmalemma of the acinar cell, but it was not seen in CVs. The ATP-diphosphohydrolase activity had a distribution similar to that of TPPase. These observations illustrated the similarity between the trans-Golgi saccules and the membrane of mature ZGs and further corroborated the view that the trans-Golgi saccules are involved in the formation of secretory granules. The interpretation of the cytochemical observations is complex, as illustrated in Fig. 3. For example,

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acid phosphatase is highly reactive in CVs, but is undetectable in mature ZGs; Trimetaphosphatase is not detectable in the trans-Golgi saccules or the GERL (the trans-Golgi network), but is present in mature granules and acinar lumen. Another example is the NADPH-phosphohydrolase which is detected in the medial saccules of the Golgi stack. It is undetectable in CVs and ZGs, but is present in the acinar lumen. It is noteworthy that some vesicles that appear to be “minigranules” do not seem to bear any type of phosphatase activities. In the parotid gland the cytochemical localizations of AcPase and TPPase in the Golgi apparatus, the GERL, and forming granules are essentially comparable to those in the pancreas. As reported by Hand and Oliver (1984), the Golgi apparatus consist of several stacks of four to six saccules, numerous vesicles, and immature secretory granules of variable sizes and densities located near the trans face. The saccules at the cis face tend to be dilated and irregular, while those at the trans face are narrower and more regular. Reaction product is usually present in one or two trans-Golgi saccules after incubation for TPPase activity. No difference was noted in the localization of reaction products when TPPase, uridine diphosphate, or inosine diphosphate was used as substrate. The GERL was identifiable as short narrow saccule adjacent to, or, more frequently, separated from, the trans-Golgi saccule. Occasionally, continuities between the GERL and an immature granule were observed. The GERL and some of the immature secretory granules contained reaction product after incubation for AcPase activity. However, the reactivity of the GERL in control acinar cells was variable, and in many instances little reaction product was present under their fixation and incubation conditions. The GERL and the immature granules were usually unreactive for TPPase. Modified cisternae of RER, lacking ribosomes on the surface adjacent to the GERL, were frequently observed paralleling the GERL and the immature granules. Perhaps one of the most significant cytochemical observations were made when these cells were stimulated. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol administration, AcPase activity was markedly increased in the GERL and the immature secretory granules compared to uninjected controls. The GERL increased, and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr and was almost complete by 16 hr after isoproterenol injection. TPPase activity, normally restricted to the trans-Golgi saccules, was frequently present in immature granules during this period. Narrow saccules resembling the GERL, occasionally in continuity with immature granules, also contained TPPase

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reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to those of control cells. These results demonstrate the dynamic nature of the Golgi apparatus and the GERL in parotid acinar cells and emphasize the close structural and functional relationship between these organelles. Along the same line Novikoff et al. (1977) also reported the effect of stimulation on AcPase in both pancreas and parotid acinar cells. In resting cells no phosphatase activity was present in mature ZGs, but high levels of activity were seen in smaller granules, located close to the Golgi apparatus, corresponding to early secretory granules. When the discharge was induced by pilocarpine, small AcPase-rich granules were seen at the apical pole of the cell. Their interpretation was that newly formed granules were lysosomes. Some recent observations by Sesso et af. (1990) have shown that there is a microvesicle budding process from the surface of CVs during conversion to mature ZGs. Along this process the cortex of the CVs, as well as vesicles that are budding, show AcPase reaction products. These observations indicate that, under resting conditions, there is a maturation process that removes AcPase and perhaps other lysosomal enzymes from the immature secretory granules. Under intense and prolonged stimulation this maturation process would be impaired, as evidenced by the presence of smaller granules containing AcPase, but the identity of these secretory vesicles must be confirmed by combined immunocytochemistry and cytochemistry.

V. Freete-Fracture Observations: Evidence That the ZG Membrane Undergoes Some Major Topographical Alterations of Protein and Lipids Fusion of the ZG membrane with the plasma membrane is normally restricted to the portion of the cell surface facing the secretory lumen. By the use of freeze-fracture techniques, De Camilli et al. (1974) compared the secretory portion of the plasma membrane to the nonsecretory portion and found some important differences. In general, after cleavage, most of the

FIG. 3 Cytochemistry of secretory vesicles in the apical cytoplasm of pancreatic acinar cells. (a) Acid phosphatase. Condensing vacuoles (CVs) are positive; ZGs and minigranules (arrow) are negative. (b) Trimetaphosphatase. Note the patchy distribution of the stain and its concentration at budding sites (arrowheads) on the ZG surface. (c) P-NADPHase. Note that CVs and ZGs are negative, whereas the acinar lumen (Lu) is highly reactive. (d) P-NADPHase precipitates (arrowhead) in the acinar lumen. Go. Golgi apparatus. Experimental conditions are as described by Beaudoin el ul. (1985).

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membrane particles are associated with the half of the membrane left frozen to the cytoplasm (the P face). The face of the half-membrane left frozen to the extracellular space (the E face) bears fewer particles per unit of area. In pancreatic acinar cells the typical P face pattern can be found only in the basal and lateral portions of the plasma membrane (including the regions close to the tight junctions and within the chambers delimited by their network). However, over the entire luminal area there are far fewer particles per unit of area of the P face. The transition is sharp across the inner continuous line of the tight junction delimiting the lumen. On the E face the number of particles is always small, with no clear difference between the luminal and lateral portions of the plasma membrane. The limiting membranes of the ZGs stored within the cells bear much fewer particles per unit of area than the membranes of other cytoplasmic organelles. The distribution is, again, asymmetrical, with more particles in the half-membrane left frozen to the surrounding cytoplasm (P face). Hence, if we look at the particle patterns, the E face of the ZG membrane resembles the E face of the whole plasma membrane, while the P face of the ZG membrane looks like the luminal plasma membrane P face. This observation is expected, since during exocytosis the P leaflet of the ZG membrane becomes continuous with the P leaflet of the luminal plasma membrane and the same occurs with the two E leaflets. Jamieson (1975) and De Camilli et al. (1976) later confirmed the polarity of the external fracture leaflet with less than half the number of particles as compared to the plasmic fracture leaflet. Cabana et al. (1981) observed a comparable distribution on purified ZGs in uitro. Indeed, ultrastructural examination of the ZG membrane by rapid-freezing and freeze-fracture techniques revealed that the E leaflet has a highly textured subparticle background with a significantly lower frequency of intramembrane particles (IMPs) (44per square micron), showing diameters of 9-18 nm and a shift to larger IMPs (12.3 nm). Two hitherto undescribed types of IMPs were found on both membrane leaflets. First, a population of 13-nm particles with an electron-lucent center, or “pore,” the most frequent type on the E face (26%),and a second population of large IMPs (15 nm) characterized by a large pore (5 nm in diameter), subdivided by a delicate crossshaped structure. Under alkaline conditions, at pH 8.2, ZG lysis occurs rapidly. Membrane ghosts thus obtained were rapidly frozen or suspended in dextran and filtered immediately. Transmission electron microscopy showed many opened ghosts with adhering amorphous material and numerous small vesicles near or still attached to openings in the ghosts. Freeze-fracture preparations also showed that granule lysis is accompanied by major alterations, suggesting a system that controls the topological distribution of membrane particles. More recently, Beaudoin et at. (1988) demonstrated that the isolated ZG

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membranes of the rat pancreas can be subfractionated on a sucrose gradient. Four discrete types of membranes corresponding to densities of I . 105, 1.085, 1.075, and 1.020 were obtained, designated types A, B, C, and D, respectively, and characterized by both morphological and biochemical criteria. Electrophoretic profiles showed that they contain the same protein bands, but in different proportions. Type A membranes comprise four major bands corresponding to 80,69,54, and 20 kDa, being in higher concentrations. Types B and C contain three major bands at 80, 54, and 20 kDa, whereas type D comprises only two major bands at 69 and 54 kDa; the latter polypeptide, corresponding to ATP-diphosphohydrolase activity, is present in all four types of membrane. Freeze-fracture of rapidly frozen membranes, followed by transmission electron microscopy (TEM), showed that type A membranes are large superimposed sheets of membranes with amorphous material between them. The surface area of these sheets corresponds roughly to the surface of intact ZGs, with a few IMPs (distributed at random or in small aggregates on large smooth fracture planes). Types B and C exhibited a totally different aspect, forming closed vesicles about the size of small ZGs, with few IMPs (distributed at random or in small aggregates on smooth fracture planes). Type D membranes were small vesicles, with no detectable IMPs on relatively smooth fracture planes. Various explanations can be suggested for the existence of different types of ZG membranes. There could be a maturation process involving changes in the ratio of lipid to protein, as well as the proportions of the various proteins; there could also be different populations of ZGs with different proportions of the same membrane proteins; the isolation procedure could have separated various domains of the in situ ZG membrane; or finally, it could have induced an artificial phase separation in the ZG membrane, followed by fragmentation. Some information regarding the alteration of architecture of the newly formed granules with time was obtained by Sesso et al. (1980), who conducted a freeze-fracture and thin-section study of CVs in rat pancreatic acinar cells in the suckling rat pancreas. The CVs showed what appeared to be a biphasic evolution. During the first stage the CVs enlarge, accumulating contents of rather low electron density. Fracture faces with irregular patterns, possibly the result of fusion (pinching off) of microvesicles with (from) the CVs, were occasionally encountered. The infrequency of such images indicates that fusion-fission during the growth stage must be a rapid event. One common type of surface irregularity is gibbosities (or convexities) in the P fracture face, with complementary images on the E fracture face. The significance of these irregularities, which are in apparent discordance with the theory of microvesicular transport, is unclear. By the end of the growing period, the CVs are large and smooth surfaced (referred to as CVd, with contents of intermediate elec-

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tron density (between that of the initial growing stage and that of the mature ZGs). The number of intercalated particles on both the large irregularly surface (CV,) and large smooth-surfaced CV (CV,) membranes is high and comparable to that of the Golgi saccule and the endoplasmic reticulum membranes. During the second stage the smooth-surfaced CVs undergo a volume reduction associated with a progressive increase in the electron density of their contents, thus becoming ZGs. Concomitant with size reduction, the number of intercalated particles in the membranes with CVt diminishes markedly. The process of membrane retrieval appears to be accomplished selectively by pinching off coated microvesicles, heavily studded with intercalated particles. More recently, Sesso er al. (19%) reported that, as the CVs mature into ZGs, they lose about 80% of their IMPS. Some preliminary results led them to suggest that lysosomal enzymes would be sorted out of CVs via mannose 6-phosphate receptors by microvesicular budding, as the CVs are converted to ZGs (Sesso et af., 1990). Using the lectin-gold technique, Kan and Bendayan (1989) studied the distribution binding sites of Helix pomatia lectin on thin sections and freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of the ZG was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex labeling by the lectin was localized on the trans saccules and the limiting membrane of the CVs. The latter appeared to be more intensely labeled than the membrane of the ZGs. Intense labeling by the lectin was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the ZG content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of lectin binding sites to the exoplasmic half of the ZG and plasma membranes. The population of ZGs was, however, heterogeneous with respect to the degree of labeling; the exoplasmic fracture face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thinsection fracture-label approach. In addition favorable profiles of thin sections of freeze-fractured ZGs showed that the labeling was not associated with the external surface of the limiting membrane, but, rather, localized over the exoplasmic fracture face. Kan and Bendayan (1989) concluded that ( 1 ) ZGs contain little lectin-binding glycoconjugates; (2) lectin binding sites are preferentially associated with the exoplasmic half of the ZG and plasma membranes, and (3) the limiting membrane of the immature CVs carries a greater number of lectin binding sites than that of the mature ZGs. The latter, in turn, constitute a heterogeneous population with respect to labeling density. These results support the current view

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that glycoconjugates are directed toward the lumen in secretory granules, but become external to the cell surface after fusion of the secretory granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas, in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with their content. Cholesterol distribution is another aspect of the architecture of the ZG membrane. Indeed, Orci et al. (1980) carried out freeze-fracture studies using filipin, an antibiotic that binds to membrane cholesterol and 3phydroxysteroid and produces deformations of the membrane. They demonstrated an asymmetrical distribution of these sterols in the plane of the membrane of both ZGs and CVs. Intriguingly, they showed a change in the polarity of filipin-induced deformations associated with the conversion of CVs into ZGs. If one assumes that the observed protuberances correspond to cholesterol molecules, then the latter would be concentrated on the P face in CVs, whereas in the mature ZGs the E leaflet would be enriched in cholesterol. When exocytosis occurs, it would end up on the E face of the luminal plasma membrane. There is no reason to believe that these observations were artifactual; therefore, this rapid transfer of cholesterol from one layer to the other raises some questions. First, at what precise moment does this transfer of cholesterol occur? Second, why does it occur? Freeze-fracture observations performed in our laboratory on isolated ZGs could provide a possible answer to some of these questions. Indeed, when we exposed ZGs to filipin, most of the ZGs showed a clear concentration of protuberances on the E leaflet, as reported by Orci et al. (1980). However, we noticed some small buddings at the surface of the isolated ZGs. These buddings were apparently filled with cholesterol, as evidenced by the protuberances and pits produced by the filipin treatment. At these sites the planar structure of the membrane was totally disrupted. From a thermodynamical viewpoint such an arrangement could greatly facilitate the transfer of cholesterol from the P layer to the E layer during the conversion of CVs to ZGs. Observations of buddings at the surface of the CVs and immature granules by scanning electron microscopy and on thin sections suggest that the same phenomenon also exists in uiuo (see Naguro and Lino, 1989). The reason that this would occur remains speculative. However, such a transfer of cholesterol from one layer to the other results in an increase in the fluidity and fusogenic properties of the P layers from both ZG and luminal plasma membranes. Information regarding the fusion process itself has been obtained by freeze-fracture studies. Indeed, De Camilli et af. (1974) examined the influence of stimulation on the distribution of IMPS in three exocrine

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glands: pancreas, parotid, and lacrimal. They found that the distribution of IMPs on the fracture faces of the luminal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMPs were rapidly cleared from the areas of granule-plasmalemma apposition in the parotid cells and especially the lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid cells they were less pronounced. Exocytotic openings were usually large, and the fracture faces of their rims were covered with IMPs. In contrast, in stimulated pancreatic acinar cells the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrow necks, and no image that might correspond to early stages of membrane fusion was revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat circular areas, also devoid of IMPS. In thin sections the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures (Tanaka et al., 1980). Finally, an internal reticulation associated with the isolated ZG membranes has been put in evidence by the freeze-fracture studies by Cabana et al. (1981). Moreover, this reticulation was seen after washing the membrane with a carbonate buffer at pH 11.O. Such a procedure removes all the IMPs and major proteins, except one from the membrane. Electrophoretic analysis led these authors to conclude that this protein was GP2. More recent results give strong support to their conclusion, as discussed in Section VII.

VI. ZG lmmunocytochemistry and the Concept of Nonparallel Secretion

Immunocytochemistry has brought some major information about the distribution of zymogens in the acinar cell of the pancreas which is related to the concept of nonparallelism, well known to pancreatologists. This concept has been the subject of debates and confrontations for many years. It is not our intention to reanimate these discussions here, but, rather, to simply point out the contribution of some immunological techniques to the explanation of the secretory process. In simple words nonparallel secretion of digestive enzymes corresponds to changes in the relative proportions of the different secretory proteins (e.g., a-amylase, chymotrypsinogen A, and lipase) which occur in response to different physiological or pharmacological stimuli.

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According to this definition, nonparallelism presupposes the existence of different pools of secretory digestive enzymes in the gland. These pools of zymogens could potentially be localized at different levels of the gland organization; thus, they could correspond to different populations of granules within cells, acini, or different areas of the gland. In addition to these possibilities, Rothman (1975) proposed the existence of a cytoplasmic pool of enzyme in the pancreas. As early as 1954, Marshall (1954) studied the localization of four enzymes in the bovine pancreas by immunofluorescence. He then reported that the distributions of chymotrypsinogen and procarboxypeptidase could be determined to a resolution of less than 0.5 pm. His results indicated that, at this level of organization, the acinar cells of the resting pancreas are alike with respect to zymogen synthesis and storage. He did not find any specialization among the cells, nor among the ZGs within each cell. Two decades later Kraehenbuhl et al. (1977) performed the first immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells and isolated ZGs. Their goal was to determine whether regional differences exist in the bovine gland with regard to the production of individual secretory proteins, and whether specialization of product handling occurs at the subcellular level. A double-antibody technique was used in which the first step consisted of rabbit F(ab')z antibovine secretory protein, and the detection step consisted of sheep F(ab')z anti-rabbit F(ab')* conjugated to ferritin. The results showed that all exocrine cells in the gland, and all ZGs and Golgi cisternae in each cell, were qualitatively alike with regard to the content of secretory proteins examined (i.e., trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggested that these secretory proteins are transported through the cisternae of the Golgi complex, where they are intermixed before copackaging in ZGs; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins and is independent of the extent of glycosylation (e.g., trypsinogen, a nonglycoprotein, versus DNase, a glycoprotein). It has long been known, on the basis of morphological and biochemical differences, that the exocrine pancreatic gland can be divided into periand teleinsular regions (Jarotsky, 1899; Sergeyeva, 1938; Hellman et af., 1962; Kramer and Tan, 1968; Malaise-Lagae et al., 1975). More recently, the enzyme profile of these regions has been investigated by Bendayan and Ito (1979) by the immunofluorescence technique, using antibodies against nine pancreatic enzymes (i.e., a-amylase, lipase, chymotrypsinogen A, trypsinogen, elastase, carboxypeptidases A and B, DNase, and RNase A). These antibodies were specific to their antigens without cross-reaction. By immunofluorescence most acinar cells of the normal rat pancreas were

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positive to the nine enzymes tested. However, an inhomogeneity in the staining pattern was found; specifically, the cells located in the periinsular region of many islets showed a brighter fluorescence than did acinar cells in the teleinsular tissue. The interesting observation emerging from this study was that not all of the acini showed the same fluorescence intensity, suggesting that the enzymatic content of the acinar cells varies quantitatively from one acinus to another. The variation in fluorescence intensity was present not only between periinsular and teleinsular acini, but also between different acini in the same region. In addition conventional light and electron microscopy have shown that the periinsular acinar cells are bigger, contain a higher concentration of ZGs, and have a larger nucleus and nucleolar volume (Jarotsky, 1899; Ferner, 1958; Hellman et al., 1962; Kramer and Tan, 1968). Moreover, in the first hours following an intravenous injection, the distribution pattern of radioactively labeled amino acid uptake showed markedly more radioactivity in the periinsular than in the teleinsular tissue (Hansson, 1959). A quantitative immunocytochemical localization of these proteins was later carried out by Bendayan et af. (1980), this time using the protein A-gold technique applied on thin sections. An increasing gradient of the labeling from the RER to the Golgi apparatus and to the ZG was found for a-amylase, carboxypeptidases A and B, chymotrypsinogen A, trypsinogen, and RNase A, while a comparable low degree of labeling in the Golgi apparatus and in the ZG was observed for DNase, lipase, and elastase. These results suggested that the nine enzymes are processed through the same intracellular compartments, but that they are concentrated to different degrees in the ZG before being released in the acinar lumen. The distributions of a-amylase and chymotrypsinogen in peri- and teleinsular cells of the rat pancreas were reinvestigated by Posthuma et al. (1986). They used ultrathin cryosections from tissue blocks consisting of tele- and periinsular tissue elements. Consecutive sections of these blocks were alternatively immunolabeled for a-amylase and chymotrypsinogen, using protein A-gold as the marker. The density of gold particles over ZGs of both peri- and teleinsular cells was measured. It appeared that the a-amylase/chymotrypsinogen labeling density ratio was significantly lower in periinsular than in teleinsular cells. This difference resulted from a lower a-amylase labeling, as well as a higher chymotrypsinogen labeling density over ZGs in periinsular cells. Their results were in agreement with those of Malaisse-Lagae et al. (1975). This immunocytochemical observation reported above provided a strong argument against the concept of the “equilibrium hypothesis” (Rothman, 1975) as a model of secretion. Immunocytochemistry has also brought some important clues regarding the packaging process. Indeed,

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Bendayan (1984) reported the concentration of a-amylase along its secretory pathway in the rat pancreatic acinar cell by high-resolution immunocytochemistry. Quantitative evaluations of the degree of labeling demonstrated an increasing intensity which progresses from the RER, through the Golgi apparatus, to the ZG and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that a-amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: the trans-most Golgi cisternae, the CVs, and the premature and mature ZGs. It is noteworthy that minigranules also transport a-amylase to the cell surface, as illustrated in Fig. 4. If immunocytochemistry has not indicated any major differences in granule composition among the granules of a given cell (see Fig. 5 ) , there would, however, be some important variations among granules isolated from the whole pancreas, as proposed by Mroz and LechCne (1986). Indeed, these authors measured the activities of both chymotrypsinogen and a-amylase in individual ZGs of the rat pancreas by micromanipulation and microfluorometric methods. The enzyme content and the ratio of a-amylase to chymotrypsinogen vaned widely among granules from the same animal. These results are compatible with short-term nonparallel bulk secretion of the two enzymes through exocytosis. The distribution of each enzyme activity in a population of granules suggests quanta1 packaging of a-amylase and chymotrypsinogen into the granules. From the immunocytochemical studies described above it is clear that ZGs all contain a-amylase and other secretory proteins, but these studies do not provide information about the turnover rate of the secretory proteins.

VII. Pancreas ZG Membrane Proteins

Meldolesi et af. (1971) and MacDonald and Ronzio (1972) were among the first to isolate the ZG fraction from pancreas acinar cells and to analyze the protein composition of their membrane. Meldolesi et al. (1971) noticed that ZG membranes were enriched in phospholipids, and Meldolesi and Cova (1972) later analyzed the profile of protein composition obtained after polyacrylamide gel electrophoresis (PAGE). They noticed a certain degree of similarity between isolated ZG membranes and a plasma membrane fraction. In the same period MacDonald and Ronzio (1974) made a comparative analysis of ZG membrane polypeptides from several species by sodium dodecyl sulfate (SDS)-PAGE. They found that these membranes contained very few proteins, among which was a common major

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glycoprotein component identified as GP2. Paquet et al. (1982) later corroborated this analysis of ZG membrane protein components of the rat pancreas by two-dimensional isoelectric focusing and SDS-PAGE. Following the identification of GP2, several groups determined its localization by immunological methods. Immunocytochemical observations by Geuze et al. (1981) showed that GP2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane and the Golgi membranes and its association with ZG membranes. On the other hand, GP2 was present in the contents of the ZG and the acinar and ductal lumena. A GP2-like glycoprotein was also found in the cannulated pancreatic secretion (Scheffer et al., 1980). The presence of GP2 in the pancreatic juice was confirmed later in both the rat and the pig pancreas (Havinga et al., 1984; Beaudoin et al., 1986a). Scheffer et al. (1980) and later Beaudoin et al. (1986a) found, by immunological techniques, that GP2 was in a sedimentable form in the pancreatic juice of the rat. Moreover, Beaudoin et al. (1986a) proposed that GP2 was associated with some microvesicles recovered in a pellet obtained by ultracentrifugation of the pancreatic juice collected under resting conditions. Recently, Rindler and Hoops (1990) reinvestigated the localization and some of the biochemical properties of GP2 in the rat pancreas and pancreatic juice. Using affinity-purified antibodies, they found it to be concentrated in the ZG and the acinar lumen. Label was also present on the apical and basolateral plasma membranes, but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties substantially reduced the staining of the basolateral surface, suggesting that their antibodies were reactive to oligosaccharides. Approximately 45% of the GP2 in the granules was not membrane associated, but appeared, instead, in the granule lumen. Parallel biochemical characterization of GP2 in isolated secretory granules demonstrated that 60% of the total GP2 fractionated with the membranes after granule lysis, while the remaining 40% was found in the content. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl phosphatidylinositol (GPI), GP2 in the content did not enter the detergent phase upon Triton X-I14 extraction; nor was it sedimentable at 200,000 g , as is characteristic of the form collected in the pancreatic juice. In addition, GP2 in the pancreatic juice was recovered in FIG. 4 Minigranules in the rat pancreas under “resting” conditions. Immunocytochemical localization shows that minigranules are filled with a-amylase. The fact that the content of these granules is more concentrated than in the lumen rules out the possibility that these are endocytic vesicles. (a) Minigranules (arrows) dispersed in the trans-Golgi area. (b) Minigranules (arrows) close to the luminal plasmalemma. Experimental methods are as described by Beaudoin ef al. (1991).

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the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. In a more recent study Beaudoin et al. (1991) reinvestigated the immunocytochemical localization of GP2 with polyclonal antibodies reactive to the protein moiety of the glycoprotein. Their immunocytochemical studies have demonstrated GP2 to be present on the membrane and in the matrix of ZGs, over the Golgi saccules, on the apical and basolateral surfaces of the plasma membrane, and in the acinar lumen. In addition this protein was observed in small vacuoles and tubular structures previously identified as “basal lysosomes” or “snakelike tubules,” and in lysosomes. Its presence in the latter group of structures involved in endocytosis suggests a possible role of GP2 in this cellular process. GP2 was readily detectable in the pancreatic juice and was totally sedimentable by ultracentrifugation, as assessed by Western blot analysis. Induced lysis of isolated ZGs also caused the release of GP2 in a sedimentable form, which, by electron microscopy, appeared as some fibrillar material. This probably corresponds to the internal reticulation found on carbonate buffer-washed ZG membrane, as observed by freeze-fracture techniques (Cabana et al., 1981). Various biochemical labeling techniques have been used to determine the localization of GP2 in the plane of the ZG membrane. No labeling of GP2 could be obtained on intact ZGs from the rat (Ronzio et al., 1978) or the pig pancreas (LeBel, 1988), indicating that it was associated with the inner layer of the ZG membrane; It was later shown by Paquette et al. ( 1986) that GP2 was associated with the membrane via a GPI linkage. Although subjected to contradictory views (see Scheffer et al., 1980; Beaudoin et al., 1991), the presence of GP2 in ZG granule lysates (40%) would suggest that a fraction of the GP2 molecules is released from the ZG membranes, as demonstrated by aqueous-phase Triton X- 1 14 partition techniques (Paquette et al., 1986; Rindler and Hoops, 1990). The absence of GP2 after high-speed centrifugation of the pancreatic juice is still puzzling, and it leads one to believe that the hydrophilic GP2 molecules would form aggregates during secretion. In this respect the immunocytochemical observations by Beaudoin e f al.

FIG. 5 ZGs (asterisks) of the rat pancreas in siru and in uitro. Immunocytochemical localization shows that all of the granules contain a-amylase in about the same proportions. High magnification does not reveal any concentration of a-amylase at the membrane surface. Isolated granules provide the same labeling and size distribution patterns as in situ. (a) a-Amylase distribution among ZGs in sitrr. (b) a-Amylase distribution in a population of isolated ZGs. (c) High magnification showing the distribution of a-amylase in a ZG matrix. Notice the ZG membrane in 5c (arrowheads).

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(1991) raise some questions regarding the origin of GP2 found in the lumen. Indeed, these authors compared the immunoreactivity of GP2 found in the acinar lumen with that in some ZG at the outset of exocytosis, and the images leave no doubt that GP2 is much more reactive in the lumen than in the corresponding ZG content. It is our opinion that this behavior is not a question of antibody accessibility, due to protein aggregation, since immunocytochemical localization of a-amylase does not show such behavior. These observations strongly suggest that some of the GP2 molecules in the lumen do not derive from ZG, but perhaps, as we previously proposed, via a paragranular pathway or a special class of ZGs (see Beaudoin and Grondin, 1991). The biochemical studies by Havinga et al. (1984) bring strong support to the latter hypothesis. Indeed, their pulse-chase experiments on isolated acinar cells showed that the incorporation of the first newly synthesized GP2 molecules into ZG membranes occurred about 60 min after the beginning of the pulse. They also demonstrated that newly made GP2 molecules reach the cell surface within the same time span. After a 6- to 8-hr chase considerably more newly synthesized GP2 molecules have reached the cell surface than would be expected from the secretion of newly synthesized zymogens. These observations strongly suggest that at least part of the GP2 molecules bypass the mature ZG compartment on their way to the plasma membrane. GP2 is the only protein that appears in discernible quantity in the plasma membrane 1-4 hr after a pulse label. Nevertheless, GP2 comprises only a small percentage of externally '251-iodinatedplasma membrane proteins. Havinga et al. concluded that GP2 has a high turnover rate at the plasma membrane level. Finally, treatment of the acinar cells with the N-glycosylation inhibitor tunicamycin does not block the intracellular transport of GP2. In these in vitro experiments GP2 is not released into the medium. We and others (Phaneuf et al., 1985; Arvan and Castle, 1987) have noticed the same phenomenon with rat pancreas acini and lobule preparations. Some molecular biology information about this protein has been reported recently by Fukuoka et al. (1990). In a carbohydrate-shift strategy amino-terminal and internal peptide sequences were obtained on glycosylated and deglycosylated forms of GP2, respectively, by gas phase sequencing. Sets of mixed oligonucleotides and the polymerase chain reaction were used to obtain a double-stranded cDNA probe, which was used to isolate overlapping cDNA clones from a dog pancreatic cDNA library constructed in XZAP-11. The sequence of these clones revealed an open reading frame which encodes a protein of 509 amino acids, containing eight N-linked oligosaccharide attachment sites. The carboxy terminus shows a 20-residue hydrophobic transmembrane domain preceded by a potential GPI attachment site. GP2, completely released from the ZG membranes with phospholipase C, showed similar immunochemical properties and

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electrophoretic mobilities compared to the form associated with ZG membranes. A similar form of GP2 was released from ZGs permeabilized with saponin. Kinetic analysis indicated two distinct pools of GP2 released from permeabilized granules. According to Fukuoka et al., these findings indicate that GP2 is a GPI-linked membrane protein, which is released from ZG membranes by GP1-anchor cleavage activity present in ZGs. In parallel to these molecular biology studies, Freedman et al. (1990) demonstrated homology between GP2 and the Tamm-Horsfall protein (THP) from the kidney. Extensive conservation of structure is demonstrated between the two proteins. Over the carboxy-terminal sequence observed between Asp-54 and Phe-530 in the rat sequence, there is 63% identity and 91% homology between rat GP2 and human THP. According to these authors, the similarities observed in molecular structure and cellular localization between GP2 and THP in the pancreas and the kidney, respectively, and the complementary information obtained from studies of GP2 (GPI-tail nature) and THP (self-aggregating property) suggest that (1) both proteins are synthesized as GPI-tailed proteins, (2) aggregation of GP2 and THP on the cisternal leaflet of trans-Golgi elements plays important roles in the assembly of ZGs and apical vesicles in the pancreas and the kidney, respectively, and (3) the release of GP2 and THP from granules and apical vesicle membranes, respectively, is required for retrieval of these membranes from the plasmalemma after exocytosis. A family of genes appears to be responsible for the expression of granule membrane assembly proteins from diverse epithelia (Freedman et al., 1990). y-Glutamyltransferase (GGT) is another glycoprotein found in the ZG membrane of the rat pancreas (Castle et al., 1985). Antibodies were produced in rabbits, using purified GGT from the rat kidney. trans-Blot experiments showed some immunoreactivity with a 58- and 30-kDa polypeptide when ZG membrane was used as the source of antigens. Battistini et al. (1990) reported the release of this protein in the rat pancreatic juice under resting and stimulated conditions. Under resting conditions in uiuo, high levels of GGT were found in the pancreatic juice, and these levels were not related to protein concentration. Under secretin infusion a relatively constant level of GGT was released, and again, there was no correlation between GGT activity and protein secretion. However, following a bolus injection of cerulein, an analog of cholecystokinin, marked and concomitant rises in protein and GGT levels were observed. Ultracentrifugation, as well as gel filtration on Sepharose 4B, demonstrated that the enzyme was not released in a soluble form. It cannot be excluded that some of the GGT in the pancreatic juice could originate from ductal cells (Yasuda et al., 1986). In addition to GGT, several other enzymes have been put into evidence in ZG membranes by cytochemical immunological and biochemical methods, including TPPase (Paquet et al., 1982), ATP-diphosphohydro-

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lase (LeBal et al., 1980; Laliberte et al., 1982), and protein disulfide isomerase (Akagi et at., 1988). Like GP2 and GGT, the latter protein is apparently secreted by the acinar cell. The hypothesis according to which phosphorylation of specific proteins could change granule membrane properties and facilitate the fusion with the plasma membrane during exocytosis led some authors (Lambert et al., 1974; Wren, 1984; MacDonald and Ronzio, 1974) to examine protein kinase activity in these membranes. An endogenous protein kinase activity was demonstrated, but these observations fail to establish any direct relationship between phorphorylation and exocytosis. A renewed interest for this hypothesis has risen recently by the detection of GTP-binding proteins (G proteins) associated with ZG membranes of the rat pancreas (Padfield et al., 1990).A 28-kDa G protein and a 25-kDa ADP-ribosylated protein were also described by Lambert et al. (1990). These authors suggested that a 28-kDa protein could be involved in protein transport between intracellular compartments along the secretory pathway andlor exocytosis. Some previous observations, using an in vitro system, support this hypothesis. Indeed, Nadin et al. (1989) developed a cell-free assay for the interaction between pancreatic ZG and plasma membranes. They showed that plasma membranes are able to trigger the release of the granule contents and that this effect is specific to pancreatic membranes. It involves membrane fusion, it requires membrane proteins, and it is stimulated by activators of G proteins, but not by Ca”.

VIII. Parotid ZG Membrane Proteins

Early attempts to characterize the proteins of the membranes of parotid secretory granules have been hampered by difficulties in obtaining membrane preparations free of secretory proteins (Castle and Palade, 1978; Wallach et al., 1975a,b). Wallach et al. (1975a) measured the a-amylase activity in a granule membrane preparation from the rat parotid gland and estimated that 10% of the protein was contributed by secretory proteins. Castle and Palade (1978) compared the proteins of secretory granules and granule membranes of rabbit parotid glands by SDS-PAGE after labeling unfractionated glands with ‘‘C-labeled amino acids. They estimated that 25-30% of the total protein in the membrane preparation are secretory proteins. Putative secretory proteins were removed by treating the membranes with saponin and sodium sulfate, conditions which caused disruption of the typical bilayer structure of the membrane. After treatment SDS-PAGE of the parotid granule membrane revealed 70 polypeptide bands. Wallach et al. (1975a) reported a simple composition of parotid

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granule membranes of the rat, whereas Casieri and Somberg (1983) found that the granule membrane contains 166 polypeptides, of which only 26 are also present in the granule content. The membrane proteins have isoelectric points between 4.75 and 6.45 and apparent molecular masses varying from 17 to 190 kDa. In a more recent study Cameron et ul. (1986) made a comparative analysis of two-dimensional isoelectric focusing: SDS-PAGE of radioiodinated granule membranes from parotid, pancreatic, lacrimal, and submandibular glands. Each profile comprises about 25 species with electrophoretic mobilities corresponding to apparent molecular masses ranging from 18 to 150 kDa. In the case of the parotid gland, the number and distribution by apparent molecular mass are consistent with the onedimensional profile shown previously using either tyrosine- or amino group-directed labeling (Cameron and Castle, 1984). Also, polypeptides of low apparent molecular mass are more prevalent than larger species in all cases. As well, the majority of granule membrane polypeptides are acidic, focusing between pH 5.0 and 7.0. This observation is consistent with the previous reports for the general nature of polypeptides from adrenal chromaffin granules (Bader and Aunis, 1983), pancreatic ZGs (Paquet et al., 1982), and parotid granules (Casieri and Somberg, 1983), although in the latter case, in which residual mitochondria1 contamination appears to be significant, the pattern is considerably more complex than the one Cameron er al. have observed. As mentioned by Cameron et ul. (1986), the most sriking observation made by comparing the profiles of the four types of membranes is the presence of apparent extensive polypeptide homology. Not only identities are suggested by overlapping mobilities for individual species, but the two-dimensional patterns of spots are also similar, if not identical, in a number of regions. More specifically, major radiolabeled species (- 10 distinct polypeptides) that coincide in isoelectric point and molecular mass are located in the range of 24-30 kDa and, in part, 2 8 5 kDa. Evidently, these common polypeptides do not correspond to incompletely removed secretory proteins, since neither radiolabeling nor protein staining of parotid granule content reveals a pattern having comparable isoelectric points and apparent molecular masses. In addition, at least four to six species in the range of 40-70 kDa exhibit common mobilities, but show much wider quantitative variations in intensity among the different types of membranes. Some of these species are observed in more than one, but not all, preparations. Finally, each sample contains unique polypeptides for which there is no counterpart in the other patterns. Peptide mapping after chymotrypsin and trypsin digestion show very clearly that membrane and content proteins are structurally unrelated and that one of the principal overlapping species (-29 kDa) is essentially

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identical in parotid and pancreatic two-dimensional polypeptide profiles (Cameron et al., 1986). In addition different enzyme activities have been associated with the ZGs of the parotid gland, using cytochemical, biochemical, and immunological approaches, including AcPase, TPPase (Hand and Oliver, 1977), GGT (Castle et al., 1985) and Ca2+ ATPase (Watson et af., 1974).

IX. ZG Membrane Lipid Composition

During exocytosis the ZG membrane fuses with the plasma membrane. This cellular process, which can be mimicked by isolated preparations in vitro (Rand and Parsegian, 1986), is in great part determined by the physicochemical properties of the lipid components of the interacting membranes. In this respect several years ago Blashcko et at. (1967) reported that chromaffin granules of the adrenal medulla, which perform a function analogous to that of the ZGs in the pancreas, contain a high proportion of lysophosphatidylcholine (lyso-PC) [ 17% of the granule lipid phosphorus (P)]. These authors suggested that, during secretion, the lyso-PC might be involved in the fusion of the granule membrane with the plasma membrane of the cell. White and Hawthorne (1970) analyzed the lipid composition of ZGs from the ox pancreas. The pancreatic ZG fraction had slightly less PC than either the mitochondria1 or microsomal fractions (41% compared with 50%), but had significantly more phosphatidylethanolamine (PE) (35% compared with 23% and 29%,respectively). No abnormally high values for Iyso-PC were obtained. The ZG membrane resembles the surface membrane in its relatively high cholesterol/phospholipid molar ratio (0.56). A corresponding value for rat liver plasma membrane is 0.53 (Coleman et al., 1967). It is noteworthy that White and Hawthorne (1970) observed hydrolysis of phospholipids during fractionation, sometimes leading to high levels of lyso-PC and lyso-PE. Therefore, they retained only those experiments in which lyso-PE was inferior to 2% of lipid P. The hypothesis that cAMP would activate phospholipase activity, which would lead to locally increased lysophospholipid formation, resulting in a fusion between the ZG and apical plasma membranes was tested by Rutten et af. (1975). cAMP added to isolated pig pancreatic ZGs leads to an increased lysis of these granules. However, the slowness of this effect renders its physiological significance dubious. In pancreatic homogenates or ZGs no stimulating effect of cAMP on lipase or phospholipase activity could be demonstrated. Isolated ZGs have a high lysophospholipid content (27% of total phospholipids), consisting of the 1- and 2-acyl forms of lyso-PC and lyso-PE. Experiments with radioactive PC indicate that the

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lysophospholipids are due to the action of endogenous (phospho)lipases during the isolation procedure. These experiments did not lend support to the hypothesis mentioned above for the mechanism of action of cAMP in pancreatic enzyme secretion. Immunocytochemical evidence for an endogenous phospholipase A2 distinct from the secreted enzyme has been obtained recently in our laboratory. Properties of ZG membrane phospholipids and their fatty acyl compositions have also been measured in the rat parotid gland by Mizuno et al. (1987). A comparison of the ZG fraction with the microsomal fraction showed that the ZG membrane had higher levels of lysophospholipids (8%) and PE (31%) and lower levels of PC (40%) and phosphatidylserine (PS) (2.1%). However, fatty acid compositions of individual phospholipid classes from the two subfractions were found to be similar to each other. Electron spin resonance analysis demonstrated that extracted phospholipids from the secretory granular fraction were more fluid than those from microsomes. Castle et al. (1981) had previously reported that rabbit parotid secretory granules had a soluble phospholipase A, which was activated by Ca2+and inhibited by EDTA. However, they inferred that this high level of lysophospholipids in ZGs does not result from enzymatic degradation during cell fractionation, since they used EDTA-containing buffer during cell fractionation, and that such lysophospholipids showed a low level in other subfractions. Thus, the higher level of lysophospholipids, as well as PE, and the lower levels of PC and PS may be characteristic properties in secretory granular membrane lipids from rat parotid glands. Considering the ZG membrane composition from the pancreas and the parotid gland, one is struck by the high level of PE, a composition which would favor the transition from a bilayer to an hexagonal configuration of the membrane. Second, it cannot be excluded that the high level of lysophospholipids, which is thought to be derived from phospholipase A activity, might contribute to the increased fluidity in the fusion area.

X. ZG Ion Transport in the Pancreas

As mentioned by Gasser et al. (1988), investigations of stimulus-secretion coupling in exocrine glands were focused mainly on the intracellular messengers that play a role in this process. Both calcium- and CAMPdependent pathways have been identified. For example, changes in cytosolic calcium have been reported in pancreatic acinar cells in response to the secretagogues cholecystokinin, bombesin and acetylcholine and its analogs (Maruyama and Petersen, 1982; Merritt and Rubin, 1985), while vasoactive intestinal peptide and secretin elevate cAMP (Kimura et a / .,

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1986; Robberecht er al., 1976; Singh, 1979). Although calcium and CAMP probably contribute to the secretory process by activation of protein kinases and selective phosphorylation of plasma andlor granule membrane proteins (Burnham et al., 1985; Spearman et af., 1984), the physiological targets and function of secretagogue-dependent phosphorylation remain speculative. In an effort to understand the underlying physiology and biochemistry of macromolecule secretion, Gasser et af. (1988) concentrated their efforts on the characterization of transport properties of the granule membrane. Regulation of the ion permeabilities of this membrane could have significant consequences in several aspects of the secretory process, particularly the accumulation and storage of secretory proteins (Johnson et al., 1981; Njus et al., 1985), fluid secretion accompanying protein release (DeLisle and Hopfer, 1986), or osmotic swelling of granules, which has been postulated to play a role in membrane fusion or fission (Cohen et al., 1980; Kachadorian et al., 1981; Zimmerberg and Whitaker, 1985). Rat pancreatic ZGs have been shown to possess an anion exchange and an anion conductance pathway. However, they typically lack cation permeabilities when isolated in a low-calcium and detergent-free environment (DeLisle and Hopfer, 1986; Gasser et al., 1988). It has been postulated that the C1conductance of the granule membrane contributes significantly to the secretagogue-stimulated C1- conductance of the plasma membrane after fusion of the granules with the plasma membrane (DeLisle and Hopfer, 1986). Secretagogue-stimulated C1- conductance of the plasma membrane has been measured by electrophysiological techniques and is thought to play an essential role in fluid secretion (Petersen, 1986). Thus, the C1- conductance of the granule membrane may have a major role in the coupling of fluid and macromolecule secretion and the determination of fluidity in the primary secretion. Gasser et al. (1988) examined the membrane permeability of rat pancreatic ZGs in uirro with granules isolated from rats in different secretory states: (1) untreated, (2) pretreated with a muscarinic antagonist, (3) pretreated with a muscarinic and an adrenergic antagonist, (4) pretreated as in the previous state and then stimulated with the secretagogue cholecystokinin 4 min before death, and ( 5 ) pretreated as in state 3 and then stimulated with the secretagogue secretin 4 min before death. Granules isolated from untreated rats had variable ionic permeabilities. In general, however, they possessed both CI- conductance and electroneutral exchange pathways, with low permeabilities to alkali metal ions. In contrast, granules from animals pretreated with secretory antagonists had low ion permeabilities to both inorganic anions (e.g., chloride) and alkali metal ions. An injection of the peptide secretagogues cholecystokinin or secretin resulted in a relatively fast (i.e., within 4 min) activation or induction of high chloride permeabilities through both C1- conductance and chloride hydroxide (or

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chloride/bicarbonate) exchange pathways. In addition the secretagogues increased the cation permeability of the granule membrane, which exhibited a distinct potassium selectivity. These results demonstrate that granules may actively participate in the secretory process and suggest that some of the physiological targets in the cascade of events leading to secretion are anion and cation transporters in the ZG membrane. The influences of secretagogues and second messengers were also examined more recently by Fuller et al. (1989). They examined ion permeability pathways in ZGs isolated from control cells and cells pretreated with the acetylcholine analog carbachol, the peptide hormone cholecystokinin, and second messengers of hormone action such as CAMPand the diacylglycerol analog 12-0-tetradecanoyl phorbol- 13-acetate (TPA). Ion and water influx rates in ZGs and consequent swelling and lysis of granules were monitored by measuring changes in the optical densities of ZG suspensions at 540 nm following additions of the electrogenic or electroneutral ionophores valinomycin and nigericin, respectively. The data show that a C1- conductance and an anion exchange pathway are both present in the granule membrane. These two pathways are activated by pretreatment of isolated cells with cholecystokinin or isolated permeabilized cells with CAMP, whereas only the C1- conductance is increased by pretreatment with carbachol or TPA. The anion transport inhibitor diisothiocyanostilbene disulfonic acid abolishes CI- conductance, but it also has no effect on the anion exchanger. Granular lysis is also inhibited by buffers of high osmotic strength. The direct application of the catalytic subunit of protein kinase A and ATP to isolated ZGs inhibits both the CIconductance and the anion exchange pathway probably mediated by phosphorylation. This result does not seem to be consistent with the observation that in isolated ZGs from CAMP-prestimulated cells both C1- conductance and the Cl- anion exchanger are activated. Fuller et al. concluded that the stimulation of pancreatic acinar cells by secretagogues results in the activation of C1- permeability pathways in the ZG membrane mediated by cAMP-protein kinase A and by a diglyceride-protein kinase Cmediated phosphorylation. It is possible that, in addition to “on” reactions, “off” reactions also exist which close the C1- channel (e.g., by dephosphorylation) and that in activation of C1- permeability pathways different sites are involved for CAMP-protein kinase A action or different protein kinases A.

XI. Cytoskeleton and ZG Movement It is well established that the cytoskeleton plays an important role in many cellular processes, including cytoplasmic transport and secretion. As re-

21 2

ADRIEN R. BEAUDOIN AND GILLES GRONDIN

ported by Bonder and Mooseker (1986), cytochalasins B and D, both of which bind the barbed end of actin microfilaments in uitro, disturb the microfilament system. Several groups have shown that these cytochalasins inhibit the protein discharge in response to secretagogues in both the exocrine pancreas and the parotid gland (Butcher and Goldman, 1974; Morisset and Beaudoin, 1977; Williams, 1977; Burnham and Williams, 1982; Busson-Mabillot et d.,1985; Stock et d . , 1978). Indeed, maximal stimulation of enzyme release from in uitro preparations of the mouse and the rat pancreas was shown to be inhibited by cytochalasin B at concentrations high enough to disrupt the network of microfilaments that underlies the luminal membrane (Bauduin et al., 1975; Williams, 1977). Cytochalasin B at these concentrations did not affect either pancreatic 45Ca2f fluxes or ATP levels (Bauduin et al., 1975; Williams, 1977). In addition Williams (1977) noted that cytochalasin B caused the disappearance of apical microvilli and considerable luminal dilation in mouse pancreatic fragments and acini. It was suggested that the apical network of microfilaments acts as a contractile ring to stabilize the luminal membrane and that loss of this structure in cytochalasin B-treated tissue leads to luminal dilation as the result of incorporation of the microvillar membrane into the luminal membrane (Williams, 1977). The finding that cytochalasin inhibits protein discharge was taken to indicate that the microfilament, and more generally, the cytoskeleton, was involved in the movement of secretory granules to the site of exocytosis (Burridge and Phillipps, 1975). These observations with cytochalasins B and D are difficult to evaluate because of the multiple effects of cytochalasin on cellular metabolism and membranes (Lin et al., 1973). Among these effects some are directly related to the intracellular messengers. In the rat parotid gland the involvement of the microfilament system in the cellular signal transmission mechanism was tested by measuring the effect of cytochalasin D (which disturbs the microfilament system) on the production of intracellular second messengers. Cytochalasin D did not affect unstimulated calcium movements (measured by the 45Caefflux technique), inositol phosphate production, or cAMP accumulation. Neither did it modify the generation of intracellular second messengers induced by activation of the cholinergic muscarinic receptor (calcium and inositol phosphates). Cytochalasin D did not affect the cAMP accumulation induced by the activation of the P-adrenergic receptor, whereas it strongly inhibited the calcium movements induced by activation of the same receptor. These data suggest that, in the rat parotid gland, calcium movemements induced by fi-adrenergic receptor stimulation need an intact microfilament system to occur, whereas the rnuscarinic pathway (via inositol triphosphate) does not. Microtubules constitute another important element of the cytoskeleton

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of exocrine glands. Markham and Cope (1976) showed that pretreatment of the rabbit parotid gland with colchicine or vinblastine sulfate resulted in a 80-85% inhibition of isoproterenol-induced granule discharge. This effect was also seen when dibutyryl CAMPwas used as the secretagogue. Several groups (Nevalainen, 1975; Seybold et al., 1975; Stock et al., 1978; Williams and Lee, 1976) showed that agents that disturb microtubules (e.g., colchicine and vinblastine) cause cellular alterations and interfere with the pancreatic secretory process. In addition to inhibiting enzyme discharge, antimicrotubular agents such as vinblastine and colchicine reduce amino acid entry and intracellular transport (Seybold et al., 1975). The localization of some cytoskeleton proteins was studied by Drenckhahn and Mannherz (1983). Actrin, myosin, and the actinassociated proteins tropomyosin, a-actinin, vinculin, and vilin were localized in acinar cells of rat and bovine pancreas, parotid, and prostate glands by immunofluorescent staining of both frozen tissue sections and semithin sections of quick-frozen, freeze-dried, and plastic-embedded tissues. Antibodies to actin, myosin, tropomyosin, a-actinin, and villin reacted strongly with a narrow cytoplasmic band extending beneath the luminal border of acinar cells. Fluorescently labeled phalloidin, which reacts specifically with F-actin, gave staining, within the cell apex, similar to that obtained with antibodies to actin, myosin, tropomyosin, a-actinin, and villin. In contrast, immunostaining with antibodies to vinculin was restricted to the area of the junctional complex. Ultrastructurally, the apical immunoreactive band corresponded to a dense web composed of interwoven microfilaments, which could be decorated with heavy meromyosin. Outside this apical terminal web antibodies to myosin and tropomyosin gave only weak immunostaining (confined to the lateral cell borders), whereas antibodies to actin and a-actinin led to a rather strong beadlike staining along the lateral and basal cell membranes, probably marking microfilament-associated desmosomes. Antivillin immunofluorescence was confined to the apical terminal web. Drenckhahn and Mannherz suggested that the apical terminal web is important for the control of transport and access of secretory granules to the luminal plasma membrane and that villin, which is known to bundle or sever actin filaments in a Ca2+dependent manner, might participate in the regulation of actin polymerization within this strategically located network of contractile proteins. Bendayan et al. (1982)in the same period also reported the immunocytochemical localization of actin by the protein A-gold technique. The labeling was found at the level of the filamentous cell web and in close association with the Golgi cisternae, CVs, and ZG-delimiting membranes, as well as with the plasma membrane. Weak labeling was also present over the dense content of the ZGs. The association of actin with different membr.anes implicates that contractile proteins might constitute structural

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membrane proteins and, thus, might play an important role in protein secretion. Intermediate filaments are other important components of the cytoskeleton. These have been observed in pancreatic acinar cells and some keratins have been seen in close association with ZGs (Bendayan, 1985). Despite all of these biochemical and morphological observations, an active role of the cytoskeleton in the migration of ZGs toward the cell apex remains to be demonstrated. With the advent of the confocal microscope and techniques for cell permeabilization, this phenomenon can be better studied. Some freeze-substitution techniques, which better preserve the integrity of the microtubules, have been applied recently on rat pancreas acinar cells and as illustrated in Fig. 6 , there is definitely a close relationship between ZGs and the cytoskeleton.

FIG. 6 Some aspects of the terminal web in the rat pancreatic acinar cell. On tangential section one can observe (a) bundles of filaments (arrow) and (b) microtubules (arrow). Both appear to be apposed to the ZG surface. Lu, Acinar lumen; Go, Golgi apparatus.

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The nature of the interaction between microtubules and the protoplasmic face of the ZG membrane is undefined. In addition to contractile proteins such as actin, there could also be some interactions with phospholipids. On the basis of their results obtained with colchicine analogs on the rat lacrimal gland, Herman et al. (1989) proposed that it is during the maturation stage that microtubules would be required. According to these authors, during this stage the secretory granules would acquire an additional property that allows eventual exocytosis. Without microtubules granule maturation would be impossible because of granular inability to acquire this property, rendering these granules incapable of exocytosis.

XII. Concluding Remarks

Early autoradiographic studies by Warshawski et al. (1963) and by Kramer and Poort (1972) on the rat pancreas have shown that newly formed granules do not mix with older granules. These findings confirm some in uivo observations by Cove11 (1928) on the mouse pancreas showing that ZGs are relatively immobile in the apical cytoplasm. However, as reported by the same authors, some movement of ZGs can be observed after pilocarpine stimulation. The fact that ZG mobility is restricted in the apex cytoplasm, together with the fact that antimicrotubular agents interacting with the cytoskeleton interfere with the secretory response, led us to believe that the cytoskeleton controls the movement of ZGs toward the cell apex. The nature of these interactions may be much more complex than a simple ZG alignment in the apical cytoplasm. The life of a ZG starts in the trans-Golgi saccule, or the so-called GERL (which is part of the trans-Golgi network) in the form of a CV, as shown by electron microscopy and cytochemistry. Both size and electron opacity of the forming CV are highly variable. The intensity and duration of stimulation are among the most important parameters regulating the aspect of CV, and they probably determine the amount of membrane available for the packaging process. Indeed, ultrastructural observations clearly show that after 3-4 hr of sustained stimulation in the rat pancreas, the amount of membrane accumulated in the trans-Golgi area is at its maximum, presumably as a result of the endocytic process. Under these conditions the need for optimization of the membrane/volume ratio is not a limiting factor; hence, the cell responds by producing smaller ZGs. However, as the pancreas gradually recovers, the pool of membrane, available for packaging secretory protein, diminishes. Under the latter condition acinar cells react by producing large

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granules, thereby optimizing the amount of membrane used per amount of material packaged. The acinar cell has developed the capacity to increase the aggregation of the secretory products in the ZG matrix to refine the packaging process. As a result of this aggregation, if water is eliminated from the ZG matrix, there would be an excess of membrane and the latter is removed by a shedding process. Such a hypothesis is supported by different observations: Indeed, freeze-fracture studies (Sesso et al., 1980) and scanning electron microscopy (Naguro and Lino, 1989) have clearly shown the presence of small blebs and buds on the ZG surface. Sesso et al., (1980) reported that some of the microvesicles shed in the cytoplasm are covered with intercalated particles. This is also accompanied by a reduction in lectin binding sites that could be attributable to glycoproteins or other glycoconjugates. Since there is no detectable AcPase in the mature ZG membrane, as demonstrated by both cytochemistry and biochemical analysis, one is led to believe that this enzyme is one of the proteins eliminated from the ZG membrane during maturation. Some unpublished observations by F. Kan, A. R. Beaudoin, and G. Grondin support the view that GP2 would be shed in the cytoplasm during maturation. These microvesicles could migrate to the lysosomal system, as suggested in Fig. 7. One can predict that, during prolonged and intense stimulation, the maturation process would be impaired. As a result one could observe some

cv

AcPase

GP2

0

TMPase

Lysosornes and Snakellke Tubules

GP2

Apical Membrane

FIG. 7 Fate of the membrane components during maturation of the condensing vacuoles (CVs) in the pancreas. During maturation acid phosphatase (AcPase), trimetaphosphatase, (TMPase), and GP2 are removed from the immature granules. These proteins could be targeted to the lysosornes or snakelike tubules (basal lysosomes). MG,Mature granules.

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AcPase in the immature secretory granules. This is indeed the case, as demonstrated in both the parotid gland (Hand and Oliver, 1984) and the pancreas by cytochemistry (Novikoff et al., 1962). We have corroborated this observation in our laboratory. We even occasionally observed some AcPase activity in the acinar lumen. Such a shedding process could provide an explanation for the fact that a fraction of the newly synthesized GP2 and amylase molecules escape from the normal granule pathway and exit from the cell much faster than the bulk of newly synthesized secretory proteins. As early as 1982, Roberge and Beaudoin proposed the existence of a paragranular pathway to account for these observations (see Beaudoin and Grondin, 1991). Finally, minigranules are, in many respects, intriguing. They could well be responsible for another type of accelerated transport of some secretory proteins to the cell surface. Their small size would allow them to escape from the constraints of the cytoskeleton which would affect larger ZG. In support of this, we recently found that GP2 is much more concentrated in these minigranules than in regular-sized ZGs. Acknowledgments We thank Anne Rousseau. Marielle Martin, and Louise Hamel for their contribution in the preparation of the manuscript. This work was supported by Natural Sciences and Engineering Research Council of Canada and Formation de Chercheurs et Aide a la Recherche Quebec.

References Akagi, S., Yamomoto, A., Yoshimori, T., Masaki, R., Ogawa, R., and Tashiro, Y. (1988). 1. Histochem. Cytochem. 36, 1069-1074. Amsterdam, A., and Jamieson, J. D. (1974). J . CellBiol. 63, 1037-1056. Amsterdam, A., Ohad, Y., and Schramm, M. (1969). J . Cell Biol. 41,753-773. Arvan, P., and Castle, J. D. (1987). J. Cell Biol. 104,243-252. Aughsteen, A. A., and Cope, G. H . (1987). Cell Tissue Res. 249,427-436. Bader, M. F., and Aunis, D. (1983). Neuroscience 8, 165-181. Battistini, B., Chailler, P., Britre, N., and Beaudoin, A. R. (1990). Life Sci. 47,2435-2441. Batzri, S . , Selinger, A., and Schramm, M. (1971). Science 174, 1029-1031. Bauduin, H., Stock, C., Vincent, D., and Grenier, J. F. (1975). J. Cell Biol. 66, 175-181. Baylis, W. M., and Starling, E. H. (1902). J. Physiol. (London) 28,325-335. Beams, H. W., and Kessel, R. G. (1968). I n r . Rev. Cyrol. 23,209-276. Beaudoin, A. R., and Grondin, G. (1987). Life Sci. 40,2453-2460. Beaudoin, A. R., and Grondin, G. (1991). J. Electron Micrusc. Tech. 17,51-69. Beaudoin, A. R., Grondin, G., Lord, A., Roberge, M., and St-Jean, P. (1983). Eur. J . Cell Bi01.29,218-225. Beaudoin, A. R., Grondin, G., Filion, M., and Lord, A. (1984). Can. J. Biochem. Cell Biol. 62, 1288-1292.

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Molecular Analysis of Plant Signaling Elements: Relevance of Eukaryotic Signal Transduction Models Klaus Palme Max-Planck-Institut fur Pflanzenziichtung, D-5000 Koln 30, Germany

I. Introduction

Vascular plants develop their highly complex organization from a structurally simple embryo. Elucidation of the underlying genetic program is one of the most keenly pursued objectives of modern plant science (Sussex et al., 1985; Steeves and Sussex, 1989; Sussex, 1989). Despite the interesting developmental features of higher plants, surprisingly little is known about the precise molecular mechanisms programming plant development. This contrasts with the burst of information gained during the past few years concerning the structure, organization, and regulation of higher plant genes (Heidecker and Messing, 1986; Schell, 1987; Okamuro and Goldberg, 1990).The central principles that determine plant gene structure and regulation have been found to be generally similar to those studied in other eukaryotes, including animals. Therefore, we expect that other fundamental regulatory mechanisms of plant cells (e.g., cell cycle control, regulation of secretory processes, and intracellular signaling processes) might be similar to those of other eukaryotic cells. Although many reviews on various aspects of plant development have been presented (Davies, 1973; Rubery, 1981; Brenner, 1981; Trewavas, 1981; Albersheim and Darvill, 1985; Schnepf, 1986; Sachs and Ho, 1986; Brummell and Hall, 1985, 1987; Gilroy et al., 1987; Poovaiah and Reddy, 1987; Poovaiah and Reddy, 1987; Owen, 1988; Ryan, 1988; Steer, 1988; Lamb et al., 1989; Morse et al., 1989; Osborne, 1989; Kende, 1989; Kutschera, 1989; West et al., 1989; Darvill et al., 1989; Bush and Jones, 1990), most reviews are restricted to specific aspects. The recent interest in molecular mechanisms of plant signaling processes asks for a unifying comparison of eukaryotic genes and proteins involved in signal perception and transduction. This may lead to rapidly testable predictions, allow lnnremnrionol Reuiru of CvtoloR?. V d . 132

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Copyright 0 1992 by Academic Press. Inc. All rights of reproduction in any form reserved.

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identification in plants of new regulatory components, using molecular techniques, and help unravel mechanisms of regulation by comparison, for example, by answering the question of whether plants share common signal transduction elements and mechanisms with other eukaryotes. In this chapter I provide an overview comparing molecular components of signaling networks which are of general importance to all eukaryotes. Analysis is concentrated on plant signaling pathways that share structural and functional elements with those of other organisms. In addition I summarize some of the current models, as well as examples of structural motifs such as nucleotide binding domains, which are used as functional units in various plant proteins. This will yield evidence for a basic concept of common modular protein structures essential for the recognition of second messengers in eukaryotic cells which have been adapted to the specific developmental needs of the plant kingdom. This chapter begins with a brief description of plant development, emphasizing specific differences from animals, and a short discussion of structural and functional properties of elements of central importance for signal transduction in eukaryotes. These introductory sections are followed by a more comprehensive discussion of their relevance for plants.

II. Advantages of Plants as Models

The plant kingdom represents a diverse group of vascular and nonvascular organisms which arose more than 400 million years ago from green multicellular algae (Raven et al., 1986). Plants have been exploited by essentially every biological discipline because of their particular developmental features and agronomical importance. As early as 1665, analysis of plants led to the detection of cells as building blocks of life, and later to the establishment of the laws of heredity, and to the discovery of the concept of transposons documenting the instability of the eukaryotic genome (McClintock, 1984; Goldberg, 1988). Hence, research with plants has provided a conceptual framework and evolutionary reference for other eukaryotes as well. The extraordinary ability of plants to adapt to a wide range of environmental conditions led to an enormous number of species in different habitats with interesting biological properties. Plants further developed biological processes such as photosynthesis, nitrogen fixation, and light control of morphogenesis. To meet the demands of changing environmental stress, they established synthetic pathways, allowing the synthesis of compounds such as phytoalexins or other defense-related chemicals. Most plant species are amenable to both laboratory and field conditions, thereby being useful systems for genetic manipulations. Ho-

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mozygous strains are easily obtained, and general methods for creating new varieties and mutants have been developed. Exploitation of Agrobacterium tumefaciens, a bacterial pathogen that infects wounded plant tissues and causes neoplasia, led to the detection of natural interkingdom DNA transfer and provided tools for the genetic engineering of plants (Kahl and Schell, 1982; Schell, 1987). Genetic transformation techniques available for most plants, including major cereals, will have a significant impact on plant-breeding programs and crop improvement. Plants represent a diverse group of morphologically heterogenous organisms. The choice of specific model plants for scientific analysis depends on the particular question to be addressed. In particular the small flowering plant Arabidopsis rhaliana of the Cruciferae has lent itself to genetic and molecular plant biologies due to its small genome, its short generation time, the presence of an extensive genetic map, and the availability of many mutants (Redei, 1975; Estelle and Somerville, 1986; Meyerowitz, 1987). As plants range from nonvascular organisms (e.g., mosses) to highly differenitated seed-producing vascular plants, they may be useful in answering questions with scientific developmental, and applied importance. Their apparent morphological simplicity and remarkable capacity to regenerate, together with their susceptibility to efficient genetic analysis, will allow us to decipher some of the major events involved in reorganization of the complex genetic program during dedifferentiation.

111. Characterization of Plant Development and Its Control

The final form of the adult plant arises by precisely controlled cell divisions in specialized tissues, the meristems, followed by cell expansion and differentiation. This contrasts with animal development, which occurs in a distinct embryonic phase and involves complex cell movements and interactions. However, complex rearrangements and interactions among different tissues, initiated by cell movements, are made virtually impossible for plant cells by the presence of a cell wall. Vascular plants lead a sedentary existence, depending on their immediate surrounding for nutrients. These features make plants particularly vulnerable to local environmental fluctuations, to which they respond by adapting their developmental program. Plastic growth responses are essential for optimal exploitation of the fluctuating supply of light or other scarce local resources in competition with neighboring plants (Trewavas and Jennings, 1986). Anatomical and in situ molecular analyses of plant structure have re-

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vealed that the various meristems, buds, leaves, and seeds, arise in repetitive succession, producing the well-documented iterative plasticity or open-ended development of many vascular plants. It is apparently only the basic pattern of each repetitive unit of the plant body which is expected to be determined by the developmental program of a plant. The adaptive developmental strategy is reflected in the organization of the plant embryo. Initially, centers of cell division are established at opposite poles of the axis. The meristems, located at the apex of the root and the shoot, form the entire plant body by repetitive production of the basic cell lineages required for formation of new tissues and organs. Thus, the apical meristems not only are crucial for the production of additional secondary meristems, but also lead to the initiation of bud primordia and to expansion of the root system. It is mainly these meristems which retain the capacity for continuous cell division throughout the life span of a plant, thereby playing a fundamental role in the control of morphogenesis and regeneration. This apparent lack of specialization has been interpreted as an evolutionary advantage for sessile organisms, which have to compensate for predation or other environmental damage by replacement of lost or damaged tissues (Trewavas, 1986a). To specify the structural and morphological features of the plant body, the spatial and temporal organization of gene expression is required. An understanding of the structure and function of plant genes controlling the pattern of cell division, the degree of cell enlargement, and the type of differentiation of particular cells will provide insight into processes regulating the morphogenesis of plants. While the general organizational principles of growth control in eukaryotes have begun to emerge from the molecular analysis of yeast and animals, our current understanding of most processes and genes determining plant development and morphogenesis is still rudimentary. It is expected that identification of the plant genes controlling cell division and differentiation and unraveling of the molecular mechanisms of their action will significantly contribute to our understanding of the control of growth responses. It will also provide the knowledge necessary for the practical control of morphogenic and regenerative events of plants in vivo. The control of plant growth and development requires external and internal factors to coordinate processes such as cell division, expansion, and differentiation. External factors include complex changes in the environment, gravity, temperature, and light. Internal control factors that affect plant growth and development are represented by plant growth regulators: the phytohormones. Interestingly, the external supply of phytohormones (e.g., by plant pathogenic bacteria or fungi) results in more or less severe developmental abnormalities. The phytohormones are represented by five major classes: auxins, gibberellins, cytokinins, abscisic

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acid, and ethylene (Davies, 1987). These substances differ from animal hormones in structure and by the wide variety of developmental responses each is able to influence (Fig. 1). Phytohormones can be transported from the site of production to their target cells, similar to animal hormones, and are active at very low concentrations, evoking both rapid and long-term responses. The observation that particular combinations of phytohormones can elicit differential responses in different target tissues suggests “cross-talking” between these phytohormones at the molecular and cellular levels. Abscisic acid, for example, a growth-inhibiting hormone found in dormant buds and fruits, prevents the growth-promoting effects of auxins and cytokinins. The pleiotropy of biological activities elicited by various combinations of phytohormones and the often antagonistic modulation of biochemical responses may point to novel regulatory pathways established in plants (Marx, 1983; Davies, 1987; Sussex, 1989; Steeves and Sussex, 1989). From the bewildering flood of often contradictory physiological data produced over several decades, questions were raised concerning (1) the limited diversity of phytohormones controlling the many susceptible physiological processes, (2) the importance of phytohormone concentrations for plant growth control, and (3) additional growth factors postulated for regulation of the great diversity of responses influenced by phytohormones and acting at various levels of hierarchy (Guern, 1987). Answers to these questions are prerequisite to a molecular understanding of plant development. Therefore, it is of crucial importance to study phytohormone perception and to identify the various steps leading to the final cellular responses. What is the structural basis for phytohormone recognition, what elements if any are involved in the transduction pathway of the primary signal, which gene families are required for signal transmission, and to what extent do they share similarity within their protein sequence to domains active in other evolutionarily conserved proteins? The dictum of Crick (1988), “if you want to understand function, study structure,” will be the essential basis for molecular studies using the available techniques and tools to dissect many of these processes. A direct comparison of information about signaling circuits in plants with those of other multicellular organisms could lead us to a clearer understanding not only of the developmental processes of higher plants, but also of the organizational principles. This knowledge will help to reveal the molecular basis of redifferentiation; that is, the potency of somatic plant cells to form complete organisms from single differentiated cells. Although several animal taxa have a sustained capacity to regenerate organs (Maden, 1985; Blau, 1988), the development of a complete organism depends in animals always on totipotent cells, present only in the early animal embryo. Thus, understanding of the structure and function of genes responsible for decisions which determine the fundamental regulatory

Plant hormone

Gibberellin

Slruclure

HO

cn, coon

Typical Concenlral ton i n Tissues

I Synlhestr

SELECTED DEVELOPMENTAL RESPONSES

Transport

young shoo1 IISSUI developing r e d s

GAl

FIG. 1 The phytohormonesand their major biological responses.

EUKARYOTIC SIGNAL TRANSDUCTION

229

processes controlling plant differentiation should also lead to generalizations on principles about development. Detailed knowledge of these processes is considered to be of fundamental importance for further progress in plant biotechnology. It is hoped that knowledge of plant-specific signal transduction, especially on the mechanisms by which phytohormones initiate and control differentiation, will make the regeneration of single, previously differentiated plant cells into complete organisms a predictable process.

IV. Different Strategies for Organizing the Body Patterns in Plants and Animals

Plants undergo morphogenesis throughout most of their life span, in stark contrast to morphogenetic processes in animals, which are almost entirely restricted to an embryonic phase. The basal body plan of animals, containing all tissues and organs at least in rudimentary form, is laid down in response to opposing morphogen gradients emanating from localized sources at each pole of the embryo. In particular the analysis of mutants and genes from Drosophila melanogaster has contributed considerably to our understanding of positional information, developmental fields, and morphogenetic gradients (Gehring, 1987; Ingham, 1988).Protein products, expressed as a morphogenetic gradient throughout the egg and the early embryo, directly dictate the spatial pattern of synthesis of many subordinate proteins, triggering inductive developmental events dependent on their concentration along the axis (Frohnhofer and Nusslein-Vollhard, 1987; Tautz, 1988; Gaul and Jackle, 1987). Several gene products involved in organizing the fate map of the embryo share similarity with certain domains of steroid hormone receptors or oncogene products, such as tyrosine kinases (Simon et al., 1989). Thus, in animals the basic body plan is established in the embryos, whereas higher plant embryos build only a root-shoot axis, including cotyledons, thus containing the rudimentary form of only a small fraction of the final body. No reproductive structures (e.g., germ line cells) develop in the primary plant embryo. After germination the seedling forms by both cell division and enlargement of preexisting cells. New meristems are produced, restricting the growth to localized areas at the tips of roots, stems, and new organs, where cell lineages are formed and new tissue is added. Meristems are thought to allow plants the continuous production of new organs and new meristematic tissues. This results generally in an indeterminate pattern of growth. All angiosperms, for example, consist of the same module structure, with the stem bud and leaf above ground and repetitions of the root meristem below ground,

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which can be repeated many times. Hence, most plant development differs radically from that of animals in that growth of the plant body is indeterminate and modular, the major organs arising in a reiterated series. It is well accepted that phytohormones play a crucial role in controlling growth in individual organs and in the plant as a whole. Although their fundamental influence on almost all aspects of plant development has been known for many years, only now are their specific roles and modes of action being studied at the molecular level (Palme et al., 1991a). In addition to the ability of phytohormones to act both at their sites of production and in distant tissues, it is evident that they elicit a wide range of responses, depending on the type of tissue or organ with which they interact. For example, auxins [indole-3-acetic acid (IAA) and related natural and synthetic analogs], the most widely studied group of phytohormones, modulate cell expansion, cell division, morphogenesis, and tropisms. They are essential for the induction of root growth, control of apical dominance, and plastic responses to environmental fluctuation, including light and gravity. They are necessary for the initiation and maintenance of tissue cultures and the control of embryo formation, as well as the micropropagation of tissues and platelets. Clearly, the remarkable pleiotropy of the many physiological and morphogenic responses related to auxin action suggests that each response elicited must be a function of the responding system, rather than the evoking substance. Nevertheless, it should be noted here that plants, in contrast to animals, lack an actively controlled circulation system. It is surprising that, despite the poorly regulated transport systems in both phloem and xylem tissues, phytohormones can be transported efficiently to their target tissues, where they elicit specific responses. Because of the pleiotropy of effects elicited in various tissues, it has been suggested that hormonal regulation in plants may call for novel regulatory principles (Trewavas, 1981; Trewavas and Cleland, 1982, 1983; Weyers, 1984; Chadwick and Garrod, 1986; Firn, 1986; Davies, 1987; Guern, 1987; Nissen, 1985, 1988). In particular novel regulatory mechanisms could be responsible for activation of the production of phytohormones in individual cells, thus allowing them to respond to specific local environmental conditions. This may be important in order for tissues to respond quickly and independently of phytohormone supply by the normal transport routes. Furthermore, this could allow, for example, particular growth responses to exploit localized changes in the environment and thus lead to optimization of resource allocation (Trewavas, 1986a). This is illustrated, for example, by the synthesis of ethylene when oxygen is depleted, production of abscisic acid in the presence of water shortage, or synthesis of cytokinins in the presence of nitrogen imbalance, resulting in metabolic

EUKARYOTIC SIGNAL TRANSDUCTION

23 1

adaptation to prevent disruption of membrane function and lipid breakdown, prevention of water loss, or decline and alteration in protein synthesis, respectively (Trewevas, 1986a). Decisions made at the level of individual cells or organs to cope with the local resource conditions result in the final adaptation and phenotypic plasticity of the plant body. Although mechanisms for the local activation of phytohormone production by external stimuli are not yet known, the responsiveness of plant cells or tissues to a particular growth factor or to combinations of different growth factors must require the presence of receptors that couple perception of the hormonal signal to metabolic events. The limited structural diversity of the five major classes of phytohormones constrasts markedly with the myriad compounds known to influence and regulate the various processes of animal development. This has led in the past to a major concern among botanists, raising the question as to whether the known plant hormones are sufficient to control the large variety of developmental processes in plants (Cannay, 1985). A substantial diversity of signals may be achieved by the antagonistic or cooperative action of groups of hormones. However, it is equally conceivable that a number of important signal molecules have not yet been detected. Support for this proposal comes from recent observations pointing to a functional role of various oligosaccharide structures (Darvill and Albersheim, 1984; Ryan, 1987, 1988; Long and Atkinson, 1990). Thus, oligosaccharides derived from the wall or from other sources could offer a considerable potential for signal complexity (Long and Atkinson, 1990; Fry, 1990). It will be fundamental to determine whether plants have evolved unique signal recognition-communication systems which regulate gene expression and function in response to the environment. Furthermore, speculation is kindled as to whether a novel plant-specific regulatory circuit, using oligosaccharides or other novel chemical messengers, will be found to rest hierarchically on a more or less strictly conserved eukaryotic cellular signaling network responsible for most of the specific communication events within plants. It will be important to know whether strong evolutionary relationships exist between various plant signal recognition elements and those of animals. As outlined earlier it is likely that plants, being more flexible in their adaptation of growth to environmental fluctuations than other organisms, have evolved novel mechanisms for the regulation of cell fate determination. Although I argue below that some elements of cellular signaling and response pathways are common to animals and plants, we should be careful not to try to force our interpretation of plant development into unjustified parallels with animal models before we know the precise areas and degree of overlap or functional relatedness.

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V. Development of Concepts

Unicellular eukaryotes have probably played an essential role in the history of life as intermediates between prokaryotic and eukaryotic phyla (Margulis and Schwartz, 1982). It is assumed that many of the present-day unicellular eukaryotes are descendants of some of the groups that proliferated during the major evolutionary transitions. Comparative analysis of all of these organisms has shed light on the molecular track, which was followed during early evolution. Traditionally, this search has been for morphological structures, but now, of course, includes molecular data. Technical advances in molecular biology, especially the use of the polymerase chain reaction (Saiki et al., 1985a,b), have provided the vast number of DNA and protein sequences in data bases, facilitating rapid similarity searches by matrix comparisons (Lipman and Pearson, 1985). Particularly useful for the systematic exploitation of phylogenetic relationships were comparisons of rRNAs (5, 18, and 28 s).rRNA sequences represent a huge supply of information for comparisons, allowing the identification of mosaic structures within domains evolving at widely different rates (Hassouna et al., 1984). By comparing 249 5 S rRNA sequences, Hori e? al. (1985) constructed a phylogenetic tree for eubacteria and metabacteria, fungi, and Proto-, Meso-, and Metazoa as well as Metaphyta, containing vascular plants. Analysis of this tree allowed the branching order among fungi, plants, and animals to be deduced (Fig. 2a). From this analysis it was concluded that red algae evolved first in early eukaryotic evolution, followed by the various fungi (Ascomycetes and Basidiomycetes). Then, approximately at the same time 400 million years ago, green plants, brown algae, and Protozoa/Oomycetes emerged within a relatively small time interval. From this tree it is clearly evident that green algae belong to the branch of green plants (Fig. 2b). It was pointed out that among green algae Charophyta (e.g., Nitella) probably were the precursors to land plants (Kumazaki et al., 1983; Lim et al., 1983). Meanwhile, similar trees have been constructed, using large stretches of 18 and 28 S rRna as phylogenetic indicators (Qu et al., 1983; Woese, 1987; Hassouna et al., 1984; Baroin et al., 1988). From all phylogenetic and distance matrix comparisons based on rRNA nucleotide sequences, it is evident that plants are clustered in the eukaryotic kingdom, together with yeast and metazoa, within relatively small distance values. The closer evolutionary relationship between metaphyta and metazoa as compared to yeast may point beyond the conservation of structural rRNAs to the conservation of even more basic elements and structures within cells. Besides the universally used genetic code, eukaryotes, including plants, may share elements of a central cellular signaling language necessary for the forma-

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a

73 1 21 2 28 24

11 2

3

13

3 5 25 28 249

Dudwead

b

Horsetail ClUbmMlS

Chlsmydomonas'

1 I 2 Knuc.

1

0.2

0.1

0

FIG. 2 (a) A phylogenetic tree constructed from 249 5 S rRNA sequences. 1/2 Knuc, Relative evolutionary distance; ---o---, uk(range of the standard error of 1/2 Knuc). Major taxon names are followed by the numbers of sequences used. (b) A phylogenetic tree of 28 5 S rRNAs from green plants. From Hori et al. (1985) with permission of the publisher.

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tion of structural and regulatory networks in cells. Despite the limited amount of molecular data available for plants, it may be reasonable to assume at present that plant cells share with all other eukaryotes the central recognition modules for commonly used signal molecules. The reasons for this belief are (1) their common evolutionary origin and (2) the fact that all cells basically use identical chemical substances to perform and control a wide variety of physiological activities. As illustrated here I believe that the concept of evolutionary homology is further based on the structural identity of many chemicals used as signal molecules or second messengers (e.g., ATP, GTP, CAMP), phosphoinositol metabolites, and calcium) in various cells requiring shared and conserved recognition modules in proteins that evolved either by convergent evolution or by gene duplication events.

VI. Molecular Elements and Mechanisms of Cellular Signaling

Multicellular organisms depend on cellular communication in order to regulate development and coordinate the formation and integration of tissues and organs. Communication among different cells, as well as information processing within a specific cell, is essential to control cell division and growth and to coordinate the various physiological activities. It has been discussed that the development and differentiation of higher animals depend on cell movement, which requires a more complex intercellular communication apparatus than that of plants. Consequently, autotrophic sessile organisms such as plants could be assumed to have more rudimentary and less complex communication systems. In general major eukaryotic communication pathways involve the secretion of chemicals, operationally termed hormones, which may interact either with the same cells (autocrine activation) or with other distantly located cells (exocrine activation). The ability of a specific cell to respond to a particular extracelMar signal depends on the presence of receptor proteins, located in the plasma membrane or the interior of the cell, being able to bind the particular hormone or signal molecule and translate the information received into a specific response. The signal molecules, for which corresponding receptors have high affinities, are usually active at very low concentrations. Most cells primed to perform specialized functions contain arrays of receptors, allowing them to respond to diverse signals. Ultimately, signals detected by receptors are translated into a limited repertoire of intracellular signals: the second messengers. Occupancy of receptors in yeast or other eukaryotes may initiate the production of active messengers, for example, the well-studied CAMP, as well as the more recently discovered

EUKARYOTIC SIGNAL TRANSDbCTION

235

messenger molecules that are derived from phosphoinositides such as arachidonic acid, inositol 1,4,5-triphosphate, and 1,2-diacylglycerol (Majerus et al., 1985; Berridge, 1984, 1987; Newton and Brown, 1986; Boss and Morre, 1989). Different second messengers are capable of regulating a vast array of physiological and biochemical processes. This can occur either by direct interaction with distinct proteins or indirectly by activating enzymes which trigger conformational changes in the final target proteins. Given the complexity of responses, the number of second messengers in eukaryotic cells appears to be surprisingly small. This indicates that probably only a limited number of widely used internal signal pathways transduce the common chemical signals to final biological responses (Fig. 3). It has become clear that animal growth factors are not necessarily limited to the control of only a single physiological activity, but rather form part of a complex cellular signaling language, in which the individual agonists and second messengers appear to be equivalent to characters of a code or an alphabet. Thus, the information content may not reside singly in the individual agonists or growth factors, but in the pattern or set of regulatory molecules to which a cell is exposed. It is conceivable that combinations selected from a number of different signal molecules not only increase the amount of information which can be transmitted, but furthermore promote error-free transmission of information in the presence of noise, owing to the redundancy of individual signal molecules or sets thereof (Shannon and Weaver, 1949; Ashby, 1956).

VII. Elements of Eukaryotic Signal Response Pathways A. Receptors Eukaryotes and bacteria constantly sense the environment and modify their gene expression in response to a wide variety of environmental stimuli, including nutrient deprivation, osmolarity changes, symbiosis, and pathogenesis (Stewart and Dahlquist, 1987; Ames et al., 1989; Forst and Inouye, 1988; Magasanik, 1988; Miller et al., 1989). In bacteria these responses are often mediated by pairs of proteins that transmit information to specific response pathways largely by protein phosphorylation to activate or deactivate target protein activities and thus physiological or functional responses. Genetic and biochemical analyses have indicated that each pair of sensor-regulator proteins belongs to a superfamily in which sensor proteins share similarities in their C-terminal domains, while their regulatory intracellular partners share similarities in their N-terminal domains (Kofoid and Parkinson, 1988; Keener and Kustu, 1988; Borkovich et al., 1989). Analysis of mutants clearly demonstrated that an extracellu-

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Receptor,

u

a

~

Second Messenger P o s s i b l e Candidates: c a l ci urn or lipids (diacylglycerol, arachidonic a c i d ) or me t a bol i t es

inactivation

-

FIG.3 C neral elements in a eukaryotic signal transduction pathway from the membrane to the nucleus.

larly located N-terminal domain of sensor proteins is required to monitor changes in levels of environmental signals (Albright et al., 1989; Bourrett et al., 1989). Changes in receptor occupancy trigger, among other responses, for example, the chemotaxis by modulating the signal output from the cytoplasmic domain (Ames and Parkinson, 1988). Commonly used elements of the various response chains are (1) a protein kinase activated by the sensor, (2) a regulator protein triggered by phosphorylation, (3) variable target proteins for regulator action, and (4) a protein phosphatase that restores the initial susceptibility of the regulator protein to activating events. Subsequently, structurally and functionally similar sensory machinery was found in eukaryotes. In these organisms cell surface receptors with binding sites for a variety of signal molecules are represented by multifunctional membrane proteins that face the external environment and effector sites that couple the binding to an intracellular event. Besides stimulus-response coupling many receptors have an additional function to transport bound signal molecules or ligands directly into cells (Goldstein et al., 1979), where endocytosis leads to their uptake and in many cases to recycling to the cell surface after ligand delivery (Brown et af., 1983).

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237

General information concerning the structure and mechanisms of action of cell surface receptors has emerged in the last few years (Hill and Kendall, 1989; Engelberg et al., 1989; Heldin and Westermark, 1989; Ullrich and Schlessinger, 1990). On the basis of sequence similarity and distinct structural characteristics, it is possible to classify these receptors into subclasses. Characteristic topological features of the subclasses include one to several membrane-spanning domains. Many of them possess large glycosylated extracellular ligand binding domains. Binding of the extracellular ligand is translated into activation of the intracellular domain. Typically, phosphorylation of endogenous substrates occurs by receptor kinases after oligomerization within the membrane (Schlessinger, 1988; Yarden and Ullrich, 1988; Williams, 1989; Ullrich and Schlessinger, 1990). Conformational changes induced in the kinases result in direct phosphorylation of effector proteins, which then effect the particular cellular responses. Such phosphorylation may result, for example, in either direct activation of ion channels (Nicoll, 1988) or desensitization of various substrates, such as G-coupled receptors (Lefkowitz and Caron, 1988). B. G Protein-Coupled Receptors

This receptor family contains members that function in visual transduction, regulation of homeostasis, and development and include known and potential oncogenes (Nathans and Hogness, 1984; Kobilka e? al., 1987; Julius e? al., 1988; Klein e? al., 1988; Hershey and Krause, 1990). Their contribution to cellular sensing mechanisms has diversified greatly during evolution. Examples of this class of receptors exist in animals and lower eukaryotes. In yeast, for example, important members of this gene family, such as STE2 and STE3 cell type-specific sterility genes, are involved in controlling the mating process. As receptors for the mating pheromones, a and a factor, they are responsible for rapid alterations in the pattern of gene expression, resulting in alteration of the cell surface and nuclear morphology required for the fusion of haploid yeast cells (Nakayama et al., 1985; Blumer e? al., 1988; Nomoto et al., 1990). The primary structures of various members of this receptor family have been proposed to contain seven transmembrane domains, each 20-28 amino acids in length (see Fig. 4 for structural features). The transmembrane domain includes conserved proline residues which may help to form the pocket necessary for ligand binding. Although hydrophobic regions are reasonably well conserved, the hydrophilic regions have strongly diverged. This has allowed adaptation of this structural theme for the recognition of a wide variety of hormones, peptides, and neurotransmitters. All of these receptors are coupled to guanine nucleotide-binding regulatory proteins that interact with effector

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enzymes inside the cell to generate a cellular response appropriate to the extracellular signal (Gilman, 1987; Stryer and Bourne ,1986). The consequence of this organization is that cells can react to diverse external signals channeled through only a few intracellular signaling pathways. C. GTP-Binding Regulatory Proteins

The GTP-binding regulatory proteins have attracted much attention in recent years. They represent a universal biomolecular switching mechanism with the potential to amplify extracellular signals and regulate or modulate intracellular processes. The G protein superfamily contains a large number of closely related members. Relevant polypeptide sequences, isolated over the last few years from a variety of organisms, are summarized in Table I; the structural and functional characteristics are indicated (Gilman, 1984, 1987; Stryer and Bourne, 1986; Neer and Clapham, 1988; Casey and Gilman, 1988; Lochrie and Simon, 1988; Hall, 1990; Bourne et al., 1990). All G proteins share the ability to bind and hydrolyze GTP, which induces a switch between two different protein conformations. The switch is generally turned on by binding of GTP and turned off by hydrolysis of GTP, but not by dissociation of GDP. Initial rate studies have demon-

E x t r a c e l lular

€2

I

n

I

El

c1

I

E3

n

c2 I ntr ace1 Iu lar

fC Terminus

I

P

I

P

FIG. 4 Schematic representation of the structure and membrane-spanning domains of a G-coupled receptor.

1

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EUKARYOTIC SIGNAL TRANSDUCTION

strated that, through a double-exchange mechanism, the bound GDP associated with the resting state is exchanged for GTP after an activation event. As a consequence of the irreversibility of GTP hydrolysis, the switch contributes to the propagation and amplification of regulatory signals, not by regulating either the rate or intensity of a specific reaction, but instead by safeguarding the unidirectional orientation of a reaction. Thus, it is not surprising that most vectorial processes (e.g., protein synthesis, transport, and secretion) utilize the GDP/GTP-induced conformational switch to link crucial steps of various pathways. The fact that G proteins share a common (de)activation mechanism depends on structural homologies. Although there is remarkable sequence conservation among the various G proteins, their activation is, nevertheless, highly specific. The subsequent modulation of effectors may be less precise, allowing several different agonists to influence common or similar pathways. Given the similarity and interchangeable nature of these proteins, it is probably only their physical arrangement in a specific cell that allows the correct response to be coupled to each signal. A widespread, but less clearly understood, feature of the different sensory systems is desensitization or adaptation, preventing a response to a subsequent challenge with the same stimulus. The most important mechanism for attenuation of the hormonal signal is posttranslational modification of the signal complex. Apparently, both receptor and G proteins can be phosphorylated by specific protein kinases, resulting in a reduced affinity for the specific ligand or prevention of coupling between the complex and their intracellular effectors. Examples are the phosphorylation of the C-terminal region of G protein-coupled receptors, leading to a blocking of coupling to its specific G protein (Lefkowitz and Caron, 19881, or the direct phosphorylation of G proteins, not only on serine, but also on tyrosine (O’Brien et al., 1987a,b; Carlson et al., 1989; Cobitz et al., 1989).

TABLE I Properties and Functions of GTP-Binding Proteins

Structural parameters

Molecular mass (kDa)

Function

Location

45-55 35 8

Allow communication between signal receptor and effector proteins by stimulation or inhibition

Nearly all cells

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D. Protein Kinases

Evidence collected during recent years suggests that the various receptor proteins linked to common intracellular signals, resulting in convergence of intracellular signal response pathways at some point inside the cell. This ability of apparently unrelated signals to trigger similar physiological events in responding cells is generally facilitated by topological similarities in signaling proteins, rather than extensive primary sequence similarities. The binding of signal molecules to receptors is believed to initiate a response pathway or cascade, in which protein kinases seem to be important elements in controlling the network of events responsible for cellular proliferation, differentiation, and metabolic control (Cohen, 1985; Hunter and Cooper, 1985; Nairn et al., 1985; Edelman et al., 1987; Hunter, 1987; Schulman and Lou, 1989; Ullrich and Schlessinger, 1990). It has been argued that all organisms may use protein kinases as major components in essential cellular regulatory circuits, similar to regulatory elements such as chips in computers (Hunter, 1987). Thus, their major input is to activate, deactivate, or amplify enzymatic activities and regulate events such as ion channel opening, receptor desensitization, and hormone release. The key feature of control by protein kinases is the reversibility of the activating or deactivating event by protein phosphatases. Phosphatases were initially studied biochemically, and mutational analysis in several organisms confirmed the crucial role of phosphatases in reversing phosphorylation in a diversity of situations, leading, for example, in Aspergillus nidulans, to an elevated mitotic index (Doonan and Morris, 1989), a block in chromosome disjunction (Ohkura et al., 1989), or a number of other defects in mitotic control (Booher and Beach, 1989). Phosphatases play a central and specific role in many other aspects of cellular physiology (Cohen, 1989; Cohen and Cohen, 1989a; Cyert and Thorner, 1989; Hunter, 1989; Tonks and Charbonneau, 1989). E. Spatial and Temporal Coordination of Cellular Communication

Second messenger activation has become an accepted concept in molecular biology, explaining the conveyance of information from the cell surface to the various intracellular compartments, where duration and intensity of the cellular response reflect the strength of the initial message. The diversity of second messengers appears to be surprisingly limited. Internal signal pathways can therefore be expected to be fairly universal. Known second messengers are capable of regulating a vast variety of physiological and biochemical processes. Several second messengers are able to elicit changes in intracellular calcium localization as a common signal leading to

EUKARYOTIC SIGNAL TRANSDUCTION

24 1

structural changes in cellular proteins. In calmodulin, for example, binding of calcium results in exposure of hydrophobic domains and subsequent interaction with other proteins. The flux of calcium across the membranes is the only variable, since the concentration of intracellular calcium remains low and constant in the range of 10 -6 M (Campbell, 1983; Carafoli, 1987). Calcium signals emanating from the plasma membrane are known to be generated by a number of mechanisms which use calcium channels that are receptor operated, voltage dependent, or regulated by second messengers. The calcium fluxes induced can be either transient or sustained, resulting in transient or sustained cellular responses (Rasmussen and Barrett, 1984; Berridge and Irvine, 1989). The proposed role of calcium as a common denominator may also explain why previously stimulated cells can retain an enhanced responsiveness that outlasts the initiating stimulus. Persistent responsiveness may be caused by oscillatory fluxes or repetitive spikes in calcium concentration, creating what has been termed a cellular memory (Goldbeter et al., 1990). Thus, in the course of evolution, all cellular systems have made extensive use of this signaling system (Miller, 1987; Holl et al., 1988; Kim and Westhead, 1989; Goldbeter et ul., 1990; Parker and Ivorra, 1990; Osipchuk et al., 1990). F. Perspective

Structural and functional correlations have greatly advanced our understanding of the modular structure of cellular signal molecules. Research in a number of different eukaryotic systems has led to a general belief that the selectivity of cellular perception systems for particular signals and the mechanisms required to provide cell-specific responses do not necessarily call for fundamentally new mechanistic elements to transduce the initial signal to their final cellular response in each case. The concept of modularization of signal recognition proteins assumes that novel proteins have evolved by the assembly of preexisting peptide units, rather than by successive single amino acid exchanges (Gilbert, 1978, 1985; Darnell and Doolittle, 1986; Brenner, 1988). We expect that proteins that recognize common signaling molecules are similar not only in unicellular eukaryotes and metazoa, but also in metaphyta. This suggests that these proteins share at least domains of identical amino acid sequence at locations which are critical for recognition and necessary to perform similar or identical functions. This further suggests that the proteins that recognize plantspecific signals may have considerable homologies to certain domains of other eukaryotic signaling proteins. If this were the case, such conserved domains could be used as diagnostic elements for the isolation and analysis of functionally related elements in plants as well as for family kinships. This approach should contribute to the solution of some of the major

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challenges facing contemporary plant science. In particular the mechanisms underlying the unique aspects of plant development and biochemical as well as physiological responses to environmental stimuli are expected to be revealed by this kind of study.

VIII. Phytohormone Perception and Cellular Responses

The wealth of information accumulated about the molecular mechanisms by which animal cells perceive and transduce hormonal signals contrasts starkly with our relative ignorance of the initial events in plant-specific signaling cascades. Analysis of hormone action in yeast or mammals has revealed two major mechanisms for hormone action: (1) activation of transcription via second messenger pathways and (2) activation of liganddependent transcription factors, the steroid receptors, which, when bound to a specific ligand, directly influence the expression of genes containing particular enhancer sequences, termed the hormone-responsive elements (Yamamoto, 1988; Evans, 1988; Beato, 1989). Over the past few years identification of the genes expressed in response to exogenously applied phytohormones has been a major goal of plant research. Current studies are aimed at the characterization of promoter sequences from such hormone-responsive genes, with a view to providing selective tools with which to dissect the mechanisms of the phytohormonal regulation of gene expression. In this section I briefly summarize progress in the structural, molecular, and functional analyses of phytohormone-activated plant genes. In the search for primary events initiated by phytohormones, both shortand long-term effects on plant growth have been studied (Hagen, 1987). A major difficulty has been to distinguish those primary hormonal responses of probable importance in growth regulation from those that are merely consequences of the primary alterations of cellular physiology. However, a number of rapid physiological responses have been noted, some with lag periods of only a few minutes after application of the stimulus (Table 11). For example, exogeneous application of auxin to hormone-depleted tissues results, within minutes, in increases in respiration rate, proton extrusion, and potassium uptake and changes in membrane potential. Growth responses such as cell elongation, however, require the completion of a series of metabolic steps to facilitate promotion and spatial control of cell wall synthesis, as well as cell division. This suggests long-term responses, with time intervals that may vary between 1 hr and several days. Alterations in the spectrum of polypeptides, as revealed by two-dimensional gel electrophoresis in both elongating and mature soybean hypocotyl sections, were observed to precede these long-term responses, becoming

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EUKARYOTIC SIGNAL TRANSDUCTION TABLE II Rapid Physiological Responses of Plant Tissues after Application of Auxin

Type of response Increase in respiration rate Decrease in ATPIADP ratio Decrease in cytosolic pH Increase in H+ secretion Increase in membrane potential Increase in K + uptake Increase in P-glucan synthase activity. P-galactosidase. cellulase Increase in cell wall extensibility Release of xyloglucan Increase in gene transcription

Response time (min)

Plant tissue

5 5 5 5-8 10-12 10-30

Coleoptiles from maize or oat Coleoptiles from maize or oat Maize coleoptile Maize coleoptile Oat, tobacco Oat, bean

10-15

Pea Oat, pea Pea stem Soybean, pea

2-20 15-20

5-30

apparent 1-3 hours after auxin application (Zurtluh and Guilfoyle, 1980). In addition cytokinins could be shown to counter effects on polypeptide patterns induced by auxins, consistent with the view that these phytohormones antagonistically modulate some physiological events. Cytokinins enhance the synthesis of enzymes such as hydroxypyruvate reductase, nitrate reductase, acid phosphatase, endopeptidase, and chromatin-bound RNA polymerase, as well as many others (Kende er al., 1971; Borris, 1967; Kulaeva, 1980; Chen and Leisner, 1985). It is not known whether some or all cytokinin-induced alterations of enzymatic activities are a result of direct transcriptional activation. Furthermore, in the few cases in which cytokinin-modulated gene expression could be demonstrated by two-dimensional gel electrophoresis, variable responses to this hormone were found in which translation of some mRNAs could be activated, reduced, or even completely suppressed in different tissues treated with identical concentrations of benzyladenine (Chen er af.,1987). This probably results from a strong influence of the particular physiological and hormonal status of the plant tissue, a difficult factor to control. Gibberellic acid (GA) and abscisic acid have response characteristics that are much better defined. In barley aleurone layers they influence the expression of a-amylase, as well as several other genes encoding hydrolytic enzymes (Ho, 1989). Recently, it was shown, using nuclear in virro run-on experiments, that GA3 induction of a-amylase is enhanced about 10-fold at the level of transcription, while abscisic acid treatment of aleurone cells leads to reversal of this GA3 effect (Jacobsen and Beach, 1985). Although GA3 induction of specific mRNAs for the various a-amylase isoforms starts slowly 1 and 4 hr after application of the hormone, inhibitor studies demonstrated that the stimulation is at the level of transcription (Ho and Varner, 1974; Ho er af., 1987; Huttly and Baulcombe, 1989).

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Interestingly, the half-life of a-amylase mRNA is more than 100 hr, suggesting that mRNA turnover is not of paramount importance for the regulation of a-amylase concentration. A number of developmental studies on seeds of diverse species have shown that abscisic acid not only counters the action of GA3at the transcriptional level, but also induces the accumulation of specific mRNAs and proteins late in embryogenesis, at a time when seeds desiccate and embryos of some species become dormant (Quatrano, 1987). Increases in levels of abscisic acid have been correlated with the expression of specific genes, mainly the late embryogenesisabundant genes and the genes responsive to abscisic acid (rub) linked to osmotic tolerance (Skriver and Mundy, 1990). In particular the expression of the rice gene rab-l6A, which is responsive to both abscisic acid and osmotic stress, contains cis-acting promoter motifs, with a consensus sequence represented by the nucleic acid sequence TACGTGGC, sufficient to confer abscisic acid regulation to the cauliflower mosaic virus (CaMV) 35 S transcript promoter (Mundy et al., 1990). Demonstration of binding of nuclear proteins to these motifs, by band mobility-shift assays and in vitro footprinting, raises the prospect that these abscisic acid response elements will serve as probes with which to isolate clones encoding hormone-activated regulatory proteins (Singh et al., 1988; Katagiri et al., 1989). Increased RNA synthesis in isolated nuclei and changes in nucleic acid metabolism initiated by auxins were observed for the first time more than 30 years ago (Silberger and Skoog, 1953; Matthysse and Phillips, 1969). Since then major efforts have been focused on elucidation of auxin-specific cis-acting promoter elements and identification of trans-acting factors regulating auxin-responsive genes. In this respect research on auxin action has been much less rewarding than analysis of abscisic acid, as it has not yet been possible to demonstrate the activation of chimeric promoters by cis-acting auxin-responsive elements. Auxin-induced changes in polypeptide abundance have been clearly observed by two-dimensional gel electrophoresis. For example, rapid changes in in vitro translation products in response to auxin application were detected in mRNA isolated from tissues from maize, pea, or soybean that were known to respond to auxin by cell elongation (Zurfluh and Guilfoyle, 1987a-c; Theologis and Ray, 1982; Theologis, 1989). Such analysis led to the isolation of a number of cDNAs encoding auxin-responsive mRNAs by differential screening of cDNA libraries (Baulcombe and Key, 1980; Walker and Key, 1982; Gantt and Key, 1983; Hagen et al., 1984; Theologis et al., 1985; McClure and Guilfoyle, 1987; van der Zaal et d.,1987; Ainley et d.,1988; Czarnecka et al., 1984,1989; Hagen et al., 1988; Alliotte et al., 1989; McClure and Guilfoyle, 1989; Takahashi and Nagata, 1990). The encoded polypeptides varied in size from 9 to 28 kDa, and analysis revealed no significant similarity either

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among several auxin-responsive genes or to other proteins present in data bases. The only exception is a recently isolated auxin-responsive cDNA from tobacco mesophyll protoplasts, the par gene, which shows 23% similarity to a protein of Escherichia coli that binds to E. coli RNA polymerase during the stringent starvation response (Takahashi et al., 1989). Although the functional significance of this finding is not clear, the presence of motifs that are conserved in heat-shock proteins may point to a requirement of these motifs to stabilize the par-encoded protein under conditions of cellular stress (T. Nagata, personal communication). The function(s) of the par protein is not yet known, but it is important to note that expression in Nicotiana tabacum of the par cDNA in antisense orientation interferes with growth, resulting in a reduction of apical dominance and in dwarfism (T. Nagata, personal communication). Attempts to define essential cisacting regulatory elements for hormone activation by analysis of various promoter sequences in auxin-responsive genes revealed several motifs [e.g., TGATAAAAG (Ainley et al., 1988)], located with comparable spacing in several of the genes identified. Deletion analysis of the par gene 5’-flanking sequences, using GUS activity in tobacco protoplasts as a reporter, showed an auxin-responsive region in a 111-bp fragment (Takahashi and Nagata, 1990). Deletion analysis of the nos promoter, which is differentially regulated by both wounding and auxin, revealed an additional 10-Z DNA element, GCACATACGT, which is also present in other auxin-regulated promoters and is thought to be a key element in auxin or wound response (An et al., 1990). Further analysis is required to demonstrate the relevance of these boxes. In particular the auxin responsiveness following transfer of these elements to plant promoters such as the minimal 35 S CaMV promoter has not yet been demonstrated. Altogether, the molecular studies described clearly demonstrate the presence of several mRNAs which are specifically and rapidly regulated by auxins. Regulation has been demonstrated at the level of transcription, using isolated nuclei from soybean (Guilfoyle, 1986; Theologis, 1986; Hagen, 1987, 1989). In uitro run-on assays showed that the rate of transcription of the pGH3 gene was increased within 5 min of auxin application. The pGHl and pGH4 genes from soybean, the pIaaa415 and plaa6 mRNAs from pea, and the SAUR gene transcripts showed a dosedependent induction by auxins, but unexpectedly also showed induction by inhibitors of protein synthesis (Theologis et al., 1985; Hagen and Guilfoyle, 1985; Guilfoyle et d., 1990). It has been argued that either the promoters of these genes or stability of their transcripts are under control of a protein that is rapidly turned over (Hagen, 1989). It has been stated that stabilization and accumulation of these mRNAs are normally prevented by a negative control mechanism active only in the absence of

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auxin (Theologis, 1989). In light of these results, it is intriguing to find that several rub gene products contain DNA binding domains or ribonuclear protein consensus sequence domains (Mundy and Chua, 1988; Bandzilius et al., 1989). One of these proteins encoded by the pMAH9 gene from maize, has been shown to bind single-stranded DNA, poly(rU), and poly(rG) at high salt concentrations (M. Pages, personal communication). These results suggest that at least some of the genes activated by abscisic acid may encode proteins that bind to RNA and may be capable of controlling developmental events at the posttranscriptional level. Although much work remains to be done, these exciting findings may point to a role for RNA-binding proteins in the stabilization of mRNAs encoded by phytohormone-activated genes. IX. Current Models and Perspectives for Phytohormone Action

In the preceding section I argued that all multicellular organisms require regulatory networks to control cell divison and organize cells into tissues. Hormonal signaling systems are essential elements for cellular communication. In responses to a molecular signal, the first step must be an interaction of the ligand with a receptor molecule, and in this regard proteins that bind the ligand acquire particular significance. In the past two decades sensitive ligand-binding techniques, including photoaffinity labeling, have proved successful in the purification and molecular characterization of many hormone and drug receptors from a variety of organisms. This led to the identification of the first G-coupled receptors, now believed to be a ubiquitous family of proteins involved in sensing a great variety of signals. Although this success is attributable in part to the development of new biochemical technologies, an additional factor of considerable importance was the close functional coupling of these receptors to well-defined functional responses, for example, the coupling of G-coupled receptors to GTP-binding regulatory proteins (Lefkowitz et al., 1983; Dohlman et al., 1987). A. Mutants Are Useful Tools t o Analyze Phytohormone Action

The lack of suitable functional assays for putative phytohormone receptors may explain why it is that, although work toward their identification began approximately when characterization of G-coupled receptors was started, we are only now beginning to discover the functions and molecular details of the proteins which have been found to bind phytohormones.

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Although plant life depends on correct interpretation of a large number of environmental and internal stimuli, it is remarkable that our present knowledge of phytohormone receptor proteins is still limited. One of the reasons has been the difficulty in devising efficient schemes for genetic selection of plant receptor mutants. This is due largely to the lack of. detailed knowledge of functional coupling between receptors and physiological responses, making mutant phenotypes difficult to predict. Precise in uitro design and in uiuo expression of dominant gain- or loss-of-function mutations in animal receptor genes can interfere with crucial signaling steps and thus cause defects severely affecting animal development. By analogy we would predict pleiotropic growth alterations for mutations interfering with central perception steps in plant hormone signaling. In the absence of precise knowledge of phytohormone perception-deficient phenotypes, selection for dramatic growth alterations represents the only criterion for isolation of putative phytohormone receptor mutants. However, any changes in central physiological pathways affecting phytohormone metabolism may result in related phenotypes. Hence, on the basis of current knowledge, selection for increased resistance to growth-inhibiting or, alternatively, growth-promoting levels of phytohormones provides the only applicable selection criterion, assuming that the corresponding hormone is essential for growth and that the rnutation will affect a step along the response pathway without leading to lethality. Using resistance to toxic concentrations of various plant hormones, several mutants resistant to abscisic acid, ethylene, or auxin analogs such as naphthylacetic acid or 2,4-dichlorophenoxyaceticacid were isolated from A . rhaliana (Mirza et al., 1984; Koorneef et al., 1984; Estelle and Somerville, 1987; Bleeker et al., 1988; Wilson et al., 1990; Guzman and Ecker, 1990). The etr locus, for example, contains a dominant mutation, resulting in elimination of several responses elicited by ethylene, including inhibition of cell elongation, promotion of seed germination, enhancement of peroxidase activity, acceleration of leaf senescence, and feedback suppression of ethylene (Bleeker et al., 1988). Analysis of saturable binding of [14C]ethylene to membranes isolated from leaf tissues of the etr mutants resulted in a 5-fold reduction in binding compared to the wild-type plant, indicating that the etr mutation may directly affect the primary receptor for this hormone. The err gene has been mapped on chromosome I from A . thaliana, thus allowing cloning by “chromosome walking” from a restriction fragment-length polymorphism fragment nearby. Using the transfer of ethylene resistance to wild-type Arabidopsis plants by gene transfer as an assay, overlapping cosmids were identified that conferred ethylene insensitivity to the wild type, suggesting that the err gene lies within a 23-kb fragment of genomic DNA. Most interestingly, structural analysis of one of the cDNA clones identified within this fragment indicates an open

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reading frame encoding a putative transmembrane protein that shares sequence similarity with a serine-type protein kinase in a subdomain that is most likely located at the cytosolic face of the plasma membrane (A. B. Bleeker, personal communication). This putative transmembrane receptor has a leucine-rich domain located in the extracellularly directed domain that is similar to those found in DNA-binding proteins. However, it was found that the gene isolated from wild-type Arabidopsis kinase could not complement the etr mutation. Mutants resistant to toxic concentrations of auxins were shown to display pleiotropic morphological changes in roots, leaves, and flowers (Estelle and Somerville, 1987). The auxin-resistant axr2 locus has been mapped to chromosome 3 and confers resistance not only to auxin, but also to other growth regulators, such as abscisic acid and ethylene (Wilson et al., 1990). These mutations may reside in a hormone receptor gene; however, it is equally conceivable that specific hormone transport systems or other cellular components of the response pathway common to several phytohormones are affected. This possibility is indicated by the phenotype of a tryptophan-requiring mutant from A . thaliana, defective in anthranilate phosphoribosyltransferase,which accumulates anthranilic acid derivatives, interfering with IAA biosynthesis (Last and Fink, 1988). Surprisingly, the observed morphological defects, including slow growth, small crinkled leaves, and reduced apical dominance resulting in bushy appearance, are similar to the auxin-resistant locus axrl (Estelle and Somerville, 1987). In addition the search for auxin auxotrophs has resulted in the identification of temperature-sensitive mutants of Hyoscyamus muticus and Nicotiana plumbaginifolia with conditional lethal phenotypes and apparently defective in IAA metabolism. Nevertheless, it is without doubt that the molecular characterization of genes identified by these various mutations will significantly contribute to our understanding of plant hormone perception. Genetically defined alterations in hormone sensitivity now provide an excellent bioassay for the functional relevance of biochemically defined hormone-binding proteins that are putative receptors. This will also help to reveal the various steps in the cascade of reactions initiated by phytohormones such as auxins. B. Phytohormone Binding Sites Are Tools in the Search for Phytohormone Receptors

1. Auxin Binding Sites Initially, the biochemical search for auxin receptors was based on analysis of high-specificity binding of radiolabeled auxins to plant membrane fractions. This approach led to the identification of three types of auxin binding

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sites. In membranes from maize, auxin binding sites were found to be located at the endoplasmic reticulum (ER), the tonoplast, and the plasmalemma and were found to have different binding affinities for different auxins (Dohrmann et al., 1978). These sites were defined in purely operational terms by auxin binding activities, and, for most, any functional correlation with known auxin responses is still lacking. Therefore, major efforts have been directed toward identification of the proteins active at these sites, as well as of their corresponding genes. It is expected that biochemical and genetic analyses will allow functions to be assigned to these binding sites. Considering the problems associated with the use of auxin binding assays, it is not surprising that it proved extremely difficult to physically and biochemically characterize these operationally defined binding proteins (for a discussion of the problems, see Venis, 1984, 1985). Photoaffinity techniques, with proven advantages over equilibrium ligand binding assays, were used in identifying auxin-binding proteins. Indeed, 5-azid0-[7-~H]-IAA(azido-IAA) could be demonstrated to be an active auxin in bioassays and to exhibit a polar transport rate equivalent to that of free IAA (Melhado er al., 1981; Jones et al., 1984; Hicks et al., 1989a). After optimization of reaction conditions, it was finally possible to demonstrate specific changes in putative auxin-binding proteins in the diageotropic (dgt) mutant of tomato (Hicks et al.,, 1989b). This mutant carries a spontaneous recessive mutation at a single locus, expresses auxin insensitivity, diagravitropic shoot growth, abnormal vascular tissue, altered leaf morphology, and blockage of lateral root branching (Zobel, 1972,1973). This correlates with the reduced responsiveness of elongation growth to applied auxin, suggesting a lesion in the primary perception of the phytohormone, similar to the defects proposed previously for the auxin-resistant mutants of A. thaliana (Bradford and Yang, 1980; Fujino et al., 1988). The photoaffinity label azido-IAA detected proteins of 40-42 kDa in plasma membranes from stems of wild-type tomato plants which were almost undetectable in stem tissue of the dgt mutant. This finding raises the prospect that, for the first time, phenotypic growth alterations can be directly related to biochemical properties of auxin-binding proteins (Hicks et al., 1989b).Application of this technology to a variety of plant tissues, including plasma membranes from zucchini (Cucurbita p e p ) or maize (Zea mays), resulted in the identification of proteins ranging from 20 to 60 kDa, depending on the tissue analyzed (Feldwisch et al., 1991;Campos et al., 1991).The 23-kDa protein identified in plasma membranes from maize coleoptiles apparently represents membrane protein which might be involved in the primary perception of IAA (Feldwisch et a f . , 1991). It can be expected that, with the help of various related auxin-specific photoaffinity ligands, it will soon be possible to identify the various auxin-binding proteins that have been proposed on the

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basis of ligand binding assays (Hertel, 1987). We can further expect that dissection of the various auxin-specific responses of cells will then be possible with the help of current molecular techniques. Of the many auxin binding sites identified, a membrane-associated binding site from maize has been the most thoroughly studied. In maize and barnyard grass, but not in other plants, it is the most abundant auxinbinding protein, and it can be released from cellular fractions highly enriched for ER (Venis, 1977). Arguments in favor of this site being a putative receptor were the specificity of binding of various auxin analogs, changes in site I binding activity correlating with physiological events (Walton and Ray, 1981), and its absence in several auxin-insensitive mutants (Narayanan et d.,1981;Poovaiah, 1982).Additionally, alterations in the degree of site I-specific auxin binding correlated with the inhibition of growth of maize mesocotyls by high-irradiance red light and with growth alterations in artichoke tubers and tobacco cell cultures (Trewavas, 1980; Walton and Ray, 1981; Vreugdenhill et al., 1981). Attempts to purify the ER-associated auxin binding sites were initially hampered by technical difficulties associated with low concentration of pH lability of ligand binding (for a discussion see Venis, 1984, 1985). Refinement of techniques, however, resulted in the purification of three related auxin-binding proteins from maize coleoptiles (Lobler and Klambt, 1985; Shimomura et al., 1986; Venis, 1987; Napier et al., 1988; Hesse et al., 1989; Palme et al., 1990b).The major protein had an apparent molecular weight of 22 kDa. The primary structure was independently deduced from different cDNAs isolated from maize (Hesse et al., 1989; Inohara et al., 1989; Tillmann et al., 1989). Meanwhile, additional members of this family were isolated from maize as well as A . thaliana (Hesse et al., 1989; Palme et al., 1991d). All proteins identified contain a N-terminal hydrophobic signal sequence of 38-41 amino acids that appears to be responsible for the uptake of these proteins into maize rnicrosomes (Fig. 5). The biochemical experiments reported by Hesse et al. (1989) and by Palme et al. (1990b) clearly established that the major auxin-binding protein is a luminal component of the ER. This observation is confirmed by the absence of hydrophobic sequences responsible for membrane insertion in all members of this auxin binding family identified so far. Furthermore, the presence of a C-terminal tetrapeptide sequence, -Lys-Asp-Glu-Leu (-KDEL), previously reported for other proteins to be a retention signal for the ER, represents further evidence for the retention of the auxinbinding protein in the lumen of the ER. The signal K/HDEL is now well recognized in eukaryotic organisms to be responsible for the retrieval of proteins from a post-ER salvage compartment by the active participation of a receptor system, and it has an apparently similar function in plants. For example, using a chimeric reporter gene with an N-terminal leader for ER uptake and a C-terminal-KDEL sequence, Iturriaga et al. (1989) were

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FIG. 5 Schematic representation of the structure and signal elements of the auxin-binding protein I from Zea mays (L.).

able to demonstrate in N . tabacum active retention of the reporter gene in the lumen of the ER only in the presence of the -KDEL sequence.

2. Abscisic Acid Binding Sites Attempts at identification of primary elements in the perception of abscisic acid in the plasma membrane resulted in affinity labeling of several proteins with molecular sizes in the range of 14-20 kDa (Hornberg and Weiler, 1984). Photoaffinity labeling was achieved by using a photoreactive a-/I unsaturated ketone moiety in radiolabeled abscisic acid as the active group. Several abscisic acid-binding proteins could be identified with this compound, but due to the limited specific radioactivity of the labeled ligand, nonspecific binding between the hydrophobic abscisic acid and plasma membrane proteins after photolysis was difficult to assess.

3. Gibberellic Acid Binding Sites Similar approaches were successful in the characterization of receptors from animals, and these techniques are being used to identify gibberellin

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receptors. Monoclonal antiidiotype antibodies directed against gibberellic acid as well as iodinated azido-gibberellic acid derivatives are presently in use for the analysis of plasma membrane proteins from barley aleurone cells (R. Hooley, personal communication). Furthermore, derivatives of gibberellic acid unable to pass the plasma membrane were shown to retain the ability to activate transcription of gibberellic acid-responsive genes (R. Hooley , personal communication). This work potentially clears the way for the eventual identification of elements responsible for regulating the expression of gibberellic acid-responsive genes. It can be hoped that work begun in several laboratories to delineate the elements involved in the primary perception of phytohormones will result in the molecular characterization of phytohormone receptors.

C. Environmental Factors Influence Phytohormone Action

1. Phytohormone Action and Light Phytohormones and light have been frequently observed to influence similar physiological processes. Light is an important environmental factor for plants, as it provides energy for photosynthesis and also influences plant differentiation. Light influences seedling growth, plastid development, primordia formation, flower induction, and dormancy (Kronenberg and Kendrick, 1986; Colbert, 1988; Silverthorne and Tobin, 1987; Nagy et al., 1988). Over the years phytochrome has become the best-characterized plant photoreceptor. The phytochrome protein, 116 kDa in size, is a soluble chromoprotein which induces morphogenetic responses due to reversible conformational changes induced by visible light (Pratt, 1982). Rapid interconversion of phytochrome between an active state, which absorbs far-red light, and a ground state, which absorbs red light, allows plant cells to perceive different wavelengths of light and mediate many light-dependent processes, in some instances by changing the transcriptional activity of the genes (Kuhlemeyer e? al., 1989). Several mutants from A . thaliana with abnormal photoresponsive characteristics have been instrumental in the analysis of phytochrome action. Long hypocotyl mutants (hy) were isolated from A . thaliana on the basis of increased hypocotyl growth relative to wild type when grown in high-photon-flux white light (Koorneef et al., 1980; Chory e? al., 1989). Of the six complementation groups isolated mutants corresponding to loci h y l , hy2, hy3, and hy6 display pleiotropic effects in adult plants, apparently caused by either structural changes or reduced concentrations of photoreversible phytochrome. In contrast hy4 and hy5 plants express photoreversible phytochrome at an apparently wild-type level (Parks et al., 1989).

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Moreover, hy4 and hy5 mutants appear to be normal in the adult state. In hy4 plants the phenotype of increased hypocotyl growth under strong illumination is transient and confined to a limited period during early development. It has been proposed that this transient hypocotyl elongation phenotype, in the presence of excess light and apparently normal phytochrome, most likely results from a modification in the transduction chain linking photoperception to morphogenetic responses (Parks et al., 1989). In light of this interpretation, it is particularly interesting that a gene from A. thaliuna, encoding a KDEL signal containing auxin-binding protein, maps to a restriction fragment-length polymorphism within 1.1 cM of the hy4 locus on chromosome 4 (Palme et al., 1991~). Phytochrome, abundant in etiolated tissue, is thought to be responsible for the switch from growth by elongation to photomorphogenesis. Two types of phytochrome responses can be defined by the amount (i.e., fluence) of red light at which they are initiated and by the ability of far-red light to reverse them. Of importance are the fully reversible low-fluence (LF) responses to far-red light, whereas the far-red light irreversible very low-fluence (VLF) effects have a much lower threshold, allowing even dim green light to induce VLF responses. Recently, a series of elegant experiments has shown that the hormonal status of coleoptile sections from oats and maize has a profound influence on red light-induced elongation growth (Shinkle and Briggs, 1984a,b, 1985; Shinkle, 1986). These experiments demonstrated a shift in the fluence response of maize coleoptile from LF to VLF growth in the presence of 6 p M IAA. This indicates an auxin-induced sensitization of elongation growth to red light, most likely as a consequence of an altered transduction of signals initiated by the far-red lightabsorbing form of phytochrome (Pf,). Although we do not fully understand the physiological events in this response cascade, there may be a functional link between transduction of the Pfr signal and auxin-induced changes in gene expression, Auxin application has been observed to induce a dramatic increase in steady levels of otherwise light-regulated transcripts [e.g., 14-fold induction of ribulose-l,5-bisphosphatecarboxylase/oxygenase (RbcS) and 20-30% induction of light harvesting chlorophyll a/b binding protein (Cab) mRNA ( J . C. Watson, personal communication; Kaufman et al., 1985; Watson, 1989)l. Notably, all transcripts analyzed exhibiting a blue light response are also induced by exogenously applied auxins. In light of these observations, it is particularly intriguing to find that an auxin-binding protein, assumed to be a receptor for this phytohormone, changes its binding characteristics for auxins in response to red light (Walton and Ray, 1981). This observation raised the question as to whether this putative auxin receptor protein may interfere in some way with the processes by which light controls gene expression. The structures of several isolated phytochrome genes have helped to under-

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stand some of the molecular properties of phytochrome (Quail, 19841, yet the molecular mechanisms of phytochrome action, especially the link between the cellular signaling network and alterations in gene expression, remains elusive. Recent studies in plants have begun to reveal the signal transduction mechanisms that connect light and hormonal perception to changes in gene expression. The involvement of calcium in phytochromemediated signal transduction was proposed some years ago after demonstration of the ability of phytochrome to modulate calcium flux in preparations of plant mitochondria (Roux et al., 1981). The role of calcium in mediating phytochrome-regulated processes was documented for the light-induced germination of fern spores (Wayne and Hepler, 1984, 1985), light-induced chloroplast rotation in Mougoetia (Dreyer and Weisenseel, 1979; Serlin and Roux, 1984), light-induced activation of calcium fluxes in corn protoplasts (Das and Sopory , 1985),and light-mediated modulation of protein phosphorylation followed by changes in gene expression (Datta et al., 1985; Gallagher et al., 1988; Lam et al., 1989).

2. Phytohormone Action and Calcium Extensive evidence, including Ca2+ mobilization studies, points to the central importance in plants of a signal transduction pathway involving both calcium- and phosphatidylinositol-derived second messengers, which may connect light and other environmental signals to the regulation of gene transcription (Rincon and Boss, 1987; Morse et al., 1987; Lawton et al., 1989; Palme et al., 1989a). It should also be stressed here that induction of calcium fluxes in plant cells is closely linked to the perception of various phytohormones, including cytokinin, abscisic acid, and auxin (Saunders and Hepler, 1981; Ettlinger and Lehle, 1988; McAinsh et al., 1990). The presence and relevance of second messengers such as 1,4,5-inositol triphosphate or 1,3,4,5-tetrakisphosphateand their rapid production in response to phytohormonal or light stimuli point to a fundamental role for these compounds in plant signaling (Boss, 1989; Ettlinger and Lehle, 1988; Gilroy et al., 1990; F. Hesse and K . Palme, unpublished observations). Perhaps the most important of their potential roles is the regulation of ion channels. Ion channels are not only important for solute transport, but apparently are also crucial elements in hormonal signaling. Technical breakthroughs have made possible the molecular characterization of ion channels. Recent results demonstrate the fundamental importance of ion channels in the survival of plant cells by adaptation to fluctuations in osmolarity in order to maintain positive turgor pressure (Schroeder and Hedrich, 1989). Fluxes of either mono- or divalent ions such as K + , Naf, or Ca2+are thought to serve as regulatory signals either by interacting with specific binding sites in regulatory proteins or by evoking responses simply by virtue of their effect on intracellular ionic strength. Even small changes

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in ion strength are supposed to modify protein conformation and thereby alter protein activity. Models involving such simple signals could account for a variety of responses that would require a minimal number of sensory systems and be able to modulate a variety of diverse intracellular functions, including effects on transcription. D. Perspective

As described in the previous section, it seems possible to dissect phytohormone signaling pathways with the use of molecular techniques. The elucidation of the pathways along which a particular phytohormone signal is transmitted from the membrane to the nucleus not only requires the identification of the primary perception elements for these phytohormones, most likely receptors, but also other elements necessary for amplification of the primary signals and the production of second messengers and various proteins responsible for the cellular responses. In the following section I discuss some plant proteins that share highly conserved primary structure motifs with other eukaryotic signaling elements.

X. Functional, Molecular, and Structural Analyses of Plant-Specific Modules for the Recognition of Second Messengers A. Guanine Nucleotide-Binding Regulatory Proteins

Guanine nucleotide-binding reguatory proteins (G proteins) are used to control a number of basic cellular functions. All G proteins share a unique GTP-binding consensus sequence which is found in a wide variety of proteins performing diverse functions and having a high affinity for GTP. Members of this family direct ribosomal protein synthesis, mediate transmembrane signaling by hormones and light, translocate nascent proteins into the lumen of the ER, guide vesicular traffic within cells, and control differentiation and cellular proliferation. It is expected that members of this apparently ubiquitous family of proteins will be shown to play an important role in plant growth control. Preliminary studies using guanine nucleotide binding assays indicated the presence of G proteins in lower eukaryotes such as Neurospora crassa as well as in plants (Hasunuma and Funadera, 1987; Hasunuma et al., 1987c; Jacobs et al., 1987; Drobak et al., 1988). Using biochemical assay, GTP binding and hydrolysis were detected in membrane fractions from monocotyledonous plants such

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as Lemna paucicostata (Hasunuma and Funadera, 1987; Hasunuma et al., 1987a), as well as dicotyledonous plants such as N. tabacum, Pisum sativum, and A. thaliana (Hasunuma er al., 1987b; Hasunuma and Funadera, 1987; Blum et al., 1989). Preliminary data indicate a phytochrome-mediated regulation of GTP-binding in Lemna (Hasunuma et al., 1987a). This may point to the use of G proteins in light perception or photoreceptor activation in plants, as in animals (Bennett and Dupont, 1985; Hasunuma et al., 1987a). This correlates well with the recent finding that light-stimulated GTPase activity is inhibited after the pretreatment of thylakoid membranes with cholera toxin (Millner and Robinson, 1989). In addition guanine nucleotides apparently regulate a protein kinase of the light-harvesting complex in pea thylakoids (Millner, 1987). The biochemical data obtained thus far provide persuasive evidence for the presence and functional importance of G proteins in plants. Here, analysis concentrates on ras proteins involved in the regulation of a wide variety of biological processes, including the control of intracellular trafficking. To search for ras-related genes in plants, primary structure motifs common to ras proteins were used for the construction of oligonucleotide probes. Direct sequence comparison of recently isolated members of the ras supergene family by multiple alignments of amino acids of the GTP binding domain is presented in Fig. 6. Comparison of amino acid sequences representing the GTP-binding domain of currently known members of the ras supergene family points to three consensus elements: GxxxxGKSsxl, DTAGQE, and lxg/NKxDL. As indicated by the threedimensional structure of the c-Ha-ras p21 oncogene (De Vos et al., 1988; Pai et al., 1989), the first two domains are involved in binding the phosphate moiety of the GTP, whereas elements located further downstream are involved in determining guanine nucleotide specificity. More extensive amino acid sequence conservation is detectable only in subfamilies. Using probes corresponding to amino aicds 5-20,53-62, and 107-1 18, a series of cDNAs and genomic clones was isolated from Z. mays and A. thaliana (Palme et al., 1989a,b, 1990a, 1991a,b; I. Moore and K. Palme, unpublished observations). Several cDNAs coding for members of the rasrelated ypt subfamily were isolated. yptml and yptm2 share, respectively, 73% and 76% similarity with YPTl from Saccharomyces cerevisiae and 79% and 85% with ypt from the mouse. Similarity to the Ha-ras or Ki-ras protein is 35% and 26% and restricted to amino acids in the GTP binding domain. The homologies are localized in four main blocks, which have all been implicated in the binding of GDP or GTP (Pai et al., 1989) (Fig. 7). The spacing of the regions of highest identity as well as the hydrophobicity demonstrates strong functional similarity for GTP binding and GTP hydrolysis within this family. Besides these blocks of identity, several additional amino acids are conserved as well. Amino acids 32-40, which

1 1 1 1 1 1 1 1 1 1 1 1 1

Pl ( - _ - - - - -P _ _2 1 I - - - - $3 ---I - - -a1 --- -- I I ---------KLVWGAGGVGKSALTIQLIQNHEWEYDPTIEDSYR-KQWIDGETCLLDI ---------KIVVVGGGGVGKSALTIQFIQSYFVDEYDPTIEDSYR-KQWIDDKVSILDI ---------KLVWGGGGVGKS€LTIQLTQSHFVDEYDPTIEDSYR-KQWIDDEVSILDI ----- ---- KLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENY-VADIEVDGKQVELAL ---------KLLLIGDSGVGKSCLLLRFADDTYTESYISTIGVDFKIRTIELDGKTIKLQI

____---__ KYIIIGDTGVGKSCLLLQFTDKWQPVHDLTMGVEFGMU4ITIDGKQIKLQI ___----__ KILIIGNSSVGKTSFLFRYADDSFTPAFVSTVGIDFKVKTIYRNQI _ _ _ _ - - KLLIIGNSSVGKTSFLLRYADDTFTPAFVSTVGIDFKVKTVYRHEKRVKLQI -__ ___----__ KFLVIGNAGTGKSCLLHQFIEKKFKDDSNHTIGVEFGQKIINVGGKYVKLQI ___----__ KLVLLGESAVGKSSLVLRFVKGQFHEFOESTIGAFZLTQTVCLDDTTVKFEI --------- KLVFLGEQSVGKTSLITRFMYDSFDNTYQATIGIDFLSKTMYLEDRTVRLQL

--------- KILLIGDSGVGKSCLLVRfVEDKFNPSFITTIGIDFKIKTVDINGKKVKLQL --------- KLLLIGNSGVGKSCLLLRFSDDTYTNDYISTIGVDFKIKTVELDGKTVKLQI K

1 1

52 52 52 52 53 53 53 53 53 53 53 53 53 62 62

G

gvGKs

f

T

f

RAs2 rho rabl rab2 rab3a rab3b rabl rab5 rab6 SECl YPTl

1

MSNEFDYLFKLLLIGDSSVGKSCFLLRFADDSYVDSYISTIGVDFKIRlVEXEGKTVKLQI

MNPEYDYLFKLLLIGDSGVGKSCLLLRFADDSYLDSYISTIGVDFKIRTVEQDGKTIKLQI

a3 ---___-----__---

YPTml YPTm2

1 I P5 I LDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHQ-YREQIKRVWSDDVPMVLVG

--I

I

- - -a-*- - - I

P4

I----- I

----

Ha-ras LDTAGQEEYSAMREQYMRTGEGFLLVYSVTSRNSFDELLS-YYQQIQRVKDSDYIPVVWG RAS 1 LDTAGQEEYSAMREQYHRNGEGFLLVYSITSKSSLDELMT-YYQQILRVKDTDYVPIVWG RAs2 WDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVFUiF--CPNVPIILVG rho WDTAGQERFRTITSSYYRGAHGIIVVYDVTDQESFNNVKQ-WLQEIDRYA-SENVNKLLVG rabl WDTAGQESFRSITRSYYRGAAGALLVYDITRRDTFNHLTT-WLEDARQHS-NSNMVIMLIG rab2 WDTAGQERYRTITTAYYRGAMGFILMYDITNEESFNAVQD-WSTQIKTYS-WDNAQ~LVG rab3a WDTAGQERYRTITTAYYRGAMGFILMYDITNEESFNAVQD-WATQIKTYS-WDNAQVILVG rab3b WDTAGQERFRSVTTSYYRGAA~LVYDITSRETYNALTN-WLTDARHLA-SQNIVIILCG rabl WDTAGQEGYHSLAPMYYRGAQAAIVWDITNEESFA"-WVKELQRQA-SPNIVIALSG rab5 W D T A G Q E R F R S L I P S Y I ~ S ~ A ~ D I T ~ S F Q Q T T K - W I D D ~ T E R - G S D V I I Mrab6 LVG WDTAGQERFRTITTAYYRGAMGIILVYDVTDERTFTNIKQ-WFKTVNEHA-NDEAQLLLVG SECl WDTAGQERFRTITSSYYRGSHGIIIVYDVTDQESMGVKn-WLQEIDRYA-TSTVLKLLVG YPTl wDTAGQEr Yr G W D T A G Q E R F R T I T S S Y Y R G A H G I I I V Y D I T D ~ S F N N V K Q - W L D E I D R Y A N - D S ~ V G YPTml WDTAGQERFRTITSSYYRGAHGIIIVYDVTDQESFNNVKQ-WLNEIDRYAS-DNVNKLLVG YPTm2

a5

a4 - P6 1 I--------I 1-1 I m C D W T - - - - - - - - - - - - - VESRQAQDLARSYGI-PYIETSAKTRQGVEDAFYTLVR- Ha-ras NXLDWNERQ-----------VSYEDGLRLAKQLNA-PFLETSAKQAINVDEAFYSLIR- PAS 1 mSDLENEKQ------------ VSYQDGLNMAKQMNA-PFLETSAKQAINVEEAFYTLAR- RAs2

- --- --

112 112 112 111 112 112 112 112 112

Ha-ras RAS 1

NKKDLRNDESTKRELHlCMKQEPVRPEDGRKINAYSYLECSAKTI(E(3VRDVFETATRmCDLTTKJ,&l------------VDYTTAKEFADSLGI-PFLETSAIWEmQSFMTXAA-

rho

121 121

rabl rab2 rab3a rab3b rabl m m - VDFQEAQSYADDNSL-LFMETSAKTSWIFMAIAK- rab5 mTD-mQ-----------VSIEEGERKAI(ELNV-MFIETSAKAGYNVKQLFRRVAA- rab6 mSD-mTRV------------ VTADQGEALAI(ELG1-PFIESSAKNDDNVNEIFFTLAK- SECl M(CDtW)=V------------ VEYDVAKEFADANKM-PFLETSALDSTNVEDAFLTMAR- YPTl NXcD V A fESAk V F m C D M M - - - - - - - - - - - -VDTSVAQAYAQEVGI-PFLETSAKESINVEEAFLAMSAA YPTml mSDLTAM(V------------VATETAKAFADEMGI -PFMETSA?CNATNVQQAFMAMAAS YPTm2

169 169

IKKSKAGSQAALERKPSNVVQMKGRPIQQEQQKSSRCCST IKDRMASQPAAANARPATVQIR-GQPVNQKTS----CCSS

112 112

112 112

m S D L E S N - - - - - - - - - - - - VKKEEGEAFAREHGL-IFMETSAKTASNVEEAFINTAKmCDmDERV------------VSSERGRQLADHLGF-EFFEASAKDNINVKQTFERLVDM(CDmEERV------------ VPTEKGQLLAEQLGF-DFFEASAKENISVRQAFERLVDNIuu)LDADm------------VTFLIDSRFAQENEL-MFLETSALTGENVEEAFMQCAR-

---- - --- - -

YPTml YPTm2

FIG. 6 Alignment of the amino acid sequences of the conserved GTP binding domain of the ras protein family. All amino acid sequences are from the PIR or European Molecular Biology Laboratory sequence data base. Dashes indicate gaps introduced for optimal alignment. Amino acids are denoted using single-letter code. The consensus line has entirely conserved amino acids in upper case and less well-conserved amino acids in lower case. Secondary structure motifs (H, helix: E. extended p structure) and loop names (LI-L9) are from De Vos e r a / . (1988) and Pai et a / . (1989).

258 c-Ha-raa Ara Mat.

YPTI YPT3

Human YPTI YPT? ne1a Y P T I Y e a ~ r YPTI 'touse Y P T I 36,.

MSYAV L P K Y I I I C D T GVCRSCLLI.0 FTDKRFOPYH D L T I C V E r G A RP!T:DhXPI RT:TIDhX?I M S I A Y L F R Y I i I C J ? GVCKSCL.L:.Q FTDXRPOPVH J:.?!CVF?GA U Y A I L F X Y l l l C D T GVCXSCLLLQ FTnKRrOPVH DLTLGVLPGA r v l l ? l . X K Q l HhPLY3I ISWLFDI MNSEVDY MSSlhPFIJY

LIKLLL:GDS LFKLLLICDS LFKLLLlGhS LFXI.LLIG3S

LOO

I

c-Hd-~aB

YPTl UOIS YPTI Human Y P T l

GVCLSCLLLR SVCXSCFLLR GVGKSCLLLR LVGXSZLLLR

FAD3SYL.DS1 FACDSWDSV FSDCTITNDY FAC?tVlESY

116

I

113

I

NLQIUMACQ USFRSI?RFI Y R C M C N . 1 ~ XLCIUDTAC.3 CSFRSITRSI Y R G M C N l V YL.QlYDTACQ ESPRSITR4V I R G M G A L L V

ERPRTITSSI Y R G W G I I I V :STIT.VDPRI R Y E V E G K N I L C I h D T A G Q ERFRTITSSY Y R Z W C L I I V ISTISLDFII X T V F L K X N Xl.ClWUTAGQ E R F P T I T S S I Y R C S 8 i C I I I V !%T!caVDF%j R T ! E - L X X T I XtQIdDTIGQ E P F R T I T S S I IU::AdZI I W

I b T l C V D I ~ IR N E O C G K T I KLOI.IMAGQ

125

I

I46 IS0

I

I

FAlNNTXSFE DHIQYREQIR RVKDSDDVPM VLVGNKCD1.A ARTVESRQAO D L N I S Y C I P YIETSAKTRQ GVLDAFYTLV REIRQHKLRK

YDITRRETF. N H U C Y L I D I RQHUIANXTL RLlGWXCUlh YDITRRETP.

NHLASWLEDA E O H I H W M T V ULICNXCDI.5

YDITRRDTF. N H L T W L E D A R Q H S N S N W I ULIGNLSDLE

HRRAVSYREG EQFAKPHGLI F X E A S U T A Q NVEEAFIKTA ATIIYXS.DG HRWIVSYEEG EOFAKEHGLV FMEAS&KT&Q NVEEAFlKT& GTIYIXIQDG SRREVXKLEG E A F A R E H t L l FVETSAXTAS W E E I F I N T I K E I Y E K I Q E G

Wais YPTl Xais YPTL Y e b 3 ~ YPTL louie Y P T I

11s

I

IS9

tOLdl

CI V l S

c-"a-re..

LNPPDESGPG CUSCK

ara XDLS

VIDVSNESYG K I VGYGGP IG LSGGRCGSTS a..cccccc XPDVSNESNG I K V G Y I V P N S SGGGAGSSSO k...CGCCS V F D I N N W l G I K I G P Q H M T N A T H I G N W G QOAGGCC

YPTL YPTI Human Y P T l

(189,

(2101 (2LOI 1212)

u a ~ a VPT2 UOL. YPTI Yeaat V P T I Mouw Y P T I

FIG. 7 Amino acid sequence similarity of ypr-related proteins from Zea mays.

are probably located within the flexible effector loop, are thought to mediate interactions with corresponding effector proteins, and therefore, these amino acids are strongly diverged from those of classical ras proteins (Pai et al., 1989). Starting from amino acid 166, the sequence to the C terminus is very diverged within the whole ras family. According to the c-Hu-rus crystal structure, the long C-terminal domain forms an a-helix, thereby separating the catalytic domain from the membrane attachment domain (De Vos et al., 1988). This long helix domain may have an important function in signal transduction. The only amino acids strictly conserved within this domain are the cysteines at the C terminus. In all ras- and ras-related proteins these cysteines are needed for palmitic acid binding or farnesylation and subsequent membrane anchoring. The conservation of these amino acids in the genes isolated here argues in favor of a membrane localization of the plant proteins. In contrast to the ras proteins, no basic amino acids are found immediately upstream of the cysteine. Moreover, downstream we find mostly hydrophilic amino acids, in contrast to other members of the ras family. These differences may indicate variations in the specificity of plant acylases, catalyzing the addition of the membrane anchor. The identification of a series of YPTl-related genes from plants raises questions about their function. In eukaryotes ypt proteins seem to serve apparently basic cellular functions. Yeast cells lacking YPTl are defective in mitosis and display severe cytoskeletal lesions (Schmitt et al., 1988). In

EUKARYOTIC SIGNAL TRANSDUCTION

259

addition the YPTl protein is required for sporulation and for starvation response (Segev and Botstein, 1987). It is now becoming clear that, at specific steps in the secretory pathway, the traffic of proteins in membrane vesicles is regulated by G proteins. Genes encoding G proteins involved in the regulation of transport between early compartments of the secretory pathway have been cloned from yeast and from mammalian cells (Zahraoui et al., 1989). The rabl protein, for example, has a homology of approximately 70% to yeast YPTl (Santos and Nebreda, 1989). Members of this rapidly growing gene family of small G proteins may be key elements in the regulation of vesicular transport and delivery of proteins to the cell surface. To obtain an evolutionary relationship of ras- related proteins, multiple sequence alignments were prepared and distances were calculated based on these alignments, by counting mismatches and adding penalties for insertions or deletions (Fig. 8). Interestingly, different algorithms, such as the Fitch-Margoliash and parsimony approaches, resulted in very similar trees for the ras family (Palme et al., 1989b, 1991b). From this tree it was deduced that ras proteins fall roughly into two major classes. The first class is represented by mammalian and yeast ras proteins, whereas the second class is grouped around the ypt proteins, including SEC4 and the rab proteins. As can be seen from the alignments in Fig. 5 , the grouping of the sequences is not based primarily on the phylogeny of species, but, rather, proposes the dominance of functional relationships within the groups. The identification of ypt-related proteins in maize and homologs in other

FIG. 8 The unrooted phylogenetic tree of ras-related polypeptide sequences.

260

KLAUS PALME

plants as well, is of particular interest. Many plant tissues respond to growth stimuli such as auxins by cell elongation (Davies, 1987). The active expansion of membranes requires regulated secretion and correct targeting of secretory vesicles. Thus, in plant cells GTP hydrolysis could facilitate the vectorial flow of membrane material to the surface, as in yeast. This would explain the high level of expression of members of the ypt gene family in elongating maize coleoptile or suspension culture cells. The work described here illustrates the presence of members of the ras gene family in plants. Future work will aim to identify specific functions for this important and newly discovered plant gene family. To gain insights into the functions of these proteins, much challenging work remains to be performed, using approaches such as the analysis of mutants and of cDNAs in transgenic plants. B. Protein Kinases Covalent coupling of phosphate with the amino acid residues serine, threonine, and tyrosine is a common cellular mechanism for regulating the activity of proteins involved in a variety of physiological processes. These include metabolic pathways, membrane transport of ions and metabolites, gene transcription, and cell division (Edelman et al., 1987; Hunter and Cooper, 1985). Within the last few years protein phosphorylation has been shown to serve general regulatory functions in prokaryotic and eukaryotic cells (Cozzone, 1984; Cohen, 1985; Albright et al., 1989). Protein kinases have been analyzed in higher plants for the last decade, but their probable importance is only now starting to be acknowledged (Trewavas, 1976; Poovaiah and Reddy, 1987; Ranjeva and Boudet, 1987). From the numerous examples analyzed the following appear to be of considerable interest: (1) Many nuclear proteins from plants are reversibly phosphorylated, with specific transitions occurring during the Gz/M phase of the cell cycle, (2) hormones such as abscisic acid, gibberellic acid, or 2,4dichorophenoxyacetic acid have been shown to affect phosphorylation to various degrees, (3) calcium is an important element in promoting phosphorylation, and (4) phosphorylation occurs in all of the cellular compartments of plants, including organelles, vacuoles, and plastids (Ralph et al., 1972, 1974; Van’t Hof, 1974; Chapman et al., 1975; Van Loon et al., 1975; Trewavas, 1976c; Palme et al., 1987; Murray and Key, 1978; Weilgat and Kleczkowski, 1981; Schafer and Kahl, 1981; Melanson and Trewavas, 1982; Kahl and Schafer, 1984; Varnold and Morre, 1985; Datta et al., 1985; Veluthambi and Poovaiah, 1984a-c). However, although indicative, many of the effects observed up to now are only broadly descriptive and do not allow precise assignment of molecular elements to mechanistic chains. A number of reasons account for this

26 1

EUKARYOTIC SIGNAL TRANSDUCTION

KOH FIG. 9 In uitro phosphorylation of microsomal membranes from Nicoriana rabacum. (A) Leaves from N. rabacum plants cv. W38 (lanes a and c ) and SR1 (lanes b and d) were homogenized, and particulate fractions were isolated and analyzed after incubation with [y-’*P]ATP by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lanes a and b, growing tissue; lanes c and d, fuIly differentiated tissue. (B) The same gel after treatment with 1 MKOH at 55°C for 2 hr.

262

KLAUS PALME

general but insufficient knowledge; for example, (1) too many different plant systems have been studied, making comparisons and reproduction of results difficult, and (2) coupling of a stimulus with a change in phosphorylation has been demonstrated only in rare cases. Further difficulties may have arisen from the specific and rapid antagonistic action of phosphatases. Recently, however, efficient control of phosphatases, both by molecular techniques and by tumor-promoting drugs such as okadaic acid or calyculin A, has become feasible (Cohen et al., 1990). These drugs will be particularly important probes for studying protein kinases previously masked by the presence of contaminating phosphatases. This will allow analysis of novel regulatory phenomena in both animal and plant cells. Phosphorylation events observed during progression of the plant cell through its cell cycle encouraged John et al. (1989) to test by functional homology for the presence of a homolog of the cell division control gene cdc2 in plants. This gene is a key participant in cell division control in other eukaryotes and encodes a protein kinase. It was thought that a plant homolog of this gene might be involved in controlling progression through the cell cycle. Using several antibodies specific for the 34-kDa cdc2 gene product ( ~ 3 4 ~ ~from " ' ) Schizosaccharomyces pombe, homologs were also identified in Chlamydomonas, A. thaliana, and Avena sativa. All shared extensive immunological similarities within the perfectly conserved region of the cdc2-related protein kinase around amino acids 42-57 (Lorincz and Reed, 1984; Hindley and Phear, 1984).The results point to the presence of a protein structurally related to the mammalian and fungal cdc2 protein kinases in taxonomically remote organisms such as algae and angiosperms. Using a cdc2 mouse monoclonal antibody, Feiler and Jacobs (1990) were able to identify a protein from peas with an apparent molecular weight of 34 kDa, having histone H 1 kinase activity. Further homologs to the yeast cdc2/28 products have been identified in alfalfa (Medicago sativa). Amino acid sequences deduced from a particular cDNA clone demonstrated high homologies (in the range of 60%) to mammalian and yeast cdc2/28 proteins (Hirt et al., 1991). Variations in cdc2-related transcripts were observed between different organs of M. satiua. After addition of the auxin analog 2,4-dichorophenoxyacetic acid to hormone-depleted alfalfa, suspension cultures resulted in accumulation of cde2-related transcripts, coinciding with the expression of the cell cyclecontrolled major histone H3 transcript. This finding suggests a function for cdc2 homologs in plants as elements in a cascade initiated by auxin and leading to the activation of cellular proliferation. Recently, stimulus-response coupling of protein kinase-mediated phosphorylation of plasma membrane proteins has been observed. Syringomycin, for example, a peptide phytotoxin produced by Pseudomonas, acts as a virulence factor in various plant diseases, including brown leaf spot of

EUKARYOTIC SIGNAL TRANSDUCTION

263

beans, holcus spot disease of maize, and bacterial cancer of stone fruits (Gross and DeVay, 1977). Treatment of yeast or plant cells with syringomycin leads to an activation of plasma membrane-associated ATPase and subsequently to increases in membrane potential and cellular pH. Ca2+sensitive phosphorylation of several plasma membrane proteins from Beta vulgaris was detected in vitro assays in response to syringomycin (Bidwai and Takemoto, 1987). A direct activation of a protein kinase by the drug resulting in phosphorylation of the proton-pumping ATPase has been suggested. Interestingly, it could be demonstrated that signals such as oligosaccharides stimulate the phosphorylation of plasma membrane proteins from various plants (Farmer et al., 1989). Polygalacturonides isolated from tomato leaf pectic polysaccharide enhance phosphorylation of a 34-kDa membrane-associated polypeptide in both the tomato and the potato. Phosphorylation of additional proteins of various sizes was demonstrated in different cultivars. Together with reports from other laboratories (Blowers and Trewavas, 1988; Grab et al., 1989; Ebel et ul., 1989; Dietrich et al., 1990), the data suggest that detection of various chemicals inducing defense reactions may involve protein phosphorylation as a common mechanism for transduction of these signals. By analogy with animal cells, it can be expected that plants possess receptor protein kinases, which contain at least one domain spanning the plasma membrane, and are involved in the primary sensing of defense-related signals. So far, only limited progress has been made toward the characterization of membraneassociated protein kinases from plants. Most thoroughly characterized is a calcium-regulated low-molecular-weight protein kinase isolated from pea bud tissue. The enzyme, having a molecular weight of 18 kDa, is enriched in plasma membrane. The highly purified protein autophosphorylates on serine within seconds, and in addition it shows rapid turnover of the phosphate in the presence of ADP (Blowers et al., 1985; Blowers and Trewavas, 1987, 1988, 1989). The unusual kinetics, along with calcium activation, reinforce the notion that it may be an important element in plant signaling. Thus, from the systematic analysis of genes encoding plantspecific protein kinases, we can expect significant contributions to the understanding of the regulatory network of plant cells coupling external stimuli to internal responses. The mammalian genome is thought to encode more than 1000 different protein kinases acting in various regulatory circuits (Hunter, 1987); we should expect a network of different protein kinases active in plant cells. Characterization commonly involves protein purification technology, which, in many cases, however, is limited by the difficulty in obtaining sufficient quantities for protein sequencing studies. Thus, many cove1 plant kinases are expected to become accessible to molecular analysis via

264

KLAUS PALME

molecular cloning techniques. Laborious and time-consuming protein purification procedures can be circumvented if conserved structural motifs in catalytic domains can effectively be used for low-stringency hybridization or polymerase chain reaction screening of gene banks (Hanks, 1987; Wilks, 1989). Comparison of a large number of primary amino acid sequences has revealed patches of sequence similarity in catalytic domains of various protein kinases (Hanks et al., 1988). Because of the conserved substrate Mg2+-ATPand the amino acids to be phosphorylated, the catalytic domains of protein kinases contain a consensus motif for ATP binding lying between the first P strand and the first a helix. This motif consists of the amino acids GxGxxGxVx,AxK-, with the third glycine often replaced by serine or alanine. The glycine residues are apparently involved in the formation of a nucleotide-binding fold, first predicted by Rossmann et al. (1974, 1975) and subsequently identified in many other nucleotide-binding proteins as well. The lysine residue in this domain is invariant in every protein kinase found and essential for binding of the Ply-phosphate of the ATP. Inactivation of this lysine by chemical modification or replacement by mutagenesis results in a complete loss of protein kinase activity. In contrast to a conserved mechanism for Mg2+-ATPbinding and catalysis, less conservation is observed in other domains responsible for the recognition of different peptide substrates. Only a few diagnostic sequence elements at variable distances could be detected. For example, in a variety of protein kinases, amino acid triplets such as RDL, DFG, and APE, with a region between DFG and APE containing the major autophosphorylating site, were detected. Further amino acid patterns preceding a glutamic acid indicate substrate specificity to serinelthreonine or tyrosine in the various families (Hunter, 1982; Saxena et al., 1987; Hanks et al., 1988). Generally, sequence similarity within different branches and clusters of related protein kinases is not more than 30-35% (Hanks et al., 1988). Nevertheless, such short stretches have been sufficient to isolate cDNAs encoding plant homologs of protein serinelthreonine kinases from Phaseolus vulgaris and Oryza sativa (Lawton et al., 1989). Using degenerate oligonucleotides corresponding to the amino acid sequence motifs DLKPEN and GTPEYLAPE, representing consensus sequences within the catalytic domain of protein serinelthreonine kinases, Lawton et al. (1989) successfully isolated clones containing all but one of the features characteristic for protein kinases. These include amino acids corresponding to G’OXGXXG’~ of the ATP binding motif (numbering corresponds to the catalytic subunit of bovine CAMP-dependent protein kinase), K72implicated in ATP binding and phosphate transfer, and D1&,N172,D184,A2M, and E208,all diagnostic for the catalytic domain and located between the domains used for screening. However, residue in the CAMPdependent protein kinase, which is essential for both CAMP-induced auto-

EUKARYOTIC SIGNAL TRANSDUCTION

265

phosphorylation and enzymatic activity, has been exchanged for serine, suggesting the loss of CAMP regulation. Analysis of evolutionary relationships revealed that both kinases fall into a branch cluster of the protein serinelthreonine kinases that contain the cyclic nucleotide-dependent protein kinases and the protein kinase C family, indicating that development of this group predated the evolutionary separation of animals from plants (Lawton et al., 1989). Similarity within the catalytic domains of yeast, animal, and plant protein kinases points to a strong selective pressure for conservation of the active modules, while allowing major differences to develop in the specific effector ligands. What type of plant circuits are expected to be regulated by protein phosphorylation? As can be seen from Fig. 9, phosphorylation of proteins can be detected in essentially all compartments of a “consensus” plant cell, indicating that, besides regulation of a variety of metabolic pathways located in the cytosol, transmembrane transduction of external stimuli such as hormones or oligosaccharides can be an important area for action of plant protein kinases. It is expected that, besides cytoskeletal structures, receptors and ion channels will be key targets. For example, calcium-dependent regulation of voltage-dependent anion channels in the plasma membrane of guard cells has recently been observed, using patchclamp techniques (Hedrich et al., 1990). Upon activation either by increasing cytoplasmic calcium concentration or by addition of phosphataseinhibiting agents such as ATP[y]S, the anion currents rose 10- to 20-fold above the inactivated state, with permeability shifting from a K+conducting state to an anion-conducting state. Results suggest that activation of the anion channel by calcium-dependent phosphorylation is a key event in the regulation of anion efflux from guard cells during stomata1 closure. C. Protein Phosphatases

Model systems used to elucidate the control of cellular functions by phosphorylation show that the role played by protein phosphatases is far from passive (Cyert and Thorner, 1989). In particular it has been found that steady-state phosphorylation is controlled through complex regulatory mechanisms involving alterations of protein phosphatase activity. The importance of these enzymes in plant signal transfer processes is evident from studies demonstrating the rapid turnover of phosphorylated proteins from Petroselineurn hortense after stimulation with an elicitor (Dietrich et al., 1990). Rapid protein phosphate turnover was also revealed within seconds by two-dimensional gel analysis of in uiuo phosphorylation of tobacco cell cultures stimulated by a variety of reagents, including plant

266

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hormones (K. Palme, unpublished observations). These studies revealed that rapid inhibition of protein phosphorylation in cell-free extracts by inhibitors such as p-nitrophenylphosphate, NaF, or NaV04 only led to partial inhibition of phosphatase activities, either because of different inhibitor specificities or because of elevated levels of various plant as compared to animal phosphatases. The latter explanation is consistent with observations by MacKintosh and Cohen (1989), who demonstrated that high levels of types 1 and 2A phosphatases existed in a variety of plant extracts. These results point to a dynamic role for protein phosphatases in the regulation of plant metabolism. The inhibition of plant phosphatases by okadaic acid, a polyether fatty acid isolated from dinoflagellates of marine sponges such as Halichondriu okaduii, provides a new method for the analysis of stimulusdependent phosphorylation events in plants. The use of okadaic acid, which inhibits both protein phosphatases 1 and 2A in a dose-dependent manner, has proved to be extremely valuable in the analysis of reversible phosphorylation in yeast, mammmals, and other organisms (Cohen and Cohen, 1989a,b). Thus, the availability of this drug now makes it possible to analyze in plants the phosphorylation of target proteins for protein kinases following stimulation (e.g., oligosaccharides or phytohormones). The sensitivity of plant phosphatases 1 and 2A, as well as fungal, yeast, and animal phosphatases, to this compound points to a high degree of structural conservation among protein phosphatases. Structural information is now available for the catalytic subunits isolated from a variety of organisms, including yeast, Aspergillus, and the mouse. Primary sequences were deduced from a series of cDNAs sequenced. Sequence analysis has revealed significant similarities between types 1 and 2 protein phosphatases, ranging from 60% to 70%, with greatest diversity at the N and C termini of the proteins. The differences in the regulation of catalytic subunits, most notably by inhibition with heat-stable peptide components, such as inhibitors 1 and 2, respectively, involve the diversified N and C termini of the proteins. Particularly striking are six highly conserved domains between phosphatases 1 and 2A, located between residues Asn-79 and Gly-90 (see Table 111.) Conservation of these domains between members of the phosphatase family, as well as between catalytic subtypes 1 and 2A and phosphatases encoded by bacteriophages (Cohen and Cohen, 1988, 1989c), suggests involvement of these domains in catalytic function. There has also been a strong selective pressure to conserve DNA sequences in both open reading frames and 3’-untranslated regions. Presently, little is known about protein phosphatases in plant cells, their substrate specificity, distribution, regulation, molecular properties, and functions. Although the precise role of plant phosphatases in signal transduction processes remains to

TABLE 111 Level of Homology between Protein Phosphatase 1 Catalytic Subunits from Arabidopsis thaliana, Rabbit, Mouse, and Yeast”

A . thaliana PPI

Rabbit PPI Mouse MI S. pombe SDS2l S. pombe DIS2+ S . cerevisiae DIS2Sl

A . thaliana PP 1

Rabbit PP I

Mouse MI

-

85.3

86.6 94.7 -

-

-

-

-

Values are expressed as the percentage of homology.

-

-

Schizosaccharomyces pombe SDS2l

86.1 86.3 86.0 -

S. pombe DIS2+ 86.2 90.5 90.0 91.6

-

Saccharomyces cerevisiae DIS2SI

85.3 91 .O 89.4 88.0 91.4 -

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be established, we expect that they represent an essential component of cell cycle and transcriptional control. Using oligonucleotides corresponding to conserved regions, cDNA clones were isolated from N. tabacurn, A. thaliana, and Brassica napus (MacKintosh et al., 1990; Nitschke et al., 1991). Analysis of the amino acid sequences deduced from these clones revealed similarity in the range of 87-89% to phosphatases from other eukaryotic organisms. Analysis of partial cDNA clones isolated from a B. napus cDNA library, using rabbit muscle protein phosphatases 1A and 2A cDNA clones as a probe, identified catalytic subunits of protein phosphatases 1A and 2A. Within the deduced polypeptide sequences overall identity is 72% and 79%, respectively (MacKintosh et al., 1990). The remarkable degree of similarity observed within the identified polypeptide sequences provides a molecular explanation for the similarity of both catalytic and regulatory properties between animal and plant protein phosphatases (MacKintosh and Cohen, 1989). It has been argued that plant protein phosphatases have a rate of amino acid sequence change which is apparently slower than that of glyceraldehyde-3-phosphatedehydrogenase, aldolase, cytochrome c, or histone H2A (MacKintosh et al., 1990). The slow rate of evolution of protein phosphatases suggests that changes in the structure of these enzymes will be detrimental for all eukaryotic organisms. This adds further evidence to our present view of the basal importance of phosphate turnover in the growth control and regulation of metabolic processes in all organisms. Molecular clones now available from various plant-specific protein phosphatases will help to discern the molecular effects. For example, mutations in the gene coding for plant protein phosphatase 1 may represent an excellent marker gene interfering with chromosome separation in plant cells. Furthermore, it can be expected that the ability to construct transgenic plants expressing this gene under regulated control will soon allow us to explore the functional and phenotypic consequences of phosphatases on the plant level.

D. Calcium-Binding Proteins

Cytosolic calcium concentrations in plants are altered by many environmental factors, including hormones, light, and gravity (for reviews see Hepler and Wayne, 1985; Poovaiah and Reddy, 1987;Marme, 1988). It was shown, for example, that auxin binding to plant cells, auxin transport, and auxin-mediated proton excretion can result in alteration of cellular calcium concentrations (Dela Fuente and Leopold, 1973; Cohen and Nadler, 1976; Ragothama et al., 1985). In epidermal cells of maize coleoptiles, auxin is responsible for the initiation of oscillations of free cytosolic calcium con-

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centrations and cytosolic pH (Brummer et al., 1984; Felle et al., 1986; Felle, 1988a,b). Calcium has been found to be essential for germination of Adiantum capillus-veneris spores, leading to the conclusion that both phytochrome and calcium were required to allow early cell cycle progression (Iino et al., 1989). In addition the perception of gravity is closely related to a series of events linking the intracellular flow of calcium with graviperception in plant cells (for a review see Roux and Serlin, 1987). Current reports indicate that, upon gravistimulation, only gravisensitive cells depolarize after induction of ion fluxes across the plasma membrane (Sievers ef al., 1984). The recent finding of pressure-sensitive ion channels in plant cells (R. Hedrich, personal communication) points to the possibility that such channels have a role in the initiation of primary responses after the activation of graviresponse through pressure by the amyloplasts on the ER complex with stathocytes (Volkman and Sievers, 1979). Recent technical advances have made it possible to visualize in situ small changes in calcium concentrations in living tissues or single cells (A. J. Trewavas, personal communication; Gehring et al., 1990). Using novel fluorescent visible wavelength probes for calcium, Gehring et al. f 1990) succeeded in demonstrating that, within minutes, light induces an increase in cytosolic calcium concentration on the shaded side of maize coleoptiles. Hence, a change in the concentration of calcium apparently precedes the specific accumulation of mRNAs correlated with the gravitropic response (McClure and Guilfoyle, 1989). A primary intracellular target for calcium is a group of calcium-modulated proteins. All members of this large family reversibly bind calcium, with dissociation constants in the micromolar to nanomolar range. Besides calmodulin members of this family include troponin C, parvalbumin, S-100, and annexins, which all share extensive similarity, at least in the domain responsible for binding calcium. Related proteins, such as calsequestrin, known to regulate calcium homeostasis in the sarcoplasmic reticulum in smooth muscle cells, have also been detected in microsomal fractions of plant cells (Chou et al., 1989). Fractionation procedures have been designed to isolate members of the annexin family from the tomato, which all show substantial similarity to sequences of known members of the annexin family from animals, sharing a Ca2+-dependent affinity for acidic phospholipid (Smallwood et al., 1990). The discovery of several elements of the calcium response chain in plant cells has indicated the fundamental importance of calcium, not only in the regulation of metabolism of plant cells, but also in the initiation of defense reactions (Hepler and Wane, 1985; Kauss, 1987). Evidence collected from a variety of plant systems indicates that some of the effects evoked by calcium are mediated by calmodulin, as in animals. Calmodulin is a heatstable acidic protein with highly conserved calcium binding domains. The

270

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monomeric protein, 148 amino acids in length, contains four calcium binding domains composed of highly conserved amino acid sequences, with homology most apparent between domains 1 and 3 and domains 2 and 4. Each calcium binding site consists of approximately 30 amino acids, in which an a-helical segment flanks a 12-residue calcium-binding loop. Each domain fits the helix-loop-helix sequence motif proposed by Kretsinger (1980) and has been termed the EF motif. After binding of calcium, calmodulin undergoes a conformational shift, which exposes hydrophobic domains and allows formation of complexes with a variety of enzymes. It is expected that, in plant cells, calmodulin could act as the primary receptor for transient pulses of calcium, as it does in other cells. Enzymes regulated by the Ca" complex in plants include NAD kinase, Ca2+ATPase, H+-ATPase, quinate : NAD' 3-oxidoreductase, and protein kinases (Poovaiah and Reddy, 1987). Activities regulated by the binding of calcium to calmodulin include the regulation of cytoskeletal components affecting cytoplasmic mobility, cell division, exocytosis, and mediation of calcium transport across the membranes. The subcellular distribution of calmodulin, mostly in the cytosol, but also partly in the particulate and nuclear fractions, correlates with its suggested functions (Collinge and Trewavas, 1989). Its localization in the plumule and root tips correlates with its involvement in the response to light and gravity (Dauwalder er al., 1986). Genes isolated from potato, barley, and tobacco encoding calmodulin will help to probe its vital role in cellular signaling (Ling and Zielinski, 1989; Jena et al., 1989; Nitschke and Palme, in preparation). Calmodulin mRNA levels are apparently precisely regulated during development. mRNA levels have been found to be elevated mostly in merktematic zones and during fruit development. In addition hormonal regulation of calmodulin mRNA levels by auxin in deachenated strawberry fruits has been found as well (Jena et al., 1989). Interestingly, the calmodulin gene expression is rapidly regulated at the level of mRNA abundance in response to flooding and touch and is also under the influence of microgravity vectors and the pressure applied to plant cells (Braam and Davis, 1990; Nitschke and Palme, in preparation). Sequence analysis of clones isolated from plants has revealed a high degree of homology between the predicted amino acid sequences of calmodulin from plant and animal taxa. A comparison of amino acid similarities between calmodulin from N . tabacum var. Wisconsin 38 and other taxa is shown in Table IV, indicating a stronger similarity to the mammalian than the yeast protein. Not surprisingly, there is much less evolutionary pressure to conserve DNA sequences. The highest levels of conservation were observed in areas of functional importance, such as the calcium binding domains, which are almost identical throughout the various phyla.

271

EUKARYOTIC SIGNAL TRANSDUCTION TABLE IV Level of Homology between Various Calmodulins, Including Spinach, and Nicotiana tabacume

Yeast Eel Bovine Wheat Chlamydomonas Spinach Nicotiana

Yeast

Eel

Bovine

Wheat

Chlamydomonas

Spinach

Nicotiuna

-

61.6

62.3 99.3 -

62.5 90.3 91.0 -

63.0 89.6 90.3 87.1 -

62.3 90.4 91.1 97.8 88.7 -

61.6 91.1 91.4 91.4 91.6 93.7

-

-

Values are expressed as the percentage of homology.

E. Outlook

Recombinant DNA techniques have provided a rich supply of structural information, allowing the consideration of structural homology in view of functional similarity. These techniques have given access to classes of molecules hardly accessible by classical biochemical techniques. Typical, for example, G-coupled receptors and ion channels, elements of intercellular communication in many eukaryotes, are usually present at very low abundance. Although novel techniques (e.g., patch-clamp analysis) have led to the identification and physical analysis of a number of channel proteins in plants (Schroeder et al., 1984; Hedrich el al., 1987; Keller et al., 1989), progress in elucidating their structure by classical biochemical techniques involving purification and molecular analysis has been slow. Now, with the advent of polymerase chain reaction-based techniques (Saiki et al., 1985a,b, 1988; Mullis and Faloona, 1987),a systematic survey of oligonucleotides deduced from conserved primary sequence elements represented in membrane-spanning domains of voltage- or ligand-gated anion, as well as cation channels (Chatterall, 1988), may provide the basis for the isolation of related genes from plants. Structural features useful for the prediction of oligonucleotides for the synthesis of DNA probes by polymerase chain reaction may include, for example, a hydrophilic probably extracellularly located domain and a cluster of four hydrophobic ahelical domains. Short patches with high similarity within the membranespanning domains of the K + , Na+, or Ca2+ channels have been detected, especially in those areas thought to function as voltage-sensor elements. Similar transmembrane topologies have been observed for rhodopsins, members of the P-adrenergic receptor family and within other members of the G-coupled receptor family, all containing seven similarly spaced hydrophobic domains that anchor these proteins in the membrane lipid bi-

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layer. Due to the recent identification of G proteins in plants, it must be expected that G-coupled receptor proteins should be present in plants as well. This receptor family could serve important functions in plants, sensing a broad array of environmental or cellular signals. Thus, we could expect that identification of a G-coupled receptor family in plants will contribute significantly to the understanding of signal perception in plants. The race is now on. With the identification of varoius conserved regions that define family memberships, researchers in many laboratories have started screening for genes with synthetic oligonucleotide probes. We can anticipate that the next few years will provide a large body of information concerning the relevance of these components to plant communication.

XI. Concluding Remarks and Future Prospects

Plants are unique in many aspects of development, cell biology, and biochemistry. Plant cells can respond in many different ways to diverse and seemingly unrelated signals. However, despite their diversity, the responses may be channeled through a relatively small number of signal transduction pathways that may end with alteration of chromatin structure and gene expression. As we probe into the nature of response elements, we should perhaps not be surprised to discover the use of common modular elements, irrespective of the nature of the activating stimulus. There are several ways to identify genes controlling plant development. Starting from mutants with abnormal developmental phenotypes, identification of the genes responsible is laborious and in most cases difficult to achieve. Arabidopsis thaliana is apparently most amenable to the analysis of mutants by ( 1 ) genome walking/jumping and complementation analysis or (2) tagging chromosomal regions by insertion of a transgene or a plant transposon. A few successful cases of gene isolation by these approaches have been documented (Feldmann et al., 1989; Koncz et al., 1989, 1990). As this overview has illustrated, a number of key developmental control genes at various levels of the cellular heirarchy contain domains that are conserved within gene families of diverse members of the eukaryotic kingdom. Protein sequence elements have provided suitable information for the design of molecular probes to screen for family members in different species and have resulted in isolation from plants of genes encoding various G proteins, protein kinases, phosphatases, and calcium-binding proteins. It can be expected that precise analysis of further protein families known to play regulatory roles in development and represented in data bases will reveal further motifs useful as probes for improved polymerase chain

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273

reaction techniques. How can the growing number of genes that can be isolated from plants be functionally analyzed? Initial steps are the identification of polypeptide products from isolated cDNAs, determination of the chromosomal localization, and establishment of expression patterns. Often, important aspects of gene function may be deduced by comparison with known genes of other eukaryotic organisms, assuming that differences lead to gain or loss of specific functions. Candidate genes may be G proteins with altered GTP hydrolase properties or protein kinases with modified residues for autophosphorylation. Structural features that determine further specificities remain to be determined. To understand the function of these plant genes, it is essential to look at the whole plant level to detect the effects of absence of function or misregulation in particular transgenic plants or mutants. A new approach to achieving somatic induction of the transcription of transgenes has proved successful, using somatic excision of transposable elements to activate a chimeric transgene after its introduction into plants (Spena et al., 1989).Currently, this may be the most suitable approach to studying the phenotypic effects of dominant genes, whose expression may preclude the production of transgenic plants by interfering with growth and development. This may help to overcome some of the limitations of reverse genetics in the analysis of lethal mutations. We have described structural and functional aspects of various elements involved in plant signaling. Extensive parallels between plants and other eukaryotes indicate that similar genetic principles apply to the kingdoms of metazoa, metaphyta, and unicellular eukaryotes. This is illustrated by the principles determining the structure, organization, and regulation of plant genes. Our present knowledge of the precise mechanisms and the modular structure of polypeptides determining plant signaling, differentiation, and development is just beginning to develop. The discovery of the first genes and structures discussed here will undoubtedly result in rapid identification and isolation of additional members of what are presumed to be gene families. Their molecular functions can be studied, using biochemical and genetic techniques. Hence, it can be expected that knowledge of modular structural elements in eukaryotic signaling molecules will contribute rapidly to our understanding of mechanisms of plant development and answer questions about (1) how differentiation is induced, (2) how developmental genes are regulated temporarily and spatially, (3) how gene expression is restricted to certain differentiated cell types, (4)how and where the products of developmental genes are localized within cells and assembled into macromolcular structures, and ( 5 ) how they contribute to the diverse responses in which they are involved. This will lead to a sophisticated understanding of the networks controlling developmental programs in plants.

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Acknowledgments I would like to thank all those who generously provided reprints and preprints of their recent work. I am grateful to Dr. J. Dangl for helpful comments on the manuscript. In particular I am indebted to Ian Moore for stimulating discussions and critical comments. The constructive criticism of J. Schell is also gratefully acknowledged.

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A Abscisic acid, eukaryotic signal transduction and, 226-227.244.247. 25 I , 254 Accessory cells, marginal zone cells and, 61-64 Acer pseudoplatanus photoautotrophic plant cell cultures and, 151

tobacco BY-2 cell line and, I , 5 Acinar cells, zymogen granules and, 177-179, 191-192,209,21 I , 214 immunocytochernistry, 196- I 9 9 pancreas ZG membrane proteins, 199, 201.203-204,206 AcPase, zymogen granules and, 188-189, 191,208,216-217 ACR-P, photoautotrophic plant cell cultures and, 131, 137 Actin cytoskeleton and. 93-94,97.99-101 zymogen granules and, 212-213.215 Adenovirus, cytoskeleton and, 92 Adhesions, cytoskeleton and, 91,98-99 Adiantum capillu-ueneris, eukaryotic signal transduction and, 269 ADP, eukaryotic signal transduction and, 263 Agrobacterium tumefaciens eukaryotic signal transduction and, 225 tobacco BY-2 cell line and. 27 Amaranthus, photoautotrophic plant cell cultures and, 137-138

Amaranthus cruentus, photoautotrophic plant cell cultures and, 131 Amaranthus powellii, photoautotrophic plant cell cultures and, 131, 167 Amino acids cytoskeleton and, 88 eukaryotic signal transduction and, 238, 242. 250, 260,270 second messengers, 256-258, 262,264 photoautotrophic plant cell cultures and, 169 cultured cells, 115, 117-1 18 herbicide effects, 163-164 photosynthesis, 145 uses, 153, 155-156 zymogen granules and, 198,204, 206, 213 a-Amylase eukaryotic signal transduction and, 243 zymogen granules and, 198-199.204. 206 Antibiotic resistance, photoautotrophic plant cell cultures and, 160 Antibiotics, zymogen granules and, 195 Antibodies cytoskeleton and. 95 marginal zone cells and antigen processing, 57-58,60-62. 65-66 B lymphocytes, 34, 37 cell types, 50 dendritic cells, 51 macrophages, 41-43.47-48 recirculating lymphocytes, 5 5 , 57

286 monoclonal, see Monoclonal antibodies photoautotrophic plant cell cultures and, 144, 153 tobacco BY-2 cell line and, 21, 23, 26 zymogen granules and, 201, 203-204, 213 Antigens cytoskeleton and, 86.95 eukaryotic signal transduction and, 250 marginal zone cells and, 31-33, 37, 51, 67-68 antigen processing, 57-67 macrophages, 41-42,45-48 zymogen granules and, 197,201,205 Aphidicolin, tobacco BY-2 cell line and, 2, 16-18,21, 23, 28 APO-P, photoautotrophic plant cell cultures and, 131, 135, 137, 139. 145 Arabidopsis, eukaryotic signal transduction and, 247 Arabidopsis thaliana eukaryotic signal transduction and, 225, 272 phytohormone action, 247-249, 252-253 second messengers, 256,262. 267-268 tobacco BY-2 cell line and, 28 Arachis hypogea, photoautotrophic plant cell cultures and, 121 Ascidia, cytoskeleton and, 97 Asparagus ofjcinalis, photoautotrophic plant cell cultures and, 121-122 Aspergillus, eukaryotic signal transduction and, 266 Aspergillus nidulans, eukaryotic signal transduction and, 240 ATP cytoskeleton and, 103 eukaryotic signal transduction and, 264 photoautotrophic plant cell cultures and, 146, 157 tobacco BY-2 cell line and, 24 zymogen granules and, 188, 193,206, 21 1 ATPase eukaryotic signal transduction and, 263, 270 zymogen granules and, 208 Atrazine, photoautotrophic plant cell cultures and, 161-164 Atropa belladonna, photoautotrophic plant cell cultures and, 137

INDEX

Autoimmune diseases, marginal zone cells and, 38-39.46 Auxins eukaryotic signal transduction and, 230, 239 binding sites, 248-251 phytohormone action, 247-248, 253-254 phytohormone perception, 243-245 plant development, 226-227 second messengers, 262. 268 photoautotrophic plant cell cultures and, 134-135

B Bacteria cytoskeleton and, 82 eukaryotic signal transduction and, 235-236 marginal zone cells and, 57-59,64, 67 Barbula unguiculata, tobacco BY-2 cell line and, I 1 B cells, marginal zone cells and, 32, 34-39, 67-68 antigen processing, 59-67 cell types, 49-50 macrophages, 43,45-46, 49 recirculating lymphocytes, 52-55 6-Benzyladenine (BA), photoautotrophic plant cell cultures and, 114, 118-1 19, 122, 133 Beta uulgaris, eukaryotic signal transduction and, 263 B lymphocytes, marginal zone cells and, 34-39.68 antigen processing, 59, 67 recirculation, 53-54 Bone marrow, marginal zone cells and, 36, 38-39,42,46,49 Brassica napus, eukaryotic signal transduction and, 268

ti

Calcium cytoskeleton and, 83, 86-88, 101, 104 eukaryotic signal transduction and, 241, 272

INDEX

phytohormone action, 254-255 second messengers, 260, 263-265, 268-27 I photoautotrophic plant cell cultures and, 148 tobacco BY-2 cell line and, 26 zymogen granules and, 206,208-210, 212-213 Calmodulin eukaryotic signal transduction and, 241, 269. 271 photoautotrophic plant cell cultures and, 149 CAM. photoautotrophic plant cell cultures and, 137, 149 Cancer, cytoskeleton and, 75-76, 104-105 genome regulation. 85 new possibilities, 90-92 reverse transformation reaction, 76, 78-79 theory, 88-89 Carbohydrate photoautotrophic plant cell cultures and. 135, 154 zymogen granules and. 204 Carbon, photoautotrophic plant cell cultures and, 109, 112, 117, 145-148 Carbon dioxide, photoautotrophic plant cell cultures and, 110, 167, 169 cultured cells. I 11-122, 125-130, 132, 134 culture initiation, 136-137 photosynthesis, 142-147 uses, 150, 154-155. 160-161 Carcinogenesis. cytoskeleton and. 76. 91 Casein hydrolysate, photoautotrophic plant cell cultures and, 128-129 Carharanrhus roseus. photoautotrophic plant cell cultures and, 130, 136, 165- 166 cdc2. tobacco BY-2 cell line and. 16 CD5 B cells eukaryotic signal transduction and, 262 marginal zone cells and, 37-39 CD8 cells, marginal zone cells and. 53 CD21, marginal zone cells and. 59 cDNA eukaryotic signal transduction and, 273 phytohormone action, 247. 250 phytohormone perception, 244-245 second messengers, 256. 260, 262, 264, 266,268

287 photoautotrophic plant cell cultures and, 151, 153 zymogen granules and, 204 Cell cycle, tobacco BY-2 cell line and, 16, 18, 21-25.28 Centromeres, cytoskeleton and, 83-84. 103 Cephalosporium aphidicolia, tobacco BY-2 cell line and, 16 Chemotherapy, cytoskeleton and, 90 Chenopodium rubrum, photoautotrophic plant cell cultures and, 167-168 cultured cells, 115-1 18 culture initiation, 135-136, 138 herbicide effects, 162-164 photosynthesis, 139. 145 uses, 149-150, 155-157, 159 Chlamydomonas reinhardtii, tobacco BY-2 cell line and, 14 Chlorophyll, photoautotrophic plant cell cultures and, 110, 167-168, 170 cultured cells, 111-122, 125-134 culture initiation, 134-136. 138 herbicide effects, 161-162 photosynthesis, 148-151, 154. 156-159 Chloroplasts eukaryotic signal transduction and, 254 photoautotrophic plant cell cultures and, 120, 122, 125, 129, 133, 168-170 genetic engineering. 160 herbicide effects, 163 photosynthesis, 139, 141-142 secondary compounds, 164-166 uses, 149-153, 156-159 tobacco BY-2 cell line and, 11, 13 CHO-KI cells, cytoskeleton and, 86, 101 cancer, 89.93 reverse transformation reaction. 77, 79-82 Cholecystokinin, zymogen granules and. 210-21 1 Cholesterol. zymogen granules and, 195. 208 Chromatin cytoskeleton and. 82. 87. 93, 105 cancer, 88-89,91 function, 102-103 eukaryotic signal transduction and, 243, 272 Chromosomes cytoskeleton and, 75,95, 103-104 genome regulation, 83-88

288 eukaryotic signal transduction and, 240, 247,253,268,272 photoautotrophic plant cell cultures and, 151, 160 tobacco BY-2 cell line and, 11, 21 Chymotrypsin, zymogen granules and, 207 Chymotrypsinogen A, zymogen granules and, 198-199 Clones cytoskeleton and, 99-100 photoautotrophic plant cell cultures and, 111, 152 tobacco BY-2 cell line and. 27 zymogen granules and, 204 Condensing vacuoles (CVs), zymogen granules and, 185, 199, 213, 215 cytochernistry, 187- 189, I9 1 freeze-fracture, 193-195 COT-P, photoautotrophic plant cell cultures and cultured cells, 132-133 culture initiation, 135 photosynthesis, 143, 145, 148 uses, 150, 159, 166 COT-PA, photoautotrophic plant cell cultures and, 132-133, 143, 145, 147, 159 CRI, marginal zone cells and, 37-38 CR2, marginal zone cells and, 37-38 Crepis capilfaris, photoautotrophic plant cell cultures and, 149 Cyclic AMP cytoskeleton and, 86, 103 cancer, 88,90 reverse transformation reaction, 77, 79-80 eukaryotic signal transduction and, 235, 264-265 zymogen granules and, 208-212 Cyclophosphamide, marginal zone cells and, 35-38 Cytisus scoparius, photoautotrophic plant cell cultures and, 118, 137, 148 Cytochalasins cytoskeleton and, 93 tobacco BY-2 cell line and, 24 zymogen granules and, 21 1-212 Cytochemistry, zymogen granules and, 178,205,208,215-217 giberrellic acid, 186-189

tNDEX

Cytokinins eukaryotic signal transduction and, 226-227,23 1,243,254 photoautotrophic plant cell cultures and, 134 Cytoplasm cytoskeleton and, 94,97,99, I04 marginal zone cells and, 3 I , 55 photoautotrophic plant cell cultures and, 155

tobacco BY-2 cell line and, 4-5.22, 24 zymogen granules and, 184, 197, 215-216 cytoskeleton, 21 1,213 freeze-fracture, 192. 1% Cytoskeleton, 75-76, 103-105 cancer, role in, 88-92 eukaryotic signal transduction and, 258, 265,270 evolution, 95-96 function, 96 development, 97 ECM, 98 focal adhesion sites, 98-99 intermediate filaments, 101 microfilaments, 100 microtubules, 99-100 nuclear matrix, 101-103 genetic regulation, 92-94 genome regulation, 82-83 information transfer system, 86 phosphorylation, 86-88 repetitive DNA sequences, 83-85 molecular biology, 94-95 reverse transformation reaction, 75-82 tobacco BY-2 cell line and, 21-25, 28 zymogen granules and, 178,211-215, 217

D

DAPI, tobacco BY-2 cell line and, 5 , 12, 17,21 DAT-P, photoautotrophic plant cell cultures and, 119-120, 135, 159 Datura innoxia, photoautotrophic plant cell cultures and, 119-120, 138, 167 Datura stramonium, photoautotrophic plant cell cultures and, 119-120, 137-138

289

INDEX

Daucus carota, photoautotrophic plant cell cultures and, 129 DCMU, photoautotrophic plant cell cultures and, 161-163 Dendritic cells, marginal zone cells and, 50-52.63-65,67 Depletion, marginal zone cells and, 35-36, 54,63 Diacylglycerol, photoautotrophic plant cell cultures and, 141 Dianthus caryophyllus, photoautotrophic plant cell cultures and, 133-134 2,4-Dichlorophenoxyaceticacid photoautotrophic plant cell cultures and, 115, 130, 134, 162 tobacco BY-2 cell line and. 2-3.25 Differentiation cytoskeleton and, 93-95, 103-105 cancer, 89,92 genome regulation, 82. 85-87 reverse transformation reaction, 76.79 eukaryotic signal transduction and, 226, 234,240,255,273 photoautotrophic plant cell cultures and, 149-150 tobacco BY-2 cell line and, 21 Digitalis purpurea, photoautotrophic plant cell cultures and. 128-129, 165 Digitoxin, photoautotrophic plant cell cultures and, 165 DNA cytoskeleton and. 75, 97, 102, 105 cancer, 91 genome regulation, 82-88 molecular biology, 95 reverse transformation reaction, 78-79,8 1-82 eukaryotic signal transduction and concepts, 232 phytohormone action, 247-248 phytohormone perception, 245-246 plants as models, 225 second messengers, 266, 270-271 marginal zone cells and, 49 photoautotrophic plant cell cultures and, 151-153, 159-160, 168-169 tobacco BY-2 cell line and, 5, 8, 10-11, 18

gene delivery, 26-27 subcellular organelles, 13-15

DNA polymerase photoautotrophic plant cell cultures and, 152-153, 159 tobacco BY-2 cell line and, 15 DNase cytoskeleton and, 86.92 zymogen granules and, 198 DNase I cytoskeleton and, 78-79,82, 104 photoautotrophic plant cell cultures and, I52 Drosophila melanogaster cytoskeleton and, 102 eukaryotic signal transduction and, 229

E ED3, marginal zone cells and, 42-43, 46,61 Electron microscopy cytoskeleton and, 88 marginal zone cells and, 55 photoautotrophic plant cell cultures and, 139 tobacco BY-2 cell line and, 12, 14-15, 23-24.26 zymogen granules and, 177-178, 186-187,203,215 Electrophoresis eukaryotic signal transduction and, 243-244 photoautotrophic plant cell cultures and, 142, 169 zymogen granules and, 196,205 Embryos, eukaryotic signal transduction and, 229-230,244 Endocytosis eukaryotic signal transduction and, 237 zymogen granules and. 182. 184, 203. 215 Endoplasrnic reticulum eukaryotic signal transduction and, 249-25 1,255,269 zymogen granules and, 188. 194 Endothelium, marginal zone cells and, 57 Energy, photoautotrophic plant cell cultures and, 109-110 Environment, eukaryotic signal transduction and, 225. 233. 272 phytohormone action. 247. 252-255 response pathways, 236, 242

INDEX

Enzymes cytoskeleton and, 78-79,92-93, 95 eukaryotic signal transduction and, 240, 243, 265, 268, 270 marginal zone cells and, 41-42 photoautotrophic plant cell cultures and, 167 cultured cells, 112. 116, 126, 132 genetic engineering, 161 photosynthesis, 142-144, 146 secondary compounds, 164-167 uses, 149-150, 154-156. 159 tobacco BY-2 cell line and, 11, 23-24 zymogen granules and, 177, 182,213, 216 cytochemistry, 191 freeze-fracture, 194 immunocytochemistry, 196-199 pancreas ZG membrane proteins, 205 parotid ZG membrane proteins, 208-209 Epithelium, cytoskeleton and, 88, 93, 100-101

Epitopes, marginal zone cells and, 47-48 ER-TR9, marginal zone cells and, 47-48, 61-62 Erythrocytes cytoskeleton and, 78, 102 marginal zone cells and, 31,42-43,45, 64,67 Escherichia coli

cytoskeleton and, 75, 82, 103 eukaryotic signal transduction and, 245 tobacco BY-2 cell line and, 15 Esterase, marginal zone cells and, 40-41, 43,45 Ethylene, eukaryotic signal transduction and, 227,23 I , 247-248 err. eukaryotic signal transduction and, 247 Euglena gracilis, tobacco BY-2 cell line and, 14 Eukaryotic signal transduction, 223-224, 272-273 body patterns, 229-232 concepts, 232-234 molecular elements. 234-235 phytohormone action, 245, 255 binding sites, 248-252 environmental factors, 252-255 mutants, 246-248 phytohormone perception, 242-246

plant development, 225-229 plants as models, 224-225 response pathways, 241-242 coordination, 241 GTP-binding proteins, 238-240 protein kinases, 240 receptors, 235-238 second messengers, 271-272 calcium-binding proteins, 268-271 G proteins, 255-260 protein kinases. 260-265 protein phosphatases, 265-268 Euphorbia, photoautotrophic plant cell cultures and, 136, 139, 146, 148, 156, 167 Euphorbia characias, photoautotrophic plant cell cultures and, 130-131, 147, 166 Evolution, cytoskeleton and, 95-96, 105 Exocytosis eukaryotic signal transduction and, 270 zymogen granules and freeze-fracture, 192, 195-196 immunocytochemistry , 199 ion transport in pancreas, 212,215 membrane lipid composition, 208 morphometry, 178. 182 pancreas ZG membrane proteins, 204-206 recycling process, 182, 184-187 Extracellular matrix, cytoskeleton and, 86-87, 92-93, 98, 102

F

Fibroblasts, cytoskeleton and, 78-80, 92, 100-101

Fibronectin, cytoskeleton and, 86,93,98 FITC, marginal zone cells and, 47-48 Flow cytometry marginal zone cells and, 37. 39 photoautotrophic plant cell cultures and, 159-160 Fluorescence eukaryotic signal transduction and, 269 marginal zone cells and, 55 photoautotrophic plant cell cultures and, I10

cultured cells, 1 I I , 115, 134 culture initiation, 136

INDEX

291

uses, 152, 158-159 tobacco BY-2 cell line and, 5 , 18, 21, 26 zymogen granules and, 198,213 Fluorescence microscopy, tobacco BY-2 cell line and, 11-13.21, 24 FMC7 antigen, marginal zone cells and, 59 Follicles, marginal zone cells and, 45, 52, 68 antigen processing, 65-66 B lymphocytes, 35-39 Freeze-fracture, zymogen granules and, 186, 191-196, 216 Fusarium, photoautotrophic plant cell cultures and, 162

G

GDP, eukaryotic signal transduction and. 239,256 Gelonin, marginal zone cells and, 62 Gelonium mult$orum, marginal zone cells and, 62 Gelsolin, cytoskeleton and, 87, 100 Gene expression cytoskeleton and, 93 eukaryotic signal transduction and, 226, 231,254,272-273 phytohormone perception, 242-243 response pathways, 235, 231 photoautotrophic plant cell cultures and, I51 Genes cytoskeleton and, 75-76, 103-104 cancer, 92 evolution, 95-96 function, 100-101 genome regulation, 82 molecular biology, 94-95 reverse transformation reaction, 78-79, 82 eukaryotic signal transduction and. 223-225, 229. 236, 273 phytohormone action, 247-249 second messengers, 259-260, 262, 268 photoautotrophic plant cell cultures and, 109-110, 169-170 Genetic engineering, photoautotrophic plant cell cultures and, 160-161

Genetic regulation, cytoskeleton and, 92-94, 96 Genome exposure, cytoskeleton and, 88, 90-92, 95, 103-105 Genome regulation, cytoskeleton and, 75-76,82-83, 103 function, 102 information transfer system, 86 phosphorylation, 86-88 repetitive DNA sequences, 83-85 reverse transformation reaction, 80 Genotypes, photoautotrophic plant cell cultures and, 138, 152 GERL, zymogen granules and, 188-189, 191, 215 Gibberellic acid, eukaryotic signal transduction and, 243-244.250-252 Glucose, photoautotrophic plant cell cultures and, 168 cultured cells, 117, 133 culture initiation, 135 uses, 154, 160, 162, 165 dlutamyltransferase (GGT), zymogen granules and, 205-206 Gtycine max, photoautotrophic plant cell cultures and, 122-128, 166 Glycoprotein, zymogen granules and, 195, 201,203. 205, 216 Glycosylation eukaryotic signal transduction and, 237 zymogen granules and, 197, 204 Golgi apparatus, zymogen granules and, 194,201,213 c ytochemistry , 186- 189 immunocytochemistry, 197-199 morphometry , 178- I79 recycling process, 184-185 Golgi saccules, zymogen granules and, 185, 188, 194, 203 Gossypium hirsufum,photoautotrophic plant cell cultures and, 132-133 GPI, zymogen granules and, 201, 203-205 GP2, zymogen granules and, 196, 201, 203-206,216-217 G phase, tobacco BY-2 cell line and, 18, 2 1-25 G protein-coupled receptors, eukaryotic signal transduction and, 237-238,240, 246,27 1-272 G proteins cytoskeleton and, 95

292

INDEX

eukaryotic signal transduction and, 255-260,272-273 zymogen granules and, 206 Granulocytes, marginal zone cells and, 50 Growth factors, eukaryotic signal transduction and, 227,235 GTP eukaryotic signal transduction and, 260, 273 tobacco BY-2 cell line and, 24 zymogen granules and, 206 GTPase, eukaryotic signal transduction and, 256 GTP-binding proteins, eukaryotic signal transduction and, 230, 255-256 regulatory proteins, 238-240, 246

H Haplopappus gracilis, tobacco BY-2 cell

line and, 16 Haptens, marginal zone cells and, 65-66 Harmin, photoautotrophic plant cell cultures and, 165 HeLa cells cytoskeleton and, 92 tobacco BY-2 cell line as, see Tobacco BY-2 cell line Helix pomatia, zymogen granules and, 194 Herbicide effects, photoautotrophic plant cell cultures and, 110, 160-164, 168 High endothelial venules, marginal zone cells and, 43, 53, 5 5 , 57,65 Homology cytoskeleton and, 87, 101-102 eukaryotic signal transduction and, 234, 239 second messengers, 259, 262, 264, 267, 27 1 zymogen granules and, 205, 207 Hormones eukaryotic signal transduction and, 227, 234 body patterns, 229-23 I phytohormone action, 246-248, 253-254 phytohormone perception, 242-244 response pathways, 238, 240-242 second messengers, 255,260, 265,268, 270

marginal zone cells and, 60 zymogen granules and, 177, 182,211 Hybridization cytoskeleton and, 94, 105 eukaryotic signal transduction and, 264 photoautotrophic plant cell cultures and, 113, 138, 151, 153, 169 tobacco BY-2 cell line and, 15 Hyoscyamus muticus, eukaryotic signal transduction and, 248 Hyoscyamus niger, photoautotrophic plant cell cultures and, 120, 137-138

I

Immune response, marginal zone cells and, 68 antigen processing, 57, 59-61,63-67 macrophages, 45,48 primary response, 64-65 secondary response, 65-67 Immunocytochemistry, zymogen granules and, 178, 191, 196-199,201,204 Immunodeficiency, marginal zone cells and, 39,59-60 Immunofluorescence cytoskeleton and, 85 zymogen granules and, 197, 213 Immunoglobulin, marginal zone cells and, 34,60,65 Immunoglobulin D, marginal zone cells and antigen processing, 59,61, 66 B lymphocytes, 35,37-39 macrophages, 46 Immunoglobulin M, marginal zone cells and, 37-39,59-60,66 Immunohistochemistry, marginal zone cells and, 39, 51, 5 5 6 3 Indole-3-acetic acid (IAA) eukaryotic signal transduction and, 230, 248-249 photoautotrophic plant cell cultures and, 114, 128-129, 135 Infection, marginal zone cells and, 57-58 Inhibitors cytoskeleton and, 93-94,97-98, 100 eukaryotic signal transduction and, 227, 230,266 phytohormone action, 247, 250

INDEX

293

phytohormone perception, 243, 245 marginal zone cells and, 40, 47, 61 photoautotrophic plant cell cultures and, I10 cultured cells, 114, 122, 125, 132-133 culture initiation, 135 photosynthesis, 143, 145-146 uses, 152-154, 159-163 tobacco BY-2 cell line and, 16, 25 zymogen granules and, 204, 209, 211-213 in siru hybridization, cytoskeleton and, 94, 105

Integrin, cytoskeleton and, 98-99 Interleukin, marginal zone cells and, 60, 68 Interleukin-I, marginal zone cells and, 59 Interleukin-la, marginal zone cells and, 51 Interleukin-2, marginal zone cells and, 35, 38,59 Interleukin-4, marginal zone cells and, 59 Interleukin-5, marginal zone cells and, 60 Interleukin-6, marginal zone cells and, 60 Intermediate filaments, cytoskeleton and, 85-86,92-93, 101-103 Intramembrane particles (IMPS), zymogen granules and, 192-193, 195-196 Irradiation, marginal zone cells and, 36, 39 Isoproterenol, zymogen granules and, 189, 213 J

Junk DNA, cytoskeleton and, 85 K

Kalanchoe blossfeldiana, photoautotrophic plant cell cultures and, 137, 149 Kanamycin, tobacco BY-2 cell line and, 27 Kidney, zymogen granules and, 205 Kinetin, photoautotrophic plant cell cultures and, 111-1 14, 119-122, 125, 128-134 Kinetochores, cytoskeleton and, 84, 103 L

Lacrimal glands, zymogen granules and, 196,215

Lamins, cytoskeleton and, 95, 101-103 Lectins cytoskeleton and, 78 zymogen granules and, 194,216 Lemna, eukaryotic signal transduction and, 255-256 Ligands, eukaryotic signal transduction and phytohormone action, 246,249-251 phytohormone perception, 242 response pathways, 237-238 second messengers, 265, 271 Light microscopy photoautotrophic plant cell cultures and, 125 zymogen granules and, 186 Light regulation, photoautotrophic plant cell cultures and, 148-149 Lipids eukaryotic signal transduction and, 271 photoautotrophic plant cell cultures and, 117-118, 139, 141, 155, 159 zymogen granules and, 193,203, 208-209 Lipopolysaccharides, marginal zone cells and, 43,45,60 Liver, marginal zone cells and, 57 Low-fluence responses, eukaryotic signal transduction and, 253 LS (Linsmaier and Skoog) medium, photoautotrophic plant cell cultures and, 118-120, 135 Lymph nodes, marginal zone cells and antigen processing, 58-60,63-65 B lymphocytes, 36-38 macrophages, 41,43,47 recirculating lymphocytes, 53, 55 Lymphocytes, see also B lymphocytes; T lymphocytes marginal zone cells and, 32, 51 antigen processing, 64-65 macrophages, 42-43,49 recirculation, 52-57 Lysophosphatidylcholine, zymogen granules and, 208-209 Lysophosphatidylethanolamine,zymogen granules and, 208-209 Ly sophosphatidylserine, zymogen granules and, 209 Lysophospholipids, zymogen granules and, 208-209

294

INDEX

Lysosornes marginal zone cells and antigen processing, 62-63 macrophages, 42.48-49 recirculating lymphocytes, 54-55 tobacco BY-2 cell line and, 26 zymogen granules and, 188, 191, 194, 203, 216

M

Macrophages, marginal zone cells and, 3 1, 40,45-48,51, 67-68 metallophilic, 40-45 turnover, 48-49 Magnesium eukaryotic signal transduction and, 264 photoautotrophic plant cell cultures and, 153 Maize, eukaryotic signal transduction and phytohormone action, 249-250, 253 second messengers, 259-260, 263, 268-269 Major histocompatiblity complex, marginal zone cells and, 45,50-51,65 Malignancy, cytoskeleton and, 101, 105 cancer, 88.90-91 reverse transformation reaction, 76-79,82 Mapping, cytoskeleton and, 95, 105 Marginal sinus of spleen, 33, 40-42, 46, 67 Marginal zone cells of spleen, 31-34. 67-68 B lymphocytes, 34-39 cell types, 49-50 dendritic cells, 50-51 function antigen processing, 57-67 recirculating lymphocytes, 52-57 macrophages. 40.45-48 metallophilic, 40-45 turnover, 48-49 reticular cells, 51-52 MECA-367, marginal zone cells and, 55,57 Medicngo sariua, eukaryotic signal transduction and, 262 Membrane proteins, zymogen granules and, 199-206 Memory cells, marginal zone cells and, 65-68

Mesophyll eukaryotic signal transduction and, 245 photoautotrophic plant cell cultures and, 130, 137, 169 photosynthesis, 139, 147 uses, 156, 158 Metallophilic macrophages, marginal. 40-46,48,5 I , 67 antigen processing, 63,66 recirculating lymphocytes, 54-55 Methylation cytoskeleton and, 95 photoautotrophic plant cell cultures and, 151

Metribuzin, photoautotrophic plant cell cultures and, 163 Microfilaments cytoskeleton and, 85-86, 100-101 tobacco BY-2 cell line and, 21-22.24-26 zymogen granules and, 212-213 Microsomes, zyrnogen granules and, 209 Microspectrophotometry, tobacco BY-2 cell line and, 17-18 Microtubules cytoskeleton and, 103 function, 97,99-101 genetic regulation, 92 genome regulation, 83-86 tobacco BY-2 cell line and, 16, 21-26 Mitochondria eukaryotic signal transduction and, 254 photoautotrophic plant cell cultures and, 113, 116, 134, 146 uses, 152-154, 157, 159 tobacco BY-2 cell line and, I I zymogen granules and, 207-208 Mitosis cytoskeleton and, 83-84, 102-104 eukaryotic signal transduction and, 240 tobacco BY-2 cell line and, 18, 25 Mitotic index, tobacco BY-2 cell line and, 1-2,4, 16-17 MOMA-1, marginal zone cells and, 41-43, 45-46, 55,63 Monoclonal antibodies eukaryotic signal transduction and, 252, 262 marginal zone cells and, 37, 51 antigen processing, 60-61 macrophages, 41-43,45-48

295

INDEX

Monocytes. marginal zone cells and, 49-50 Morindu lucidu. photoautotrophic plant cell cultures and, 129, 165 Morphogenesis, eukaryotic signal transduction and, 226 body patterns, 229-230 phytohormone action, 248-249. 252-253 Morphology cytoskeleton and, 75-76.78, 88,98. 104 eukaryotic signal transduction and, 225-226.237 marginal zone cells and, 33 tobacco BY-2 cell line and. 5, 28 zymogen granules and, 184, 187, 193, 197, 214 Morphometry. zyrnogen granules and, 178-182. 185 Mougoetiu, eukaryotic signal transduction and, 254 M phase, tobacco BY-2 cell line and, 17-18,22-23,25,28 mRNA cytoskeleton and, 94.97 eukaryotic signal transduction and, 243-246,253,269-270 photoautotrophic plant cell cultures and, 134, 149-151 MS (Murashige and Skoog) medium, photoautotrophic plant cell cultures and, 115, 117-121, 128, 130-132, 156 Glycine max, 125, 127 Nicotiuna, 1 11, I 13 Mutagenesis, photoautotrophic plant cell cultures and, 162-163 Mutagens, cytoskeleton and. 91, 105 Mutation cytoskeleton and. 76. 91-92, 95-96. 104 eukaryotic signal transduction and, 225, 229.236, 272-273 phytohorrnone action, 246-250, 252-253 marginal zone cells and, 65 photoautotrophic plant cell cultures and, 110, 168-169 cultured cells, 128 culture initiation, 138 uses, 161, 163 tobacco BY-2 cell line and, 27

N Naphthaleneacetic acid (NAA), photoautotrophic plant cell cultures and, 118-119, 122, 125, 130, 132- I34 Nicotiana, 11 1-1 14 Nicotiana photoautotrophic plant cell cultures and, 115, 138-139, 167 tobacco BY-2 cell line and, 2 Nicotiana glutinosu, photoautotrophic plant cell cultures and, 113 Nicotiana plumbaginijolia eukaryotic signal transduction and, 248 photoautotrophic plant cell cultures and, 110, 167-168 cultured cells, 114-1 15 culture initiation, 137-138 photosynthesis, 143-144, 146 uses, 162-164 Nicotiana syluestris, photoautotrophic plant cell cultures and, 161 Nicotiana tabacum eukaryotic signal transduction and, 245, 25 I , 256,268, 270-271 photoautotrophic plant cell cultures and, 110, 167-168, 170 cultured cells, I 1 1-1 15 culture initiation, 134, 137 genetic engineering, 160 herbicide effects, 161, 163-164 photosynthesis, 139, 141, 145, 147-148 secondary compounds, 166 uses, 149, 151, 153, 157, IS9 tobacco BY-2 cell line and, 2 , 2 8 Nitrate reductase (NR), photoautotrophic plant cell cultures and, 155-156, 169 Nitrogen eukaryotic signal transduction and, 23 1 photoautotrophic plant cell cultures and, 155-156, 159 NPT I1 genes, tobacco BY-2 cell line and, 27 NTG-P, photoautotrophic plant cell cultures and, 135, 139, 143 Nuclear matrix, cytoskeleton and, 83, 88, 92.94, 101-103 Nucleoids, tobacco BY-2 cell line and, 5 , 8, 11-13

INDEX

Nucleotides eukaryotic signal transduction and, 234, 238,255-260,264-265 photoautotrophic plant cell cultures and, 152 Nucleus cytoskeleton and, 101, 103, 105 genome regulation, 82, 84-86 reverse transformation reaction, 78-79,82 tobacco BY-2 cell line and, 5 , 12, 17-18

0 Okadaic acid, eukaryotic signal transduction and, 266 Oncogenes cytoskeleton and, 75, 83, 102 eukaryotic signal transduction and, 229, 237,256 Opsonization, marginal zone cells and, 67 Organelles, subcellular, tobacco BY-2 cell line and, 12-15 Oxygen, photoautotrophic plant cell cultures and cultured cells, 119-122, 125, 127-130, 133-134 Nicotiana, 112-1 14 photosynthesis, 145-148 uses, 154, 156, 158, 162

P Pancreas, zymogen granules and, 177, 215, 217 cytochemistry, 187-188 cytoskeleton, 212-214 freeze-fracture, 193-194, 196 immunocytochemistry, 196-199 ion transport in pancreas, 209-21 1 membrane lipid composition, 208 morphometry, 178- 182 pancreas ZG membrane proteins, 199-206 parotid ZG membrane proteins, 207-208 recycling process, 182, 184-187 Paraquat, photoautotrophic plant cell cultures and, 161-162

par gene, eukaryotic signal transduction and, 245 Parotid gland, zymogen granules and, 177, 217 cytochemistry, 187-189, 191 cytoskeleton, 2 12-213 freeze-fracture, 196 membrane lipid composition, 209 morphometry, 178-182 recycling process, 186 Parotid membrane proteins, zymogen granules and, 206-208 Pathogens, marginal zone cells and, 32, 38,64 Peganum harmala, photoautotrophic plant cell cultures and, 118, 134, 139, 165 PEPcase, photoautotrophic plant cell cultures and, 170 cultured cells, 116-118, 120-121, 131-133 culture initiation, 137 Glycine max, 125-127 Nicotiana, 112 photosynthesis, 144- 145 uses, 154-155, 158-159 Peptides eukaryotic signal transduction and, 238, 24 1 photoautotrophic plant cell cultures and, 153 zymogen granules and, 207,211 Penarteriolar lymphocyte sheath (PALS), marginal zone cells and, 31-32 antigen processing, 61,63-67 B lymphocytes, 36 dendritic cells, 50-51 macrophages, 43,48 recirculating lymphocytes, 52-54 Peninsular cells, zymogen granules and, 180, 198 PH eukaryotic signal transduction and, 250, 263 photoautotrophic plant cell cultures and, 155, 157 tobacco BY-2 cell line and, 4, 26 Phagocytosis, marginal zone cells and, 31-32.67-68 antigen processing, 57, 61, 64 macrophages, 40-41,43,45,48

INDEX

Phellodendron amurense, photoautotrophic plant cell cultures and, 137 Phenotype cytoskeleton and, 77, 88, 90,93, 101 eukaryotic signal transduction and, 268, 212-213 body patterns, 231 phytohormone action, 247-248, 253 marginal zone cells and, 43. 50 antigen processing, 59,66 B lymphocytes, 36-37,39 Phosphatases, eukaryotic signal transduction and, 240, 262, 265-268, 272 Phosphate eukaryotic signal transduction and, 264 tobacco BY-2 cell line and, 4-5 Phospholipase A, zymogen granules and, 209 Phospholipids, zymogen granules and, 199, 208-209,215 Phosphorylation cytoskeleton and, 104 function, 99-102 genome regulation, 83, 86-88 molecular biology, 95 reverse transformation reaction, 77 eukaryotic signal transduction and, 254, 273 response pathways, 236-237, 240 second messengers, 260, 262-266 photoautotrophic plant cell cultures and, 154 tobacco BY-2 cell line and, 25 zymogen granules and, 206, 210-211 Photoautotrophic plant cell cultures, 109-111, 167-170 cell biology, 157-160 cultured cells, 11 1-134 culture initiation, 134-138 differentiation, 149- 150 genetic engineering, 160-161 herbicide effects, 161-164 light regulation, 148-149 membrane properties, 156-157 metabolic regulation, 154-156 molecular biology, 150-154 photosynthesis, 138- 148 secondary compounds, 164-167 Photomixotrophic plant cell cultures, 109, 169

297 cultured cells, 113-1 14, 116, 118, 122, 125, 128 culture initiation, 134, 137 herbicide effects, 161-163 photosynthesis, 139, 141 secondary compounds, 164-167 uses, 149, 151-154, 156 Photorespiration, photoautotrophic plant cell cultures and, 132, 145-147, 161, 169 Photosynthesis eukaryotic signal transduction and, 252 photoautotrophic plant cell cultures and, 109-111, 138-148, 170 cultured cells, 116-119, 121, 128-131, 133-134 culture initiation, 134, 137 herbicide effects, 161-162, I64 Nicotiana, 111, 114 secondary compounds, 164-166 uses, 150-151. 154, 156, 160 tobacco BY-2 cell line and, I I Photosystem 1 (PSI), photoautotrophic plant cell cultures and, 113, 141-142, 146 Phragmoplasts, tobacco BY-2 cell line and, 16,21-24,28 Phylogenetic relations, eukaryotic signal transduction and, 232-234,259 Phytochrome eukaryotic signal transduction and, 252-254,256,269 photoautotrophic plant cell cultures and, 148- 149 Phytohormones, eukaryotic signal transduction and binding sites, 248-252 environment, 252-255 mutants, 246-248 perception, 242-246 plant development, 226-23 1 response pathways, 242 Phytophthora megasperma, photoautotrophic plant cell cultures and, 166 Pisum sativum, eukaryotic signal transduction and, 256 Plant signaling elements, see Eukaryotic signal transduction Plasma cells, marginal zone cells and, 39,43

298 Plasmalemma eukaryotic signal transduction and, 249 zymogen granules and, 186, 196,206 Plasma membrane eukaryotic signal transduction and, 235, 24 I phytohormone action, 248, 251-252 second messengers, 263, 269 zymogen granules and, 182, 185,201, 208, 213 Plastids eukaryotic signal transduction and, 252, 260 photoautotrophic plant cell cultures and, 113, 161, 165, 169 uses, 151-152, 160 tobacco BY-2 cell line and, 5, 8, 10-15 Polymerase chain reaction eukaryotic signal transduction and, 232, 264,271-273 zymogen granules and, 204 Polymerization cytoskeleton and, 94, 100-101 tobacco BY-2 cell line and, 24 Polypeptides cytoskeleton and, 101 eukaryotic signal transduction and, 239, 243-244, 263, 268, 273 photoautotrophic plant cell cultures and, 142, 153 zymogen granules and, 199,205-208 Pol ysaccharides cytoskeleton and, 98 marginal zone cells and, 67 antigen processing, 57-64, 66 macrophages, 45-48 Populus, tobacco BY-2 cell line and, 2 Preprophase band, tobacco BY-2 cell line and, 16,22,24-25 Proliferation eukaryotic signal transduction and, 255, 262 marginal zone cells and, 66 Prolifin, cytoskeleton and, 100 Propidium iodide, tobacco BY-2 cell line and, 17-18 Propizamide, tobacco BY-2 cell line and, 18, 22-24, 28 Protein cytoskeleton and, 101, 103-104 cancer, 88-89,91-92

INDEX

function, 97-100 genetic regulation, 93-94 genome regulation, 82-85.87 molecular biology, 95 reverse transformation reaction, 77 eukaryotic signal transduction and, 223, 227,272 body patterns, 229, 231 concepts, 232,234 molecular elements, 234-235 phytohormone action, 246-255 phytohormone perception, 244-246 response pathways, 236-242 second messengers, 255-271 marginal zone cells and, 48, 61, 63, 65 photoautotrophic plant cell cultures and, 110, 168 cultured cells, 117-118 culture initiation, 134 herbicide effects, 163 uses, 149-153, 155-156 tobacco BY-2 cell line and, 13, 16, 21, 25,27 zymogen granules and, 177, 180, 184, 188,215-216 c ytoskeleton, 212-2 14 freeze-fracture, 193, 196 immunocytochemistry , 196, 198- 199 ion transport in pancreas, 210 pancreas ZG membrane proteins, 199-206 parotid ZG membrane proteins, 206-208 Protein kinases eukaryotic signal transduction and, 248, 272-273 response pathways, 236,240-241 second messengers, 250-265 tobacco BY-2 cell line and, 25 Protooncogenes, cytoskeleton and, 88, 91-92, 102, 104 Protoplasts photoautotrophic plant cell cultures and cultured cells, 114 culture initiation, 137-138 uses, 150, 158-163, 167 tobacco BY-2 cell line and, 12, IS, 24-26 psbA gene, photoautotrophic plant cell cultures and, 62-164, 168 Pseudomonas

eukaryotic signal transduction and, 262

299

INDEX

tobacco BY-2 cell line and, 3 Psoralea bituminosa, photoautotrophic plant cell cultures and, 137

R

Radiosensitivity, marginal zone cells and, 37-38 ras, eukaryotic signal transduction and, 256, 259 rDNA, photoautotrophic plant cell cultures and, 160, 169 Receptors eukaryotic signal transduction and. 235-238.247-252.272 marginal zone cells and, 46-48, 61, 67-68 Recirculating lymphocytes, marginal zone cells and, 52-57 Recombination, tobacco BY-2 cell line and, 27 Red blood cells cytoskeleton and, 78, 102 marginal zone cells and, 31.42-43,45, 64,67 Repetitive DNA sequences, cytoskeleton and, 83-85 Replication cytoskeleton and, 97 photoautotrophic plant cell cultures and, 152 tobacco BY-2 cell line and, 13-16 Reticular cells of spleen, 31-32. 51-52 Reverse transformation reaction, cytoskeleton and, 75-82, 104-105 cancer. 89-92 Rhodotorula, tobacco BY-2 cell line and, 3 RNA eukaryotic signal transduction and, 244, 246 photoautotrophic plant cell cultures and. 152 tobacco BY-2 cell line and, 26 RNA polymerase cytoskeleton and, 87 eukaryotic signal transduction and, 243. 245 photoautotrophic plant cell cultures and. 151

Rough endoplasmic reticulum, zymogen granules and, 178-179, 185, 189, 198- I99 rRNA eukaryotic signal transduction and, 232, 234 tobacco BY-2 cell line and, 15 RuBPcase, photoautotrophic plant cell cultures and, 169-170 cultured cells, 116-118, 120-121, 129, 131-133 culture initiation, 137-138 Glycine max, 125-127 Nicotiana, 1 12 photosynthesis, 142-145, 147 uses. 150-151, 154, 158, 166 Ruta graueolens, photoautotrophic plant cell cultures and, 115, 135

S Saccharomyces cerevisiae cytoskeleton and, 99 tobacco BY-2 cell line and, 16 SB-P cells, photoautotrophic plant cell cultures and, 168-169 cultured cells, 122, 125-128, 132 culture initiation, 135, 138 photosynthesis, 139, 143-145, 147-148 secondary compounds, 166 uses. 149-153, 155-156, 158-160 SBI-P cells, photoautotrophic plant cell cultures and, 169 cultured cells, 125-128 photosynthesis, 144-145. 148 uses, 159-160 Scanning electron microscopy marginal zone cells and, 3 I , 33 zymogen granules and, 195, 216 SDS-PAGE, zymogen granules and, 199. 201,206-207 Second messengers eukaryotic signal transduction and, 234-235,241-242.271-272 binding proteins. 255-260, 268-271 phytohormone action, 254-255 protein kinases, 260-265 protein phosphatases, 265-268 zymogen granules and, 212

300 Secretory granules, zymogen granules and, 217 c ytochemistry , 186- 189, 19I cytoskeleton, 2 12-2 13, 2 15 freeze-fracture, 195 morphometry, 179-182 pancreas ZG membrane proteins, 201 parotid ZG membrane proteins, 206 Secretory vesicles, eukaryotic signal transduction and, 260 Sequences cytoskeleton and, 95,99, 102, 104 cancer, 92 genome regulation, 83, 87 eukaryotic signal transduction and, 234, 272 phytohormone action, 250-251 phytohormone perception, 242, 244-246 response pathways, 237, 239-240, 242 second messengers, 258,262-264.266. 268, 271 photoautotrophic plant cell cultures and, I53 tobacco BY-2 cell line and, 15 zymogen granules and, 207 Sheep erythrocyte receptor (SER), marginal zone cells and, 42-43.46 Signal transduction eukaryotic, see Eukaryotic signal transduction photoautotrophic plant cell cultures and, 149, I51 Sinus-lining cells, marginal zone cells and, 53.55-57 Solanurn nigrum, photoautotrophic plant cell cultures and, 160 Solanurn tuberosurn, photoautotrophic plant cell cultures and, 128 Soybean eukaryotic signal transduction and, 245 photoautotrophic plant cell cultures and, 152-153, 158, 160, 167, 169 tobacco BY-2 cell line and, 10 S phase, tobacco BY-2 cell line and, 17-18,21,28 Spinacia oleracea, photoautotrophic plant cell cultures and, 119, 135 Spleen, marginal zone cells of, see Marginal zone cells of spleen Splenectomy, marginal zone cells and, 57-58

INDEX

Starch, photoautotrophic plant cell cultures and cultured cells, 117, 119-120, 130-131 photosynthesis, 141 uses, 150, 158 Steroid receptors, eukaryotic signal transduction and, 229,242 Subcellular organelles, tobacco BY-2 cell line and, 12-15 Sucrose, photoautotrophic plant cell cultures and cultured cells, 116-119, 121, 128-130, 132-133 culture initiation, 135, 137-138 genetic engineering, 160- 161 Glycine max, 122, 125 herbicide effects, 161-162 secondary compounds, 165 uses, 149-150, 154-155, 157-158 Sugar, photoautotrophic plant cell cultures and, 109-110, 168 cultured cells, 115, 120, 130-131, 134 uses, 150, 154, 161

T

T cell receptors, marginal zone cells and, 65 T cells, marginal zone cells and, 67-68 antigen processing, 57, 59, 62,64-65 dendritic cells, 50-51 macrophages, 42-43,45 recirculating lymphocytes, 52-55 Teleinsular cells, zymogen granules and, 180, 198 Thiamine, photoautotrophic plant cell cultures and, 135 THP, zymogen granules and, 205 TI-1 antigens, marginal zone cells and, 45,57 T1-2 antigens, marginal zone cells and, 45. 48,57-64,66,68 Ti plasmids, tobacco BY-2 cell line and, 27 Tissue specificity, cytoskeleton and, 78, 82 T lymphocytes, marginal zone cells and, 50, 54, 67-68 TNP-Ficoll, marginal zone cells and, 59-63 TNP-KLH, marginal zone cells and, 58, 61-62 Tobacco BY-2 cell line, 1-2, 27-28 cell elongation, 25-26

301

INDEX

cytoskeleton changes, 21-25 gene delivery, 26-27 growth, 3-1 1 origin of, 2-3 subcellular organelles, 12-15 synchronization of cells, 16-20 Tobacco mosaic virus. tobacco BY-2 cell line and, 26 TPA cytoskeleton and, 90-91, 103 zymogen granules and, 21 1 TPPase, zymogen granules and, 188-189, 191,206,208 Transcription cytoskeleton and cancer, 88 function, 97. 102 genetic regulation, 93-94 genome regulation, 82, 87 reverse transformation reaction, 78 eukaryotic signal transduction and phytohormone action, 252-253, 255 phytohormone perception, 243-245 response pathways, 242 second messengers, 260, 262, 268 photoautotrophic plant cell cultures and, 149- 15 1 tobacco BY-2 cell line and, 14 Trans-Golgi apparatus, zymogen granules and. 188 Trans-Golgi network, zymogen granules and, 189,205,215 Trans-Golgi saccules, zymogen granules and, 189, 215 Translocation eukaryotic signal transduction and, 255 photoautotrophic plant cell cultures and, 168 tobacco BY-2 cell line and, 24 Transmission electron microscopy, zymogen granules and, 192-193 Triacylglycerol, photoautotrophic plant cell cultures and, 141 Triazine, photoautotrophic plant cell cultures and, 162-164, 168-169 Trypsin, zymogen granules and, 207 Tubulin cytoskeleton and function, 97, 99-102 genetic regulation, 93-94 tobacco BY-2 cell line and, 24 Turnover, marginal zone cells and, 48-49

U

Ultraviolet light, photoautotrophic plant cell cultures and, 149

V Vacuoles, condensing, see Condensing vacuoles Very low-ff uence responses, eukaryotic signal transduction and. 253 Vesicles, zymogen granules and cytochemistry, 188-189, 191 freeze-fracture, 192- I93 pancreas ZG membrane proteins. 205 recycling process, 184, 186 Vimentin, cytoskeleton and, 87,93-94, 101-102

x Xenopus, cytoskeleton and, 97

Y

Yeast, eukaryotic signal transduction and, 234,237,258-260,266-267 YF'Tl, eukaryotic signal transduction and, 258-259

z Zea mays, photoautotrophic plant cell cultures and, I37 Zymogen granules, 177-178,215-217 cytochemistry 186-19 1 cytoskeleton, 21 1-215 freeze-fracture, 191- I96 immunocytochemistry, 196- 199 ion transport in pancreas, 209-21 I membrane lipid composition, 208-209 morphometry, 178-183 pancreas ZG membrane proteins,

.

199-206

parotid ZG membrane proteins, 206-208 recycling process, 182, 184-187

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