Integrative Plant Biochemistry, Volume 40 (Recent Advances in Phytochemistry)

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phytochemistry recent advances in phytochemistry volume 40

Integrative Plant Biochemistry

IN PHYTOCHEMISTRY PHYTOCHEMISTRY RECENT ADVANCES IN of the Phytochemical Society of of North America Proceedings of General Editor: John T. Romeo, University of of South Florida, Tampa, Tampa, Florida Volumes in in the the Series: Recent Volumes Volume 33

Phytochemicals in Human Health Protection, Nutrition, and Plant Defense Proceedings of of the the Thirty-eighth Thirty-eighth Annual Meeting of of the the Phytochemical Society America, Pullman, Society of of North North America, Pullman, Washington, Washington, July, July, 1998 1998

Volume 34

Evolution of Metabolic Pathways Proceedings Annual Meeting Proceedings of of the the Thirty-ninth Thirty-ninth Annual Meeting of of the the Phytochemical Phytochemical Society America, Montreal, Society of of North North America, Montreal, Quebec, Quebec, Canada, Canada, July, July, 1999 1999

Volume 35

Regulation of Phytochemicals by Molecular Techniques Proceedings Annual Meeting Proceedings of of the the Fortieth Fortieth Annual Meeting of of the the Phytochemical Phytochemical Society America, Beltsville, Society of of North North America, Beltsville, Maryland, Maryland, June, June, 2000 2000

Volume 36

Phytochemistry Phytochemistry in the Genomics and Post-Genomics Eras Proceedings Proceedings of of the the Forty-first Forty-first Annual Annual Meeting Meeting of of the the Phytochemical Phytochemical Society of North America, Oklalohoma City, Oklahoma, August, 2001 2001 Society of North America, Oklalohoma City, Oklahoma, August,

Volume 37

Integrative Phytochemistry: From Ethnobotany to Molecular Ecology Proceedings of of the the Forty-second Annual Meeting of of the the Phytochemical Society of of North America, Mérida, Merida, Yucatán, Yucatan, Mexico, July, 2002

Volume 38

Secondary Metabolism in Model Systems Proceedings of of the the Forty-third Annual Meeting of of the the Phytochemical Society of North America, Peoria, Illinois, August, 2003 of

Volume 39

Chemical Ecology and Phytochemistry Phytochemistry of Forest Ecosystems Proceedings of of the the Forty-fourth Annual Meeting of of the the Phytochemical Society of of North America, Ottawa, Ontario, Canada, July, 2004

Volume 40

Biochemistry Integration of Plant Biochemistry Proceedings of of the the Forty-fifth Annual Meeting of of the the Phytochemical Society of of North America, LaJolla, California, July, July, 2005

Cover design: Biosynthetic pathway of sorgoleone showing the incorporation pattern obtained 13C-labeled with 13 C-labeled substrates substrates (see Figure Figure 7.2). 7.2). Photomicrograph Photomicrograph of Sorghum bicolor bicolor roots roots showing showing sorgoleone-rich exudates secreted from the and closer view of a root hair sorgoleone the root hairs and (see Figure 7.1). exuding at the the tip (see 7.1).

phytochemistry recent advances in phytochemistry volume 40

Integrative Plant Biochemistry Edited by

John T. Romeo University of South Florida Florida Tampa, Florida, USA

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PREFACE The publication of this volume marks the 40 th anniversary of Recent Advances in Phytochemistry. The series (> 13,000 cumulative pages) has been essentially a history of the origins of Phytochemistry - where we have been and where we are, as well as where we appear to be going. Early volumes were entitled simply "Recent Advances in Phytochemistry" with specific titles appearing later in the series as our symposia became increasingly focused on annual topics. A cursory look at the list of titles in the series (see accompanying) will demonstrate that while we have occasionally drifted into broader areas, such as chemical ecology, medicinal chemistry, and increasingly molecular biology, we have remained grounded with our roots in natural products or secondary metabolism. With the "omics" revolution of the 1990s, the phytochemical pioneers were slowly but inexorably supplanted by molecular biologists and geneticists able to answer more definitively the metabolic, evolutionary, and the functional ecological questions which have often absorbed us. The distinctions between secondary and primary chemistry have now blurred to an extent that the term plant biochemistry seems poised to subsume the term "phytochemistry". The 45 th annual meeting of the Phytochemical Society of North America (PSNA) was held Julyl3-August 3, 2005 in La Jolla, California, USA. The meeting was hosted by the Salk Institute for Biological Studies. The theme of the meeting was - Integrative Plant Biochemistry as we Approach 2010. As articulated by Joseph Noel, the local organizer, the design was: " to celebrate the past accomplishments of the PSNA and its focus, the growing importance of phytochemistry and plant biochemistry to the public, and to set a course for the future, by linking the past with the present and attracting a wider breath of scientists and disciplines to the society." To this end, various symposia were organized that focused on: 1. Metabolic Networks; 2. Temporal/Spatial regulation of Metabolism; 3. Biosynthesis/Regulation of Signaling Molecules; 4. Translation Opportunities in Plant Biochemistry; 5. Lipids, Fatty Acids and Related Molecules; and 6. Conservation/ Divergence in Enzyme Function. Those who chose to contribute to this volume are grouped as possible to reflect these various foci. While the chapters by O'Connor and McCoy, Panaccione et ah, and Samanani and Facchini all center on alkaloids (terpene indole, ergot fungal, and a variety of types (tropane, pyrrolizidine, quinolizidine and benzylisoquinoline), respectively, the focus differs; O'Connor and McCoy mainly discuss biosynthesis, enzymology, and structural diversity of TIAs with an eye on substrate specificity and future exploitation of these diverse compounds as Pharmaceuticals; Panaccione et al. address ergot alkaloid-associated gene clusters that seem to have a common genetic

v

vi

PREFACE

and biosynthetic origin in fungi. Gene knockout analyses and characterization of mutants are determining functions in pathways for gene products. Future applications in agriculture and medicine are inevitable; Samanani and Facchini concentrate on specialized plant structures where alkaloids are compartmentalized. Bifunctional or multifunctional enzymes are being targeted to alternative subcellular compartments where they may interact with differing substrates to produce unique products. Knowledge of the spatial distribution of transcripts, enzymes, and products within and between cells is ultimately necessary for the regulation of these processes. The chapter by Hamberger et al. exemplifies the necessity of examining metabolic networks. Most aromatic compounds and their precursors are synthesized via the shikimate and its branched pathways. Focusing on the genomes of Arabidopsis, rice, and poplar, their expression data support functional classifications of gene families that encode shikimate pathway enzymes. Comparative data are highlighting promising candidate genes for encoding unknown enzymes in the pathway. Crop improvement is an obvious long-range goal. The lipid papers by Fridman et al. and by Schultz et al. discuss recent progress and potential in this area of plant chemistry. The former use tomato glandular trichomes as a model system. The divergence of primary metabolism (fatty acid biosynthesis) to specialized methylketone biosynthesis is being examined. By combining quantitative trait loci (QTL) analyses with transcriptosome and proteome analysis of the glandular trichomes, the molecular factors regulating this divergence will be determined. Schultz et al. remind us that although anacardic acids, related to salicylic acid, have been studied for over four decades, relatively little is known of their physiological functions in plants, although much is known regarding their bioactivity. This area is reviewed with an emphasis on utilization in agriculture and medicine. Using geranium trichomes, they have found an active lipid metabolism dominated by anacardic acid metabolism, and they suggest a combination primary lipid metabolism and polyketide synthesis. Cook et al. concentrate on a single molecule, the benzoquinone, sorgoleone. Forming greater than 80% or root exudates of sorghum, sorgoleone is phytotoxic and implicated in crop allelopathy. Identification of the putative genes responsible for its synthesis and also the encoded enzymes is leading to manipulation of the pathway in planta. EST analysis with real-time PCR has led to identification of a number of enzymes that are preferentially expressed in root hair cells. Ultimately this may become an allelopathic tool for agriculture. Page and Nagel focus on terpenophenolics of the Cannabaceae that include two fascinating groups - the psychoactive cannabinoids in Cannabis sativa (marijuana) and the bitter acids of Humulus lupulus (hops) that provide flavoring, preservation, and putative healthpromoting effects. While the structures of the terpenopheolics vary, the biosynthetic pathways by which they are formed in glandular trichomes display a common pattern of polyketide formation, prenylation, and cyclization/decoration. By using trichome-

PREFACE PREFACE

vii

target EST analysis, several of the responsible genes have been identified, and the role of polyketide synthases, prenyltransferases, and other enzymes are being unraveled with an eye to future bioengineering of these economically important crops. The chapters by Jayasimha et al., Yang et al., and Bowers and Zhao focus on biosynthesis and regulation by signaling molecules. The Texas Tech group led by Nes (Jayashimah et al.) focuses on molecular recognition in the production and processing of phytosterols. Through evolutionary analyses of sterol methyl transferases, differences are being used to uncover the problem of structure and its relationship to function. Not only enzyme structure and membrane requirements, but also the recognition elements of proteins that utilize sterols as regulatory or "sparking" molecules have controlled sterol evolution. The group led by Pichersky (Yang et al.) at Michigan State concentrates on the methylation and demethylation of signaling molecules that regulate the production of floral volatiles. Methylation appears to be involved in cell-to-cell transport or even long-distance transport, and methylation/demethylation may be responsible for deactivating/activating them. Bowers and Zhao study auxin homeostasis and demonstrate that a complicated network of multiple pathways and genes are responsible for regulating auxin levels in response to environmental and environmental signals. The roles of the separate pathways involved remain obscured. The Normanly group (Calio et al.) focus their work on auxin and the recent advances in the understanding of its biosynthesis from both biological and chemical perspectives. Evidence for "cross-talk between the auxin homeostasis pathways and other hormonal networks is accumulating, and additional metabolic profiling is needed for understanding these interactions. New analytical tools are making this easier. Finally, a number of important methodological approaches and innovative techniques were discussed at the meeting that have been summarized by Mclntosh as "translational" opportunities. The development of a Natural Products Repository in Mississippi, USA will help the address long-standing problem of sample availability by making the sharing of samples and phytochemical information easier by making them available through a real and virtual network. The "omics" revolution has spawned not only new technologies but new ways of thinking about phytochemical exploitation, and some innovative ones are briefly highlighted. Each chapter in this volume concludes with a short summary that also addresses the expected future directions of the work. These short pieces will no doubt serve not only as a stimulus to further reading, but also to encourage intellectual debate and creative thinking as metabolic engineering and direct applications become increasingly feasible. The Phytochemical Society of North America expresses it thanks to the local organizing committee at the Salk Institute and to those who organized the various symposia that resulted in this compendium. The authors who contributed the

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PREFACE

excellent reviews, mostly in a timely fashion, deserve special kudos. Another nod of thanks and appreciation to Darrin T. King, who with his usual collection of professionalism, magical skills, and good humor that he brought to the last 7 volumes, met some special challenges this time. Celia Mclntosh was especially helpful in the dual task of summarizing a number of presentations and providing historical information. It is both with sadness and hope that we close the door on Recent Advances in Phytochemistry. My tenure as Editor-in-Chief for the past 12 years has been longer than anticipated. It marks the transition and progression of the dramatic integration of classical phytochemistry into molecular plant biology. It seems appropriate now to note and look backward with thanks to the gatekeepers who created this legacy: Tom Mabry, V.C. Runeckles, Tony Swain, Frank Loewus, Eric Conn, and Helen Stafford. It is evident that phytochemistry as we approach 2010 has become a mainstream discipline in ways that many of its guardians never envisioned. Exciting days are ahead!

John T. Romeo Editor-in-Chief 1994-2006 University of South Florida

RECENT ADVANCES IN PHYTOCHEMISTRY 1

Recent Advances in Phytochemistry

21

2

Recent Advances in Phytochemistry

22

3

Recent Advances in Phytochemistry

23

Plant Nitrogen Metabolism

4

Recent Advances in Phytochemistry

24

Biochemistry of the Mevalonic Acid Pathway to Terpenoids

5

Structural and Functional Aspects of Phytochemistry

25

Modern Phytochemical Methods

Terpenoids: Structure, Biogenesis, and Distribution

26

Phenolic Metabolism in Plants

The Chemistry and Biochemistry of Plant Hormones

27

Phytochemical Potential of Tropical Plants

6 7

Phytochemical Effects of Environmental Compounds Opportunities for Phytochemistry in Plant Biotechnology

8

Metabolism and Regulation of Secondary Plant Products

28

Genetic Engineering of Plant Secondary Metabolism

9

Phytochemistry As Related to Disease and Medicine

29

Phytochemistry of Medicinal Plants

10

Biochemical Interaction Between Plants and Insects

30

Phytochemical Diversity and Redundancy in Ecological Interactions

11

The Structure, Biosynthesis, and Degradation of Wood

31

Functionality of Food Phytochemicals

12

Biochemisry of Plant Phenolics

32

Phytochemical Signals and PlantMicrobe Interactions

13

Topics in the Biochemistry of Natural Products

33

Phytochemicals in Human Health Protection, Nutrition, and Plant Defense

14

The Resource Potential in Phytochemistry

34

Evolution of Metabolic Pathways

15

The Phytochemistry of Cell Recognition and Cell Surface Interactions

35

Regulation of Phytochemicals by Molecular Techniques

16

Cellular and Subcellular Localization in Plant Metabolism

36

Phytochemistry in the Genomics and Post-Genomics Eras

17

Mobilization of Reserves in Germination

37

Integrative Phytochemistry: From Ethnobotany to Molecular Ecology

18

Phytochemical Adaptations to Stress

38

Secondary Metabolism in Model Systems

19

Chemically Mediate Interactions Between Plants and Other Organisms

39

Chemical Ecology and Phytochemistry of Forest Ecosystems

20

The Shikimic Acid Pathway

40

Integrative Plant Biochemistry

IX ix

This Page is Intentionally Left Blank

CONTENTS

1. Terpene Indole Alkaloid Biosynthesis Sarah E. O'Connor and Elizabeth McCoy

1

2. Pathways to Diverse Ergot Alkaloid Profiles in Fungi Daniel G. Panaccione, Christopher L. Schardl, Christine M. Coyle

23

3. Compartmentalization of Plant Secondary Metabolism Nailish Samanani and Peter J. Facchini

53

4. Comparative Genomics of the Shikimate Pathway in Arabidopsis, Populus trichocarpa and Oryza sativa: Shikimate Pathway Gene Family Structure and Identification of Candidates for Missing Links in Pehnylalanine Biosynthesis Bjorn Hamberger, Jiirgen Ehlting, Brad Barbazuk, and Carl J. Douglas 5. Tomato Glandular Trichomes as a Model System for Exploring Evolution of Specialized Metabolism in a Single Cell Eyal Fridman, Takao Koezuka, Michele Auldridge, Mike B. Austin, Joseph P. Noel, Eran Pichersky 6. Anacardic Acid Biosynthesis and Bioactivity David J. Schultz, Nalinie S. Wickramasinghe, and Carolyn M. Klinge 7. Molecular and Biochemical Investigations of Sorgoleone Biosynthesis Daniel Cook, Franck E. Dayan, Agnes M. Rimando, Zhiqiang Pan, Stephen O. Duke, and Scott R. Baerson 8. Biosynthesis of Terpenophenolic Metabolites in Hop and Cannabis Jonathan E. Page and Jana Nagel

xi xi

85

115

131

157

179

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CONTENTS

9. Engineering Pathway Enzymes to Understand the Function and Evolution of Sterol Structure and Activity Pruthvi Jayasimha, C. Bryson Bowman, Julia M. Pedroza, and W. David Nes

211

10. Methylation and Demethylation of Plant Signaling Molecules Yue Yang, Marina Varbanova, Jeannine Ross, Guodong Wang, Diego Cortes, Eyal Fridman, Vladimir Shulaev, Joseph P. Noel, and Eran Pichersky

253

11. Recent Advances in Auxin Biosynthesis and Conjugation Amber Kei Bowers and Yunde Zhao

271

12. Auxin Biology and Biosynthesis Jessica Calio, Yuen Yee Tarn, and Jennifer Normanly

287

13. Translational Opportunities in Plant Biochemistry Cecilia A. Mclntosh

307

Index

319

Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter One

TERPENE INDOLE ALKALOID BIOSYNTHESIS Sarah E. O'Connor* and Elizabeth McCoy Department of Chemistry Massachusetts Institute of Technology 77 Massachusetts Avenue Cambridge, MA, 02139; (617) 324-0180 *Author for correspondence, email: soc(g),mit.edu

Introduction Terpene Indole Alkaloids Early Enzymes of Terpene Indole Alkaloid Biosynthesis Strictosidine Synthase Tryptamine Substrate Specificity Strictosidine Synthase Secologanin Substrate Specificity Identified Enzymes After Strictosidine Synthase Structural Diversity of Terpene Indole Alkaloids Turnover of Strictosidine Analogs by Strictosidine Glucosidase Summary and Future Directions

2 2 3 4 7 8 8 12 13

2

O’CONNOR O 'CONNOR and McCOY

INTRODUCTION Microbes and plants have evolved and produce a large array of complex secondary metabolites. "3 Secondary metabolites- natural products- are some of the most structurally interesting molecules found in nature, and also serve as effective pharmaceutical agents. Alkaloids, nitrogen containing compounds produced by higher plants, are arguably some of the most chemically complex molecules found in nature, and exhibit a diverse array of potent biological activities.4 However, the biosynthetic pathways of these molecules are more challenging to elucidate than the polyketide and peptide pathways found in microbial organisms.5 First, eukaryotic plant hosts are slower growing and developmentally more complex than prokaryotes, and genomic sequence data of medicinal plants are unavailable. Furthermore, the genes of a given metabolic pathway are physically clustered on the genome of a microbial organism; once a fragment of the metabolic pathway has been identified, it is a relatively straightforward process to sequence the adjoining DNA that contains the remainder of the pathway. Plant pathways are generally not clustered, meaning that each plant enzyme of a pathway must be individually isolated and cloned independently of one another. For these reasons, despite the medicinal importance of plant natural products, plant pathways are not as well understood as bacterial natural product pathways. To date, comparatively few enzymes involved in plant alkaloid biosynthesis have actually been cloned, though more enzymes have been purified. Many plant enzymes are characterized by "reverse genetics" in which the enzymes are isolated from plants or plant cell culture by traditional biochemical chromatography techniques.6 More recently, plant cDNA libraries have been screened for the presence of P450 enzyme or acetyl transferase homologues.7'8 If the appropriate DNA libraries or arrays are available, cDNA subtraction techniques can be used to compare the differences in gene expression between alkaloid producing and nonproducing plants.9 These sequencing and proteomics technologies have been sufficiently refined such that they can be used to address the challenges associated with plant pathways. Therefore, although study of plant alkaloid pathways remains a challenging prospect, modern technologies are beginning to transform the study of plant secondary metabolism.

TERPENE INDOLE ALKALOIDS The terpene indole alkaloids, produced primarily by the Apocynaceae and Rubiaceae, effectively illustrate the complexity of alkaloid biosynthesis. The terpene indole alkaloids are a particularly diverse class of natural products, comprising approximately 3000 members that possess a range of chemical structures and a

TERPENE INDOLE ALKALOID ALKALOID BIOSYNTHESIS TERPENE INDOLE BIOSYNTHESIS

3

wealth of biological activities (Fig. l.l). 10 ' 11 A number of terpene indole alkaloids are used as anti-cancer, anti-malarial, and anti-arrhythmic agents (Fig. l.l). 4 In the US, vinblastine (Velban) and vincristine (Oncovin) are used clinically to treat cancers including Hodgkin's disease,13 non-Hodgkin's lymphoma,14 and Kaposi's sarcoma. 15 Notably, 500 kilograms of the plant Catharanthus roseus are required to 16 produce 1 gram of vincristine (a yield of 0.0002%), and total synthesis of this compound is not practical on an industrial scale.17

H

H3CO2CS Ajmalicine (hypertension treatment)

OH

Yohimbine (adrenergic receptor blocker)

Ajmaline (cardiac arrythmia treatment)

OAc H3CO Strychnine (poison)

''CO 2 CH 3 OH CHO

Vincristine (anti-cancer)

Figure 1.1: Representative terpene indole alkaloids.

EARLY ENZYMES OF TERPENE INDOLE ALKALOID BIOSYNTHESIS The first few steps of TIA biosynthesis are well known and are outlined in Figure 1.2. Terpene indole alkaloids are derived from tryptophan,18 which is decarboxylated to yield tryptamine.19 The involvement of the monoterpene iridoid secologanin 20"24 has been established. Strictosidine (S stereochemistry at C5) is a common intermediate for all terpene indole alkaloids. 25"30 The enzymes that catalyze these first steps of terpene indole alkaloid biosynthesis are known and have been cloned. Tryptophan decarboxylase, a pyridoxal dependent enzyme,31'32 converts tryptophan to tryptamine. Strictosidine synthase catalyzes the stereoselective Pictet-Spengler condensation 33'34 of

4

O’CONNOR O'CONNOR and McCOY

Strictosidine Synthase

;

V

PHO H3CO2C

/^ OH

Strictosidine

Strictosidine Deglucosidase

O

H 3 CO 2 C

H 3 CO 2 C

Figure 1.2: The early steps of terpene indole alkaloid biosynthesis. Strictosidine synthase sets the stereochemistry at C5.

tryptamine (1, Fig. 1.2) and the aldehyde secologanin (2) to yield strictosidine (3). 35 ' 47 Secologanin is a natural product in its own right, and a few of the enzymes responsible for secologanin biosynthesis have also been isolated.48"50 Previous studies with strictosidine synthase from the plants Catharanthus roseus and Rauwolfia serpentina have reported Km values of 20-200 uM for tryptamine (no reported Km for secologanin) and a range of Vmax values. A limited number of alternate substrates have been tested with strictosidine synthase, including Nsubstituted tryptamine, tryptophan, phenylethylamine, tyramine, and a variety of iridoid aldehydes.36 Strictosidine Synthase Tryptamine Substrate Specificity51 The Pictet-Spengler cyclization is critical for the biosynthesis of thousands of alkaloids. We sought to expand our understanding of strictosidine synthase by systematically probing the electronic and steric requirements of the indole substrate and quantifying the steady state kinetics for each of these substrates. Both the 3-(2aminoethyl)-benzofuran (4) 52 and benzothiophene (5) 53 analogs have been assayed. Precedence exists for benzofuran and benzothiophene heterocycles with interesting biological properties.54'55 Both compounds are turned over by strictosidine synthase

TERPENE INDOLE ALKALOID ALKALOID BIOSYNTHESIS TERPENE INDOLE BIOSYNTHESIS

5

Table 1.1: Substrates tested with strictosidine synthase and the resulting products. Amine Substrate

Aldehyde Substrate

Strictosidine Analog

5X = MeO 2 C

no reaction N Me

7 R4 = F; R5, R6, R7 = H 8 R5=F; R4, R6, R7 = H 9 R6=F; R4, R5, R7 = H 10 R7=F; R4, R5. R6 = H

11 R4 = Me; R7 = H 14 R7 =Me; R4 = H MeO 2 C

no reaction 12 R5 = Me; R6 = H 13 R6 = Me; R5 = H

MeO 2 C

no reaction MeO

P-GIc MeO

MeO

H MeO,C

NH2 19X = CH 20 X = N

no reaction

L A ,.0-Glc /L MeO2C v

o

21 R = /-butyl 22 R = butyl

,.0-Glc 23 R = ethyl 24 R = allyl

no reaction

O’CONNOR O'CONNOR and McCOY

6

in the presence of the aldehyde substrate secologanin 2, and a single diastereomer is observed, indicating that enantioselective enzymatic catalysis is not compromised (Table 1.1). The alternate heterocycles are turned over by strictosidine synthase at a diminished rate relative to the tryptamine 1 substrate. Although the low activity of the thiophene substrate precluded a quantitative comparison of 4 and 5, the rate of reaction of benzothiophene 5 is significantly slower than benzofuran 4. Notably, no chemical reaction of 3-(2-aminoethyl)-benzofuran (4) and 3-(2-aminoethyl)benzothiophene (5) occurred at 40 mM concentration under mild acidic conditions, demonstrating that the enzyme can catalyze product formation with relatively chemically inactive substrates with complete enantioselective control. Since the Pictet-Spengler cyclization is inherently dependent on an electron rich aminoethylarene substrate,34 the decreased electron density of the benzofuran and benzothiophene rings may cause the slower rate compared to indole. Alternatively, strictosidine synthase may utilize a specific hydrogen bonding interaction to the indole nitrogen. The benzofuran 4 exhibits a Km value close to that of tryptamine, but displays a significantly reduced kcat, suggesting that the electron deficient nature of the heterocylic ring is slowing catalysis (Table 1.2). jV-methyl tryptamine 6 (Table 1.1) was not a competent substrate, suggesting that the enzyme tolerates only small steric perturbations at the indole nitrogen. To further explore the effect of electron density on catalysis, the indole ring was substituted with electronwithdrawing substituents (fiuoro=F) at each of the indole ring positions (Table 1.1). Substitution with a fluoro moiety results in a decrease in kcat in each case, suggesting that the enzymatic reaction is inherently dependent on the electron density of the substrate. Table 1.2: Kinetic parameters for the most highly active amine strictosidine synthase substrates. kcat and Km were measured using a purified E. coli preparation of strictosidine synthase. K m 0iM)

kcat (min" 1 )

kcat/Kn, (M'Vs- 1 )

1 4 7

7.4 7.7 42

0.9 0.023 0.35

2,030 50 139

8 9

7.1 8.9

0.043 0.056

101 105

10

13

0.11

141

11 14

80 198

0.19 0.29

40 24

15

1,200

0.096

1.3

Substrate

TERPENE INDOLE ALKALOID ALKALOID BIOSYNTHESIS BIOSYNTHESIS TERPENE INDOLE

7

Early qualitative studies, performed after strictosidine synthase was first isolated, indicated that some substitution on the indole ring was tolerated.36 To rigorously quantify the effect of indole ring substitution on catalysis, each position of the indole ring was systematically substituted with a methyl group (compounds 1114, Table 1.1), and the kinetic parameters of active substrates were measured. In general, reactivity of substrates with methyl substitutions in the 4 (compound 11) and 7 (compound 14) indole positions was significantly higher than substrates with substitutions at the 5 (compound 12) and 6 (compound 13) positions (Tables 1.1 and 1.2). The Km for the 4-substituted tryptamine analog 11 was approximately 2-fold lower than the Km for 7-substituted compound 14, while substrates with methyl moieties in the 5 and 6 positions- compounds 12 and 1 3 - were completely inactive. Substitution with a hydroxyl group in the 5 position (compound 15, Table 1.1 and Table 1.2) did yield an active substrate, although the Km was the highest measured in this series- a 60-fold increase compared to the native substrate tryptamine. Therefore, the binding pocket of the enzyme can better tolerate a hydrophilic hydroxyl substituent than a hydrophobic methyl group at the 5 position. The 2-pyrrole-3-ethylamine analog (19) has been shown to undergo a nonenzymatically catalyzed Pictet-Spengler reaction.56 Surprisingly, this smaller substrate along with the isosteric histamine (20) were not turned over by strictosidine synthase, indicating that the benzyl moiety is absolutely required for recognition by the enzyme (Table 1.1). It was previously established that tryptophan, phenylethylamine, and tyramine are not accepted by strictosidine synthase,3 and since pyrrole substrates are also not tolerated, we conclude that the basic indole framework is required for recognition by this enzyme. Interestingly, the only other sequenced "Pictet-Spenglerase" (norcoclaurine synthase), which utilizes tyrosine derived amine and aldehyde substrates, exhibits no sequence homology to strictosidine synthase.57 Strictosidine Synthase Secologanin Substrate Specificity Several naturally occurring iridoid terpenes had been previously shown to fail to serve as competent aldehyde substrates in place of secologanin. Therefore, we modified two of the key functional groups of the secologanin substrate to assess the aldehyde substrate requirements. A streamlined gram-scale isolation protocol of secologanin from a local source of Lonicera tatarica enabled a semisynthetic approach to yield secologanin derivatives. Olefin cross metathesis was used to introduce a variety of alkyl groups at the vinyl position of secologanin {i.e., compounds 21 and 22, Table 1.1). Since a reduced version of secologanin, in which the vinyl group is hydrogenated to yield a saturated single C-C bond, had been previously shown to be a competent substrate,36 we were optimistic that this position could be derivatized. However, our assays indicated that bulkier groups at the vinyl position completely prevented turnover (Table 1.1). In contrast, fraws-esterification

8

O’CONNOR O 'CONNOR and McCOY

at the methyl ester with larger alkyl groups58 gave substrates 23 and 24 that were turned over by the enzyme to yield the corresponding strictosidine analogs, suggesting that this is a more promising position for derivatization (Table 1.1).

IDENTIFIED ENZYMES AFTER STRICTOSIDINE SYNTHASE In the first enzymatic step after strictosidine formation, the glucose of strictosidine is enzymatically hydrolyzed to reveal a reactive hemi-acetal (Fig. 1.2). In essence, the glucose moiety is serving as a protecting group to mask a reactive species, a strategy that is utilized in other plant biosynthetic pathways such as the cyanogenic glucosides and the glucosinolates.2 The dedicated glycosidase, strictosidine-(3-glucosidase has been isolated and cloned from Catharanthus roseus and Rauwolfia serpentina. 59"63 Based on its amino acid sequence, strictosidine glucosidase is predicted to be a type 1 beta glycosyl hydrolase with a retaining mechanism.64 Some biochemistry of the glucosidase from the plants C. roseus and R. serpentina has been investigated.5 >65 Km values of -100-200 uM for strictosidine, a pH optimum of 5-8.5, and a range of Vmax values have been reported for this enzyme. Figure 1.3 summarizes much of what is known about the enzymes of alkaloid biosynthesis that act after strictosidine deglycosylation.6 In ajmaline biosynthesis, at least eight enzymes are predicted to catalyze the subsequent steps after strictosidine deglycosylation. Two of these enzymes have been cloned,66'6 and the remainder have been either purified or detected in crude cell extracts.68"75 The pathway for ajmaline biosynthesis is arguably the best-characterized terpene indole alkaloid pathway. Five enzymes are predicted to catalyze the transformations leading from tabersonine (23) to vindoline (24).76 Three of these enzymes have been cloned,77'80 and the remainder have been partially purified.81"84

STRUCTURAL DIVERSITY OF TERPENE INDOLE ALKALOIDS A number of examples of the terpene indole alkaloid classes, each arising from rearrangement of strictosidine, are shown in Figure 1.4. How the wealth of TIA structures each derive from the deglycosylated strictosidine intermediate remains one of the most fascinating problems in secondary metabolism. Extensive feeding studies and biomimetic syntheses executed in the 1960's and 1970's yielded chemical information about how this branching process might occur. A figure summarizing some of the conclusions is presented below (Fig. 1.5). After deglycosylation of strictosidine (3), the resulting aglycone (25) opens to form an intermediate often referred to as a dialdehyde (26). The resulting aldehyde then reacts with the secondary amine to form a six membered ring to yield

TERPENE INDOLE ALKALOID ALKALOID BIOSYNTHESIS TERPENE INDOLE BIOSYNTHESIS

9

H 3 CO vindoline

Figure 1.3: This scheme summarizes much of the current knowledge of TIA enzymes that act after the deglycosylation of strictosidine. A. Ajmaline biosynthesis. SB, sarpagan bridge enzyme; PAE, polyneuridine aldehyde reductase; VS, vinorine synthase; VH, vinorine hydroxylase; VR, vomilenine reductase(s); AE, 17-O-acetyl-ajmalan acetylesterase; NMT, norajmaline-Af-methyltransferase. Only polyneuridine aldehyde reductase and vinorine synthase have been cloned. B. Vindoline biosynthesis from tabersonine. T16H, tabersonine16-hydroxylase; HTOM, 16-hydroxytabersonine-16-O-methyltransferase; NMT, Nmethyltransferase; D4H, desacetoxyvindoline-4-hydroxylase; DAT, desacetylvindoHne Oacetyltransferase. Tabersonine-16-hydroxylase, desacetoxyvindoline-4-hydroxylase and desacetylvindoHne O-acetyltransferase have been cloned.

10 10

O'CONNOR and McCOY O’CONNOR and McCOY

MeO2C

MeO2C corynanthe (ajmalicine)

corynanthe (yohimbine)

= \

CO2Me aspidosperma (tabersonine)

CO2Me iboga (catharanthine)

MeO. MeO

strychnos (strychnine)

C9 iboga (ibogaine)

quinoline (quinine)

C9 corynanthe (ajmaline)

Figure 1.4: Representative classes of the terpene indole alkaloids. The name of the alkaloid class is given in parentheses below the name of the molecule. Each of these alkaloids is derived from the common intermediate strictosidine.

dihydrocorynanthe aldehyde (27). Dihydrocorynanthe aldehyde can undergo ally lie isomerization and enolization to produce either the enol (28) or keto (29) forms of dehydrogeissoschizine. Dehydrogeissoschizine can be reduced by an oxidoreductase enzyme to yield geissoschizine (30), which is an intermediate that may also play a role in TIA biosynthesis.85'87 It is likely that the enol form of dehydrogeissoschizine 28 will undergo 1,4 conjugate addition to produce the heteroyohimbine cathenamine (33). Early biomimetic syntheses support the hypothesis that cathenamine can be produced from dehydrogeissoschizine.8 An equilibrium between cathenamine and 89 90 dehydrogeissoschizine has also been observed. ' Stereoselective reduction of cathenamine will yield ajmalicine 34, and further oxidation will yield serpentine 35.91"94 Cathenamine 9 is the major product isolated after incubation of strictosidine with strictosidine-P-glucosidase in vitro. 60 Therefore, an enzymatic pathway to the corynanthe skeleton from strictosidine appears to be relatively straightforward.

TERPENE ALKALOID BIOSYNTHESIS TERPENE INDOLE ALKALOID BIOSYNTHESIS

11 11 32

OH

HO N N H Me

ajmaline 30 31

MeO2C

3 N H

25 NH H

O-Glc O

NH H

N H

MeO2C

OH

NH H

N H

O MeO2C

N H

O N H

OH MeO2C

29

O

36

33

N H CH2OH CO2Me

N H

MeO2C

MeO2C

N

N H

N H

OH

MeO2C

39

N H H

CHO

N H

N H

OH

CO2Me

N

28

27

26

CHO

N H

N H

cathenamine

N H

N H

O

N H

MeO2C

MeO2C

O

37

34 N

40

N H N H

aspidosperma

CH2OH CO2Me

N H

N

H N H

O

ajmalicine

H

MeO2C

MeO2C

OH

43

44 N H MeO N

HO

N H

35

N

41

38 N

OAc CO2Me

N H

N H

CO2Me

vindoline

N H CO2Me

iboga 45

HO N

N H MeO2C MeO

42

serpentine

N

H O

MeO2C

corynanthe

N H

MeO2C

N H

OAc CO2Me OH

N H

OH

corynanthe (yohimbine type)

N

N

H

H

CO2Me

vinblastine

Figure 1.5: Summary of some of the key rearrangements of the intermediates of the terpene indole alkaloid biosynthetic pathway to yield the corynanthe, aspidosperma, and iboga skeletons. The pathways are hypothesized from evidence derived from feeding experiments, biomimetic syntheses and transformations with crude cell extracts.

12 12

O’CONNOR O 'CONNOR and McCOY

The enzymatic conversion of deglycosylated strictosidine to the other classes of alkaloids remains less clear. For yohimbine (38) formation, a direct biosynthetic route could involve homoallylic isomerization of the keto dehydrogeissoschizine 29 followed by 1,4 conjugate addition.95 The structurally more complex aspidosperma, strychnos, and iboga alkaloids may each be derived from the corynanthe alkaloids. This hypothesis is indirectly supported by observation that the corynanthe alkaloids are produced early in the lifetime of the Catharanthus roseus plant, with the aspidosperma and iboga alkaloids appearing after the plant ages.96 Deglycosylated strictosidine can rearrange to form the strychnos, aspidosperma, and iboga alkaloids. Although the details of the pathway are not absolutely certain, it is generally agreed that dehydrogeissoschizine (28) can rearrange to form a strychnos-like intermediate termed preakkumacine (39).97 Stemmadenine (40) could then in turn rearrange to form an acrylic ester (41) that could serve as a common intermediate for the aspidosperma (i.e., tabersonine 43) and the iboga skeletons (i.e., catharanthine 42). A few of the key experiments and hypotheses are described in the following selected references.20'97"117 (Some of these findings are reviewed115'118"121). The branching among these enzymes is in part controlled by the species of plant. While the corynanthe, iboga, and aspidosperma alkaloids are observed in Catharanthus roseus plants, strychnine (Strychnos nux vomica) and ajmaline (Rauwolfia) are not. Moreover, the aspidosperma and iboga alkaloids appear to be concentrated in the ariel portions of C. roseus, while the corynanthe appear primarily in the roots.122 The coexistence of multiple pathways-the corynanthe, aspidosperma, and iboga- makes Catharanthus an intriguing system to monitor alkaloid biosynthesis.

TURNOVER OF STRICTOSIDINE ANALOGS BY STRICTOSIDINE GLUCOSIDASE Chemical synthesis of complex natural products is often impractical on a commercial scale, and isolation of these compounds from the environment can be an expensive and low yielding process. Furthermore, isolation procedures provide limited opportunities to modify the chemical and biological properties of the natural product. Understanding the enzymes that catalyze natural product synthesis may enable production in more tractable host organisms and may also facilitate reprogramming of biosynthetic pathways to produce "unnatural" natural products with potentially improved pharmacological activities. Natural products from polyketide,123'124 non-ribosomal peptide,125 terpene,126 and saccharide 27 biosynthetic pathways have been heterologously expressed in organisms that are faster growing or easier to culture. Although metabolic engineering has proven remarkably successful in polyketide biosynthetic and nonribosomal peptide pathways, ' ' in terpene indole alkaloid biosynthesis, the backbone of strictosidine is significantly rearranged

TERPENE INDOLE TERPENE INDOLE ALKALOID ALKALOID BIOSYNTHESIS BIOSYNTHESIS

13

over the course of several steps, whereas polyketides and nonribosomal peptides are synthesized by an iterative, "assembly-line" process, in which a linear chain is successively elongated.130 Can a "non-modular" pathway process unnatural substrates to yield novel alkaloids? While initial results suggest that strictosidine synthase can produce a range of strictosidine analogs, it remains to be established whether these alternate intermediates can be processed by the downstream terpene indole alkaloid machinery to produce novel, biologically active alkaloids. In the next step of the pathway, a dedicated glucosidase hydrolyzes the glycosidic linkage of strictosidine to yield cathenamine 33 (Fig. 1.5).59'60 To evaluate whether cathenamine derivatives could be enzymatically produced from the corresponding strictosidine analogs, we incubated all enzymatically generated strictosidine derivatives (Table 1.1, column 4) with the second enzyme of the pathway, strictosidine-P-glucosidase. All strictosidine derivatives were processed by strictosidine glucosidase as monitored by the disappearance of the strictosidine derivative peak by HPLC. These results suggest that the substrate specificities of strictosidine synthase and glucosidase are sufficiently complementary to produce a variety of terpene indole alkaloid intermediate analogs.

SUMMARY AND FUTURE DIRECTIONS The terpene indole alkaloids are a diverse family of plant-derived compounds that exhibit numerous potent pharmaceutical properties. Strictosidine synthase catalyzes a Pictet-Spengler reaction in the first step in the biosynthesis of terpene indole alkaloids to generate strictosidine. This (3-carboline intermediate is next turned over by strictosidine glucosidase to yield a reactive intermediate that rearranges to form the fused 5-ring cathenamine structure. Subsequent enzymes are responsible for converting this early intermediate into a structurally diverse set of alkaloids. We have systematically probed the substrate requirements for strictosidine synthase and shown that the enzymatically generated unnatural strictosidine intermediates are processed by the glucosidase to yield analogs of the corynanthe type. As more enzymes in the terpene indole alkaloid pathways are cloned, the substrate specificity of these enzymes can also be evaluated to determine whether this biosynthetic pathway can potentially be used to produce novel alkaloid derivatives.

14 14

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ACKNOWLEDGMENTS I gratefully acknowledge my students and post-docs for their help and also for the American Chemical Society, the Smith Family Medical Foundation, 3M and the Beckman Foundation for supporting this work. REFERENCES 1. 2. 3. 4. 5. 6. 7.

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93.

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LAFLAMME, P., ST. PIERRE, B., DE LUCA, V., Molecular and biochemical analysis of a madagascar periwinkle root specific minovincinine-19-hydroxy-Oacetyltransferase., Plant Physiol., 2001, 125, 189-198. ST. PIERRE, B., LAFLAMME, P., ALARCO, A., DE LUCA, V., The terminal Oacetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer., Plant J., 1998,14, 703-713. VAZQUEZ-FLOTA, F., DE CAROLIS, E., ALARCO, A., DE LUCA, V., Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don., Plant Mol. Biol, 1997, 34, 935-948. RODRIGUEZ, S., COMPAGNON, V., CROUCH, N. P., ST. PIERRE, B., DE LUCA, V., Jasmonate induced epoxidation of tabersonine by a cytochrome P450 in hairy root cultures of Catharanthus roseus., Phytochemistry, 2003, 64, 401-409. DETHIER, M., DE LUCA, V., Partial purification of an N-methyltransferase involved in vindoline biosynthesis in Catharanthus roseus., Phytochemistry, 1993, 32, 673-678. ST. PIERRE, B., DE LUCA, V., A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus. Plant Physiol, 1995,109, 131-139. CACACE, S., SCHRODER, G., WEHINGER, E., STRACK, D., SCHMIDT, J., SCHRODER, J., A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations., Phytochemistry, 2003, 62, 127-137. STOCKIGT, J., Biosynthesis in Rauwolfia serpentina. In: The Alkaloids, vol. 47 (ed. CORDELL, G. A.), Academic Press, San Diego, 1995, pp. 115-172. PFITZNER, A., STOCKIGT, J., Partial purification and characterization of geissoschizine dehydrogenase from suspension cultures of Catharanthus roseus., Phytochemistry, 1982, 21, 1585-1588. STOCKIGT, J., HOFLE, G., PFITZNER, A., Mechanism of the biosynthetic conversion of geissoschizine to 19-epi-ajmalicine in Catharanthus roseus., Tetrahedron Lett, 1980,21, 1925-1926. KAN-FAN, C , HUSSON, H. P., Isolation and biomimetic conversion of 4,21dehydrogeissoschizine., J. Chem. Soc. Chem. Comm., 1979, 1015-1018. EL-SAYED, M., CHOI, Y. H., FREDERICH, M., ROYTRAKUL, S., VERPOORTE, R., Alkaloid accumulation in Catharanthus roseus cell suspension cultures fed with stemmadenine., Biotech. Lett., 2004, 26, 793-798. HEINSTEIN, P., HOFLE, G., STOCKIGT, J., Involvement of cathenamine in the formation of N-analogues of indole alkaloids., Planta Med., 1979, 37, 349-357. KAN-FAN, C , HUSSON, H. P., Stereochemical control in the biomimetic conversion of heteroyohimbine alkaloid precursors. Isolation of a novel key intermediate., J. Chem. Soc. Chem. Comm., 1978, 618-619. HUSSON, H. P., KAN-FAN, C , SEVENET, T., VIDAL, J.-P., Structure de la cathenamine intermediaire cle de la biosynthese des alcakloides indoliques., Tetrahedron Lett., 1977,22, 1889-1892. BROWN, R. T., LEONARD, J., SLEIGH, S. K., One-pot biomimetic synthesis of heteroyohimbine alkaloids., J. Chem. Soc. Chem. Comm., 1977, 636-638.

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94. BROWN, R. T., LEONARD, J., Biomimetic synthesis of cathenamine and 19epicathenamine, key intermediates to heteroyohimbine alkaloids., J. Chem. Soc. Chem. Comm., 1979, 877-879. 95. KAN-FAN, C, HUSSON, H. P., Biomimetic synthesis of yohimbine and heteroyohimbine alkaloids from 4,21-dehydrogeissoschizine., Tetrahedron Lett., 1980,21, 1460-1463. 96. QURESHI, A. A., SCOTT, A. I., Biosynthesis of indole alkaloids: Sequential precursor formation and biological conversion in Vinca rosea., Chem. Comm., 1968, 948-950. 97. SCOTT, A. I., QURESHI, A. A., Biogenesis of Strychnos, Aspidosperma and Iboga alkaloids. The structure and reactions of preakuammicine., J. Amer. Chem. Soc, 1969, 91, 5874-5876. 98. WENKERT, E., Biosynthesis of indole alkaloids. The Aspidosperma and Iboga bases., J. Amer. Chem. Soc, 1962, 84, 98-102. 99. WENKERT, E., WICKBERG, B., General methods of synthesis of indole alkaloids. IV. A synthesis of dl-eburnamonine.,./ Amer. Chem. Soc, 1965, 87, 1580-1589. 100. BATTERSBY, A. R., BYRNE, J. C, KAPIL, R. S., MARTIN, J. A., PAYNE, T. G., ARIGONI, D., LOEW, P., The mechanism of indole alkaloid biosynthesis., Chem. Comm., 1968, 951-953. 101. BATTERSBY, A. R., BURNETT, A. R., HALL, E. S., PARSONS, P. G., The rearrangement process in indole alkaloid biosynthesis., Chem. Comm., 1968, 15821583. 102. QURESHI, A. A., SCOTT, A. I., Biogenetic-type synthesis of Iboga alkaloids: Catharanthine., Chem. Comm., 1968, 947. 103. QURESHI, A. A., SCOTT, A. I., Interconversion of corynanthe, aspidosperma and iboga alkaloids a model for indole alkaloid biosynthesis., Chem. Comm., 1968, 945946. 104. KUTNEY, J. P., CRETNEY, W. J., HADFIELD, J. R., HALL, E. S., NELSON, V. R., WIGFIELD, D. C, Studies on indole alkaloid biosynthesis., J. Amer. Chem. Soc, 1968, 90,3566-3567. 105. KUTNEY, J. P., EHRET, C, NELSON, V. R., WIGFIELD, D. C, Studies on indole alkaloid biosynthesis., J. Amer. Chem. Soc, 1968, 90, 5929-5930. 106. SCOTT, A. I., CHERRY, P. C, QURESHI, A. A., Mechanisms of indole alkaloid biosynthesis. The Corynanthe-Strychnos relationship., J. Amer. Chem. Soc, 1969, 91, 4932-4933. 107. BATTERSBY, A. R., HALL, E. S., The intermediacy of geissoschizine in indole alkaloid biosynthesis: Rearrangement to the strychnos skeleton., Chem. Comm., 1969, 793-794. 108. BATTERSBY, A. R., BHATNAGAR, A. K., Evidence from synthesis and isolation concerning the rearrangement process in indole biosynthesis., Chem. Comm., 1970, 193-195. 109. BATTERSBY, A. R., GIBSON, K. H., Further studies on rearrangement during biosynthesis of indole alkaloids., Chem. Comm., 1971, 902-903. 110. SCOTT, A. I., Regio and stereospecific models for the biosynthesis of the indole alkaloids, prologue and commentary., J. Amer. Chem. Soc, 1972, 94, 8262.

TERPENE INDOLE ALKALOID ALKALOID BIOSYNTHESIS BIOSYNTHESIS TERPENE INDOLE

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111. BROWN, R. T., SMITH, G. F., POISSON, J., KUNESCH, N., Indole alkaloid rearrangements in acetic acid., J. Amer. Chem. Soc, 1973, 95, 5778. 112. SCOTT, A. I., REICHARDT, P. B., SLAYTOR, M. B., SWEENY, J. G., Mechanisms of indole alkaloid biosynthesis., Bioorg. Chem., 1971, 1, 157-173. 113. SCOTT, A. I., LEE, S.-L., Biosynthesis of the indole alkaloids. A cell free system from Catharanthus roseus.,J. Amer. Chem. Soc, 1975, 97, 6906-6908. 114. LEE, S. L., HIRATA, T., SCOTT, A. I., Indole alkaloid biosynthesis in Catharanthus roseus: involvement of geissoschizine and 19-epiajmalicine., Tetrahedron Lett., 1979, 8, 691-694. 115. KUTNEY, J. P., BECK, J. F., EHRET, C , POULTON, G., SOOD, R. S., WESTCOTT, N. D. Studies on indole alkaloid biosynthesis., Bioorg. Chem., 1971, 1, 194-206. 116. BROWN, R. T., LEONARD, J., Reversible trapping of labile 21dehydroheteroyohimbines as 21-cyano adducts., Tetrahedron Lett., 1977, 48, 42514254. 117. BATTERSBY, A. R., BURNETT, A. R., PARSONS, P. G., Alkaloid biosynthesis. Partial synthesis and isolation of vincoside and isovincoside. Biosynthesis of the three major classes of indole alkaloids from vincoside., J. Chem. Soc, C, 1969, 1193-1200. 118. SCOTT, A. I., Biosynthesis of the indole alkaloids., Ace Chem. Res., 1970, 3, 151157. 119. SCOTT, A. I., QURESHI, A. A., Regio and stereospecific models for the biosynthesis of the indole alkaloids., Tetrahedron, 1974, 30, 2993-3002. 120. BATTERSBY, A. R., Biosynthesis [of alkaloids]. II. Terpenoid indole alkaloids., Alkaloids, 1971,1,31-47. 121. ZENK, M. H., Enzymatic synthesis of ajmalicine and related indole alkaloids., J. Nat. Prod, 1980, 43, 438-451. 122. BONZOM, M.-P., ANDARY, C , GUEIFFIER, A., ROUSSEL, J.-L., GARGADENNEC, A., Effect of a tertiary amine on alkaloid accumulation in Catharanthus roseus., Phytochemistry, 1997, 46, 235-239. 123. PFEIFER, B. A., ADMIRAAL, S. J., GRAMAJO, H., CANE, D. E., KHOSLA, C , Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli., Science, 2001, 291, 1790-1792. 124. KIM, B. S., SHERMAN, D. H., REYNOLDS, K. A., An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases., Prot. Eng. Des. Select., 2004, 17, 277-284. 125. SCHNEIDER, A., STACHELHAUS, T., MARAHIEL, M. A., Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping., Mol. Gen. Genetics, 1998, 257, 308-318. 126. DEWICK, P. M., The biosynthesis of C5-C25 terpenoid compounds., Nat. Prod. /te/?., 2002,19, 181-222. 127. HOFFMEISTER, D., WILKINSON, B., FOSTER, G., SIDEBOTTOM, P. J., ICHINOSE, K., BECHTHOLD, A., Engineered urdamycin glycosyltransferases are broadened and altered in substrate specificity., Chem. Biol, 2002, 9, 287-295.

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128. TSOI, C. J., KHOSLA, C , Combinatorial biosynthesis of 'unnatural' natural products, the polyketide example., Chem. Biol, 1995, 22, 355-362. 129. O'CONNOR, S. E., WALSH, C. T., LIU, F., Biosynthesis of epothilone intermediates with alternate starter units, Engineering polyketide-nonribosomal interfaces., Angew. Chem. Intl. Ed, 2003, 42, 3917-3921. 130. STAUNTON, J., WEISSMAN, K. J., Polyketide biosynthesis: A millennium review., Nat. Prod. Rep., 2001,18, 380-416.

Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Two

PATHWAYS TO DIVERSE ERGOT ALKALOID PROFILES IN FUNGI Daniel G. Panaccione,1* Christopher L. Schardl,2 Christine M. Coyle1 'Division of Plant & Soil Sciences West Virginia University Morgantown, WV 26506-6058 Department of Plant Pathology University of Kentucky Lexington, KY 40546-0312 * Author for correspondence, email: [email protected]

Introduction Significance of Ergot Alkaloid-Producing Fungi Ergot Fungi in the Genus Claviceps Ergot Alkaloid-Producing Endophytes of Grasses Aspergillus fumigatus Different Sets of Ergot Alkaloids via Diverging Paths from a Common Origin Overview of Diverging Pathways and Associated Gene Clusters Shared Early Pathway Steps Clavines in Different Ergot Alkaloid Pathways Ergopeptines Simple Amides of Lysergic Acid Diversification of Ergot Alkaloid Profiles within Producers Gene Cluster Differences and Evolution Potential Origins of Ergot Alkaloid Gene Clusters Differences in Cluster Composition and Arrangement Codon Usage Bias Summary and Future Directions

23

24 24 24 25 26 27 27 33 36 39 41 43 43 43 44 45 46

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PANACCIONE, et al. PANACCIONE,etal.

INTRODUCTION The ergot alkaloids are a large, complex family of mycotoxins that originate from prenylated tryptophan and are modified and further processed into different sets of related alkaloids in several different fungi. Ergot alkaloids may be classified into three groups: clavines, ergopeptines, and simple amides of lysergic acid. Clavines are generally simpler ergot alkaloids from the earlier steps of the ergot pathway, some of which provide a pathway to lysergic acid. The ergopeptines are nonribosomally synthesized peptides containing lysergic acid and a modified threemembered peptide ring. Simple amides of lysergic acid contain an amide linkage to a simple amine, a single amino acid, or a modified single amino acid. Ergot alkaloids have a long history of association with agricultural problems and human suffering. This chapter will focus on the ergot alkaloids produced by three different groups of fungi: the ergot pathogens in the genus Claviceps, fungi in the genus Neotyphodium that grow endophytically in grasses, and Aspergillus fumigatus, a common saprophyte and opportunistic human pathogen. Claviceps spp. and Neotyphodium spp. are members of the family Clavicipitaceae (clavicipitaceous fungi) in the order Hypocreales. In contrast, A. fumigatus is an imperfect fungus derived from the distantly related ascomycete order Eurotiales. We will introduce the three groups of fungi and briefly consider the significance of ergot alkaloids in these different producers. The majority of the chapter will focus on the ergot alkaloid pathway and how it differs among different producers, as well as on recent data on genes involved in ergot alkaloid production. These molecular genetic studies provide a means for further elucidating the pathway and investigating the significance of ergot alkaloids.

SIGNIFICANCE OF ERGOT ALKALOID-PRODUCING FUNGI Ergot Fungi in the Genus Claviceps The genus Claviceps contains ovarian pathogens of rye (Secale cereale), wheat (Triticum aestivum), sorghum (Sorghum vulgare), and other wild and cultivated grasses. These fungi attack flowering plants, colonize the pistils, and produce hard, dark sclerotia (called ergots) in place of the developing seed. High concentrations of ergot alkaloids can accumulate in sclerotia, which may then be harvested with the grain and, unless removed, ingested by humans or livestock. The contamination of rye and other grain crops with ergot alkaloid-rich sclerotia of the ergot fungus Claviceps purpurea was responsible for gangrenous and convulsive forms of ergotism known as St. Anthony's fire.1"3 This toxicosis periodically plagued people from the dawn of recorded history until diets were diversified and the screening of grain to remove ergot sclerotia became a common practice over the past

PATHWAYS PATHWAYS TO DIVERSE DIVERSE ERGOT ERGOT ALKALOID ALKALOID PROFILES PROFILES

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few centuries. Noteworthy epidemics of ergot poisoning have been postulated by some historians to have contributed to the Salem witch trials, the French revolution,1 inciting the crusades,5 and thwarting the campaign of Peter the Great of Russia to conquer the Ottoman Empire.6 Different ergot alkaloids act as partial agonists or antagonists at a variety 5hydroxytryptamine (5-HT or serotonin), dopamine, and a-adrenaline receptors types and subtypes.7'8 Collectively, ergot alkaloids affect nervous, circulatory, reproductive, and immune systems, leading to high or low blood pressure, muscle contractions, reduced fertility, reduced lactation, disturbances in sleep/wake cycles, lowered immune response, hallucinations, and dry gangrene of the extremities.7"10 In controlled doses, some ergot alkaloids and their semisynthetic derivatives are used medicinally, but their lack of specificity for individual monoamine receptors often results in unpredictable and undesirable side effects (most famously exemplified by LSD). Other species of ergot fungi, each accumulating a different portfolio of ergot alkaloids, are associated with other monocot hosts. Claviceps paspali and Claviceps fusiformis cause ergot grain replacement diseases of Paspalum spp. and pearl millet {Pennisetum glaucum), respectively. At least three species of Claviceps produce ergot diseases of sorghum worldwide, but only one of these species, Claviceps africana, is known to produce significant quantities of ergot alkaloids.11"13 Sorghum ergot, in general, is a problem because of losses in seed production and because the sticky honeydew associated with ergot infection interferes with the harvest of the grain. Sorghum ergot caused by C. africana has the added threat of causing toxicoses in animals to which the contaminated sorghum is fed.14 Ergot Alkaloid-Producing Endophytes of Grasses Several asexual fungi in the genus Neotyphodium (and occasionally their sexual relatives in the genus Epichloe) produce ergot alkaloids. These fungi live symbiotically as endophytes with their grass hosts, which include species that are important as forage and turf.15"17 They typically grow in the intercellular spaces, and in the vegetative plant they often are most concentrated near the bases of pseudostems. During flowering, the endophytes colonize the floral meristem, a hyphal pad extensively colonizes the seeds beneath the seed coat, and some hyphae are incorporated into the embryos. Thus, the next generation of grass seedling comes pre-inoculated with its endophyte. Sexual endophytes differ in that in some infected tillers the fungus erupts though the plant epidermis to form a stroma in which sexual reproduction will occur. During this sexual phase, these particular endophytes are capable of plant-to-plant dispersal. Endophytes may have significant effects on their host plants and on herbivores, parasites, and competitors that interact with their host plants. Some endophytic fungi are highly desirable from an agronomic perspective because they

26

PANACCIONE, et al. PANACCIONE,

increase host fitness and stress tolerance.15"22 However, endophytes also are associated with a variety of livestock toxicoses. In the U.S.A., particular attention has been paid to the toxicosis associated with the symbiosis of Neotyphodium coenophialum with tall fescue (Lolium arundinaceum = Festuca arundinacea = Schedonorus phoenix). Symptoms of tall fescue endophyte toxicosis, several of which resemble historical ergotism, include the following: weight loss or poor weight gain; low fertility; poor milk production; inability to control body temperature; low immunity; and, a dry gangrene of the limbs and tail. '' 15' ' 4 Several carefully performed experiments have indicated that ergot alkaloids produced by N. coenophialum are major factors in the toxicosis.24" 6 However, which ergot alkaloid(s) among the complex ergot profile produced by N. coenophialum are primarily responsible is not clear.24'26 Potential contribution of any ergot alkaloid to the agronomically beneficial traits associated with endophyte presence is unknown. Other endophytes in which ergot alkaloids have been well characterized include the hybrid Neotyphodium lolii x Epichloe typhina {Neotyphodium sp. Lpl) from perennial ryegrass {Lolium perenne), and an endophyte tentatively named 'Neotyphodium inebrians' from Achnatherum inebrians. Neotyphodium sp. Lpl produces a portfolio of ergot alkaloids similar to that produced by N. coenophialum, including the ergopeptine ergovaline. In contrast, the Ach. inebrians/N. inebrians symbiota have high levels of simple amides of lysergic acid and no detectable ergopeptines.28 Aspergillus fumigatus The common saprophyte and opportunistic human pathogen A. fumigatus produces a set of ergot alkaloids that differs significantly from those produced by any clavicipitaceous fungus. Aspergillus fumigatus was originally reported to produce ergot alkaloids in fermentation cultures.29'30 We have recently demonstrated that A. fumigatus accumulates large quantities of ergot alkaloids in or on its conidia. ' Quantities measured on environmentally relevant substrates were approximately 1% of the mass of the conidium. Because of the association of ergot alkaloids with its conidia, A. fumigatus presents the potential that ergot alkaloids can be encountered through the involuntary route of inhalation. As a thermotolerant saprophyte, A. fumigatus is sometimes associated with compost heaps, and when those compost heaps are on the scale of municipal compost heaps, the number of conidia encountered can be quite high.32'33 In culture, the fungus sporulates abundantly; a typical 8 5-mm diameter culture on potato dextrose agar contains approximately 60 billion conidia.31 Even with their relatively small diameter of 2.8 \xm per conidium, an aligned chain of conidia from one such culture would extend for over 100 miles.

PATHWAYS PATHWAYS TO DIVERSE DIVERSE ERGOT ERGOT ALKALOID ALKALOID PROFILES PROFILES

27

Aspergillus fumigatus is the species most frequently associated with invasive aspergillosis and is the most common agent of airborne mycoses in humans.34 Any role for conidium-associated ergot alkaloids in human disease has not yet been investigated. Such studies should be facilitated by comparisons of wild-type isolates with the ergot alkaloid-deficient gene knockout mutants.35

DIFFERENT SETS OF ERGOT ALKALOIDS VIA DIVERGING PATHS FROM A COMMON ORIGIN Overview of Diverging Pathways and Associated Gene Clusters Information on the ergot alkaloid pathway comes from several sources. Some of the intermediates in the pathway have been identified because they accumulate to measurable levels in different ergot alkaloid producers. More information has been obtained through in vitro feeding experiments with C. purpurea and C. fusiformis and isolated or synthesized candidate precursor molecules.2'9'36 A few of the early intermediates are postulated based on their context. Recent analyses of gene clusters associated with known ergot alkaloid pathway genes in C. purpurea and A. fumigatus have provided further clues on the nature of the pathway.35' 9 The pathways drawn in Figs. 2.1 and 2.4 are primarily derived from those summarized by Floss and Groger & Floss,9 based on the work of several laboratories, and that in Fig. 2.3 is based on the data of Barrow et al40 We will attempt to integrate genetic data with previous chemical work in our description of the pathways. Immediately below is an overview of the pathways and their major differences among ergot alkaloid-producing fungi. This overview section is followed by sections with more detailed descriptions of the pathways to different groups of ergot alkaloids. The early steps of the ergot alkaloid pathway (Fig. 2.1) are proposed to be shared among all ergot alkaloid-producing fungi. Alternate pathways diverge after these common early steps to yield different sets of ergot alkaloids in different ergot alkaloid producing fungi. Aspergillus fumigatus differs from most clavicipitaceous fungi in that it produces a set of clavine alkaloids with saturated D rings (the last to form of the four rings in the ergoline ring system) (Fig. 2.2) and is not known to produce the more complex lysergyl alkaloids (lysergic acid or its peptide or amide derivatives). Claviceps africana, a sorghum ergot fungus, also produces a set of ergot alkaloids containing a saturated D ring—referred to as dihydroergot alkaloids—but follows a different pathway beyond festuclavine to produce the ergopeptine dihydroergosine as its pathway end product (Fig. 2.3). Other clavicipitaceous fungi, including C. purpurea and several Neotyphodium spp. accumulate some subset of a complex group of A8,9 clavines, A9,10 clavines, ergopeptines, and simple amides of lysergic acid illustrated in Fig. 2.4.

PANACCIONE, PANACCIONE, et al.

28

DMATrp synthase

DMATrp

CH3 from AdoMet

N-methyl DMATrp

Neotyphodium spp. infected plants only

6,7-secolysergine jj H—'

chanoclavine aldehyde

Ring and position labeling referred to in text

dihydro chanoclavine aldehyde

Jj

i

festuclavine end of spur in Neotyphodium spp.

// isochanoclavine aldehyde

agroclavine

and C. purpurea

to A. fumigatus pathway (Fig. 2) and C.africana pathway (Fig. 3)

to Claviceps spp. and Neotyphodium spp. pathway (Fig. 4)

Figure 2.1: Early steps of the pathway proposed to be shared among all ergot alkaloid-producing fungi, and hypothetical branch point to fungus-specific pathways. Dashed arrows indicate the uncharacterized shunt to 6,7-secolysergine. The boxed inset shows the labeling of rings and positions within ring D that are referred to in the text. Abbreviations: DMAPP = dimethylallylpyrophosphate; Trp = tryptophan; DMATrp = dimethylallyltrpytophan; AdoMet = S-adenosylmethionine.

PATHWAYS TO DIVERSE ERGOT ALKALOID ALKALOID PROFILES PATHWAYS PROFILES

//

festuclavine festuclavine

'

fumigaclavine B

dihydroelymoclavine

dihydolysergic acid

fumigaclavine A

lysergyl peptide synthetase

dihydroergosine //

fumigaclavine C \

Figure 2.2 (Left, above): Final steps in the biosynthesis of ergot alkaloids of A. fumigatus (continued from Fig. 2.1). Figure 2.3 (Right, above): Final steps in the biosynthesis of ergot alkaloids of C. africana (continued from Fig. 2.1).

29

30 30

PANACCIONE, et et al. al.

lysergic acid a-hydroxyethylamide

ergine

eroopeptines R1 = side chain for: o,

••

a l a

v a l

2 £ val ergovaline ergocornine ™ .g leu ergosine a-ergocryptine gj E phe ergotamine ergocristine

ergopeptines

PATHWAYS PATHWAYS TO DIVERSE DIVERSE ERGOT ERGOT ALKALOID ALKALOID PROFILES PROFILES

31

Figure 2.4 (Previous): Final steps in the biosynthesis of ergot alkaloids in several Claviceps spp. and Neotyphodium spp. (continued from Fig. 2.1). Figure shows alkaloids produced collectively but not individually by several Claviceps spp. and Neotyphodium spp. See text for details of profiles of individual fungi. Uncharacterized shunts are indicated with dashed arrows. The table of ergopeptines indicates six common ergopeptines that can be produced by substitution of the side chains of two amino acids at position Rl and three amino acids side chains at position R2.

Comparison of ergot alkaloid gene clusters of A. fumigatus and C. purpurea supports the hypothesis of a shared early portion of the pathway followed by different terminal branches.35'41 The functions of three genes in the ergot alkaloid pathway have been defined by gene knockout analyses. These include genes encoding lysergyl peptide synthetase subunit 1 (LPS1) in Neotyphodium sp. Lpl, 42 lysergyl peptide synthetase subunit 2 (LPS2) in C. purpurea, and 43 dimethylallyltryptophan (DMATrp) synthase in Neotyphodium sp. Lpl and A. fumigatus. Details of the demonstrated roles of each of these genes in the ergot alkaloid pathway are described below. These three genes are clustered in the genome of C. purpurea^9 along with several other genes that encode functions that could theoretically be involved in the ergot alkaloid pathway (Fig. 2.5). The accumulation of mRNA of several of these genes has been studied in cultured C. purpurea. Transcripts are found at higher levels in ergot alkaloid-producing cultures compared to cultures in which ergot alkaloid production is repressed by the addition of high levels of potassium phosphate. ' The A. fumigatus DMATrp synthase gene {dmaW) is found among a cluster of genes, several of which appear to be orthologs of genes from the C. purpurea ergot cluster.35 Additional genes, some potentially encoding steps unique to the A. fumigatus pathway are also found in the A. fumigatus cluster (Fig. 2.5; Table 2.1). The potential functions of genes in these clusters are discussed in the relevant sections below.

32

PANACCIONE, et al. PANACCIONE, Claviceps purpurea cpps3 PS

cp0X3

OYE

cpps2 lysergyl PS2

P450-1

Tat

«-

cppsi lysergyl PS1

cpox2 cpoxi DH FAD dmaW ox/red ox/red orfB orfA (cpd1)

»

O rtE

diox

^

^

»

orfC diox

•»

cpps4 lysergyl PS1

— = ~1 kb Aspergillus fumigatus methyl transf

DH ox/red 1

FAD monoox

hyp 1 A.n.

diox pseudo

cinnamoyl red

majfac

OYE

orfA

pda P450

DH uratf ox/red 2 tf 1 psp-1

alt dmaW cpox2 prenyl DH transf ox/red

lovA P4S0

tf 2

OAc transf

xyl/ ospH arab

cat

hyp 2 A.n.

cpoxi FAD dmaW ox/red

to teloinere long arm chrom. 2

orfB

^-

Figure 2.5: Genes clustered with ergot alkaloid biosynthesis genes in C. purpurea and A. fumigatus. The C. purpurea cluster is described by Haarmann et al.39 and was redrawn from that source. The A. fumigatus cluster is derived from the cluster described by Coyle and Panaccione35 and was extended (first line under A. fumigatus) to show several additional flanking genes that have not been investigated and are only associated with ergot alkaloids based on their position in the genome. Sequence data for A. fumigatus were obtained from The Institute for Genomic Research (http://www.tigr.org). Arrows indicate the orientation of transcription. Black arrows represent genes that are shared between the two clusters. White arrows represent genes found in C. purpurea but not in A. fumgiatus, and grey arrows represent genes from A. fumigatus that are not found in the C. purpurea cluster as presently defined. The arrow marked with a slash (diox pseudo) indicates a sequence with numerous frame shifts and stop codons. Descriptions and designations of proteins deduced from A. fumigatus open reading frames (not intended as gene names) are provided in Table 2.1. Names for C. purpurea genes begin with "cp" and are italicized. Abbreviations: A.n., Aspergillus nidulans; aid, aldehyde; alt, alternate; cat, catalase; DH, short chain alcohol dehydrogenase; diox, dioxygenase; DS(3H, double-stranded P helix; hyp, hypothetical; lovA, associated with lovastatin biosynthesis; maj fac, major facilitator superfamily transporter; monoox, monooxygenase; OAc transf, Oacetyl transferase; ox/red, oxidoreductase; orf, open reading frame; OYE, old yellow enzyme oxidoreductase; P450, cytochrome P450 monooxygenase; pda, associated with pisatin demethylase activity; prenyl transf, prenyl transferase; pseudo, pseudogene; PS, peptide synthetase; PSP, parasitic phase specific flippase; tf, transcription factor; ura, associated with uracil biosynthesis; xyl/arab, xylanase/arabinase. The scale applies to both clusters.

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Shared Early Pathway Steps The first committed step in the ergot alkaloid pathway is the prenylation of tryptophan catalyzed by DMATrp synthase, an enzyme whose role has been well established in clavicipitaceous fungi43'44 and A. fumigatus.35'45 The DMATrp synthase-encoding gene dmaW was first isolated from C. fusiformis, and its activity was demonstrated by heterologous expression in Saccharomyces cerevisiae. 4 Homologs were identified in several clavicipitaceous fungi, and gene knockout and complementation experiments demonstrated the central role of dmaW in the ergot alkaloid pathway.43 Recently, an ortholog of dmaW was identified in A. fumigatus, embedded among several genes with similarity to those in the ergot cluster of C. purpurea. Knockout of this gene in A. fumigatus eliminated all ergot alkaloids, demonstrating a common genetic and biosynthetic origin for the ergot alkaloid pathways of these two divergent lineages. The second step in the ergot alkaloid pathway is the 7V-methylation of DMATrp. There are indications that the gene labeled orfB (Fig. 2.5) encodes an Nmethyl transferase. The product of orfB aligns well with a class of hypothetical proteins designated COG4301. The best-conserved amino acid sequence motif within the COG4301 family matches an TV-methyl transferase. In A. fumigatus, a more recognizable methyl transferase gene is loosely clustered with the homologs of ergot alkaloid biosynthesis genes (Fig. 2.5), though this gene more closely resembles (9-methyl transferases (Table 2.1) and is spatially separated from the other ergot alkaloid biosynthesis genes. Planned gene knockouts and complementations should clarify the role of each gene. Following the iV-methyl transferase step is a series of oxidation and reduction steps (Fig. 2.1). Genes encoding oxidoreductases/dehydrogenases are shared in the gene clusters of A. fumigatus and C. purpurea (Fig. 2.5; Table 2.1) and may encode enzymes required for some of these steps. The presence of a catalase-like gene in each cluster is intriguing. One possibility is that the product of this gene functions catalytically (perhaps peroxidatively) in the pathway. A second possibility is that it provides a protective function from reactive intermediates that may be formed by some early step in the pathway (though a step requiring such protection is not obvious).

Table #.1: Sequences closely linked to dmaW ofAspergillusfumigatus 34 PANACCIONE, et al. Closest matching protein (and other relevant descriptive match)a A. fumigatus Description / Accession Accession number designation*5 number methyl transf XM_.751025 EAA57700 BAA861O3 hyp 1 A.n. XM__751026 EAA64940 XM..751027 EAA58440 maj fac T52148 aid red / XM__751028 Q9UUN9 cinnamoyl red NP_593981 XM__751029 EAA60072 uratf NP_594497 NP_791407 E value0 40/2e-32 39/4e-35 21/6e-08 45/e-102 24/3e-13 28/2e-20 35 / 3e-20 31 / le-62 28/4e-13 38/3e-27

tfl PSP-1 XM_.751033

XM..751031 XM..751032

XM .751030

34 / le-68 36/3e-19

tf2

XM._751035 XM..751036 XM 751037

XM..751034

DSpH xyl/arab hyp 2 A.n. DH ox/red 1

DH ox/red 2

EAL85291 AAL30767

55/e-144 27 / 5e-07 35 / le-23 50 / 6e-2 50/e-142 61 /le-134 27/2e-13 32/le-18 31/3e-15

XM 751038

Deduced protein description (Organism) hypothetical protein (Aspergillus nidulans) O-methyltransferase (Aspergillus parasiticus) hypothetical protein (Aspergillus nidulans) hypothetical protein (Aspergillus nidulans) transport protein holl; major facilitator (Candida albicans) aldehyde reductase II (Sporidiobolus salmonicolor) putative cinnamoyl-coA reductase (S. pombe) hypothetical protein (Aspergillus nidulans) putative transcriptional regulatory protein (S. pombe) oxidoreductase, short-chain dehydrogenase (Pseudomon. syringae) putative transcription factor (Aspergilllus fumigatus) parasitic phase-specific protein 1, self-protection, flippase (Coccidioides posadasii) hypothetical protein (Aspergillus nidulans) putative transcriptional regulatory protein (S. pombe) hyp. protein: double-stranded P helix (Magnaporthe grised) xylosidase; arabinosidase (X. axonopodis pv. citri) hypothetical protein (Aspergillus nidulans) hypothetical protein (Aspergillus nidulans) related to short chain alcohol dehydrogenase (Trichodesmium sp.) hypothetical protein (Magnaporthe grised) FAD monoox., maackiain detox. (Nectria haematococca) EAA66337 NP_594497 EAA51259 NP_642848 EAA66975 EAA64052 ZP00072819 EAA49815 ACC49410

FADmonoox

s

I

PATHWAYS TO DIVERSE ERGOT ALKALOID PROFILES XMJ751O39

35 33/3e-12 32 / 5e-07

dioxpseudo

XM_751040

AAK01512 AAG27131

OYE

50 / 5e-99 40 / 3e-70 37/8e-41 37/2e-29

XM_751041

XP 323805 NP_015154 AY836771 NP_929597

orfA

30/4e-47 31/4e-28 27 / le-39

XM_751042 EAA58471 Q12645 AAP81206 61/6e-81

pda P450

CAB39316

46 / 8e-76

XMJ751O43

XP_325231

hypothetical protein, hydroxylase domains (P. aeruginos) fum3p, 2-ketoglutarate-dep. dioxygenase {Gibberella moniliformis) hypothetical protein (Neurospora crassa) old yellow enzyme, NADPH dehydrogenase (S. cervisiase) hypoth, prot, ergot cluster {Clavicepspurpurea) hypothetical protein, epimerase domains (Photorhabdus luminescens) hypothetical protein {Aspergillus nidulam) P450, pisatin demethylase activity (Nectria haematococca) dimethylallyltryptophan synthase, ergot pathway {Neotyphodium sp. Lpl)

XM_751044 cytochrome P450 monooxygenase (lovA) {Neurospora crassa)

cpox2 ox/red, short chain dehydr., ergot cluster {Claviceps purpurea)

alt dmaW prenyl transf

XM_751045

cpox2 DH ox/red XM_751046

30/3e-50 23/4e-ll 55 / le-155 44/le-112 63/le-152

OAc transf

40 / le-93

lovA P450 EAA66317 BAC65220 AJ703808 AAF01463 AAP81207

XM_751047

CAB39328

cat

hypothetical protein {Aspergillus nidulam) trichothecene 3-O-acetyltransferase {Fusarium oxysporuni) catalase, ergot cluster {Clavicepspurpurea) catalase isozyme P {Ajellomyces capsulatus) DMATrp synthase, ergot pathway {N. coenophialum)

XM_751048

50/le-101 29 / 3e-33

dmaW FAD-containing oxidoreductase, ergot cluster {Claviceps purpurea)

AY836772 XP 323696

XM_751049 hypothetical protein, ergot cluster {Claviceps purpurea) hypothetical protein {Neurospora crassa)

cpoxl FAD ox/red XM 751050

a If closest match was to a hypothetical protein, the closest descriptive match also was provided; if closest match was to unpublished sequence, the next closest match also was provided. correspond to open reading frames identified in Fig. 5. percent amino acid sequence identity and number of similar matches expected in the database by chance b

c

s ©

§

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PANACCIONE, PANACCIONE, et al.

Clavines in Different Ergot Alkaloid Pathways Divergence of Saturated D Ring from Unsaturated D Ring Ergot Alkaloids Chanoclavine is the earliest pathway intermediate that accumulates in most ergot alkaloid producers. We hypothesize a common pathway through chanoclavine and that its aldehyde derivative is the point at which alternate pathways diverge. The proposed branch point illustrated in Fig. 2.1, dividing ergot alkaloids with unsaturated D rings (A8,9 clavines, A9,10 clavines, and all lysergyl derivatives) from the those with saturated D rings {e.g., festuclavine, fumigaclavines, and dihydroergot alkaloids), is supported by several lines of evidence. Barrow et al40 detected chanoclavine, festuclavine, dihydroelymoclavine, dihydrolysergic acid, and dihydroergosine in C. africana. (This fungus was originally identified as Sphacelia sorghi, the name given to the anamorph of Claviceps sorghi, but later was recognized to be an isolate of C. africana.)11 Agroclavine was not detected in C. africana, and feeding labeled agroclavine to cultures resulted in poor incorporation of label into dihydroergosine. Conversely, feeding labeled festuclavine resulted in efficient labeling of dihydroelymoclavine and dihydroergosine. Based on these data, a pathway from festuclavine through dihydroelymoclavine and dihydrolysergic acid to dihydroergosine was proposed (Fig. 2.3).40 The spectrum of alkaloids detected in A. fumigatus—chanoclavine, festuclavine, and the three fumigaclavines—also supports reduction of the relevant double bond prior to D ring closure (Fig. 2.2).31'35'41 Neither agroclavine nor any other tetracyclic ergot alkaloid containing a double bond in the D ring has been detected in A. fumigatus or C. africana. Analysis of genomic sequence data provides a possible mechanistic basis for the origin of the festuclavine derivatives (including the fumigaclavines and dihydroergot alkaloids). The presence of a gene encoding an "old yellow enzyme" (OYE) in the A. fumigatus ergot gene cluster (Fig. 2.5, Table 1) suggests that reduction of the double bond may occur at the chanoclavine aldehyde step (Fig. 2.1). Old yellow enzymes reduce a double bond occurring in trans conformation relative to an aldehyde or ketone.46 A putative OYE-encoding gene also is found in the C. purpurea ergot cluster (Fig. 2.5), where it is called cpox3.39 The presence of this gene in the C. purpurea cluster casts some doubt that it encodes the enzyme that functions as suggested above, since C. purpurea accumulates mainly ergot alkaloids with unsaturated D rings. However, festuclavine and its isomers are detectable as a minor component in C. purpurea sclerotia and Neotyphodium spp.-infected plants.27'36'4 >48 Perhaps C. purpurea has an old yellow enzyme for which chanoclavine aldehyde is a relatively poor substrate or with which another oxidoreductase catalyzing formation of agroclavine more effectively competes.

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Clavines with a Saturated D Ring The predicted A. fumigatus-specific pathway from festuclavine to fumigaclavine C (Fig. 2.2) requires an additional hydroxylation step, an 0-acetyl transferase step, and a prenyl transferase step. The A. fumigatus ergot gene cluster contains three monooxygenases that have not been found in the C. purpurea cluster (Fig. 2.5), making them candidates to encode the enzyme that catalyzes the oxidation of festuclavine to fumigaclavine B. (Caution must be used in such comparisons of clusters. Since the genome of C. purpurea has not been completely sequenced, orthologs of certain genes may be present in the genome but outside of the known ergot alkaloid gene cluster.) The A. fumigatus cluster also contains an excellent match for an O-acetyl transferase, an activity that would be required to acetylate fumigaclavine B to fumigaclavine A. The predicted prenyl transferase that would use fumigaclavine A as substrate presents an interesting puzzle. The arrangement of the prenyl group relative to the ergoline ring system in fumigaclavine C (Fig. 2.2) is typical of the product of a "reverse" prenylation reaction.49'50 The prenyl group is "reversed" relative to the product of a "normal" prenylation, exemplified by the prenylation of tryptophan by DMATrp synthase in the first step of the ergot alkaloid pathway (Fig. 2.1). An analogous reverse prenylation of the 2 position of the indole ring in paraherquamide biosynthesis was analyzed in labeled tracer studies by Stocking et al.50 They hypothesized that the reverse prenyl transferase presented dimethylallylpyrophosphate (DMAPP) to the indole co-substrate in an upside-down, or reverse, orientation relative to that used by normal prenyl transferases, permitting a facially nonselective SN' attack on the olefinic n system of DMAPP. The pyrophosphate moiety of DMAPP was hypothesized to be anchored in the active site of the enzyme. Genes encoding enzymes responsible for reverse prenylation have not been characterized. The A. fumigatus ergot alkaloid cluster contains a second prenyl transferase-like gene (labeled alt dmaW in Fig. 2.5; Table 2.1) whose deduced product has 25% amino acid sequence identity with the product of the DMATrp synthase-encoding gene dmaW.35 Based on this low but significant sequence identity and its presence in the ergot alkaloid gene cluster, we speculate that the product of this gene may catalyze the reverse prenylation of fumigaclavine A to fumigaclavine C. Interestingly, Penicillium paxilli contains a divergent dmaW homolog with no described function,51 and alkaloids (such as oxaline, neooxaline, aurantiamine, and roquefortine C) produced by some Penicillium spp. contain the same reverse prenyl group at the identical position on the indole ring system as observed on fumigaclavine C.52 Additional genes from the A. fumigatus cluster that may be peripherally involved in the pathway include those with coding capacity for transcription factors,

38

PANACCIONE, et al. PANACCIONE,

a major facilitator transporter, and a flippase-like, self-protection protein (PSPl)(Fig. 2.5; Table 2.1). The biosynthesis of ergot alkaloids in the C. africana pathway (Fig. 2.3) is hypothesized to proceed with enzymes analogous (perhaps homologous) with those described below for the more typical clavicipitaceous fungi. Festuclavine (which can be considered to be dihydroagroclavine) would be oxidized to yield dihydroelymoclavine. A second oxidation at the same carbon would yield dihydrolysergic acid, which would be incorporated into dihydroergosine. Outside of the labeled precursor feeding studies that established these connections,40 no investigations of the basis for the differences in alkaloids in C. africana and C. purpurea have been reported. Clavines with an Unsaturated D Ring The unsaturated clavines common in clavicipitaceous fungi other than C. africana (Figs. 2.1 and 2.4) can be divided into two groups. One group provides a direct path to lysergic acid, which is then incorporated into ergopeptines and simple amides of lysergic acid. The second group includes products of presumed shunts off this main pathway to produce alternate products. In the direct path to lysergic acid, two additional monooxygenases (believed to be cytochromes P450) are required to catalyze the successive oxidations of agroclavine and elymoclavine.9'53"55 In the oxidation of elymoclavine, the doublebond shift (from the A8,9 position in elymoclavine to the A9,10 position in lysergic acid) may occur concomitantly with oxidation or spontaneously after oxidation.9'3 '56 Putative cytochromes P450 encoded by genes that are part of the ergot alkaloid clusters of C. purpurea or Neotyphodium spp. and that are not present in the A. fumigatus genome are excellent candidates for these late pathway monooxygenases. One such gene, designated P450-1 (Fig. 2.5) has already been identified in C. purpurea^' 9 and does not have a close ortholog in the A. fumigatus cluster or genome.35 Whereas agroclavine and elymoclavine can provide a path to lysergic acid in some fungi, in some other ergot alkaloid-producing fungi, agroclavine and elymoclavine are pathway end products that are not further modified.36'47 For example, the ergot fungus from pearl millet, C. fusiformis, produces elymoclavine but not lysergic acid and has the monooxygenase activity associated with agroclavine oxidation but not that associated with oxidation of elymoclavine.53"55 Clavines that accumulate as apparent shunt product off the central ergot alkaloid pathway have a variety of structures and biosynthetic origins (Figs. 2.1 and 2.4). Setoclavine (along with its 8-epimer isosetoclavine) and penniclavine (also found with its 8-epimer isopenniclavine) are readily formed in vitro from agroclavine and elymoclavine, respectively, through the activity of non-specific peroxidases such as horseradish peroxidase or through activities found in complex mixtures such as

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plant cell extracts.57 When supplied to endophyte-free perennial ryegrass via the transpiration stream, agroclavine and penniclavine were oxidized to setoclavine/isosetoclavine and penniclavine/isopenniclavine, respectively.27 These data indicate that when found in plants infected with clavicipitaceous fungi, setoclavine and penniclavine may be products of both fungus and plant catalysis. The clavine alkaloid 6,7-secolysergine (Fig. 2.1) likely represents the product of a shunt from the early, shared portion of the pathway. However, it has been detected only in plants with the endophytes Neotyphodium sp. Lpl and N. coenophialum?1 The nature of the shunt pathway or even its origin off the central pathway is not known. In Neotyphodium sp. Lpl, 6,7-secolysergine appears to provide a means to regulate the flow of intermediates through the central pathway. Levels of this apparent early pathway shunt product increased when the pathway was blocked further downstream by knockout of the lysergyl peptide synthetase 1encoding gene ipsA?1 The clavine intermediates from the central pathway appeared to be maintained at wild-type levels in this knockout strain despite the knockout, while levels of 6,7-secolysergine increased approximately two-fold. Ergopeptines Ergopeptines are the most abundant ergot alkaloids in sclerotia of C. purpurea and as such were the first ergot alkaloids isolated and studied. They are nonribosomally synthesized peptides containing D-lysergic acid and three L-amino acids. The sequence of the three amino acids determines the name of the ergopeptine (Fig. 2.4). For example, ergovaline contains L-alanine, L-valine, and L-proline attached to D-lysergic acid; ergotamine differs by substitution of L-phenylalanine for L-valine. Several other common ergopeptines that can be assembled with variation at two amino acid positions are indicated in Fig. 2.4. Ergopeptines are produced by lysergyl peptide synthetase complexes. Peptide synthetases are multifunctional enzymes in which sequential reactions are catalyzed by a series of connected catalytic domains from amino to carboxy terminus of the peptide synthetase.5 '5 There are catalytic domains for adenylation (the activation of the amino acid or carboxylic acid by adenylation), thiolation (the binding of the activated amino acid as a thioester to enzyme-bound 4'phosphopantetheine), and condensation (the formation of peptide bonds between adjacent thioesterified amino acids). Some peptide synthetases contain additional tailoring domains, e.g., domains for epimerization, N-methylation, or reduction of amino acids,58'59 but those do not pertain to the biosynthesis of ergopeptines. Lysergyl peptide synthetase is different from all other known eukaryotic peptide synthetases in that it is composed of two separately encoded polypeptides as opposed to being a single gene product.38'60 Lysergyl peptide synthetase 2 (LPS2) activates D-lysergic acid by adenylation before binding it as a thioester. In this state, D-lysergic acid is prepared for peptide bond formation with the first amino acid of

40

PANACCIONE, PANACCIONE, et al.

the tripeptide moiety of the ergopeptine. This first condensation also appears to be catalyzed by LPS2 through a condensation domain found near its carboxy terminus.38 Lysergyl peptide synthetase 1 (LPS1) activates by adenylation and binds as thioester the three amino acids of the ergopeptine. It forms the peptide bonds between the three amino acids, and also catalyzes the lactam bond between the second and third amino acids, a reaction that releases the lysergyl peptide lactam from the lysergyl peptide synthetase complex.60'61 The final step in the synthesis of ergopeptines is catalyzed by a monooxygenase (believed to be of the cytochrome P450 type), which uses molecular O2 to hydroxylate the a-carbon of the lysergyl peptide lactam. This reactive intermediate is hypothesized to cyclize spontaneously to form the final cyclol ring of the ergopeptines (Fig. 2.4).62 The different ergopeptines observed within and between ergopeptineproducing species may result from either of two strategies, or perhaps a combination both. Hypothetically, different versions of LPS1, each containing adenylation domains with distinct substrate specificities, could each determine the sequence of an individual ergopeptine. Alternatively, versions of LPS1 with low amino acid substrate specificity might accept more than one type of amino acid at one or more adenylation domains, thus generating a family of different ergopeptines after several cycles of peptide synthesis. Haarmann et al39 compared LPS1-encoding genes from isolates of C. purpurea known to accumulate primarily ergotamine in one case and ergocristine in the other. They found variation at amino acid residues critical to determination of amino acid substrate specificity, indicating that, at least in some cases, individual LPS1-encoding genes are associated with specific ergopeptines. This finding does not preclude the possibility that in some other cases, considering the diversity of ergopeptines described and the limited number of LPS1-encoding genes detected by Southern hybridization,42 ergopeptines found at lower concentrations may result from occasional acceptance of alternate amino acids by adenylation domains in certain versions of LPS1. The nature of the peptide synthetase involved in forming the dihydroergopeptine dihydroergosine in C. africana has not been investigated directly. However, Riederer et al60 demonstrated that the lysergyl peptide synthetase 2 of C. purpurea has similar Kms for dihydrolysergic acid and lysergic acid, and that the lysergyl peptide synthetase complex forms dihydroergopeptine alkaloids when supplied with the dihydrolysergic acid as substrate. Moreover, when fed dihydrolysergic acid, ergotamine-producing cultures of C. purpurea produced dihydroergotamine in addition to ergotamine. These observations and the precursor feeding studies of Barrow et al40 raise the possibility that the pathways of C. purpurea and C. africana may differ at very few steps, potentially just those resulting in the formation festuclavine and dihydroelymoclavine. Interestingly, C. fusiformis lacks the gene encoding LPS1 42 and does not produce ergot alkaloids beyond elymoclavine. 6'47 Aspergillus fumigatus also lacks

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the lysergyl peptide synthetase genes as expected, since it does not produce ergopeptines.3 Simple Amides of Lysergic Acid Some of the fungi that produce ergopeptines also accumulate one or more simple amides of lysergic acid. These include C. purpurea, which accumulates the simple amides ergonovine and lysergyl-alanine,27'3 >47 and Neotyphodium sp. Lpl and N. coenophialum, which accumulate ergine and lysergyl-alanine.27'64 For some other ergot alkaloid-producing fungi, simple amides appear to be end products of their ergot alkaloid pathways. The Ach. inebrians-N. inebrians symbiotum accumulates ergine and ergonovine but has not been reported to produce ergopeptines.28 Similarly C. paspali accumulates ergonovine, ergine, and lysergic acid othydroxyethylamide but no known ergopeptine.47 The biosynthesis of simple amides of lysergic acid has not been studied extensively. As such, it has not been determined whether there is a common branch point from the central ergot alkaloid pathway for the various simple amides or whether different amides are derived via shunts from different points in the pathway. The genetic and biochemical data described below are more consistent with some simple amides being derived directly from lysergic acid and others being derived from the lysergyl peptide lactam or its hydroxylated intermediate (Fig. 2.4). Little is known about the involvement of specific genes and enzymes in the biosynthesis of lysergic acid amides. The knockout of the LPS 1-encoding gene ipsA in Neotyphodium sp. Lpl resulted in the elimination of the simple amides ergine and lysergyl-alanine in addition to ergovaline.27'42 These data demonstrate that either the activities or products of LPS 1 are required for production of these particular simple amides of lysergic acid. Considering that intermediates in nonribosomal peptide synthesis are covalently bound to their peptide synthetases,58'59 it would appear unlikely for ergine and lysergyl-alanine to be direct products of the lysergyl peptide synthetase. An alternate possibility that is consistent with the genetic and biochemical observations is that the lysergyl peptide lactam (Fig. 2.4), which is the immediate product of the lysergyl peptide synthetase complex, ° is involved in the pathway to these simple amides. Hydrolysis of the branched peptide bond between alanine and the diketopiperizine ring would release lysergyl-alanine. The branched peptide bond, with its two electron-withdrawing carbonyl groups, would appear to be particularly sensitive to hydrolysis to yield lysergyl-alanine. The possibility that lysergyl-alanine measured in extracts of ergopeptine-producing fungi may be a product of hydrolysis of the lysergyl peptide lactam during extraction cannot be excluded. However, prior incubation of sclerotia of C. purpurea in 0.5 M acetic acid or 0.5 M ammonium carbonate (conditions that should promote such a hydrolysis) did not change the ratio of lysergyl-alanine to total ergopeptines measured in subsequent extracts (D.G. Panaccione, unpublished data).

42

PANACCIONE, et al. PANACCIONE,

Ergine could theoretically be produced via a process analogous to that used to produce peptides with carboxy-terminal amides, such as peptide hormones in mammals and insects and the antibiotic myxothiazol in myxobacteria.65'66 This process involves oxidation at the a-carbon of the amino acid found at what will be the cleavage point, followed by dealkylation of the alcohol amide, via a lyase reaction, to reveal the terminal amide. By analogy with this process, the a-carbon of alanine in the lysergyl peptide lactam would be hydroxylated by a monooxygenase activity and the hydroxylated intermediate cleaved through a lyase activity to release ergine. Hydroxylation at this carbon is known to occur during the production of ergopeptines, and the intermediate spontaneously cyclizes to yield the lactam ring in the ergopeptines (Fig. 2.4).62 In the production of peptides with carboxy-terminal amides in mammals, the relevant oxidase and lyase activities are encoded on a single polypeptide and typically post-translationally processed into separate catalytic units. In fruit fly (Drosophila melanogaster) and honey bee {Apis mellifera), the monooxygenase and lyase activities are encoded separately. One possible explanation to account for the production of ergine in some ergot alkaloid producers but not in others is that the lyase activity may be present, or highly expressed, only in ergine producers. Alternatively, ergine producers may have a separate monooxygenase that strictly hydroxylates (leading to ergopeptine production) and a competing oxidase/lyase enzyme for oxidative cleavage to the terminal amide. The pharmacologically significant simple amide ergonovine has been proposed to be produced by reduction of the carbonyl group of a lysergyl-alanine intermediate. Precursor feeding studies showed that alanine served as a precursor to ergonovine.2'9'36'56 Interestingly, Haarmann et al29 report the presence in the ergot gene cluster of a single-module peptide synthetase that contains a reductase domain. Although this gene has not yet been functionally analyzed, it is tempting to speculate that by interaction with the lysergic acid-activating peptide synthetase (LPS2) it could produce a complex capable of synthesizing lysergyl-alanine and reducing it to ergonovine. The production of lysergic acid a-hydroxyethylamide (Fig. 2.4) also requires alanine as a precursor. ' The resulting peptide is presumably hydroxylated at the acarbon of alanine, in a reaction similar to that described above for the lysergyl peptide lactam. The specifics of this reaction and the subsequent decarboxylation have not been studied. Flieger et al67 reported that ergine can be derived from lysergic acid a-hydroxyethylamide by incubation in an aqueous ammonia solution. Such an origin could be invoked to account for the presence of ergine in extracts of fungi such as C. paspali that also accumulate lysergic acid a-hydroxyethylamide, but this does not explain its presence in Neotyphodium spp., which lack lysergic acid ahydroxyethylamide. Incubation of sclerotia of C. purpurea (containing ergopeptines and ergonovine but not lysergic acid a-hydroxyethylamide) under similar alkaline conditions prior to extraction did not yield ergine (D.G. Panaccione, unpublished

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data). The data indicate that ergine may result from more than one process. Determination whether its production results from enzymatic activity in some cases will require further investigation. Diversification of Ergot Alkaloid Profiles within Producers The diverging pathways described above account for major differences in ergot alkaloid profiles among different fungi. Diversification of alkaloid profiles also occurs within individual fungi in each major branch. Many ergot alkaloidproducing fungi accumulate a complex profile of ergot alkaloids that includes pathway end products as well as accumulating intermediates and/ or products of shunts off the central ergot alkaloid pathway.36'41'47 One means of diversification is the utilization of one or more shunt pathways. Neotyphodium sp. Lpl in perennial ryegrass utilizes shunts to 6,7-secolysergine, setoclavine, lysergyl-alanine, and ergine on it way to producing ergovaline.27 Conversely, in most isolates of C. purpurea, the shunt to ergonovine is the only significant diversion on the path to producing ergopeptines. Accumulation of intermediates provides another mechanism of ergot alkaloid profile diversification.41 The apparent inefficiency in converting one intermediate to the next may be due to differences in activities and/ or concentrations of enzymes that catalyze successive steps or it may be due to compartmentalization or secretion of some portion of the intermediate pool for the next step in the pathway. Examples of accumulating intermediates include chanoclavine in Neotyphodium sp. Lpl, 7'41 festuclavine and fumigaclavine A in A. fumigatus^ and dihydroelymoclavine in C. africana. One interpretation of the diversification of profiles by accumulation of intermediates and shunt products is that these different alkaloids may provide some benefit to the producing fungus that differs from those conferred by the ultimate pathway end product.41 An alternate interpretation is that the various alkaloids all have similar activities and, thus, there is little pressure to evolve an efficient pathway. The former interpretation is favored by studies that demonstrate different biological activities for different ergot alkaloids.41

GENE CLUSTER DIFFERENCES AND EVOLUTION Potential Origins of Ergot Alkaloid Gene Clusters The discovery of similar gene clusters associated with the production of similar families of secondary metabolites from divergent lineages of fungi raises interesting questions about the origins of the gene clusters and the corresponding biosynthetic pathways. One explanation for the phylogenetically discontinuous distribution of the pathway is that horizontal transfer of the ergot gene cluster may

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have occurred between producing fungi or their ancestors after the divergence of the lineages. An alternate explanation is that the ability to produce some rudimentary ergot alkaloid was present as an ancestral trait. This ability may have been lost in many intervening lineages and retained and developed differently in the remaining ergot alkaloid-producing fungi. A third possibility, invoking convergent evolution, is that a functioning pathway did not exist prior to divergence, but the same clustered genes were later recruited into the different lineages to form the shared portion of the pathway. In any of these scenarios, it is interesting to consider whether different sets of ergot alkaloids accumulate in different fungi because the rudimentary cluster (or genetic raw material) was present in different genetic backgrounds with differing capacities to finish the intermediates, or whether pathways to different end products developed because of the differing pressures applied in the different niches that the fungi occupy. Differences in Cluster Composition and Arrangement The presence of several clustered genes with high sequence identity, including placement of introns, within the ergot clusters of A. fumigatus and C. purpurea (Fig. 2.5), provides evidence that the clusters are homologous. However, at least six recombination events must have occurred, since transfer or divergence, to account for the differences in the arrangement of the shared genes. Moreover, the interspersion of fungus-unique genes that are involved (e.g., the lysergyl peptide synthetase genes in C. purpurea), or likely involved (numerous other genes in both clusters) in the fungus-unique pathways presents another interesting question about pathway and cluster evolution. If the clusters were passed by horizontal transfer, then additional recombination events would have been required to bring fungusspecific genes from elsewhere in the genome to the acquired cluster. Conversely, genes initially present or acquired but not used would have had to have been deleted to eliminate them from the pathway. An alternate possibility is that an ancestral cluster containing the genes currently observed in both clusters (a hypothetical metacluster) was selectively modified over time in both lineages to yield the current divergent clusters. One observation providing evidence of fungus-specific tailoring of the cluster is that the apparent fum3p-like dioxygenase-encoding gene in A. fumigatus (Fig. 2.5; Table 1) is, in fact, a pseudogene. The reading frame is irreconcilably closed. Two apparently open coding sequences similar to this pseudogene are found in the C. purpurea cluster.39 Their function is unknown but transcripts from these genes are co-regulated with those of other genes in the ergot cluster. The presence of such a remnant in the A. fumigatus cluster provides evidence of fungus-specific modification of some originally different biosynthetic capability. Its presence could be consistent with either the horizontal transfer theory or the selective retention and divergence theory. The observation does suggest that if the clusters were passed by

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horizontal gene transfer, then the A. fumigatus lineage was the recipient rather than the donor. It is interesting to note that the A. fumigatus ergot cluster is located within 50 kb of the telomere on the long arm of chromosome 2. Subtelomeric regions of chromosomes in fungi and other eukaryotic microorganisms are proposed to be rich in genes conferring niche-specific adaptations.68"71 These subtelomeric regions may have exceptionally high frequencies of recombination.69'70 The relative chromosomal position of the ergot alkaloid biosynthetic genes of C. purpurea or other clavicipitaceous fungi is not known. Codon Usage Bias Differences in codon usage bias may be helpful in identifying genes that have been acquired by horizontal gene transfer. We investigated whether genes in the A. fumigatus ergot cluster had codon usage bias more like that of other A. fumigatus genes or more like those in the C. purpurea ergot cluster. Seven A. fumigatus genes that have apparent homologs in the C. purpurea ergot alkaloid cluster (refer to Fig. 2.5, Table 2.1) were studied for biases in the third position of codon families. (The dioxygenase genes were omitted due to the lack of an open reading frame in A. fumigatus and the presence of two different versions in C. purpurea.) The only readily apparent codon usage bias in the A. fumigatus copies of the shared ergot cluster genes was toward G in the third position of codons that had a G versus A option only. This includes codons for glutamic acid, glutamine, lysine, and a subset of the leucine and arginine codons. This preference also was seen in the coding sequences of a random sampling of seven A. fumigatus genes not associated with the ergot alkaloid cluster. Conversely, the seven C. purpurea genes that have homologs in the A. fumigatus cluster did not display this same codon usage bias (Fig. 2.6). When the data for these five codon sets were pooled, the proportion of codons that contained a G in the third position was significantly lower (P<0.01; Tukey-Kramer HSD test) in the C. purpurea ergot cluster genes than in the A. fumigatus ergot cluster genes or the random sampling of A. fumigatus genes. The means for the two A. fumigatus gene sets were not significantly different. No other codon usage biases were apparent in any of the data sets. The G+C content for these same three sets of seven genes also was investigated, and no significant differences were identified. The data do not support a recent transfer of the ergot alkaloid biosynthesis genes between A. fumigatus and C. purpurea.

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E

R(CGR)

K

L(UUR)

Amino Acid Figure 2.6: Histogram indicating the percentage of codons for the indicated amino acids (single amino acid code used) in which G as opposed to A appears in the third position of the codon. For arginine and leucine codons, only the subset of codons for which A versus G is the sole possible difference (i.e., CGR, with R representing purine, for arginine, and UUR for leucine) were considered. Key: C.p. (white bars) represents the seven C. purpurea ergot alkaloid cluster genes with orthologs in the A. fumigatus cluster; A.f. ergot (black bars) represents the seven A. fumigatus ergot alkaloid cluster genes with orthologs in the C. purpurea cluster; and, A.f. other (grey bars) represents an arbitrary sample of seven A. fumigatus genes retrieved from GenBank that have no association with the ergot alkaloid gene cluster.

SUMMARY AND FUTURE DIRECTIONS Ergot alkaloid-producing fungi collectively produce a diverse array of ergot alkaloids by following different pathways that branch after a series of shared steps from a common origin. Individual fungi produce complex profiles of ergot alkaloids that contain the final product of their pathway and, often, the products of one or more

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shunts along the pathway and certain intermediates that accumulate to relatively high levels. The discovery and analysis of ergot alkaloid-associated gene clusters has supported the idea that the different ergot alkaloids have a common genetic and biosynthetic origin. Comparison of clusters is beginning to provide information on the evolution of the pathways. Determination of the organization of ergot biosynthetic genes in additional ergot alkaloid producers, for example the Neotyphodium spp. and ergot fungi such as C. fusiformis and C. paspali, that produce different profiles of ergot alkaloids should provide valuable new information for this purpose. Molecular methods for manipulation of the ergot alkaloid pathway have been established in three different ergot alkaloid producers, and the functions of several genes in the pathway have been demonstrated by gene knockout analyses.35'38'42'43 The ergot alkaloid gene clusters have provided numerous new candidate genes that will likely encode enzymes that catalyze additional steps in the ergot alkaloid pathways. Gene knockout analysis and subsequent characterization of the knockout mutants provides a strategy to determine the functions in the pathway of the gene products. Mutants derived from such analyses also should be useful tools for determination of the contribution of specific alkaloid or groups of alkaloids to the ecological success of the producing fungus. Moreover, the ability to alter and control the ergot alkaloid pathway may have application in medicine, where ergot alkaloid have great activity but low receptor specificity, and agriculture, where toxicoses are associated with ergot-alkaloid producing endophytes.

ACKNOWLEDGMENTS Research in the authors' laboratories was supported by USDA NRI grants 2001-35319-10930 and 2005-35318-16184. We thank Sarah O'Connor (MIT) for helpful suggestions regarding ergine biosynthesis. Preliminary sequence data from the A. fumigatus genome were obtained from The Institute for Genomic Research web site at http://www.tigr.org. Sequencing of the A. fumigatus genome was funded by the National Institute of Allergy and Infectious Disease grant U01 AI 48830 to David Denning and William Nierman, the Wellcome Trust, and Fondo de Investigaciones Sanitarias.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Three

COMPARTMENTALIZATION OF PLANT SECONDARY METABOLISM Nailish Samanani and Peter J. Facchini* Department of Biological Sciences University of Calgary Calgary, Alberta, Canada T2N1N4 * Author for correspondence, e-mail: pfacchin(S>ucalgary.ca

Introduction Specialized Structures Glandular Trichomes Resin Ducts S-Cells Laticifers Compartmentalization of Alkaloid Pathways Tropane Alkaloids Monoterpenoid Indole Alkaloids Pyrrolizidine Alkaloids Quinolizidine Alkaloids Benzylisoquinoline Alkaloids Cellular Compartmentalization Subcellular Compartmentalization Metabolic Complexes Summary and Future Directions

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54 54 54 56 57 57 58 58 59 62 63 64 65 69 71 72

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INTRODUCTION The spatial distribution of transcripts, enzymes, and biosynthetic products within and between cells is an important component of regulation for eukaryotic metabolic processes. Many metabolic processes in primary metabolism are compartmentalized, enabling the separation of incompatible and competing reactions, and concentrating enzymes and metabolites. For example, cellular compartmentalization is achieved in C4 plants that fix atmospheric CO2 into C4 acids in the chloroplast-containing mesophyll cells. The C4 acids are decarboxylated and donated to Rubisco in the bundle sheath cells that surround the vascular tissue, thus creating a higher concentration of CO2 in these cells than in the photosynthetic cells of C3 plants. The enrichment of CO2 allows C4 plants to sustain higher rates of photosynthesis and lower rates of photorespiration than C3 plants. Subcellular compartmentalization is utilized during cellular respiration in eukaryotic cells. Glycolysis, the aerobic catabolism of glucose, takes place in the cytoplasm. The product of glycolysis, pyruvic acid, is then further catabolized in the mitochondria to carbon dioxide and water with the release of ATP through the mitochondriallocalized tricarboxylic acid (TCA) cycle and the electron transport chain. Finally, Calvin cycle enzymes are organized into a multienzyme complex, which appears to be loosely associated with thylakoid membranes near the sites of ATP and NADPH synthesis.1 The assembled complex results in enhanced activities of component enzymes such as Rubisco, and may also play a role in the regulation of some pathway enzymes by light.1 Many secondary compounds that play a role in plant defense are sequestered in specialized cells or structures, likely serving to protect the plant against the toxicity of such products. Furthermore, gene transcripts and proteins involved in the biosynthesis of these metabolites are often localized to specialized compartments. The fundamental questions that require investigation regarding the spatial distribution of metabolic processes remain the same for secondary metabolism as for primary metabolism. These questions are centered on the number and types of cells or organelles involved, the types and quantities of the metabolites that are present, and the factors involved in controlling the temporal and spatial relationships among gene expression, enzyme activity, and product localization.

SPECIALIZED STRUCTURES Glandular Trichomes Glandular trichomes, epidermal appendages found on the stems and leaves of many plants, store and secrete secondary metabolites in a species- and cultivarspecific fashion. The accumulation of these toxic compounds at the plant's surface

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may provide a first line of defense against insects, pathogens, and herbivores. Members of the Lamiaceae, including basil and mint, possess peltate glandular trichomes on the surface of their leaves. Peltate glands consist of a stalk cell attached to the leaf, four to eight secretory cells attached to the stalk cell, and a subcuticular oil sac above the secretory cells.3'6 Secondary products accumulate within the subcuticular pocket of the peltate glandular trichomes. Damage or physical pressure to the tissue rupture the sacs and release their toxic contents. The essential oils of peppermint (Mentha x piperita) and spearmint (Mentha spicatd) are localized in peltate glandular trichomes. Evidence of monoterpene biosynthesis within the secretory cells of peltate glandular trichomes prompted the development of a protocol for isolating and purifying these cells from the trichomes.7'8 The purified secretory cells provided an enriched source of monoterpene biosynthetic transcripts and enzymes, and enabled the cloning of nearly all of the genes of the (-)-menthol pathway.9 Geranyl diphosphate synthase (GPPS), a prenyl transferase that condenses the primary precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) to produce 4S'(-)-limonene, and the subsequent enzyme, 4S(-)-limonene synthase that catalyzes the first committed step in the pathway were immunocytochemically localized to the stroma of peppermint leucoplasts of secretory stage peltate glandular trichomes. ' This result was not unexpected since the primary precursors of monoterpene biosynthesis, IPP and DMAPP, are derived via the plastid-localized methylerythritol 4-phosphate (MEP) pathway, in higher plants, rather than the cytosol-localized mevalonate pathway.12"' (4S)-(-)-Limonene acts as a common precursor in the biosynthesis of menthol in peppermint and carvone in spearmint.15 The cytochrome P450-dependent 3-hydroxylase that catalyzes the regiospecific hydroxylation of limonene in the biosynthesis of menthol has a typical iV-terminal membrane insertion sequence and was localized exclusively in the microsomal fraction of oil gland cells, predicting an association with the endoplasmic reticulum (ER).16"17 The equivalent 6-hydroxylase that directs (45)-(-)limonene towards the biosynthesis of carvone was localized to smooth endoplasmic reticulum membranes.10 In menthol biosynthesis, (-)-isopiperitenol dehydrogenase (IPD) catalyzes the oxidation of isopiperitenol to isopiperitenone. The localization of IPD to the mitochondrial matrix was unexpected due to its unclear iV-terminal targeting information. Pulegone reductase, the enzyme that catalyzes the penultimate step in menthol biosynthesis by the reduction of a A4'8 double bond, was localized to the cytosol by immunocytochemical analyses.10 These studies indicate that diverse subcellular compartments are involved in monoterpene biosynthesis in mint. The intracellular transport of monoterpenes during their biosynthesis and their secretion into the subcuticular pocket against their concentration gradient likely requires specialized lipid carriers and transfer proteins. The peltate glands of sweet basil (Ocimum basilicum) produce and store monoterpene, sesquiterpene, and phenylpropene volatiles.18 Relative amounts of

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these compounds vary between cultivars. Examination of the glandular trichomes of O. basilicum revealed a correlation between the levels of transcripts for eight genes encoding distinct terpene synthases with relative levels of specific terpenes.18 For example, cultivars that produce the oxygenated monoterpenes (i?)-linalool and geraniol contained (/?)-linalool synthase and geraniol synthase, respectively, as the most abundant transcripts in their glandular trichomes. Peltate glands have also been identified as the major site of phenylpropene accumulation in O. basilicum leaves. Enzyme activities leading to phenylpropanoid biosynthesis were localized almost exclusively to the peltate glands in leaves of the sweet basil line SW, which produces primarily eugenol, and line EMX-1, which produces primarily methylchavicol.19 Analysis of an expressed sequence tag (EST) database from sweet basil leaf peltate glands showed that expression of known genes for the phenylpropanoid pathway accounted for 13% of the total ESTs.19 Furthermore, 14% of the cDNAs encoded enzymes are involved in the biosynthesis of S-adenosyl-methionine, which is required for the biosynthesis of many phenylpropanoids. The importance of flavonoid metabolism in alfalfa (Medicago sativd) glandular trichomes and its association with pest resistance has also been suggested. Alfalfa varieties with a high density of glandular trichomes on their stems and with increased resistance against the potato leafhopper (Empoasca fabae) have recently been introduced.20 ESTs from an alfalfa glandular trichome cDNA library support the production of flavonoids as the most abundant secondary compounds in these trichomes.21 Several ESTs were found to correspond to enzymes for the core phenylpropanoid, flavonoid and isoflavonoid pathways. Resin Ducts Oleoresin, a complex mixture consisting primarily of monoterpenes and diterpenes and a smaller proportion of sesquiterpenes, is accumulated in specialized anatomical structures found in conifers. These structures include the simple resin blisters of fir (Abies spp.), and the more complex resin-filled, interconnected canals of spruce (Picea spp.) and pine (Pinus spp.).3'22 The volatile monoterpenes and sesquiterpenes evaporate after exposure to the atmosphere, whereas the diterpene acids seal the wound site by polymerization.23 Oleoresin terpenes may be involved as constitutive or inducible defenses. Oleoresin sequestered in pre-formed resin ducts is released during the initial stages of attack by stem-boring insects and repels the pests by intoxication or formation of physical barriers.24 Insect attack, fungal elicitation, wounding, and treatment with methyl jasmonate can induce the formation of new traumatic resin ducts (TRD) in phloem and xylem tissue. " Tissue-specific differentiation of TRDs in Norway, White and Sitka spruce is accompanied by the induction of enzyme activities for prenyltransferases and terpene synthases, as well as terpenoid accumulation.28'30'31 The formation of TRDs has been shown to

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correspond to the area of potential insect larvae development and appears to be synchronized with the time of insect emergence from their eggs.32 S-Cells All members of the Brassicacea accumulate glucosinolates, a diverse group of sulfur containing compounds. In these plants, glucosinolates have been detected in the vacuoles of all organs33 and may serve as a sink for nitrogen and sulfur.34 Enzymatic hydrolysis of glucosinolates by thioglucoside glucohydrolases (myrosinases) results in the formation of isothiocyanates, which have a toxic effect on most cells via alkylation reactions and likely function in plant defense responses against microorganisms and insects. Energy disruptive X-ray imaging and cell sap analysis were used to localize glucosinolates to groups of S-cells containing high sulphur content.36 The Scells were situated between the phloem of every bundle and the endodermis in Arabidopsis flower stalks. Interestingly, transgenic Arabidopsis plants carrying pglucuronidase (GUS) and green fluorescent protein (GFP) reporter genes fused to the Arabidopsis myrosinase gene promoter showed that expression was restricted to guard cells and phloem tissues distinct from the S-cells. This result was consistent with the immunocytochemical localization of myrosinase to these cells. In the phloem of Arabidopsis flower stalks, myrosinase-containing cells were localized between sieve elements and S-cells. This juxtaposed compartmentalization of myrosinase-containing cells and S-cells next to the phloem sieve elements may be ideally situated to protect the phloem and its contents against microbial and insect attack. Upon mechanical damage, infection, or pest attack, cellular breakdown would allow the enzymatic hydrolysis of glucosinolates and the formation of toxic products. Laticifers Laticifer is a general term applied to a large and heterogenous group of cell types. Found in over 900 genera of plants, laticifers typically function as internal secretory systems. Laticifers from various species are primarily classified as either articulated, developing from cells whose common end walls have broken down, or nonarticulated, originating from single cells in the embryo. Both types produce a copious and often milky cytoplasm, known as latex, which exudes from the laticifers when the plant is damaged. Natural rubber (cw-l,4-polyisoprene) accumulates in the laticifers of many plants. Transcripts encoding enzymes involved in rubber biosynthesis are highly expressed in the latex of Hevea brasiliensis,37 the only plant from about 2000 rubberproducing species that produces commercially viable quantities of rubber. Although natural rubber is an important raw material in commercial, defense, and transportation industries, its role in the plant is not well understood. However, the

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latex of Hevea brasiliensis likely plays a role in plant defense, since it also accumulates high levels of transcripts from plant defense- or stress-related genes,38 and hydrolytic enzymes.39 Various alkaloids also accumulate in laticifers. Chemical indicators have identified laticifers and specialized parenchyma cells as the sites of alkaloid accumulation in Catharanthus roseus.40 Epifluorescence microscopy showed the random distribution of unbranched, nonarticulated laticifer cells throughout the mesophyll of C. roseus leaves.41 Larger autofluorescent idioblast cells, which are morphologically related to laticifer cells, also occurred around the mesophyll cells of C. roseus.41 Both laticifer and idioblast cells are also associated with the biosynthesis of alkaloids in different tissues of C. roseus plants.42'43 In opium poppy, articulated laticifers are present in all organs of the mature plant,44 occurring exclusively in the phloem region of a vascular bundle and consisting primarily of large vesicles into which alkaloids are sequestered. These vesicles are thought to originate from dilations of the rough and smooth ER during maturation.44'45 However, it has also been suggested that the vesicles result due to subdivision of the main vacuole of laticifer initials during maturation.46 Opium poppy laticifers were previously thought to be the site of alkaloid biosynthesis.47"49 However, only one of the known enzymes involved in alkaloid biosynthesis was identified among nearly 100 proteins isolated from the cytosolic and vesicular fractions of opium poppy latex.50 Furthermore, recent investigations have localized alkaloid biosynthesis to cell types other than the laticifers.51"53 These data can be reconciled by recognizing that latex harvested by the crude lancing of flower capsules or stems is subjected to a considerable amount of contamination from the adjacent cells, especially phloem sap due to the turgour pressure of sieve tubes. The detection of soluble enzymes in crude seed capsule exudates50'54 and the absence of membrane-bound enzymes55'56 support this suggestion. Opium poppy latex also contains abundant low molecular mass proteins, named major latex proteins (MLPs). The localization and function of the MLPs remain to be determined. In vitro, MLPs precipitate when exposed to alkaloids and various phenolic compounds, which are both released by laticifers upon wounding.46'57 Therefore, wounding might cause the coagulation of MLPs in order to restrict excessive loss of the latex.

COMPARTMENTALIZATION OF ALKALOID PATHWAYS Tropane Alkaloids Tropane alkaloids (TPA) occur mostly in the Solanaceae and include the anticholinergic drugs atropine, hyoscyamine, and scopolamine, as well as the narcotic, topical anesthetic cocaine. TPAs are typically produced near the root apex

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but accumulate within the vacuoles of leaves and roots.58 Tropane alkaloid biosynthesis in plants begins with the decarboxylation of ornithine and/or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC) to form the polyamine putrescine. The activities of ODC and ADC are dependent on physiological and developmental conditions and are regulated separately. ODC activity primarily regulates cell division in actively growing tissues, whereas ADC activity is associated mainly with secondary metabolic processes, cell extension, and stress responses.59 Putrescine A'-methyltransferase (PMT) catalyzes the first committed step in TPA biosynthesis by a SAM-dependent iV-methylation of putrescine.60 GUS activity was localized to the root pericycle in transgenic Atropa belladonna plants carrying a PMT promoter-GUS fusion (Fig. 3.1A).61 Tropinone is the first branch-point intermediate with a tropane ring in the TPA pathway. Two related dehydrogenases, tropinone reductase I (TR-I) and tropinone reductase II (TR-II), stereospecifically reduce the 3-keto group of tropinone to produce tropine and y-tropine, respectively. Hyoscyamine 6phydroxylase (H6H) catalyzes the hydroxylation of hyoscyamine to 6p hydroxyhyoscyamine and its subsequent epoxidation to scopolamine.62 Similar to PMT, the enzyme63 and transcripts6 for H6H were localized to the pericycle of A. belladonna roots. Transformations of A. belladonna, Hyoscyamus niger, and Nicotiana tabacum with an H6H promoter-GUS fusion showed that cell-specific expression of the H6H gene only occurred in scopolamine-producing plants.65 In contrast to PMT and H6H localizations, A. belladonna TR-I was immunolocalized to root endodermis and outer cortex, whereas TR-II was immunolocalized to the pericycle, endodermis, and outer cortex.66 Similarly, in H. niger transgenic hairy roots, TR-I and TR-II promoters activated transcription of reporter genes in the root endodermis and pericycle.67 Immunolocalization of TR-I to cells other than the pericycle suggests the translocation of intermediates between the pericycle and endodermis in scopolamine biosynthesis. PMT localization to the pericycle would allow access to putrescine and arginine precursors from the phloem. Scopolamine produced by H6H in the pericycle could be translocated to the leaves via the xylem (Fig. 3.1 A). Monoterpenoid Indole Alkaloids Enzymes involved in the biosynthesis of the powerful anti-tumor, terpenoid indole alkaloids (TIAs) vinblastine and vincristine have been localized to at least three different cell types and five different subcellular compartments in the Madagascar periwinkle, Catharanthus roseus.6S Precursors for the biosynthesis of TIAs contain an indole moiety derived from tryptophan and an iridoid moiety derived from the monoterpenoid geraniol. The IPP and DMAPP required for geraniol

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SAMANANI and FACCHINI SAMANANIandFACCHINI Pericycle

Phloem

D Endodermis

Cortex

Pericycle Xylem

Xylem Pericycle p--

Immature endodermis Parenchyma

Fig. 3.1: Alkaloid biosynthetic pathways are associated with a diverse variety of cell types. The tissue-specific localization (shaded) of enzymes and/or gene transcripts are depicted for the biosynthesis of tropane alkaloids in Atropa belladonna and Hyoscyamus niger roots (A), monoterpenoid indole alkaloids in Catharanthus roseus leaves (B), pyrrolizidine alkaloids in Senecio vernalis roots (C), pyrrolizidine alkaloids in Eupatorium cannabinum roots (D), benzylisoquinoline alkaloids in Papaver somniferum vascular bundles (E), and protoberberine alkaloids in Thalictrum flavum roots (F).

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biosynthesis are derived via the plastid-localized MEP pathway, rather than the cytosol-localized mevalonate pathway.11"13 Tryptophan decarboxylase (TDC) and the P450-dependent enzyme monooxygenase geraniol 10-hydroxylase, CYP76B6 (G10H) catalyze the decarboxylation of tryptophan and the hydroxylation of geraniol en-route to tryptamine and secologanin biosyntheses, respectively. Geraniol is converted to loganin, the direct precursor for secologanin, by the P450-dependent hydroxylase activity of a secologanin synthase, CYP72A1 (SLS).69 Strictosidine is the common precursor for the biosynthesis of all TIAs and results from the condensation of tryptamine and secologanin by strictosidine synthase (STR). Strictosidine is hydrolyzed by strictosidine (3-D-glucosidase (SGD). The subsequent biosynthetic enzymes that catalyze the reactions leading to tabersonine formation have not been identified. Tabersonine is converted to vindoline via a series of six enzymatic reactions, which include hydroxylation at C-16, 16-0 methylation, hydration of the 2, 3-double bond, iV-methylation, hydroxylation at C-4 and 4-0acetylation.70'71 The first of these reactions is catalyzed by tabersonine 16hydroxylase (T16H) and the last two reactions are catalyzed by desacetoxyvindoline 4-hydroxylase (D4H) and acetyl coenzyme A (CoA):deacetylvindoline 4-0acetyltransferase (DAT), respectively. The coupling of vindoline with catharanthine and further elaboration of the resulting product produces the dimeric alkaloids vinblastine and vincristine. Vindoline, vinblastine and vincristine accumulate in specialized cells of the shoot mesophyll, the laticifers and idioblasts.41'42 Due to their pharmaceutical importance and low content, a considerable amount of research has been conducted on the control mechanisms involved in the biosynthesis of vinblastine and vincristine in C. roseus plants, including compartmentalization of the biosynthetic enzymes and the corresponding transcripts. TDC and STR are most abundant in C. roseus roots, but also occur in aerial organs.72 In contrast, T16H, D4H, and DAT are restricted to young leaves and other shoot organs.73"75 D4H and DAT transcripts and enzymes only occur in the laticifer and idioblast cells of aerial organs, which also accumulate TIAs (Fig. 3.IB). However, the steps involving TDC, SLS, and STR activities occur several cell layers away in the epidermis of these organs.42'69'70 Furthermore, the expression of three genes involved in the MEP pathway, along with G10H expression were co-localized to the internal phloem parenchyma of young C. roseus aerial organs, a specialized additional internal phloem developed by this plant during its histogenesis.77 Interestingly, transcripts for C. roseus Protein S (CrPS) were also localized to the internal phloem parenchyma cells of developing leaves.78 CrPS is a member of the ot/p hydrolase superfamily and may have a putative role in the signal transduction pathway triggering monoterpenoid indole alkaloid biosynthesis. These results suggest the translocation of biosynthetic intermediates from the internal phloem to the epidermis and from the epidermis to the alkaloid-accumulating laticifer and idioblast cells. It has been noted that cell cultures that do not accumulate vindoline recover this ability upon differentiation of shoots. The high degree of

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compartmentalization of metabolic reactions to specialized cell types may, in part, explain the inability of de-differentiated C. roseus cell cultures to produce TIAs. Subcellular compartmentalization also occurs in TIA biosynthesis. Tryptophan is converted to tryptamine by TDC in the cytosol.43'80 However, the cytochrome P450-dependent enzymes, G10H and SLS, are endomembrane bound.69'81 The putative association of G10H with provacuolar membranes has also been noted.82 Tryptamine from the cytosol must be transported across the tonoplast into the vacuole where STR couples it to secologanin to form strictosidine.83 SGD may be bound to the external side of the tonoplast,80 however, in vivo localization studies have shown SGD to be associated with the ER. Further downstream in TIA biosynthesis, the P450-dependent enzyme that catalyzes the 16-hydroxylation of tabersonine, T16H, is associated with the ER.73 Furthermore, while NMT was found to be associated with thylakoid membranes.43'85 D4H and DAT were associated with the cytosol.86'87 Finally, the non-specific peroxidases necessary for coupling vindoline to catharanthine are located in the vacuole.88 The discrete compartmentalization of strictosidine and SGD may be comparable to cyanogenic glucosides and their specific glucosidases. Upon cell damage, SGD would rapidly convert strictosidine into the aglucon, which has been shown to have antimicrobial activity. It should be noted, however, that it is not known whether SGD occurs in epidermal or laticifer/idioblast cells. Pyrrolizidine Alkaloids The constitutively expressed pyrrolizidine alkaloids (PAs) comprise a diverse group of over 350 structures with scattered occurrence in certain higher plant taxa. Their occurrence is mostly restricted to the Asteraceae {e.g., the genera Senecio and Eupatorium), many genera of the Boraginaceae, a few genera of the Fabaceae, and certain genera of the Orchidaceae.90 The necine base of PAs is thought to originate from ornithine or arginine, whereas the necic acid moiety originates from aliphatic amino acids such as isoleucine.90 For most herbivores, PAs are strong feeding deterrents and liver toxins in vertebrates.91 In plants of Senecio species, senecionide TV-oxide is synthesized in the roots and transported to the preferential sites of storage, the inflorescences and peripheral tissues of the leaves and stems, via the phloem. In some species of Senecio, 60 to 80% of the PAs are found in the inflorescences, which represents concentrations 10- to 30-fold higher than in the vegetative organs of the plant.92 In vegetative organs, they are found mostly in the epidermal and subepidermal layers. Accumulation in reproductive and peripheral tissues of the plant is consistent with their role as defense compounds. Remarkably, specialized herbivorous insects from diverse taxa have evolved adaptations to not only overcome the barriers posed by these toxic alkaloids but also to obtain and use them for their own defense against predators.90

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The first pathway-specific enzyme involved in the biosynthesis of homospermidine, a precursor to the necine base moiety of pyrrolizidine alkaloids, is homospermidine synthase (HSS). Sequence analysis of HSS revealed amino acid and nucleic acid identities of 79% and 83%, respectively, to deoxyhypusine synthase (DHS), an enzyme catalyzing the first step in the activation of a translation initiation factor (eIF5A) essential for eukaryotic cell proliferation.93 Evidence suggests that in pyrrolizidine alkaloid-producing plants the HSS gene evolved from the DHS gene via gene duplication.93 RNA-blot, reverse transcriptase-PCR and immunolocalization analyses showed that although DHS is constitutively expressed in the shoots and roots of Senecio vernalis (Asteraceae), HSS expression is root specific.94 Furthermore, HSS expression is restricted to distinct groups of endodermal and adjacent cortical cells opposite the phloem (Fig. 3.1C). Thus, the expression pattern of HSS in S. vernalis has undergone a drastic change from the ancestral DHS gene. In contrast, HSS expression in Eupatorium cannabinum was observed to occur uniformly in all cells of the root cortex parenchyma, but not in the endodermis and exodermis (Fig. 3.ID).95 These data, along with the scattered occurrence of pyrrolizidine alkaloids in several plant families, suggest the independent chance recruitment of HSS from the ubiquitous DHS gene in various angiosperm families. Quinolizidine Alkaloids Quinolizidine alkaloids (QAs) are found mainly within the Leguminoseae and especially in the subfamily Papilionaceae.96 They are biosynthesized from lysine through the decarboxylated intermediate cadaverine, which undergoes oxidative cyclization to form the QA skeleton. Ester-type QAs exist as esters of acetic acid, tiglic acid, />coumaric acid, ferulic acid, and benzoic acid, and are thought to represent biosynthetic end products for transport and storage. Compared to the information available for other alkaloids, little work exists on the compartmentalization of QAs. Studies using purified leaf chloroplasts of Lupinus polyphyllus localized the first two enzymes of QA biosynthesis, lysine decarboxylase and 17-oxosparteine synthase, to the chloroplast stoma where lysine biosynthesis also takes place.97 However, activities of two acyl-CoA-dependent acyltransferases involved in subsequent reactions were shown not to be associated with the chloroplast fractions in L. albus and L. hirsutus green seedlings.98 13aHydroxymultiflorine/13a-hydroxylupanine O-tigloylase (HMT/HLT) activity was localized to the mitochondrial matrix, whereas epilupinine O-p-coumaroyltransferase activity was concentrated in a different subcellular compartment. Likely due to restrictions imposed by weak enzyme activities during purification, the isolated cDNA for HMT/HLT represents the first cloned gene encoding a biosynthetic enzyme for QAs.99 In correlation with enzyme activity, RNA blot analysis of this acyltransferase suggested the gene was expressed in the roots and hypocotyls of L.

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albus." The protein sequence deduced using the HMT/HLT cDNA clone showed the presence of a mitochondrial translocation signal." However, a cleavable mitochondrial pre-sequence was not found. A peroxisomal translocation signal was also found at the C-terminus of the protein. Thus, the subcellular location of this acyltransferase remains unclear but it appears that the quinolizidine nucleus is synthesized in the chloroplast and subsequent modifications can only occur after biosynthetic intermediates are transported out of the chloroplast. QAs likely accumulate in the vacuoles of epidermal cells where their defensive properties would best be utilized. Benzylisoquinoline Alkaloids More than 2,500 benzylisoquinoline (BIA) compounds have been identified in plants, many of which possess potent pharmacological activity.100 Included in this group are the analgesic morphine, the muscle relaxant papaverine, and the antimicrobial agent sanguinarine. Despite a diversity of pharmacological effects, all BIAs share a common origin that involves the condensation of two aromatic rings, both of which are derived from tyrosine. Common intermediates in this pathway have been studied intensively at the chemical and enzymological levels. These studies have primarily focused on three branches of the pathway leading to the protoberberine (e.g., berberine), benzophenanthridine (e.g., sanguinarine), and morphinan (e.g., morphine) alkaloids. The biosynthetic and structural relationships between these three important classes of BIAs are outlined in Figure 3.2. The first steps in the biosynthesis of all BIAs involve the decarboxylation and ortho-hydroxylation of tyrosine to yield dopamine. Concomitantly, decarboxylation and deamination of tyrosine yields 4-hydroxyphenylacetaldehyde (4-HPAA). More than one route from tyrosine to dopamine and 4-HPAA is thought to occur, but this has not been confirmed in vivo.101 The only enzyme purified from Eschscholzia californica (California poppy) and Thalictrum flavum (meadow rue),102 and cloned, from Papaver somniferum (opium poppy),103'104 is a tyrosine/dopa decarboxylase (TYDC). Dopamine and 4-HPAA are condensed by the stereospecific enzyme (S)norcoclaurine synthase (NCS) to form fSJ-norcoclaurine, the central alkaloid intermediate in the biosynthesis of all BIAs in higher plants. (S)-Norcoclaurine is converted to (S^)-reticuline through a series of reactions catalyzed by 6-0methyltransferase (6OMT), an JV-methyltransferase, a P450-dependent hydroxylase (CYP80B1), and a 4'-<9-methyrtransferase (4'OMT). 68 (5)-Reticuline is a key branch-point intermediate in the biosynthesis of most benzylisoquinoline alkaloids, including those with a morphinan, benzophenanthridine, or protoberberine nucleus. In P. somniferum and a few other select species of the genus Papaver, (S)reticuline is converted to its (7?j-epimer by 1,2-dehydroreticuline synthase (DRS) and 1,2-dehydroreticuline reductase (DRR) and then diverted toward the biosynthesis of the morphinan alkaloids. The P450-dependent monooxygenase salutaridine

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synthase (STS) converts (i?)-reticuline to salutaridine, which is hydroxylated and acetylated by salutaridine:NADPH 7-oxidoreductase (SOR) and salutaridinol Oacetyltransferase (SAT), respectively, to yield salutaridinol 7-O-acetate.55'56107 Spontaneous rearrangement of salutaridinol 7-O-acetate produces thebaine, which is oxidized to codeinone, via neopinone, and subsequently reduced by codeinone reductase (COR) to yield codeine.108 Finally, morphine is produced by the demethylation of codeine. Alternatively, thebaine can be demethylated to oripavine, which is oxidized to morphinone and subsequently reduced by COR to yield morphine. The first committed step in protoberberine, benzophenanthridine, and protopine alkaloid biosynthesis is catalyzed by the berberine bridge enzyme (BBE). In opium poppy and other members of the Papaveraceae and Rutaceae, BBE converts (S)-reticuline to (5)-scoulerine as the first committed step in the benzophenanthridine branch pathway. The P450-dependent monooxygenases cheilanthifoline synthase (CFS) and stylopine synthase (SPS) catalyze the formation of two methylenedioxy bridges, which result in the conversion of (5)-scoulerine to (5)-stylopine.U0 (S)-stylopine is converted to (S)-c«-./V-methylstylopine by tetrahydroprotoberberine czs-iV-methyltransferase (TNMT). Subsequently, (S)-cis-Nmethylstylopine is hydroxylated by the P450-dependent enzyme ./V-methylstylopine 14-hydroxylase (MSH). The resulting reaction product tautomerizes to protopine, which is hydroxylated by protopine 6-hydroxylase (P6H) to yield dihydrosanguinarine, which is subsequently oxidized by dihydrobenzophenanthridine oxidase (DBOX) to yield sanguinarine. In T. flavum and other members of the Ranunculaceae and Berberidaceae, (S)adenosylmethionine:scoulerine-9-0-methyltransferase (SOMT) catalyzes the transfer of the S-methyl group of SAM to the 9-hydroxyl group of fS)-scoulerine to produce fiSj-tetrahydrocolumbamine,112 which is converted by the P450-dependent enzyme canadine synthase (CYP719A1) to (5)-canadine via methylenedioxy bridge formation.113 Oxidation of (5)-canadine to berberine is catalyzed by an irondependent (S)-canadine oxidase (CDO) in Coptis japonica and Thalictrum minus, or a flavinylated (5)-tetrahydroprotoberberine oxidase (STOX) in Berberis stolonifera. Cellular Compartmentalization ofBIAs In the opium poppy, many biosynthetic enzymes are preferentially active in certain organs. For example, TYDC transcripts and NCS activity are abundant in the roots and stems, whereas only low levels occur in other organs.104'105 CYP80B1 mRNA levels are also highest in stems, followed by roots, leaves, and floral tissues.114 Salutaridine synthase (STS) and salutaridine:NADPH 7-oxidoreductase (SOR), which convert (i?)-reticuline to salutaridine and salutaridinol, respectively, in the pathway leading to morphine, are abundant in roots and shoot organs. '

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SAMANANI and FACCHINI SAMANANIandFACCHINI

coa kJ^-o

V-^-o

SPS

(S)-Cheilanthifoline

(S)-Stylopino

TNMT

SAT H3CO,

MSH (S>-c/s-W-M9thylstylopine

Protopine

P6H

"**o DihydrosanguinarinG

DBOX

Sanguinarlne

spontaneous

^X^O

6-Hydroxyprotopine

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Fig. 3.2 (previous): Biosynthesis of the benzylisoquinoline alkaloids berberine, sanguinarine, and morphine in opium poppy. Enzymes for which corresponding molecular clones have been isolated are shown in bold. Abbreviations: TYDC, tyrosine decarboxylase; NCS, norcoclaurine synthase; 60MT, norcoclaurine 6-O-methyltransferase; CNMT, coclaurine iV-methyltransferase; CYP80B1, ./V-methylcoclaurine 3'-hydroxylase; 4'0MT, 3'-hydroxy-./V-methylcoclaurine 4'-0-methyltransferase; 70MT, reticuline 7-0-methyltransferase; BBE, berberine bridge enzyme; SOMT, scoulerine 9-O-methyltransferase; CYP719A1, canadine synthase; STOX, (5)-tetrahydroxyprotoberberine oxidase; CFS, cheilanthifoline synthase; SPS, stylopine synthase; TNMT, tetrahydroprotoberberine cis-Nmethyltransferase; MSH, N-methylstylopine 14-hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase; DRS, 1,2-dehydroreticuline synthase; DRR, 1,2-dehydroreticuline reductase; STS, salutaridine synthase; SOR, salutaridine:NADPH 7oxidoreductase; SAT, salutaridinol-7-(9-acetyltransferase; COR, codeinone reductase.

Enzyme activity for codeinone reductase (COR), which catalyzes the penultimate step in morphine biosynthesis, is present throughout the plant, especially in shoot organs.103'10 This correlates with the accumulation of morphine in the laticifers of opium poppy, with the greatest abundance found in aerial organs. In contrast, transcript levels of BBE, which is specifically involved in sanguinarine biosynthesis, are highest in roots but are also found in shoot organs, even though sanguinarine does not accumulate in these organs.114 Low levels of BBE enzyme activity were also detected in shoots,109 suggesting that sanguinarine or a late intermediate of the pathway might be translocated from the aerial organs to the roots of opium poppy. Recent studies using immunofluorescence labeling and in situ transcript localization have led to an interesting controversy regarding the cellular location of BIA biosynthesis in opium poppy. Enzymes and transcripts for CYP80B1, BBE, and COR have been localized to sieve elements and their companion cells, respectively.52 A subsequent study has described the localization of BIA biosynthetic enzymes to parenchyma cells that purportedly surround laticifers within the vascular tissues.53 These cells were not identified as sieve elements since sieve plates were not detected. However, the possibility exists that the enzymes were localized to immature sieve elements lacking a sieve plate. A total of seven BIA biosynthetic enzymes have now been localized to sieve elements, which were irrefutably identified using a monoclonal antibody specific for an H+-ATPase isoform only found in the sieve elements (R. Bourgault, N. Samanani, J. Alcantara, K. Zulak, and P. Facchini, unpublished results). Transcripts for BIA biosynthetic genes appear to

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be localized to companion cells in all of the above studies. Thus, BIA biosynthetic genes appear to be transcribed and translated in the companion cells of the phloem and, subsequently, transported to the adjacent sieve elements, which are incapable of performing basic transcriptional and translational processes (Fig. 3.IE). In the sieve elements, the membrane bound P450-dependent enzyme CYP80B1, and the membrane-associated BBE,115 could interact with the sieve element reticulum (SER), a form of ER in the sieve elements that has also been proposed to function in the trafficking of proteins through the plasmodesmata that connect sieve elements with companion cells.116 The co-sedimentation of CYP80B1, BBE, and sanguinarine with the ER marker calreticulin on a sucrose density gradient suggests that the entire biosynthetic pathway occurs in association with the ER in the opium poppy. The co-localization of BBE, COR, and SAT to sieve elements suggests that both morphine and sanguinarine biosynthesis occurs in the same cell type.52'53 The localization of seven biosynthetic steps to the sieve elements provides support for the role of sieve elements beyond the transport of solutes and information macromolecules, as previously thought. In contrast to the requirement for three cell types in the biosynthesis and accumulation of alkaloids in the plant, the biosynthesis and accumulation of sanguinarine in cell cultures of opium poppy apparently occurs within a single dedifferentiated cell.115 Berberine is a constitutive secondary metabolite with potent antimicrobial properties. It confers protection against Gram-positive bacteria, Gram-negative bacteria, and other microorganisms,117 a general inhibitor of DNA and protein synthesis, and the predominant alkaloid in T. flavum } n Berberine content is highest in the oldest parts of T. flavum rhizomes, petioles, and roots.119 In the rhizomes, this alkaloid accumulates throughout the pith, cortex, and rib parenchyma,119'120 which is in stark contrast to the accumulation of berberine biosynthetic gene transcripts for nine consecutive steps, from TYDC to CYP719A, in the protoderm of leaf primordia.120 The biosynthesis and accumulation of berberine in the roots of T. flavum appears to be under developmental control. The youngest parts of the root near the apical meristem do not accumulate berberine. However, biosynthetic gene transcripts for nine consecutive steps have been localized to the root apex, primarily in immature endodermal cells prior to the formation of a casparian strip (Fig. 3.IF). 120 Transcript accumulation was also observed in pericycle and cortical cells. Protoberberine alkaloids appear to accumulate in the endodermal cells of T. flavum roots after the onset of secondary growth.119'120 As secondary growth of the root progresses, the endodermis remains intact, despite a rapid expansion of the stele, and emerges as the outer-most layer of the mature root. In the oldest parts of the root, berberine also accumulates in the pericycle cell layers.119 Therefore, it appears that the endodermis and, subsequently, the pericycle provide a protective outer barrier of cells containing potent antimicrobial alkaloids. The abundant accumulation of berberine in T. flavum roots and rhizomes, which are surrounded by a multitude of soil-borne pathogens, supports its role as a plant defense compound.

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Overall, the spatial separation of the sites of biosynthesis from the sites of alkaloid accumulation suggests the occurrence of intracellular transport of alkaloids in opium poppy plants and T. flavum rhizomes, although immunolocalization studies are required to confirm the cellular location of the biosynthetic enzymes in T. flavum. The movement of proteins from companion cells to sieve elements is well established.116 However, the mechanisms involved in the translocation of alkaloids from sieve elements to laticifers, in the case of opium poppy, or possibly from the protoderm of leaf primordia to the pith, cortex and rib parenchyma of rhizomes, in the case of T. flavum, are not known. Possible mechanisms may include apoplastic transport across plasma membranes and cell walls or symplastic transport via connections between different cell types. An ATP-binding-cassette (ABC) multidrug-resistant transporter associated with xylem parenchyma and capable of transporting berberine and other low molecular weight molecules has been isolated from C. japonica}21 Several homologs of ABC transporters are present in opium poppy (K. Zulak and P. Facchini, unpublished results), but require functional characterization. The symplastic movement of alkaloids would require the presence of functional plasmodesmata between participating cell types. In the opium poppy, immunolocalization of callose, which lines plasmodesmatal pores, suggests the existence of symplastic connections between these cell types (D. Bird and P. Facchini, unpublished results). Subcellular Compartmentalization ofBIAs Several enzymes involved in BIA biosynthesis are associated with a subcellular compartment other than the cytosol. Based on density gradient fractionation, BBE, CFS, SPS, MSH, and P6H of the sanguinarine branch appear to be localized to microsomes with a density of 1.11 to 1.14 g ml'1.110>m-122>123 with the exception of BBE, these non-cytosolic enzymes are P450-dependent and are thus, integral endomembrane proteins. Although BBE is not an integral membrane protein, it is initially targeted to the ER, after which it is sorted to a vacuolar compartment where sanguinarine also accumulates in response to elicitor treatment.124 Due to the association of BIA biosynthetic enzymes with endomembranes, it has been speculated that specialized alkaloid-synthesizing vesicles are present in alkaloidproducing cells.12 Similar vesicles have been reported from cell cultures of Berberis spp., Thalictrum glaucum, and C. japonica.122'12 However, the proposed existence of a unique organelle involved in BIA biosynthesis, distinct from the ER, 122 should be reconsidered. Elicitor treatment of cultured opium poppy cells results in the simultaneous activation of alkaloid biosynthetic gene expression and dilation of the ER.115 CYP80B1, BBE and sanguinarine were co-localized with the ER in these cell cultures. Most notably, immunogold labeling showed the association of CYP80B1

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and BBE with dilated ER. The large dilated vesicles, which were formed from lamellar ER upon elicitor treatment, fused with the central vacuole (Fig. 3.3). The localization of CYP80B1, BBE, and sanguinarine to the ER of opium poppy cell cultures may be extrapolated to the SER in sieve elements of the plant. It is noteworthy that the alkaloid-containing vesicles of opium poppy laticifers are thought to result from dilations of the ER.45'46 The sequestration of alkaloids to vacuolar compartments within laticifers and the localization of sanguinarine biosynthesis to the ER U1 suggest symplastic transport from the sieve element reticulum into the dilated ER of the laticifer through plasmodesmata desmotubules of opium poppy. The ER is contiguous through plasmodesmata and permits the movement of small molecules, such as dextrans.126 The presence of functional plasmodesmata between sieve elements and laticifers would eliminate the requirement for multiple membrane transporters for the different alkaloids that accumulate in the opium poppy.

Vesicles

I

Multivesicular body

Vacuole Fig. 3.3: Sanguinarine biosynthesis is restricted to endomembranes in cultured opium poppy cells. The induction of sanguinarine biosynthesis in opium poppy cell cultures results in extensive dilations of the ER accompanied by the accumulation of an electron-dense flocculent material within the ER and vacuoles. Electron-dense, ER-derived vesicles seem to fuse with the central vacuole. The co-localization of CYP80B1, BBE and sanguinarine to the ER by sucrose density gradient analysis and of CYP80B1 and BBE to the ER vesicles by immunogold labeling has also been observed in these cells.

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METABOLIC COMPLEXES The organization of biosynthetic enzymes into macromolecular complexes allows the direct transfer, or channeling, of biosynthetic intermediates from the catalytic site of one enzyme to another. Spatial organization of enzymes in this way would result in the sequestration of highly reactive or potentially toxic intermediates, allow metabolic reactions to proceed more rapidly under low substrate concentrations, control metabolic flux between multiple branch pathways that often function at the same time within the same cell type, and may overcome problems associated with the lack of substrate specificity of enzymes. The enzymes of phenylpropanoid biosynthesis were suggested to function as multienzyme complexes over thirty years ago.127 Since then, strong evidence has accumulated in support of this hypothesis for the early stages of the general phenylpropanoid pathway, the early stages of the flavonoid and isoflavonoid pathways, the late stages of condensed tannin biosynthesis in Onobrychis viciifolia,m and for cyanogenic glycoside biosynthesis in sorghum.129 In the opium poppy, the co-localization of seven enzymes to the parietal region of the sieve elements is consistent with the possible assembly of metabolic channels for BIA biosynthesis in association with the SER.52 In further support of this suggestion, the RNAi-mediated silencing of COR genes in transgenic opium poppy plants shut down the morphine-specific branch pathway and resulted in the accumulation of (S)-reticuline, rather than codeinone.130 The suggestion that COR is present in the laticifers, whereas other morphinan branch pathway enzymes are not,53 would not be consistent with a possible multi-enzyme complex. The phenylpropanoid pathway is unique to plants and involves the conversion of phenylalanine to a large number of secondary compounds including lignins, sinapate esters, stilbenes, and flavonoids. Many roles have been described for phenylpropanoids in plant metabolism, including growth, defense, protection from UV light, and reproduction.131 Although transcriptional controls are involved in response to biotic and abiotic stresses, the operation of several branches of the pathway within one cell requires additional control mechanisms, including the subcellular organization of pathways. The first committed step in phenylpropanoid biosynthesis is the conversion of phenylalanine to fraMS-cinnamic acid by phenylalanine ammonia lyase (PAL). Transcriptional activation of PAL results after microbial infection or treatment of plant cells with microbial elicitors.132'133 The hydroxylation of trans-cirmamic acid to 4-coumaric acid is the second step in the phenylpropanoid pathway, being catalyzed by a cytochrome P450-dependent monooxygenase, cinnamic 4-hydroxylase (C4H). PAL and C4H inductions are often coordinated.134 Although PAL is considered to be an operationally soluble enzyme, various studies have suggested that PAL and C4H activities co-localize on membranes of the ER.135'136 Evidence for the existence of a

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metabolic channel between PAL and C4H has been presented.137 Similarly, immunolocalization, co-immunoprecipitation, affinity chromatography, and yeast two-hybrid-based evidence also exists for the co-localization of the enzymes of the flavonoid pathway in Arabidopsis to the ER.138'139 Isoflavone 0-methyltransferase (IOMT), which mediates the second branchspecific step in the biosynthesis of the anti-microbial isoflavonoid medicarpin, has been co-localized with the ER-associated cytochrome P450-dependent 2hydroxyisoflavanone synthase, the entry point enzyme into the pathway. Metabolic channeling at the entry point in this pathway may help prevent the dehydration of the unstable intermediate 2, 4', 7-trihydroxyisoflavanone to daidzein, a poor precursor of the phytoalexin medicarpin. The association of IOMT with a metabolic complex is thought to capture the unstable intermediate and channel it toward phytoalexin biosynthesis.139 Based on these studies, models for the protein complexes have been proposed where cytochrome P450-dependent proteins provide scaffolds for the self-assembly of soluble enzymes into complexes.1

SUMMARY AND FUTURE DIRECTIONS A recent bioinformatics analysis of the Arabidopsis genome predicted that members of 239 protein families were targeted to different subcellular compartments.141 Bifunctional or multifunctional enzymes targeted to alternative subcellular compartments may interact with different substrates to produce unique products. This may partially explain the observed diversity of plant secondary products. Broad enzyme specificities have been observed for O-methyltransferases, glucosyltransferases, P450-dependent monooxygenases, polyketide synthases, and monoterpene synthases.141 The retargeting of Arabidopsis plastidial and cytoplasmic fatty acid desaturases affected the regiospecificities of these enzymes. 42 The function of some secondary metabolic enzymes may, similarly, be influenced by their metabolic context through varied compartmentalization. Targeting of recombinant TDC, a cytosolic enzyme, to the chloroplast, cytosol, and ER of tobacco showed that TDC function, stability, and accumulation were significantly and differently affected.143 Evidence from flavonoid research suggests that the differential targeting of a pathway may be used to control the accumulation of endproducts in distinct cellular locations.144 Flavonoid biosynthesis in plants was thought previously to occur exclusively in the cytoplasm although flavonoids could accumulate in distinct subcellular compartments in different tissues. However, at least two of the enzymes of flavonoid biosynthesis occur in the nuclei of Arabidopsis cells, where the flavonoids also accumulate. Although much progress has recently been made toward the deciphering of the compartmentalization of secondary product metabolism, a comprehensive understanding of the spatial relationships between transcripts, enzymes, and biosynthetic products requires further research in several important areas. The

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identities, compartment-specific expression and function of transcription factors involved in the coordinated regulation of pathway enzymes and other metabolic components require elucidation for many of the systems studied. Mechanisms of intra- and inter-cellular translocation of pathway intermediates or secondary compounds also require identification. Finally, a thorough understanding of the subcellular organization of the biosynthetic machinery involved in the metabolism of model systems, such as the opium poppy, will definitively resolve current controversies.51'53

ACKNOWLEDGEMENTS Research in our laboratory is funded by grants from the Natural Sciences and Engineering Research Council of Canada. P.J.F. is the Canada Research Chair in Plant Biotechnology. REFERENCES 1. 2. 3. 4.

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methyl transfer enzymes of alkaloid biosythesis in opium poppy, Plant J., 2003, 36, 808-819. GERARDY, R., ZENK, M.H., Formation of salutaridine from (#)-reticuline by a membrane-bound cytochrome P-450 enzyme from Papaver somniferum, Phytochemistry, 1993, 32, 79-86. GERARDY, R., ZENK, M.H., Purification and characterization of salutaridine NADPH 7-oxidoreductase from Papaver somniferum, Phytochemistry, 1993, 34, 125-132. NESSLER, C.L., ALLEN, R.D., GALEWSKY, S., Identification and characterization of latex-specific proteins in opium poppy, Plant Physiol. 1985, 79, 499-504. WINK, M., Plant secondary metabolites from higher plants: biochemistry, function and biotechnology, in Biochemistry of Plant Secondary Metabolism, Annual Plant Reviews (M. Wink ed.), Sheffield Academic, Sheffield, 1999, 2, 1-16. HASHIMOTO, T., YAMADA, Y., Tropane alkaloid biosynthesis: regulation and application, in Proceedings of the Seventh Annual Pennsylvania State Symposium in Plant Physiology (B.K. Singh, H.E. Flores, and J.C. Shannon, eds.), American Society of Plant Physiologists Press, Rockville, MD, 1992, pp. 122-134. HIBI, N., FUITA, T., HATANO, M., HASHIMOTO, T., YAMADA, Y., Putrescine iV-methyltransferase in cultured roots of Hyoscyamus albus, Plant Physiol., 1992, 100, 826-835. SUZUKI, K., YAMADA, Y., HASHIMOTO, T., Expression of Atropa belladonna putrescine N-methyltransferase gene in root pericycle, Plant Cell Physiol., 1999, 40, 287-297. HASHIMOTO, T., YAMADA, Y., Alkaloid biogenesis: molecular aspects, Annu. Rev. Plant Physiol. Plant Mol. Biol, 1994, 45, 257-285. HASHIMOTO, T., HAYASHI, A., AMANO, Y., KOHNO, J., IWANARI, H., USUDA, S., YAMADA, Y., Hysoscyamine 6p-hydroxylase, an enzyme involved in tropane alkaloid bosynthesis, is localized at the pericyle of the root, J. Biol. Chem., 1991,266,4648-4653. SUZUKI, K., YUN, D-J., CHEN, X.Y., YAMADA, Y., HASHIMOTO, T., An Atropa belladonna hoscyamine 6p-hydroxylase gene is differentially expressed in the root pericycle and anthers, Plant Mol. Biol., 1999, 40, 141 -152. KANEGAE, T., KAJIYA, H., AMANO, Y., HASHIMOTO, T., YAMADA, Y., Species-dependent expression of the hyoscyamine 6p-hydroxylase gene in the pericycle, Plant Physiol., 1994,105, 483-490. NAKAJIMA, K., HASHIMOTO, T., Two tropinone reductases, that catalyze opposite stereospecific reductions in tropane alkaloid biosynthesis, are localized in plant root with different cell-specific patterns, Plant Cell Physiol, 1999, 40, 10991107. NAKAJIMA, K., OSHITA, Y., KAYA, M., YAMADA, Y., HASHIMOTO, T., Structures and expression patterns of two tropinone reductase genes from Hyoscyamus niger, Biosci. Biotechnol. Biochem., 1999, 63, 1756-1764.

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SAMANANI and FACCHINI SAMANANIandFACCHINI FACCHINI, P.J., Alkaloid biosynthesis in plants: Biochemistry, cell biology, molecular regulation, and metabolic engineering applications, Annu. Rev. Plant Physiol. Plant Mol. Biol, 2001, 52, 29-66. IRMLER, S., SCHRODER, G., ST-PIERRE, B., CROUCH, N.P., HOTZE, M , SCHMIDT, J., STRACK, D., MATERN, U., SCHRODER, J., Indole alkaloid biosynthesis in Catharcmthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase, Plant J., 2000, 24: 797-804. DE LUCA, V., BALSEVICH, J., TYLER, R.T., EILERT, U., PANCHUK, B.D., KURZ, W.G.W., Biosynthesis of indole alkaloids: Developmental regulation of the biosynthetic pathway from tabersonine to vindoline in Catharanthus roseus, J. Plant Physiol, 1986,125, 147-156 DE LUCA, V., FERNANDEZ, J.A., CAMPBELL, D., KURZ, W.G.W., Developmental regulation of enzymes of indole alkaloid biosynthesis in Catharanthus roseus, Plant Physiol., 1988, 86, 447-450. PASQUALI, G., GODDIJN, O.J.M., DE WAAL, A., VERPOORTE, R., SCHILPEROORT, R.A., HOGE, J.H., MEMELINK, J., Coordinated regulation of two indole alkaloid biosynthetic genes from Catharanthus roseus by auxin and tWcxtors, Plant Mol. Biol, 1992,18, 1121-1131. ST-PIERRE, B., DE LUCA, V., A cytochrome P-450 monooxygenase catalyzes the first step in the conversion of tabersonine to vindoline in Catharanthus roseus, Plant Physiol, 1995,109, 131-139. VAZQUEZ-FLOTA, F.A., DE CAROLIS, E., ALARCO, A.M., DE LUCA, V., Molecular cloning and characterization of deacetoxyvindoline 4-hydroxylase, a 2oxoglutarate dependent dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don, Plant Mol. Biol, 1997, 34, 935-948. ST-PIERRE, B., LAFLAMME, P., ALARCO, A.M., DE LUCA, V., The terminal O-acetyltransferase involved in vindoline biosynthesis defines a new class of proteins responsible for coenzyme A-dependent acyl transfer, Plant J., 1998, 14, 703-713. DE LUCA, V., ST-PIERRE, B., The cell and developmental biology of alkaloid biosynthesis, Trends Plant. Sci., 2000, 4: 168-173. BURLAT, V., OUDIN, A., CURTOIS, M., RIDEAU, M., ST-PIERRE, B., Coexpression of three MEP pathway genes and gerniol 10-hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during the biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites, Plant J., 2004, 38, 131-141. LEMENAGER, D., OUELHAZE, L., MAHROUG, S., VEAU, B., ST-PIERRE, B., RIDEAU, M., AGUIRREOLEA, J., BURLAT, V., CLASTRE, M., Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus, J. Exp. Bota., 2005, 56, 1221-1228. CONSTABEL, F., GAUDET-LAPRAIRIE, P., KURZ, W.G.W., KUTNEY, J.P., Alkaloid production in Catharanthus roseus cell cultures. XII. Biosynthetic capacity of callus from original explants and regenerated shoots, Plant Cell Rep., 1982, 1, 139-142.

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SAMANANI and FACCHINI SAMANANIandFACCHINI MOLL, S., ANKE, S., KAHMANN, U., HANSCH, R., HARTMANN, T., OBER, D., Cell-specific expression of homospermidine synthase, the entry enzyme of the pyrrolizidine alkaloid pathway in Senecio vernalis, in comparison with its ancestor, deoxyhypusine synthase, Plant Physiol., 2002,130, 47-57. ANKE, S., NIEMULLER, D., MOLL, S., HANSCH, R., OBER, D., Polyphyletic origin of pyrrolizidine alkaloids within the Asteraceae. Evidence from differential tissue expression of homospermidine synthase, Plant Physiol., 2004, 136, 40374047. OHMIYA, S., SAITO, K., MURAKOSHI, I., Lupine alkaloids, in The Alkaloids, Vol. 47, Academic Press, New York, NY, 1995. WINK, M., HARTMANN, T., Localization of the enzymes of quinolizidine alkaloid biosynthesis in leaf chloroplasts of Lupinus polyphyllus, Plant Physiol., 1982, 70, 74-77. SUZUKI, H., KOIKE, Y., MURAKOSHI, I., SAITO, K., Subcellular localization of acyltransferases for quinolizidine alkaloid biosynthesis in Lupinus, Phytochemistry, 1996, 42, 1557-1562. OKADA, T., HIRAI, M.Y., SUZUKI, H., YAMAZAKI, M., SAITO, K., Molecular characterization of a novel quinolizidine alkaloid O-tigloyltransferase: cDNA cloning, catalytic activity of recombinant protein and expression analysis in Lupinus plants, Plant Cell Physiol., 2005, 46, 233-244. SCHMELLER, T., LATZ-BRUNING, B., WINK, M., Biochemical activities of berberine, palmitine and sanguinarine mediating chemical defense against microorganisms and herbivores. Phytochemistry, 1997, 44, 257-266. RUEFFER, M., ZENK, M.H., Enzymatic formation of protopines by a microsomal cytochrome P-450 system of Corydalis vaginans, Tetrahedron Lett., 1987, 28, 53075310. MARQUES, I.A., BRODELIUS, P.E., Elicitor-induced Z-tyrosine decarboxylase from plant cell suspension cultures, Plant Physiol., 1988, 88, 46-51. FACCHINI, P.J., DE LUCA, V., Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy, J. Biol. Chem., 1994, 269, 26684-26690. FACCHINI, P.J., PENZES-YOST, C , SAMANANI, N., KOWALCHUK, B., Expression patterns conferred by tyrosine/dihydroxyphenylalanine decarboxylase promoters from opium poppy are conserved in transgenic tobacco, Plant Physiol., 1998,118,69-81. SAMANANI, N., FACCHINI, P.J., Isolation and partial characterization of norcoclaurine synthase, the first committed step in benzylisoquinoline alkaloid biosynthesis, from opium poppy, Planta, 2001, 213, 898-906. HIRATA, K., POEAKNAPO, C , ZENK, M.H., 1,2-Dehydroreticuline synthase, the branch-point enzyme opening the morphinan biosynthetic pathway, Phytochemistry, 2004, 65, 1039-1046. GROTHE, T., LENZ, R., KUTCHAN, T.M., Molecular characterization of the salutaridinol 7-Oacetyltransferase involved in morphine biosynthesis in opium poppy Papaver somniferum, J. Biol. Chem., 2001, 276, 30717-30723.

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108. UNTERLINNER, B., LENZ, R., KUTCHAN, T.M., Molecular cloning and functional expression of codeinone reductase: the penultimate enzyme in morphine biosynthesis in the opium poppy Papaver somniferum, Plant J., 1999,18, 465-475. 109. STEFFENS, P., NAGAKURA, N., ZENK, M.H., Purification and characterization of the berberine bridge enzyme from Berberis beaniana cell cultures, Phytochemistry, 1985, 24, 2577-2583. 110. BAUER, W., ZENK, M.H., Two methylene bridge forming cytochrome P-450 dependent enzymes are involved in (5^-stylopine biosynthesis, Phytochemistry, 1991,30,2953-2961. 111. TANAHASHI, T., ZENK, M.H., Elicitor induction and characterization of microsomal protopine-6-hydroxylase, the central enzyme in benzophenanthridine alkaloid biosynthesis, Phytochemistry, 1990, 29, 1113-1122. 112. MUEMMLER, S., RUEFFER, M., NAGAKURA, N., ZENK, M.H., 5-Adenosyl-Lmethionine: (5)-scuolerine 9-O-methyltransferase, a highly stereo- and regiospecific enzyme in tetrahydroprotoberberine biosynthesis, Plant Cell Rep., 1985, 4, 36-39. 113. IKEZAWA, N., TANAKA, M., NAGAYOSHI, M., SHINKYO, T., SAKAKI, T., INOUYE, K., SATO, F., Molecular cloning and characterization of CYP719, a methylenedioxy bridge-forming enzyme that belongs to a novel P450 family, from cultured Coptis japonica cells, J. Biol. Chem., 2003, 278, 38557-38565. 114. FACCHINI, P.J., PARK, S.-U., Developmental and inducible accumulation of gene transcripts involved in alkaloid biosynthesis in opium poppy, Phytochemistry, 2003, 64, 177-186. 115. ALCANTARA, J., BIRD, D.A., FRANCESCHI, V.R., FACCHINI, P.J., Sanguinarine biosynthesis is associated with the endoplasmic reticulum in cultured opium poppy cells after elicitor treatment, Plant Physiol., 2005, 138, 173-183. 116. VAN BEL, A.J.E., KNOBLAUCH, M., Sieve element and companion cell: the story of the comatose patient and hyperactive nurse, Aust. J. Plant Physiol., 2000, 27, 477-487. 117. ISAWA, K., NANBA, H., LEE, D.U., KANG, S.I., Structure-activity relationships of protoberberines having antimicrobial activity, Planta Med., 1998, 64, 748-751. 118. VELCHEVA, M., DUTSCHEWSKA, H., SAMUELSSON, G., The alkaloids of the roots of Thalictrum flavum L., Ada Pharm. Nord., 1992, 4, 57-58. 119. SAMANANI, N., YEUNG, E.C., FACCHINI, P.J., Cell type-specific protoberberine alkaloid accumulation in Thalictrum flavum, J. Plant Physiol., 2002, 159, 1189-1196. 120. SAMANANI, N., PARK, S-U., FACCHINI, P.J., Cell type-specific localization of transcripts encoding nine consecutive enzymes involved in protoberberine alkaloid biosynthesis, Plant Cell, 2005,17, 915-926. 121. SHITAN, N., BAZIN, I., DAN, K., OBATA, K., KIGAWA, K., UEDA, K., SATO, F., FORESTIER, C , YAZAKI, K., Involvement of CjMDRl, a plant multidrugresistance-type ATP-binding cassette protein, in alkaloid transport in Coptis japonica, Proc. Natl. Acad. Sci. USA, 2003, 100, 751-756. 122. AMANN, M., WANNER, G., ZENK, M.H., Intracellular compartmentation of two enzymes of berberine biosynthesis in plant cell cultures, Planta, 1986,167, 310-320.

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138. SASLOWSKY, D., WINKEL-SHIRLEY, B., Localization of flavonoid enzymes in Arabidopsis roots, Plant J., 2001, 27, 37-48. 139. WINKEL-SHIRLEY, B., Flavonoid biosynthesis. A colourful model for genetics, biochemistry, cell biology, and biotechnology, Plant Physiol., 2001, 126, 485-493. 140. LIU, C-J., DIXON, R.A., Elicitor-induced association of isoflavone Omethyltransferase with endomembranes prevents the formation and 7-O-methylation of daidzein during isoflavonoid phytoalexin biosynthesis, Plant Cell, 2001, 13, 2643-2658. 141. SCHWAB, W., Metabolome diversity: too few genes, too many metabolites?, Phytochemistry, 2003, 62, 837-849. 142. HEILMANN, I., PIDKOWICH, M.S., GIRKE, T., SHANKLIN, J., Switching desaturase enzyme specificity by alternate subcellular targeting, Proc. Natl. Acad. Sci. USA, 2004, 28, 10266-10271. 143. FIORE, S.D., LI, Q., LEECH, M.J., SCHUSTER, F., EMANS, N., FISCHER, R., SCHILLBERG, S., Targeting tryptophan decarboxylase to selected subcellular compartments of tobacco plants affects enzyme stability and in vivo function and leads to a lesion-mimic phenotype, Plant Physiol, 2002, 129, 1160-1169. 144. SASLOWSKY, D.E., WAREK, U., WINKEL, B.S., Nuclear localization of flavonoid enzymes in Arabidopsis, J. Biol. Chem., 2005, 280, 23735-23740.

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Chapter Four

COMPARATIVE GENOMICS OF THE SHIKIMATE PATHWAY IN ARABIDOPSIS, POPULUS TRICHOCARPA AND ORYZA SATIVA: SHIKIMATE PATHWAY GENE FAMILY STRUCTURE AND IDENTIFICATION OF CANDIDATES FOR MISSING LINKS IN PHENYLALANINE BIOSYNTHESIS Bjorn Hamberger,1 Jurgen Ehlting,2 Brad Barbazuk,3 and Carl J. Douglas1* department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada 2 Institute for Plant Molecular Biology, Centre National de la Recherche Scientifique, 67000 Strasbourg, France 3 Donald Danforth Plant Science Center, St. Louis, Missouri 63132, USA * Author for correspondence, email: [email protected]. ca Introdution 86 Skimimate Pathway in Plants 87 The Poplar Genome 89 Global Annotation of Poplar and Rice Shikimate Pathway Genes 91 Identification and Analysis of Shikimate and Candidate Shikimate Genes .... 91 Gene Family Topologies 91 Expression of Annotated Genes 93 Pre Chorismate Pathway 95 DHS genes 95 DHQS genes 95 DHQD/SDH genes 96 £ £ genes 99 EPSPS genes 99 GS genes 100 Post Chorismate Pathway 100 CM Genes 100 Candidates for Genes Encoding Prephanate Aminotransferase (PNT) 101 Candidates for Genes Encoding Arogenate Dehydratase (ADT) 107 Summary and Future Directions 110 85

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INTRODUCTION The great majority of aromatic compounds and their precursors are synthesized via the shikimate and its numerous branch pathways. This pathway, found in bacteria, fungi, and plants, connects primary carbohydrate metabolism with the biosynthesis of the three aromatic amino acids tryptophan, tyrosine, and phenylalanine. These three amino acids, essential for the animal diet, serve in plants as precursors for innumerable secondary metabolites ranging from alkaloids to phenylpropanoids.1 Shikimate pathway derived metabolites such as flavonoids, monolignols, and soluble and wall-bound phenolics represent the main metabolic sink for phenylalanine after its entry into the phenylpropanoid pathway, catalyzed by the combined actions of phenylalanine ammonia-lyase (PAL) and cinnamate-4hydroxylase (C4H) to generate /7-coumarate. It is estimated that 20% of the total carbon fixed in plants growing under standard conditions flows through this pathway,2 and the myriad of shikimate-derived compounds play vital roles in plant defence against biotic and abiotic stresses, plant development and structure, and plant-environment interaction. Most of the genes and corresponding enzymes in the shikimate pathway have been characterized in model plants such as Arabidopsis and tomato, and there is evidence from these and other plant systems that activity of the pathway, as measured by changes in gene expression, is coordinated with demand for phenylalanine entry into phenylpropanoid metabolism.3"6 However, the genes encoding two key steps in phenylalanine biosynthesis, prephenate aminotransferase (PNT) and arogenate dehydratase (ADT) have yet to be identified, and the specific corresponding enzymes remain unknown. Advances in plant structural genomics have the potential to allow comparative genomic studies on the nature and evolution of gene families, especially for genomes that have been completely sequenced such as Arabidopsis thaliana (Arabidopsis)? Oryza sativa (rice),8 and Populus trichocarpa (poplar).9 However, the great majority of genes in sequenced genomes are of unknown specific function, and functional genomics approaches such as global studies on mRNA abundance that reveal the transcriptome (the set of expressed genes at a given time or state) can help to assign functions to the large numbers of genes. A number of functional genomic approaches are possible, including in silico measurements of Expressed Sequence Tag (EST) abundance10 and microarray expression profiling.11 Using a combination of these approaches, the full set of genes necessary for monolignol biosynthesis has been identified in Arabidopsis}2'n We have applied comparative and functional genomics approaches to classify the likely full complement of shikimate pathway genes in poplar, the first woody perennial plant with a full genome sequence, and the third full angiosperm genome sequence to be completed. The characterization of the complete set of shikimate pathway genes in this alternative model plant was complemented by an analogous analysis of the shikimate pathway genes in the rice genome. The combination of

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genomic data from all three sequenced species in phylogenetic reconstructions, together with biochemical characterized of bona fide prechorismate and chorismate mutase enzymes in the skikimate pathway from Lycopersicon esculentum (tomato), allowed us to elucidate the likely full extent of gene families encoding most shikimate pathway enzymes in poplar and to make comparisons of gene family structure between these taxa. Using combined comparative and functional genomics data from poplar and Arabidopsis, we were able to build on previous inferences from Arabidopsis functional genomics studies11'12 to infer the likely identities of the orthologous poplar and Arabidopsis genes that encode enzymes for the remaining two uncharacterised steps in phenylalanine biosynthesis, PNT and ADT.

SKIMIMATE PATHWAY IN PLANTS The steps of the shikimate pathway and branch pathways leading to tyrosine and phenylalanine biosynthesis are illustrated in Figure 4.1. The first seven steps of the pathway, the prechorismate pathway, is common for tryptophan, tyrosine, and phenylalanine, and these steps are well supported, with genes of every family cloned and enzymes characterized in several plants.14 Chorismate represents a metabolic node with one branch catalyzed by anthranilate synthase leading to tryptophan biosynthesis (not shown in Fig. 4.1). Chorismate mutase (CM) is specific for the biosynthesis of tyrosine and phenylalanine and has been characterized in tomato and Arabidopsis}5'16 The last enzymatic steps in plants, subsequent to chorismate synthase, which is required for tyrosine and phenylalanine biosynthesis, are distinct from those characterised in most bacteria and fungi. As illustrated in Figure 4.1, plants employ a pathway leading from prephenate via arogenate to phenylalanine and tyrosine respectively. To generate the arogenate intermediate, a transamination reaction catalyzed by prephenate aminotransferase (PNT) is employed. PNT activity was detected in Nicotiana sylvestris (flowering tobacco),17 and a purified protein fraction with enzymatic activity was described in Sorghum bicolor and characterized to be highly specific for prephenate and L-Aspartate as the amino-donor substrate in Anchusa officinalis (common bugloss),19 although neither a cDNA nor a gene has been isolated. Arogenate dehydrogenase (AD), catalyzing the conversion of arogenate into tyrosine was identified and characterized in Arabidopsis?® Arogenate dehydratase (ADT) represents the final step in phenylalanine biosynthesis. The enzyme activity was described in Sorghum bicolor by Siehl and Conn,21 but again, cDNA clones and the corresponding arogenate dehydratase genes have still to be identified and characterized.

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Shikimate Pathway to Tyrosine and Phenylalanine OH O OH OH „.<>.

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Figure 4.1: Overview of the shikimate pathway leading to phenylalanine and tyrosine biosynthesis. Individual metabolic intermediates starting from erythrose-4-phosphate and phsophoenolpyruvate are shown. The central intermediate chorismate at the branch point between tryptophan and phenylalanine/tyrosine pathways is boxed. Enzymes catalyzing individual steps are numbered as follows: (1) 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, (2) 3-dehydroquinate synthase, (3) dehydroquinate dehydratase/shikimate dehydrogenase, (4) dehydroquinate dehydratase/shikimate dehydrogenase, (5) shikimate kinase, (6) 5-enolpyruvylshikimate-3-phosphate synthase, (7) chorismate synthase, (8) chorismate mutase, (9) prephenate aminotransferase, (10) arogenate dehydrogenase, (11) arogenate dehydratase, (12) prephanate dehydratase (13) aromatic aminotransferase. The pathway in grey catalyzed by (12) and (13) is found in yeast and other fungi, but not plants.

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THE POPLAR GENOME An international consortium led by the U.S. Department of Energy Joint Genome Institute (JGI) generated the whole-genome sequence for Populus trichocarpa (western black cottonwood), and this sequence is publicly available at http://genome.jgi-psf.org/Poptrl/Poptrl.home.html. This is the first complete genome sequence for a tree, and the third complete angiosperm plant genome available, after Arabidopsis and rice. Populus (poplar) is a highly polymorphic outcrossing undomesticated species, and analysis of the genome revealed a relatively recent (-60-65 million years ago) whole-genome duplication that is shared among modern taxa in Salicaceae (poplars and willows), while a second, older duplication (-100-120 mya) appears to be shared with the Arabidopsis lineage. The recent genome duplication was a major event in the evolutionary history of the Salicaceae lineage and has allowed the patterns of retention and loss of duplicated genes to be studied at the whole genome level (G. Tuskan et ah, unpublished). These patterns of loss and retention can provide information on gene function and specialization within gene families, since following a genome-wide duplication event, paralogous gene pairs may be lost by deletion or accumulation of mutations (non-functionalization) or diversify functionally by neofunctionalization (one gene achieves a new function), or subfunctionalization (the original function is split in time or space between the two copies). All of these cases may be expected upon close examination of gene family structure and expression in poplar, and in addition, recent (often tandem) gene duplications may have occurred. The poplar genome contains approximately 45,000 genes predicted by gene finding programs, and on average there are 1.33 putative poplar homologs for each Arabidopsis gene (G. Tuskan et ah, unpublished). Thus, comparisons of the structure and evolution of selected gene families, such as those encoding shikimate pathway enzymes, between poplar and Arabidopsis can help illuminate the functions and evolution of poplar and Arabidopsis gene family members, while comparisons with rice can identify family members and functions that are common to monocot and eudicot angiosperm lineages.

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Table 4.1: Annotated Arabidopsis, rice, and other shikimate pathway genes Gene annotation1 ArathDHSl ArathDHS2 ArathpDHS Lyces DHS1 Lyces DHS2 OrysaDHS3 Orysa DHS1 Orysa DHS2

Gene model2 At4g39980 At4g33510 Atlg22410 GI:410486 GL584778 Os07g42960 Os03g27230 Os08g37790

ArathDHQS LycesDHQS OrysapDHQS

At5g66120 GI: 18654278 Os09g36800

ArathDHQD LycesDHQD OrysaDHQDl OrysaDHQD2 OrysaDHQD3

At3gO635O GI:3169883 Os01g27750 Os01g27780 Osl2g34870

ArathSKl ArathSK2 ArathSK3 ArathSK4 LycesSK OrysaSKl OrysaSK3 OrysaSK2 OrysaSKL

AT2G21940 AT4G39540 AT3G26900 AT2G35500 GI: 114200 Os02g51410 Os04g54800 Os06gl2150 Osl0g42700

ArathEPSPSl ArathEPSPS2

At2g45300 Atlg48860

LycesEPSPS OrysaEPEPS

GI:66619 Os06g04280

1

Gene annotation1 ArathCS LycesCSl LycesCS2 OrysaCS

Gene model2 Atlg48850 GI:410482 GI:410484 Os03gl4990

ArathCMl LycesCM OrysaCMl OrysaCM2 OrysaCM3 OrysaCM4

At3g29200 GI:5732018 Os01g55870 Os02g08410 Os08g34290 Osl2g38900

ArathAGT2L ArathAGT21/PNT2 ArathpAGT2L OrysaPNT2 OrysaPNTl OrysaPNTL

At4g39660 At2g38400 At3g08860 Os05g39770 Os03g21960 Os03g07570

ArathASP2 ArathASP3 ArathASP4 OrysaPNT3 LupanAATPl

At5gl9550 At5g 11520 Atlg62800 Os01g55540 GI:388896

ArathADTl ArathADT2 ArathADT3 ArathADT4 ArathADT5 ArathADT6 Orysa ADT1 Orysa ADT2 Orysa ADT3 Orysa ADT4 Orysa ADT5

Atlgll790 At3g07630 At2g27820 At3g44720 At5g22630 AtlgO825O Os03gl7730 Os04g33390 Os07g49390 Os09g39230 Os09g39260

See legends to Figures 4.4 and 4.5 for abbreviations; Arath, Arabidopsis thaliana; Orysa, Oryza sativa; Lyces, Lycopersicum esculentum 2 Gene models available at TAIR (Arabidopsis),TIGR (rice), and GenBank (tomato)

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GLOBAL ANNOTATION OF POPLAR AND RICE SHIKIMATE PATHWAY GENES Identification and Analysis ofShikimate and Candidate Shikimate Genes We used the set of characterized genes encoding the first seven steps of the shikimate pathway as well as the annotated Arabidopsis homologs (Table 4.1) to identify all potential poplar orthologs with reciprocal BLAST searches of the poplar genome assembly (Joint Genomics Institute, Populus trichocarpa v. 1.1; http://genome.jgi-psf.org/Poptrl/Poptrl.home.html'). BLAST results corresponded to loci anchored to poplar linkage groups, or to genetically unanchored sequence scaffolds. The automatically annotated models for a given locus were evaluated, annotated manually, and are shown in Table 4.2. Corresponding rice homologs for all gene families (Table 4.1) were identified in the rice genome using the rice genome annotation at The Institute for Genome Research (TIGR; http://www.tigr.org/tdb/e2kl/osal/). The deduced protein sequences for the individual gene families were aligned (Dialign2)22 and manually optimized. To reconstruct phylogenetic trees, maximum likelihood analyses with 100 bootstrap replicates were carried out using PhyML v2.4.4.23 Ehlting et al. reported the identification of 6 prephenate aminotransferase (PNT) candidate genes in the Arabidopsis genome based on the presence of an aminotransferase domain (PF00155) and an expression pattern during primary stem development consistent with a function related to phenylpropanoid biosynthesis.1 Three putative arogenate dehydratase (ADT) candidate genes were identified in an analogous approach according to their expression profiles, similarity to prephenate dehydratase from yeast, and predicted chloroplastidic localization.12 Poplar and rice PNT and ADT candidate sequences were initially retrieved from the databases based on low stringency BLAST searches with all family members previously inferred as Arabidopsis PNT and ADT genes. Gene Family Topologies The gene families of the individual enzymatic steps in the shikimate pathway are far better defined and less complex than those found within the phenylpropanoid pathway, where large numbers of genes encoding enzymes related to true phenylpropanoid enzymes are detected.12'13 Two representative unrooted phylogenetic trees area shown in Figure 4.2, for genes encoding 3-dehydroquinate synthase (DHQS) and shikimate kinase (SK). The shikimate kinase gene family was an exceptional case, in which a distant outgroup of unknown function contained one poplar, one Arabidopsis, and two rice genes (ArathSK4, PoptrSKL, OrysaSKLl, and

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Table 4.2: Annotated poplar shikimate pathway genes Poplar annotation PoptrDHSl PoptrDHS2 PoptrDHS3 PoptrDHS4 PoptrDHS5p

Poplar Gene Model eugene3.00050687 estExt fgenesh4_pm.C LG II0453 gwl.V.2754.1 eugene3.00290110 gwl. VII. 1743.1

PoptrDHQS

estExt_fgenesh4_pm.C_570024

PoptrDHQDl PoptrDHQD2 PoptrDHQD3 PoptrDHQD4 PoptrDHQD5

eugene3.00100327 gwl.XIII.1215.1 estExt Genewisel vl.C 700420 estExt_fgenesh4_pg.C_LG_XIV0743 eugene3.00130283

PoptrSKl PoptrSK2 PoptrSKL PoptrSK3 PoptrSK4p

estExt_fgenesh4_pg.C_LG_VII0602 estExt_fgenesh4_pg.C_880051 eugene3.0003 0693 gwl.V.3099.1 fgenesh4_pg.C_LG_II000575

PoptrEPSPSl PoptrEPSPS2p

eugene3.00021350 gwl.XIV.746.1

PoptrCSl PoptrCS2

estExt_Genewise 1 _v 1 .C JLGX2626 grail3.0049006403

PoptrCMl PoptrCM2 PoptrCM4p PoptrCM3

fgenesh4_pg.C_LG_XVII000439 eugene3.00101527 gwl.VIII.2241.1 fgenesh4_pg.C_LG_XVIII000491

PoptrADTl PoptrADT2 PoptrADT3 PoptrADT4 PoptrADT5p

eugene3.00660027 estExt_Genewisel_vl .C_LG_IX0680 eugene3.00110045 estExt_fgenesh4_pg.C_LG_IV0012 eugene3.00081851

PoptrpPNTl estExt_fgenesh4_pm.C_LG_XVI0476 PoptrpPNT2 gwl.VI.969.1 PoptrPNTLl estExt fgenesh4_pm.C LG VII0215 PoptrPNTL2p gwl.V.3014.1 PoptrpPNT3 estExt_fgenesh4_pg.C_LG_VI 1672 PoptrPNTL3 estExt fgenesh4_pg.C LG VI1671 PoptrPNT4 estExt fgenesh4 pm.C LG XVIII0241 See legends to Figures 4.4 and 4.5 for abbreviations 2 Gene models available at http://genome.jgi-psf.org/Poptrl/Poptrl .home.html

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OrysaSKLl). The DHQS tree (Fig. 4.2) is representative of other shikimate pathway families, which consist of single subgroups, in this case with single genes in each taxon. For both the SK and DHQS trees, tomato representatives with functional data were included, which help to verify the functionality of the annotated genes. The topology of most shikimate pathway gene families stands in sharp contrast to phenylpropanoid pathway families where tree topologies are complex and outgroups with sequence, but not functional, similarity are common. As discussed below, the average number of clones identified for genes of the shikimate pathway, excluding specific steps for tryptophan and tyrosine biosynthesis, is lower compared to the phenylpropanoid pathway. This could probably be explained by the higher degree of functional specialization that phenylpropanoid related genes exhibit compared to the shikimate pathway genes whose activity is more generally required. The overall similarity within the families may also reflect the fact that, in addition to feeding carbon into the biosynthesis of specialized plant natural products, the shikimate pathway is central to primary metabolism, with plants, bacteria, and fungi sharing most steps. Thus, there appears to have been little evolutionary diversification of enzymes encoding plant-specific functions in the shikimate pathway, as in the more complex groups of enzymes related to phenylpropanoid metabolism. Expression of Annotated Genes Functional annotation of gene family members is aided by data on expression patterns of individual gene family members. For example, the annotation of the set of Arabidopsis phenylpropanoid genes involved in monolignol biosynthesis depended on analysis of EST abundance from different sources13 and microarray expression profiling along a developmental gradient of lignification in the Arabidopsis inflorescence stem.12 After annotating the complete sets of poplar phenylpropanoid genes (B. Hamberger, C. Souza, and M. Ellis, unpublished; G. Tuskan et ah, unpublished), and shikimate pathway genes described here, we determined the relative numbers of EST sequences specific to these sets in each of numerous libraries constructed from various specific poplar tissues and organs tissues, using data on poplar EST abundance from Sterky et al. A summary of this analysis is shown in Figure 4.3. This analysis indicates that the demand for high gene expression of both the shikimate and phenylpropanoid genes correlates similarly in both pathways with the tissues related to lignification. Shikimate and phenylpropanoid gene EST clone abundance dominates in the cambial zone, tension wood, and wood cell death related tissue. This reflects the primary metabolic function of both pathways in monolignol precursor biosynthesis, suggesting that correlation of expression of genes in both pathways with lignin deposition is a useful aid to functional annotation in poplar, as it is in Arabidopsis. '

94

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100

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Figure 4.2: Representative un-rooted phylogenetic reconstruction of genes encoding dehydroquinate dehydratase (DHQS) and shikimate kinase (SK). Predicted proteins from all annotated Arabidopsis (Arath), poplar (Poptr), rice (Oryza), and representative tomato (Lyces) genes in each family were aligned and phylogenetic reconstructions performed as described in the text. Numbers refer to bootstrap values, 100 repetitions.

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PRECHORISMATE PATHWAY DHS Genes Regulation of the abundance of 3-deoxy-D-arabino-heptulosonate 7phosphate synthase (DHS), which catalyzes the initial step of the shikimate pathway, is reported to be regulated at the transcriptional level,24 and induction of gene expression was similar to PAL mRNA induction in response to UV exposure in parsley.25 DHS is encoded by three gene family members in Arabidopsis. ArathDHS3, which has not been previously characterized, is expressed at increasingly higher levels over the course of fiber differentiation, while ArathDHS2 is expressed at higher levels only in mature stem sections. Our analysis (Fig. 4.4) shows that poplar has four DHS genes, making it the first organism with more than three isoforms; only Morinda citrifolia (noni) and Arabidopsis actually contain three DAHP synthase isoenzymes. The reason for the higher number of DHS genes in poplar is unclear. Both of the poplar genes with closest homology to ArathDHS3, PoptrDHSl and PoptrDHS2, are almost exclusively expressed in lignifying tissues (9/10 clones). The tomato gene LycesDHS2 in the same subclass responds to elicitor treatment and is transcriptionally induced after pathogen treatment, whereas the second tomato LycesDHSl did not show any response. This phenomenon likely reflects subfunctionalization on expression level after gene duplication, and is in tomato found again in the chorismate synthase family. One poplar gene, PoptrDHS4 with low expression in wood forming tissues (1 clone) falls into the same DHS subgroup as the Arabidopsis candidate ArathDHS2, a group of enzymes with seemingly a more specialized expression patterns and perhaps functions. DHQS Genes In all species analysed so far, including poplar, 3-dehydroquinate synthase (DHQS) is encoded by a single copy gene (Fig. 4.3 and 4.4). Expression of the Arabidopsis ArathDHQS gene is only weakly induced in concert with lignification during primary stem development.12 In cultured tomato cells, the abundance of LycesDHQS transcripts increased 9-fold within 4 to 5 h of elicitor treatment,28 apparently to meet the higher demand of metabolic activity. In poplar, only one out of five DHQS clones was found in wood related tissues (tension wood). Thus, similar to Arabidopsis and in contrast to other shikimate pathway genes, genes encoding this enzyme do not appear to be upregulated in concert with demand for phenylpropanoids.

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DHQD/SDH Genes The single gene encoding the dual function enzyme dehydroquinate dehydratase/shikimate dehydrogenase in Arabidopsis, ArathDHQD/SDH, was not significantly up-regulated during the course developmental lignification with genes encoding enzymes of monolignol biosynthesis,12 and could, therefore, apparently already meet the metabolic demand with the basal expression level. In tomato the elicitor inducible expression of the LycesDHQS gene resembles that of DHS and DHQS.29 In poplar, previous studies showed that SDH and PAL enzyme activities are co-ordinately increased in old and mid-aged leaves in response to ozone exposure,30 suggesting that the gene could be responsive to metabolic demand for phenylalanine. With five DHQD/SDH genes (and EST expression support for four of them) the number of gene family members in poplar is significantly higher compared to Arabidopsis (Fig. 4.4). Based on EST numbers, PoptrDHQDl is the most highly expressed gene, and is widely expressed in multiple tissues not all related to lignin biosynthesis, similar to ArathDHQD. Phylogenetic analysis showed that three poplar genes, PoptrDHQD3-5, occur in a branch outside of the main DHQD/SD group, which is devoid of Arabidopsis or rice homologs (not shown). This clade, however, is not specific for poplar, as it shares a tobacco member. Interestingly, the small number of EST clones found here was exclusively from the cambial zone and the active cambium, tissues where no PoptrDHQDl EST clones were found, suggesting non-overlapping expression patterns and potential subfunctionalization of these clades of poplar DHQD/SDH genes. Both 3-dehydroquinate and shikimate may play a role as quinate precursors. Quinate, a carbon reservoir and UV protectant, is essential for the biosynthesis of chlorogenic acid, an abundant natural product in poplar, tobacco, and other plants which is linked to pathogen defense and the biosynthesis of intermediates in the phenylpropanoid pathway. The high number of DHQD genes in poplar could, therefore, reflect subfunctionalization of duplicated gene family members recruited to meet increased needs for specific natural products in this lineage.

COMPARATIVE COMPARATIVE GENOMICS GENOMICS OF OF THE THE SHIKIMATE SHIKIMATE PATHWAY PATHWAY

97 97 co

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Figure 4.3: Relative distribution of ESTs specific to annotated poplar shikimate and phenylpropanoid genes. The percentages of the total shikimate and total phenylpropanoid ESTs in each tissue or organ, relative to the total number of ESTs is given, based on the data of Sterky et al. (2004).10

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Figure 4.4: Gene annotation in the pre-chorismate branch of the shikimate pathway. The pathway with individual steps is given at the left. Total numbers of annotated genes encoding each enzyme family in poplar (P. trichocarpa), Arabidopsis (A. thaliana), rice (O. sativa), and tomato (L. esculentum) is given. DHS, 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase; DHQS, 3-dehydroquinate synthase; DHQD/SDH, 3-dehydroquinate dehydratase/shikimate dehydrogenase; SK, shikimate kinase; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase; CS, chorismate synthase.

00

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COMPARATIVE GENOMICS GENOMICS OF THE SHIKIMATE SHIKIMATE PATHWAY COMPARATIVE PATHWAY

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SK Genes The overall topology of the phylogenetic shikimate kinase (SK) tree consists of three distinct clades, with the one closer to the main group devoid of rice members (Fig. 4.2). Among the four Arabidopsis genes with homology to a functionally characterized shikimate kinase from tomato, x ArathSKl and ArathSK4 (sharing low overall similarity) displayed an expression pattern similar to that of monolignol biosynthesis genes, while ArathSK2 was only expressed at higher levels in the most mature stem sections.12 In contrast, ArathSK3 was transcriptionally downregulated during primary stem development. In cultured tomato cells treated with fungal elicitors, transcript accumulation of the tomato LycesSK gene was strongly and rapidly activated, more so than mRNAs for genes encoding the enzymes preceding or following this step.26 This is consistent with transcriptional regulation of this step according to metabolic demand for phenylalanine required for natural product biosynthesis seen in heat-treated Arabidopsis cell cultures. Our annotation showed that poplar has five SK genes, one likely a pseudogene (PoptrSK4p) and one, PoptrSKL falling into the same distant branch as the ArathSK4 (Fig. 4.2), PoptrSKL, and PoptrSK3 are supported as expressed genes by ESTs, but no expression data based EST abundance is available for either. PoptrSKl and PoptrSK2 on the other hand are closely related to ArathSKl and ArathSK3, respectively. The number of EST clones for both is low, but one clone from each gene was found in libraries from wood related tissues. The expression of SK genes in poplar, therefore, seems to be regulated in a manner distinct from Arabidopsis and tomato SK genes (highly up-regulated in concert with activation of phenylpropanoid metabolism), possibly as a result of functional specialization of gene family members in poplar. EPSPS Genes The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the target of the herbicide Glyphosate, which competes with the substrate of the native enzyme, inhibiting its activity. Both Arabidopsis and poplar genomes contain duplicated EPSPS genes (Fig. 4.4), apparently the result of independent duplication events in their lineages, with both copies being retained in Arabidopsis. Close inspection of the gene model for PoptrEPSPS2 suggests a frame shift mutation in the first exon of this gene, indicating that it is not under selection for function, and is likely a pseudogene. An unusually high degree of divergence from its paralog PoptrEPSPSl might indicate a higher mutation rate for this gene, supporting this interpretation. Similarly, rice and tomato appear to have only single EPSPS genes. Expression data for the Arabidopsis EPSPS genes during stem development is not available, since elements specific to these genes were not present on the array employed by Ehlting et al. Similar to tomato EPSPS,32 PoptrEPSPSl is widely

100 100

HAMBERGER, et ah al. HAMBERGER,

expressed, with clones found in libraries derived from tension wood, bark, the apical shoot, petioles, flower, and the shoot meristem. CS Genes Chorismate synthase (CS) catalyzes the trans-1,4 elimination of 5enolpyruvylshikimate 3-phosphate to generate chorismate, which is the last common precursor in the biosynthesis of aromatic compounds in bacteria, fungi, and plants. In Arabidopsis, only a weak increase in transcripts of the single ArathCS gene could be detected during stem development.12 It appears that this single copy gene obviously meets all requirements for the biosynthesis of secondary metabolites and aromatic amino acids. In both poplar and tomato, two genes coding for CS were found that most likely have evolved independent functions after duplication. Functional diversification of the poplar genes is supported by the finding that PoptrCSl is predominantly expressed in the cambial zone and tension wood, whereas a single clone for the PoptrCS2 was found in the active cambium. A similar subfunctionalization was reported in tomato, where only LycesCSl responded to elicitor, whereas transcript levels of LycesCS2 remained unchanged, analogous to the LycesDHS21

POST CHORISMATE PATHWAY CM Genes Chorismate mutase (CM) catalyzes the first step of the shikimate branch pathway specific to phenylalanine and tyrosine biosynthesis (Fig. 4.1 and 4.5). Among the three CM genes in Arabidopsis}5'16 only ArathCMl was found to be highly expressed in coordination with lignification, while the expression levels of both ArathCM2 and ArathCM3 were unaltered,12 a finding in keeping with the observation that only ArathCMl is responsive to wounding and pathogen attack.16 In tomato, only one gene, LycesCM is reported, for which there is no evidence for transcriptional control.33 With the exception of ArathCM2, CM genes in Arabidopsis16 and tomato33 are localized to the plastid. We found three CM gene genes in the poplar genome (Fig. 4.5), whose expression is each supported by the presence of ESTs. However, no information on PoptrCM EST abundance in different tissues could be found in the data of Sterky et al. ° PoptrCM2 is predicted to be plastid localized, and PoptrCMS likely cytoplasmatic. This finding is consistent with the existence two distinct clades of chorimate mutases. Phylogenetic reconstructions (not shown) indicate that ArathCM2, LycesCM, and PoptrCMS, all plastidic forms, group together, while a second branch is formed by the plastidic chorismate mutases, for which there is evidence of allosteric regulation.

COMPARATIVE GENOMICS GENOMICS OF THE SHIKIMATE SHIKIMATE PATHWAY COMPARATIVE PATHWAY

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Candidates for Genes Encoding Prephenate Aminotransferase (PNT) Prephenate aminotransferase (PNT) catalyzes the conversion of prephenate to arogenate, the last step of the shikimate pathway in common to tyrosine and phenylalanine biosynthesis (Fig. 4.1 and 4.5).17'19 However, gene(s) encoding PNT have not been identified. Six potential PNT candidate genes were identified in Arabidopsis based on the presence of an aminotransferase domain and up-regulation in concert with lignin biosynthesis and deposition (Fig. 4.6).12 Our and others analyses show that these candidates represent distinct phylogenetic families of genes encoding aminotransferase enzymes in Arabidopsis. We used the Arabidopsis genes in reciprocal BLAST searches of the poplar genome to identify potential poplar orthologs, performed parallel analyses of the rice genome, and reconstructed phylogenetic trees for all PNT candidate families. We then examined the expression data available for the poplar homologs to determine which, if any of them, showed expression consistent with a role in phenylalanine biosynthesis (i.e., higher expression in wood forming tissues where a heavy metabolic demand for phenylalanine entry into lignin biosynthesis would be expected). PNT1 (Atlg34060) Alliinase Family Two poplar homologues were identified. Although the Arabidopsis gene showed low similarity to the aminotransferase domain pfamOO155, this motif was absent in the poplar proteins. Both poplar candidates were unrepresented in the database of EST abundance,10 and EST support was only found for the fgenesh4_pg.C_LG_II000599 model, likely reflecting a low or specialized expression, both of which are inconsistent with a putative PNT function in phenylalanine biosynthesis. Nevertheless, it is interesting to note that the closest characterized homologue within this family is an alliinase gene with cysteine sulfoxide lyase activity found in onion and garlic. PNT2 (At2g38400) Putative Alanine-Glyoxylate Pyruvate Aminotransferase (AGT) Family

Aminotransferase,/Beta-Alanine-

The members of this family show high similarity to the aminotransferase motif pfam00202, a characteristic of the class-Ill pyridoxal-phosphate-dependent aminotransferase family. The Arabidopsis PNT2 candidate (ArathAGT3, At2g38400) is strongly activated in concert with lignin biosynthesis in developing stems (Fig. 4.6). n A phylogenetic reconstruction of this family is shown in Figure 4.7, and includes two additional Arabidopsis genes (At4g39660 and At2g38400), which are not up-regulated during lignification and fiber development. These genes, together

102

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AD

PNT candidate

CM

Enzyme Family

2

3

2

3

2

3

1

3

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3

1

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1

1

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1

Gene number P. trichocarpa A. thaliana 0. sativa L. esculentum

HAMBERGER, et al.

prephenate

[PNT]

arogenate

[ADT] COOH

phenylalanine

Figure 4.5: Gene annotation in the post-chorismate branch of the shikimate pathway. The pathway with individual steps is given at the left. Total numbers of annotated genes encoding each enzyme family in poplar (P. trichocarpa), Arabidopsis {A. thaliana), rice (O. sativa), and tomato (L. esculentum) is given. CM, chorismate mutase; PNT, prephanate aminotransferase, ADT, arogentate dehydratase, AD, arogenate dehydrogenase. Activities of the enzymes in brackets are known, but the genes have not been identified.

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with the PNT2 candidate were described by Liepmann and Olsen34 as the closest homologs to the rat {Rattus norvegicus) glyoxylate aminotransferase 2 (AGT2) gene, and are annotated as AGT2-like (AGT2L). However, testing for AGT activity failed to support this enzymatic function.34 Four members of this gene family were identified in poplar, with EST support and abundance data for two. As shown in Figure 4.6, of the five ESTS representing PoptrPNTLl (estExt_fgenesh4_pm.C_LG_VII0215), three are expressed in wood-forming tissues (active cambium), while two are found on other libraries (and cold stressed leaves). ESTs specific to PoptrPNTl (estExt_fgenesh4_pm.C_LG_XVI0476) are found primarily in wood forming tissues (the cambial zone, wood cell death, dormant cambium), similarly to known poplar phenylpropanoid genes (Fig. 4.6). Figure 4.6 also shows that this pattern of expression {i.e., the percentage of ESTs found in libraries made from wood-related tissues) is similar to that of poplar genes deduced to be involved in lignin biosynthesis (B. Hamberger and C. Douglas, unpublished). No expression support could be found for the other two candidate genes, PoptrPNT2, PoptrPNTL2p, with the latter apparently being a pseudogene coding for a truncated protein. With the exception of the rice PNT-like OrysaPNTL and the poplar PoptrPNTL2p, all other predicted proteins share a conserved C-terminal PTS1 peroxisomal targeting sequence, hence are potentially peroxisomal aminotransferases. A reconstruction of the PNT2/AGT2L phylogenetic tree is shown in Figure 4.7. The expression differences between PNT2 gene family members are as well reflected in tree topology. PoptrPNTLl falls into one clade with ArathAGT2L, while PoptrPNTl PoptrPNTl groups with the Arabidopsis PNT candidate ArathPNT2 (At2g38400), both expressed in coordination with lignification. Three rice representatives of the PNT2/AGT2L gene family were found, two of which group with the ArathPNT2/PoptrPNTl clade (Fig. 4.7), and one falling into the PNTL group, suggesting that both both clades are conserved in angiosperms. Taken together with data from the literature,34 it seems unlikely that the PNT2/AGT2L family plays a role in the photorespiratory glyoxylate metabolism. Phylogenetic and expression data from both Arabidopsis and poplar support a role for ArathPNT2 and PoptrPNTl to encode peroxisomal aminotransferases with non- photorespiratory enzymatic roles and potential prephenate aminotransferases, one of the last missing steps in phenylalanine biosynthesis and a link between the shikimate and the phenylpropanoid pathway. PNT3 (At2g20610) Putative Aminotransferase Family Four poplar homologs were identified within the PNT3 family, all with expression support and three with EST abundance data. All poplar genes fall into a poplar-specific clade,and it seems likely that the three expressed members are

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ArathPNT candidates

AtSg22630 At1g0t250 ADT3

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ArathADT candidates

At1g34060 PNT1 At2g38400 PNT2/AGTL At2g2C610 PNT3 At1g628O0 PNT4/ASP4 At2g24850 PNT5 At4g11280 PMT6 AMg33380 At3g61510 A13g48730 At2g3O37O A13g04520 At5gQ4620

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Figure 4.6: Expression of prephenate aminotransferase (PNT) and arogenate dehydratase (ADT) candidates in Arabidopsis and poplar. Arabidopsis expression was measured by microarray analysis, as reported by Ehlting et al.n in sections taken from the top to the bottom of Arabidopsis inflorescence stems, a gradient of increasing lignin biosynthesis. Up and down regulated genes are indicated, with the relative degree of up or down regulation indicated by lighter shades on the grey scale. Relative expression of poplar genes was deduced from EST abundance reported by Sterky et al.]0 in libraries constructed from multiple tissues and organs, including woody tissues with a high commitment to lignin biosynthesis.

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the result of a recent duplication as they are closely linked in the poplar genome. The expression of the poplar genes varies widely, is mostly non-overlapping, and no indication of high expression in wood related tissues could be found. Therefore, despite an expression pattern of the Arabidopsis genes that shows some correlation with lignin biosynthesis (Fig. 4.6), a function of these genes in phenylalaninphenylalanine/lignin biosynthesis related aminotransferase seems unlikely. This finding is consistent with report that the Arabidopsis gene encodes an CS-lyase involved in glucosinolate that plays a role in auxin homeostasis.39 PNT4 (Atlg62800) Putative Aspartate Aminotransferase Family (ASP) Family The Arabidopsis PNT candidate PNT4 (Atlg62800) has been previously annotated as ASP4, a member of the Arabidopsis aspartate aminotransferase (ASP) gene family.35 While its expression pattern over the course of stem development is only partially consistent with a role in phenylalanine biosynthesis, (Fig. 4.6),12 we used information from related poplar genes as a further test of its potential to encode a PNT. As shown in Figure 4.8, three poplar and one rice PNT4 homologue were identified, together with three Arabidopsis ASP genes that fall into the PNT4 cluster. Prediction of the localization for PoptPNT3PoptPNT3 and the closely related PoptrPNTL4 shows weak support for cytoplasmatic localization. In spite of the strikingly high number of EST clones for two of the three poplar PNT4 candidates (PoptrPNT3 and PoptrPNT4), there was no support for restricted expression in lignifying tissues, and instead partially overlapping broad expression profiles in almost all tissues (including but not biased towards wood forming tissues) were observed. Thus, the Arabidopsis PNT4/ASP4 gene is the only one in the family with evidence for preferred expression in concert with demand for phenylalanine during lignification (Fig. 4.8). Based on these expression profiles and high similarity to characterized Arabidopsis aspartate aminotransferases (ArathASP2 and ArathASPS) whose expression does not vary over the course of stem development (Fig. 4.8), these three poplar isoforms are debatable candidates for PNTs. By implication, rather than encoding a PNT, PNT4/ASP4 may function to provide aspartate for protein synthesis, while ASP2 serves to synthesize aspartate used as a nitrogen transport amino acid in the phloem.36 Other PNT Candidates Poplar homologs for the two remaining PNT candidate families (PNT5, At2g24850; PNT6, At4gll280), both exclusively expressed in the Arabidopsis middle stem sections (Fig. 4.6)12 were identified. However, these were not further considered since none of the poplar homologs showed expression related to lignifying tissues, as determined by EST abundance in the data of Sterky et al.10

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'

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ArathAGT2UPNT2 At2g38400

PoptrPNTI PoptrPNT2

Orysa PNT2

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Figure 4.7: Un-rooted phylogenetic reconstruction of genes encoding the enzymes related to the Arabidopsis PNT candidate PNT2/AGL2L (At2g38400). Predicted proteins from all annotated Arabidopsis (Arath), poplar (Poptr), and rice (Orysa), in the family were aligned and phylogenetic reconstructions performed as described in the text. Numbers refer to bootstrap values, 100 repetitions. Arabidopsis genes in bold italics are upregulated during the courses of fiber development and lignification; poplar genes in bold italics have EST support.

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Summary of Data on PNT Candidate Genes Identification of poplar and rice homologs of the Arabidopsis PNT candidate candidate genes, phylogenetic analyses, and expression patterns were used to support potential prephenate aminotransferase candidates in both poplar and Arabidopsis. All gene families examined have members from all three species with sequenced genomes, suggesting that each family encodes enzymes of conserved function in plants. Among all six gene families examined, overlap in expression patterns of Arabidopsis and poplar PNT homologs was found only for one member of the PNT2/AGTL family, strengthening the assumption that the PNT2 family could play a role in prephenate metabolism related to the shikimate pathway. However, the apparent peroxisomal localization of the enzymes encoded by these poplar and Arabidopsis genes will need to be taken into account when rationalizing their potential roles in phenylalanine biosynthesis. Candidates for Genes Encoding Arogenate Dehydratase (ADT) Arogenate dehydratase (ADT) activity, catalyzing the last step of the shikimate pathway and specific only for phenylalanine synthesis, was detected in N. sylvestris? However, no cDNA clone has been identified yet, and no gene coding for this enzyme has been found. Six ADT candidate genes were identified in the Arabidopsis genome by similarity to bacterial and fungal prephenate dehydratase, the biochemically closest characterized reaction, and were suggested to be the plant analogs, arogenate dehydratase.38 ArathADT5, ArathADT6, and possibly ArathADT3 displayed expression patterns consistent with a role in phenylalanine biosynthesis.12 Five poplar and five rice ADT homologs were identified and integrated into the phylogenetic tree (Fig. 4.9). At least three distinct clades dominate the ADT family tree, one lacking rice members. Recent species-specific duplications are evident for all three plants, e.g., the PoptrADTl and PopADT2 pair, closest to the potentially phenylpropanoid related ArathADT6 and ArathADT3 pair. Strikingly, the poplar PoptrADTl IPoptrADT2 pair shows the highest number of EST clones within the family, and both genes are expressed largely in non-overlapping tissues with the exception of tension wood and wood cell death, where both are highly expressed. It is likely that the differences in expression are due to subfunctionaliztion in most tissues, whereas a higher expression in lignifying tissue requires the coordinated expression of both genes to fulfill the high metabolic demand. Thus, PoptrADTl and PopADT2 are candidates for encoding actual ADT enzymes required for the last step of phenylalanine biosynthesis, along with the Arabidopsis ArathADTS and ADT6 and rice OrysaADT2, OrysaADT4, and OrysaADT5 homologues.

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ArathASP4 At1g62800

0.1

ArathASP3 HAMBERGER, et al. At5g11520

Orysa ASP

PoptrpASPI ArathASPI At2g30970

PoptrASP3

ArathASP2 At5g 19550

PoptrASP2

Figure 4.8: Un-rooted phylogenetic reconstruction of genes encoding the enzymes related to the Arabidopsis PNT candidate PNT4/ASP4 (Atlg62800). Predicted proteins from all annotated Arabidopsis (Arath), poplar (Poptr), and rice (Orysa), in the family were aligned and phylogenetic reconstructions performed as described in the text. Numbers refer to bootstrap values, 100 repetitions. Arabidopsis genes in bold italics are upregulated during the courses of fiber development and lignification; poplar genes in bold italics have EST support.

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Arath ADT4

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ArathADT3 PoptrADT2 PoptrADTI

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COMPARATIVE GENOMICS OF THE SHIKIMATE PATHWAY

Arath ADT1

0.1 OrysaADT4 OrysaADT5

Arath ADT2

Orysa ADT1

Orysa ADT3

Figure 4.9: Un-rooted phylogenetic reconstruction of genes encoding the enzymes related to the Arabidopsis ADT candidates. Predicted proteins from all annotated Arabidopsis (Arath), poplar (Poptr), and rice (Orysa), in the family were aligned and phylogenetic reconstructions performed as described in the text. Numbers refer to bootstrap values, 100 repetitions. Arabidopsis genes in bold italics are upregulated during the courses of fiber development and lignification; poplar genes in bold italics have EST support.

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HAMBERGER, et ah al. HAMBERGER,

The other poplar ADTs, PoptrADT3, and PoptrADT4 are most highly expressed together in the apical shoot. Given their position within the family it is likely that both, collectively with PoptrADTl provide phenylalanine for protein biosynthesis in this metabolically highly active tissue rather then lignin biosynthesis. SUMMARY AND FUTURE DIRECTIONS We reconstructed the whole shikimate biosynthetic pathway, the central pathway of plant metabolism controlling metabolic flux from carbohydrate metabolism into the phenylpropanoid pathway, on the genomic level. Given that we focused on fully sequenced genomes, all families of the three species rice, Arabidopsis, and poplar should be represented in their full extent. We also included expression data for Arabidopsis and poplar that support functional classifications, providing an unprecedented view of the structure and expression of the gene families that encode shikimate pathway enzymes. This comparative data also allowed us to build on previous observations to highlight promising candidate genes that encode yet unknown enzymes for missing links in shikimate pathway required for phenylalanine biosynthesis (PNT and ADT candidates). In the future, it will be necessary to functionally test PNT and ADT candidate genes for their hypothesized biochemical functions, and to characterize the enzymes. This may be achieved by heterologous expression of the candidate recombinant enzymes in E. coli or yeast expression systems. Yeast is a particularly attractive system to study the enzymes specific to phenylalanine biosynthesis, since yeast mutants impaired in this ability are available. Thus, it may be possible to reconstruct the late steps of phenylalanine biosynthesis in yeast, using candidate Arabidopsis or poplar genes, resulting in the rescue of phenylalanine auxotrophy in impaired strains. Once identified, these novel plant enzymes required for phenylalanine biosynthesis could be of value in crop improvement, for example as targets for new herbicide development analogous to glyphosate, which targets the shikimate enzyme EPSPS synthase. ACKNOWLEDGEMENTS We thank our colleagues Margaret Ellis, Brian Ellis, and Clarice Souza (UBC) for collaboration on annotation of poplar and rice gene sets, and the U.S. Department of Energy Office of Energy Biosciences and Joint Genomics Institute for sponsoring a poplar genome annotation jamboree at which this work was started. This work was partially supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to CJD.

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HAMBERGER, et ah al. HAMBERGER, RITLAND, K., ELLIS, B.E., BOHLMANN, J., DOUGLAS, C.J., Global transcript profiling of primary stems from Arabidopsis thaliana identifies candidate genes for missing links in lignin biosynthesis and transcriptional regulators of fiber differentiation, Plant J, 2005, 42, 618-40. RAES, J., ROHDE, A., CHRISTENSEN, J.H., VAN DE PEER, Y. BOERJAN, W., Genome-wide characterization of the lignification toolbox in Arabidopsis, Plant Physiol, 2003,133, 1051-71. SCHMID, J., AMRHEIN, N., Molecular organization of the shikimate pathway in higher plants, Phytochemistry, 1995, 39, 737-749. EBERHARD, J., EHRLER, T.T., EPPLE, P., FELIX, G., RAESECKE, H.R., AMRHEIN, N., SCHMID, J., Cytosolic and plastidic chorismate mutase isozymes from Arabidopsis thaliana: Molecular characterization and enzymatic properties, Plant J., 1996,10, 815-21. MOBLEY, E.M., KUNKEL, B.N., KEITH, B., Identification, characterization and comparative analysis of a novel chorismate mutase gene in Arabidopsis thaliana, Gene, 1999,240, 115-23. BONNER, C.A., JENSEN, R.A., Novel features of prephenate aminotransferase from cell cultures of nicotiana silvestris, Arch. Biochem. Biophys., 1985, 238, 23746. SIEHL, D.L., CONNELLY, J.A. and CONN, E.E., Tyrosine biosynthesis in sorghum bicolor: Characteristics of prephenate aminotransferase, Z Naturforsch [C], 1986, 41, 79-86. DE-EKNAMKUL, W., ELLIS, B.E., Purification and characterization of prephenate aminotransferase from anchusa officinalis cell cultures, Arch. Biochem. Biophys., 1988,267,87-94. RIPPERT, P., MATRTNGE, M., Molecular and biochemical characterization of an Arabidopsis thaliana arogenate dehydrogenase with two highly similar and active protein domains, Plant Mol. Biol, 2002, 48, 361-8. SIEHL, D.L., CONN, E.E., Kinetic and regulatory properties of arogenate dehydratase in seedlings of sorghum bicolor (1.) moench, Arch. Biochem. Biophys., 1988,260,822-9. MORGENSTERN, B., Dialign 2: Improvement of the segment-to-segment approach to multiple sequence alignment, Bioinformatics, 1999,15, 211-8. GUINDON, S., GASCUEL, O., A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood, Syst. Biol, 2003, 52, 696-704. DYER, W.E., WEAVER, L.M., ZHAO, J.M., KUHN, D.N., WELLER, S.C., HERRMANN, K.M., A cdna encoding 3-deoxy-d-arabino-heptulosonate 7phosphate synthase from solanum tuberosum 1, J. Biol. Chem., 1990, 265, 1608-14. LOGEMANN, E., TAVERNARO, A., SCHULZ, W., SOMSSICH, I.E., HAHLBROCK, K., Uv light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley, Proc. Natl. Acad. Sci., C/5^,2000, 97, 1903-7. GORLACH, J., RAESECKE, H.R., RENTSCH, D., REGENASS, M., ROY, P., ZALA, M., KEEL, C, BOLLER, T., AMRHEIN, N., SCHMID, J., Temporally distinct accumulation of transcripts encoding enzymes of the prechorismate pathway

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in elicitor-treated, cultured tomato cells, Proc. Natl. Acad. Sci. USA, 1995, 92, 3166-70. GORLACH, J., RAESECKE, H.R., ABEL, G., WEHRLI, R., AMRHEIN, N., SCHMID, J., Organ-specific differences in the ratio of alternatively spliced chorismate synthase (Iecs2) transcripts in tomato, Plant J., 1995, 8, 451-6. BISCHOFF, M., ROSLER, J., RAESECKE, H.R., GORLACH, J., AMRHEIN, N., SCHMID, J., Cloning of a cdna encoding a 3-dehydroquinate synthase from a higher plant, and analysis of the organ-specific and elicitor-induced expression of the corresponding gene, Plant Mol. Biol, 1996, 31, 69-76. BISCHOFF, M., SCHALLER, A., BIERI, F., KESSLER, F., AMRHEIN, N., SCHMID, J., Molecular characterization of tomato 3-dehydroquinate dehydrataseshikimate:Nadp oxidoreductase, Plant Physiol, 2001,125, 1891-900. CABANE, M., PIREAUX, J.C., LEGER, E., WEBER, E., DIZENGREMEL, P., POLLET, B., LAPIERRE, C, Condensed lignins are synthesized in poplar leaves exposed to ozone, Plant Physiol, 2004,134, 586-94. SCHMID, J., SCHALLER, A., LEIBINGER, U., BOLL, W., AMRHEIN, N., The in-vitro synthesized tomato shikimate kinase precursor is enzymatically active and is imported and processed to the mature enzyme by chloroplasts, Plant J., 1992, 2, 375-83. GASSER, C.S., WINTER, J.A., HIRONAKA, CM., SHAH, D.M., Structure, expression, and evolution of the 5-enolpyruvylshikimate-3 -phosphate synthase genes of petunia and tomato, J. Biol. Chem., 1988, 263, 4280-7. EBERHARD, J., BISCHOFF, M., RAESECKE, H.R., AMRHEIN, N., SCHMID, J., Isolation of a cDNA from tomato coding for an unregulated, cytosolic chorismate mutase, Plant Mol. Biol, 1996, 31, 917-22. LIEPMAN, A.H., OLSEN, L.J., Alanine aminotransferase homologs catalyze the glutamate:Glyoxylate aminotransferase reaction in peroxisomes of Arabidopsis, Plant Physiol, 2003,131, 215-27. SCHULTZ, C.J., CORUZZI, G.M., The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments, Plant J., 1995, 7, 61-75. SCHULTZ, C.J., HSU, M., MIESAK, B., CORUZZI, G.M., Arabidopsis mutants define an in vivo role for isoenzymes of aspartate aminotransferase in plant nitrogen assimilation, Genetics, 1998, 149, 491-9. JUNG, E., ZAMIR, L.O., JENSEN, R.A., Chloroplasts of higher plants synthesize 1phenylalanine via 1-arogenate, Proc. Natl. Acad. Sci. USA, 1986, 83, 7231-5. HSIEH, M.H., GOODMAN, H.M., Molecular characterization of a novel gene family encoding act domain repeat proteins in Arabidopsis, Plant Physiol, 2002, 130, 1797-806. MIKKELSEN, M.D., NAUR, P., HALKIER, B.A, Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis. Plant J., 2004, 37, 770-777.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Five

TOMATO GLANDULAR TRICHOMES AS A MODEL SYSTEM FOR EXPLORING EVOLUTION OF SPECIALIZED METABOLISM IN A SINGLE CELL Eyal Fridman,a'b* Takao Koezuka,a Michele Auldridge,c Mike B. Austin,0 Joseph P. Noel,c Eran Picherskya "Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA b

The Robert H. Smith Institute of Plant Sciences and Genetics in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem P.O.Box 12, Rehovot 76100, Israel 'Structural Biology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA *Author for correspondence, e-mail: fridmane(g),agri.huji.ac.il Introduction 116 Identification of the Methylketone Pathway in the Glands 118 In silico Analysis of the Gland EST Database 118 MKS1 - a Key Enzyme in Biosynthesis of Methylketones in the Glands ... 120 The Origin and Structure of MKS1 122 Genetic Dissection of the Methylketone Biosynthetic Pathway 124 Analysis of Interspecific Populations for Methylketone Content 124 Abundance of MKS1 in the F2 Population 125 Summary and Future Directions 127

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INTRODUCTION Plants employ a plethora of mechanisms to bring their sessile life cycle to fruition. Being surrounded by a dynamic biotic and abiotic environment, plants have evolved the ability to synthesize an enormous array of chemicals that help them deal with challenges and attract beneficial agents. More than 100,000 compounds are known to be synthesized by various plants, and the real number is probably much higher. With the limited number of genes in the genome of each plant species, a single plant has only a fraction of the genes encoding the enzymes for the synthesis of this repertoire of specialized (or secondary) compounds. A major challenge for plant biologists is to elucidate the interspecies and intraspecies genetic variation underlying such enormous chemical diversity within the plant kingdom. The key to obtaining high resolution in correlating genotypes with phenotypes, with the help of bioinformatics tools, is the availability of both genetic and phenotypic diversity. Since phenotypic outcome is a result of the action of the products of many genes working together, the two main levels of genetic variation are the gene and the genome. A good biological system for studying the relationship between genotype and phenotype should provide us with large number of allelic variants for each gene, and a way to test the outcome of different combinations of alleles in many loci in the genome. In the case of chemical analysis, the tissue examined should be self-contained and easy to isolate, to allow a direct correlation of gene expression to the enzymatic function without the confounding effects of metabolism in adjacent tissues. One such system is the glandular trichomes in tomato, consisting of a single type of cells that serve as a major site of production and secretion of several types of specialized compounds (Fig. 5.1). There are additional attributes that make the tomato glandular trichomes an attractive system to study specialized metabolism: (i) The availability of a large collection of related species representing enormous biodiversity of alleles and phenotypes. In fact, the cultivated tomato represents only a small portion of the gene pool in tomato, while most of the alleles are found in related wild species that are known to synthesize many specialized metabolites in their glands;1'2^'/) The availability of a collection of interspecific crosses, together with a highly saturated genetic map. This allows for the testing of the effect of allelic combinations in many loci on the phenotype;3'4 (Hi) High gland density, which provides sufficient material for RNA, protein and chemical analysis; (iv) Amenability to genetic manipulation, hence providing the opportunity to test function of genes inplanta.

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A

B Acyl sugars (2,3,4-O-Tri-IsobutyrylGlucose)

Terpenes (Germacrene B)

Methylketones (2-Tridecanone) Fig. 5.1: Glandular trichomes of tomato synthesize and store various compounds. (A) Scanning electron microscope of the abaxial surface of a mature leaf of S. habrochaites. The four-celled glandular trichomes (arrow and in inset) predominante. (B) Selected compounds found in the glands of tomato species.

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The specialized metabolites known to be synthesized in tomato glands include terpenes5 and acyl sugars,6 as well as methylketones. In this chapter, we describe the use of tomato glandular trichomes to study methylketone biosynthesis in the wild species Solarium habrochaites f. glabratum. These compounds (Fig. 5.2B) are synthesized by many plants, but the glands of this wild tomato species are particularly rich in these compounds. 2-Tridecanone (2TD) and 2-undecanone (2UD) have been shown to be potent agents for control of a variety of insects including tobacco hornworm (Manduca sexta), spotted spider mite (Tetranuchus urticae), green peach aphids {Myzus persicae), the corn earworm Heliothis zea, and the Colorado potato beetle Leptinotarsa decemlineata.7'8 We have applied a biochemical genomics approach to elucidate the hitherto unknown pathway, and have identified a key enzyme in the pathway, methylketone synthase 1 (MKS1). In addition, we have begun to explore the 3-dimensional structure of MKS1 to understand its catalytical mechanism by comparing it to its paralogs and orthologs. To zoom out to the entire pathway leading to the synthesis of methylketones, we have begun dissecting it through QTL analysis, using interspecific crosses to identify additional factors involved in the regulation of methylketone synthesis.

IDENTIFICATION OF THE METHYLKETONE PATHWAY IN THE GLANDS In silico Analysis of the Gland EST Database The four-celled glandular trichomes on the leaves of the wild tomato S. habrochaites f. glabratum had previously been recognized as the site of accumulation of methylketones.9 We showed that methylketone content correlates well with the presence and density of glands on leaves as well as on other green parts of the plant. ° For example, brushing treatments of leaves and stems, causing removal of the majority of the glands, resulted in a large decrease in methylketone content in these organs. Furthermore, isolated glands contained much higher levels of methylketones compared to the levels of these methylketones in whole leaves (including glands), supporting the conclusion that the glands are the main, and perhaps exclusive, site of methylketone storage in the plant.10 With the demonstration of glandular trichomes as the major site of methylketone storage, we applied the single-cell EST database approach to determine if these glands might also be the site of synthesis of methylketones and, if so, to identify the genes and enzymes involved in their biosynthesis. Analysis of EST databases constructed from a single type of cell in conjunction with other types of data, such as metabolic profiling, is a powerful method to identify major biochemical pathways operating in that cell. We and others have previously used this approach to identify

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genes important in the terpene and phenylpropene pathways by constructing and comparing EST databases from peltate gland cells of mint and several basil varieties.11"15 Gene identification in these studies was based on prevalence of specific sequences, comparisons with known sequences encoding enzymes of similar function (either in term of substrates or type of reactions), metabolic profiling of the plant material, and enzymatic assays of candidate proteins.16 To examine whether the methylketones in S. habrochaites f. glabratum are derived from fatty acid biosynthesis or degradation, we constructed an EST database from the glands of PI126449. As a reference, we analyzed the closely related wild species S. habrochaites accession LAI 777 for which a trichome EST database had been reported previously (TIGR, http://www.tigr.org/tdb/Igi/). Analysis of isolated glands from both accessions showed that PI 126449 contain mainly 2TD with lesser amounts of 2UD and 2PD (Fig. 5.2), while LAI777 glands contain mostly sesquiterpenes, with no detectable methylketones.10 The prevalence of cDNAs encoding enzymes involved in fatty acid biosynthesis and degradation was compared between the two EST databases. The PI 126449 database contains a total of

ll

12

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15

16

17

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Fig. 5.2: Analysis of the main volatiles in isolated glandular trichomes of S. habrochaites f. Glabratum (PI 126449). Glands were isolated from young leaves, and their volatiles were extracted with hexane and injected into a gas chromatograph-mass spectrometer (GC-MS). 2UD, 2-Undecanone; 2TD, 2Tridecanone; 2PD, 2-Pentadecanone.

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approximately 5,500 ESTs, while the LA1777 database contains about 2,500. Most of the plant genes encoding both the enzymes of the degradative pathway (which occurs in the peroxisomes) and the enzymes of the biosynthetic pathway (which occurs in the plastids; Fig. 5.3A) have been molecularly characterized,17' 8 so this analysis was relatively straightforward. One of the most striking results of this analysis was the high abundance of the cDNA for the acyl carrier protein (ACP) in the PI126449 EST database. ACP is a small (app. 9.5 kDa) acidic protein that functions as an essential co-factor in fatty acid synthesis, acyl-ACP desaturation,19 and plastidic acyl-transferase reactions.20 We identified two distinct contigs in PI 126449 that encode two isoforms of the protein (in 3:1 ratio), which together account for 0.9% of the total transcripts in the glands. The LAI777 EST database, on the other hand, contained only 1 cDNA (accession number AW616614; 0.04% of total cDNAs) encoding ACP (Fig. 5.3B). With two exceptions, cDNAs for all other enzymes of the biosynthetic pathway were highly represented in the PI 126449 database compared to their representation in the LAI777 database. The two exceptions are cDNAs for the p-hydroxyacyl-ACP dehydratase (no cDNA in PI126449, 0.04% in LA1777) and enoyl-ACP reductase (0.05% in PI126449, 0.08% in LAI777). MKS1 -A Key Enzyme in Biosynthesis of Methylketones in the Glands The most abundant "unknown" sequence in the PI 126449 database (107 ESTs, 2% of total ESTs, ranked third in abundance after a putative secretory carrier membrane protein and S-adenosyl-L-homocysteinase) encoded a 29 kDa protein, with an open reading frame (ORF) containing 265 amino acids. Based on our in silico analysis of the EST database, and the sequence similarity of this cDNA with other plant esterases, we hypothesized that this gene, designated Methylketone Synthase 1 (MKS1), hydrolyzes Cn (3-ketoacyl-ACPs (intermediates in fatty acid biosynthesis; Fig. 5.3A) to give Cn-i methylketones, respectively. Since the (3ketoacyl-ACP substrates are unstable and not commercially available, we developed a method to synthesize such compounds, and tested this hypothesis.10 MKS1 was expressed in E. coli, and the purified protein was used to catalyze in vitro reactions in which C12, Ci4; and Ci6 P-ketoacyl-ACPs were hydrolyzed and decarboxylated to give Cn, Co, and C15 methylketones, respectively . Furthermore, levels of MKS1 transcript, protein, and enzymatic activity were correlated with levels of methylketones and gland density in a variety of tomato accessions and in different plant organs.10

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co2

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Fig. 5.3: In silico analysis of the enzymes of fatty acid biosynthesis pathway in S. habrochaites gland EST databases. (A) Scheme of the fatty acid pathway (FAS). The (3-ketoacyl-ACP substrate of MKS1 is boxed. (B) Comparison of the abundance of ESTs encoded by each of the FAS genes in the PIl 26449 (PI; 5,500 ESTs total) and LA1777 (LA; app. 2,500 ESTs total).

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Although there are three methylketones found in different quantities in the glands (Cn, Co, and C15; Fig. 5.2), MKSl showed similar specificity with the three |3-ketoacyl-ACP substrates leading to the synthesis of these respective methylketones. Furthermore, while in S. habrochaites f. glabratum accession LA0407 the ratio between the 2-tridecanone to 2-undecanone is the opposite of that found in PI126449,8 the sequence of the LA0407 allele of MKSl is identical to that of MKSl from PI 126449 (Fridman and Pichersky, unpublished data). This intraspecies phenotypic diversity in the absence of MKSl diversity suggests that the variation in the abundance of the different methylketones in the glands (and the real turn-over rate of the enzyme) may be determined by the availability of the substrate rather than the specificity of MKSl for the different substrates. The Origin and Structure of MKSl The protein encoded by MKSl share sequence similarity with a large group of plant proteins, all considered to be part of the a/(3-hydrolase family. ' These enzymes share a conserved a/(5 core domain, and catalyze the hydrolysis of various different substrates. This family in plants contains only a few proteins with proven function, including the tomato methyl jasmonate esterase (LeMJE),22 the alkaloid biosynthetic enzyme polyneuridine aldehyde esterase (PNAE) from Rauvolfia serpentine^ the recently identified tobacco methyl salycilate esterase,24 and the Hevea brasiliensis (Brazil nut) hydroxynitrile lyase (HNL).25 To gain more insights into the origin of MKSl and its biochemical and biological functions, we aligned MKS 1 protein sequence with other members of the a/p-hydrolase family in plants, and modeled its three-dimensional (3D) structure, based on the available resolved structure of for HNL and SABP2. Interestingly, although MKSl shares more than 40% sequence identity with other members of the a/p-hydrolase, it does not share two of the three residues that form the conserved catalytic triad in HNL and SABP2 (Fig. 5.4A). One of these residues, a serine, was found to be crucial for SABP2's esterase activity,23 but it is replaced by alanine (Ala87) in MKSl. A second member of this catalytic triad in SABP2, an aspartate, is replaced by asparagine. Only the third member of the catalytic triad, a histidine (His244), is the same in both enzymes. Nevertheless, structural modeling of MKSl based on the previously resolved 3D structure of SABP2 as well as HNL clearly show that it is structurally similar to these two proteins, with an overall conserved structure of six-stranded parallel (3-sheet that is flanked on both sides by six helices (Fig. 5.4A).

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B

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Fig. 5.4: Structure-function analysis of MKSl. (A) Overall ribbon of S. habrochaites f. glabratum MKSl 3-dimensional model is shown superimposed on the experimentally determined structure of Hevea brasiliensis hydroxynitrile lyase (Hb_HNL). The three MKSl residues corresponding to the Hb HNL catalytic triad are indicated in darker grey. (B) The effect of substitutions in the catalytic triad of MKS1 on the methylketone synthesis. Conversion of P-ketopalmitoyl to 2pentadecanone (indicated with arrow) is similar in the S. habrochaites f. glabratum MKSl (wild type; WT) and in the Ala87Ser mutant, but the activity of the His243Ala mutant is very low.

Although two of the three residues of the putative catalytic triad are not conserved, the pocket of the catalytic site is observed with the three residues appearing in the same position in all three proteins. Docking of the P-ketoacyl-ACP substrate into the MKSl model showed that this pocket can accommodate a varying chain length of the acyl group (data not shown), in agreement with the previous biochemical characterization that showed similar specificity of MKSl towards the three P-ketoacyl-ACP.10 To further explore the mechanism for hydrolysis of the thioester bond of the P-ketoacyl-ACP substrate, we created several mutants in the catalytic triad and tested the methylketone production of the mutated isozymes compare to the wild type enzyme. The replacement of Ala87 to serine (Ala87Ser) did not change the esterase activity (conversion of P-ketopalmitoyl-ACP to 2pentadecanone) of the enzyme, whereas the His244Ala mutant had very low activity

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(Fig. 5.4B). These results suggest that although MKS1 shares a common origin with other members of the a/p-hydrolase family, it has evolved a modified mechanism for ester hydrolysis (the catalytic nucleophile is probably not serine as it is in SABP2). Future experiments will be designed based on the actual 3D structure of MKS1 to refine the enzymatic mechanism of MKS1 in respect to ester hydrolysis and to examine if it also possesses a decarboxylating activity responsible for the production of the final product of methylketone from the free |3-ketoacid, which is the direct product of the hydrolysis, or whether the decarboxylation reaction proceeds nonenzymatically.

GENETIC DISSECTION OF THE METHYLKETONE BIOSYNTHETIC PATHWAY Analysis of Interspecific Populations for Methylketone Content The in silico analysis showed that the abundance of transcripts of most genes for the biosynthesis of methylketones (through the fatty acid biosynthetic pathway) are elevated in the glands of methylketones-producing PI 126449 plants. It is likely that these genes are coordinately regulated by a common trans-activating factor. A similar situation has been observed in petunia, where ODORANT, a member of the R2R3-type MYB family, regulates the genes of the shikimate pathway that are involved in the biosynthesis of precursors of floral volatiles.26 In addition, the low turnover rate of MKS1 in vitro10 suggests that in plant a additional protein factors may influence the rate of methylketones formation. An obvious candidate for such a function is the acyl carrier protein (ACP), which is a part of the substrate. In our in vitro assays, we used spinach ACP, but the enzyme may work faster with S. habrochaites ACPs. Several previous kinetic characterizations of fatty acid biosynthetic enzymes {e.g., acetyl-CoA carboxylase and malonyl-CoA:ACP transacylase) and polyketide synthases, some of which use substrates containing ACP moieties, have reported either low in vitro V max values or high Km values27 that could not explain the observed rate of polyketide or fatty acid synthesis in the organism. In some cases, the ACP moiety was directly tied to the low Vmax values. Yet another possible explanation is the absence of a multienzyme complex in vitro that might form in vivo. The fatty acid biosynthetic complex in the chloroplast is an example of such a complex in which the product of one reaction is generated immediately adjacent to the active site of the next enzyme in the reaction sequence, thus achieving substrate channeling. To test these hypotheses and to search for additional components that regulate the quantity and quality (e.g., chain length) of the methylketones in the glands, we have begun to genetically dissect the pathway by analyzing an interspecific population. We have obtained an F2 population by crossing the

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cultivated tomato (S. lycopersicum, cultivar M82) to S. habrochaites f. glabratum PI 126449 and selfing the Fl. Analysis of this F2 population has allowed us to test plants with various combinations of the wild species alleles at multiple loci. The F2 plants were planted in pots in the greenhouse, together with the parental lines. We sampled young leaves and determined their volatile content by dipping them in the organic solvent MTBE,12 and injecting the extract directly to a GC-MS. The variation of the methylketone content in the PI 126449 F2 population was significant (Fig. 5.5A). The majority of the plants showed similar phenotype to the cultivated parent M82, e.g., no detectable methylketones in the leaves. Some plants had intermediate levels (0.5-4.5 mg/g) between the two parental lines and approximately 10% of the plants had higher methylketone content (>4.5 mg/g) than the wild species. The continuous variation and skewed segregation towards low levels of 2TD observed in the F2 population is characteristic of a quantitative trait, regulated by multiple genes (thus defined as quantitative trait loci, or QTL). The transgressive segregation, e.g., progenies having higher levels of 2TD than the parental line (PI 126449), is also indicative of multiple loci controlling the trait, as it was shown previously for other quantitative traits in interspecific crosses of tomato.30 Abundance ofMKSl in the F2 Population We next tested the levels of MKS1 protein in these tissues using an antiMKS1 polyclonal antibody.10 Soluble protein from leaves of 12 plants, equally representing three different levels of 2TD were extracted and analyzed by Western blotting. One group of four plants (Lanes 2-5 in Fig. 5.5B) had no 2TD, a second group of four plants contained 2TD in the range of 1.5-2 mg/g fresh weight (lanes 69), and a third group of 4 plants contain >5 mg/g fresh weight of 2TD (lanes 10-13). In addition, we included leaf protein samples from the two parental lines. As expected based on our earlier study of MKS1, the leaves of the wild species (lane 14) had high levels of MKS1, while the cultivated M82 had no protein (lane 1). Progenies with high levels of 2TD also showed high levels of MKS1, in comparable amounts to PI126449. The levels of the protein varied among the progenies with medium levels of 2TD. Most significantly, of the four F2 progeny that showed no detectable 2TD in their leaves, only one plant did not have MKS1 protein in the leaves (lane 2), while the other three plants showed varying levels of MKS1, comparable to the leaves of plants with medium and high levels of 2TD (lanes 3-5).

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Fig. 5.5: Levels of 2TD and MKSl in parental and segregating F2 population. (A) Frequency distribution of the 2TD levels in F2 segregating population derived from the cross of M82 (S. lycopersicum) and PI 126449 (S. habrochaites f. glabratum). The averages of methylketone content of the parental lines and the F2 population is indicated with arrows below. (B) Protein immunoblot of the levels of MKSl protein in young leaves from F2 progenies with high (black bars; lanes 10-13), medium (hatched bar; lanes 69), and no (dotted bar; lanes 2-5) 2TD content in the leaves. The parental lines, M82 and PI 126449, are shown in lane 1 and 14, respectively.

These results indicate that the presence of MKSl is by itself not sufficient for the biosynthesis of methylketones in the glands, and that there are other factors needed to achieve production of these compounds. If MKSl were the single factor regulating the trait, then low or absent levels of MKS enzymatic activity or lack of

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expression of the gene in the cultivated species would cause a gaussian-like shape of the segregation of the methylketone content phenotype in the F2 progenies, which was not observed. The observed results could be explained if the MKS1 enzyme encoded by the allele of the cultivated species has similar activity to the wild species' allelic product but an upstream enzyme in the pathway (Fig. 5.3A), or alternatively a protein co-factor (like ACP) that acts with MKS1, is lacking. The observed results, thus, call for further study to find the nature of the additional factors determining the biosynthesis of methylketones. Specific issues to be examined include: (i) What is the allelic status of MKS1 in these plants {e.g., are they homozygous for the wild species or cultivated alleles, or heterozygous)? (ii) What is the relative activity of the cultivated form of MKS1, compare to the wild species isoenzyme, towards the P-ketoacyl-ACPs? (Hi) Are there significant differences in the expression of MKS 1 between the two alleles? SUMMARY AND FUTURE DIRECTIONS The tomato glandular trichomes function as a site of biosynthesis as well as the site of storage of specialized metabolites. We have shown that this single type of cell is an ideal system for high-resolution study of hitherto unknown pathways, such as methylketone biosynthesis. Once isolated, these cells can be analyzed on different levels including metabolic profile, transcriptosome, and proteome. In conjunction with the availability of a rich collection of wild species of tomato, and advances in the sequencing of the entire tomato genome (website as follows: http://www.sgn.cornell.edu/help/about/tomato sequencing.html). this system provides a powerful tool for associating genetic variation with phenotypic diversity, both on the gene and genome levels. We aim to further study the mechanisms that underlie the divergence of primary metabolism (fatty acid biosynthesis) to specialized metabolism (methylketones) in these cells. Molecular factors that regulate this divergence will be identified by combining quantitative trait loci (QTL) analysis of interspecific crosses, derived from crosses between wild species and the cultivated tomato, with transcriptosome and proteome analysis of the glandular trichomes. The three dimensional structure of methylketone synthase 1 (MKS1) will be determined to decipher the mechanism of the hydrolysis and decarboxylation of fatty acids Pketoacyl-ACP intermediates to methylketones. This integrated approach will develop an infrastructure that will be used to import natural insecticide compounds into the cultivated varieties, and will set a model on how to explore specialized metabolism in other plants.

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ACKNOWLEDGMENTS This project is supported by the National Research Initiative of the USDA Cooperative State Research, Education, and Extension Service Grant 2004-3531814874 to E.P.

REFERENCES 1. MILLER, J.C., TANKSLEY, S.D., RFLP analysis of phylogenetic-relationships and genetic-variation in the genus Lycopersicon, Theor. Appl. Genet, 1990, 80, 891-897. 2. KENNEDY, G.G., Tomato, pests, parasitoids, and predators: Tritrophic interactions involving the genus Lycopersicon, Annu. Rev. Entomol, 2003, 48, 51-72. 3. TANKSLEY, S.D., MCCOUCH, S.R., Seed banks and molecular maps: Unlocking genetic potential from the wild, Science, 1997, 277, 1063-1066. 4. FRIDMAN, E., CARRARI, F., LIU, Y.S., FERNIE, A.R., ZAMIR, D., Zooming in on a quantitative trait for tomato yield using interspecific introgressions, Science, 2004, 305, 1786-1789. 5. FRELICHOWSKI, J.E., JUVIK, J.A., Sesquiterpene carboxylic acids from a wild tomato species affect larval feeding behavior and survival of Helicoverpa zea and Spodoptera exigua (Lepidoptera : Noctuidae), J. Econ. Entomol, 2001, 94, 1249-1259. 6. GHANGAS, G.S., STEFFENS, J.C., UDPglucose: fatty acid transglucosylation and transacylation in triacylglucose biosynthesis, Proc. Natl. Acad. Sci. USA, 1993, 90, 9911-9915. 7. KAUFFMAN, W.C., KENNEDY, G.G., Inhibition of Campoletis sonorensis parasitism of Heliothis zea and of parsitoid development by 2-tridecanone mediated insect resistance of wild tomato,./ Chem. Ecol, 1989,15, 1919-1930. 8. ANTONIOUS, G.F., Production and quantification of methyl ketones in wild tomato accessions, J. Environ. Sci. Health Part B-Pesticides Food Contaminants and Agricultural Wastes, 2001, 36, 835-848. 9. LIN, S.Y.H., TRUMBLE, J.T., KUMAMOTO, J., Activity of volatile compounds in glandular trichomes of Lycopersicon species against two insect herbivores, J. Chem. Ecol, 1987, 13, 837-850. 10. FRIDMAN, E., WANG, J., IIJIMA, Y, FROEHLICH, J.E., GANG, D.R., OHLROGGE, J., PICHERSKY, E., Metabolic, genomic, and biochemical analyses of glandular trichomes from the wild tomato species Lycopersicon hirsutum identify a key enzyme in the biosynthesis of methylketones, Plant Cell, 2005, 17, 1252-1267. 11. LANGE, B.M., WILDUNG, M.R., STAUBER, E.J., SANCHEZ, C, POUCHNIK, D., CROTEAU, R., Probing essential oil biosynthesis and secretion by functional evaluation of expressed sequence tags from mint glandular trichomes, Proc. Natl. Acad. Sci. USA, 2000, 97, 2934-2939. 12. GANG, D.R., WANG, J., DUDAREVA, N., NAM, K.H., SIMON, J.E., LEWINSOHN, E., PICHERSKY, E., An investigation of the storage and biosynthesis of phenylpropenes in sweet basil, Plant. Physiol, 2001,125, 539-555.

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13. GANG, D.R, LAVID, N , ZUBIETA, C, CHEN, F , BEUERLE, T., LEWINSOHN, E., NOEL, J.P., PICHERSKY, E., Characterization of phenylpropene O-methyltransferases from sweet basil: facile change of substrate specificity and convergent evolution within a plant O-methyltransferase family, Plant Cell, 2002,14, 505-519. 14. IIJIMA, Y., GANG, D.R., FRIDMAN, E., LEWINSOHN, E., PICHERSKY, E., Characterization of geraniol synthase from the peltate glands of sweet basil, Plant Physiol, 2004, 134, 370-379. 15. IIJIMA, Y., DAVIDOVICH-RIKANATI, R., FRIDMAN, E., GANG, D.R., BAR, E., LEWINSOHN, E., PICHERSKY, E., The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil, Plant Physiol, 2004,136, 3724-3736. 16. FRIDMAN, E., PICHERSKY, E., Metabolomics, genomics, proteomics, and the identification of enzymes and their substrates and products. Curr. Opin. Plant Biol., 2005,8, 242-248. 17. MEKHEDOV, S., DE ILARDUYA, O.M., OHLROGGE, J., Toward a functional catalog of the plant genome. A survey of genes for lipid biosynthesis, Plant Physiol., 2000,122, 389-402. 18. GRAHAM, LA., EASTMOND, P.J., Pathways of straight and branched chain fatty acid catabolism in higher plants, Prog. Lipid Res., 2002, 41, 156-181. 19. MCKEON, T.A., STUMPF, P.K., Purification and characterization of the stearoyl-acyl carrier protein desaturase and the acyl-acyl carrier protein thioesterase from maturing seeds of safflower, J Biol. Chem., 1982,257, 12141-12147. 20. FRENTZEN, M., HEINZ, E., MCKEON, T.A., STUMPF, P.K., Specificities and selectivities of glycerol-3-phosphate acyltransferase and monoacylglycerol-3-phosphate acyltransferase from pea and spinach chloroplasts, Eur. J. Biochem., 1983, 129, 629636. 21. NARDINI, M., DIJKSTRA, B.W., Alpha/beta hydrolase fold enzymes: the family keeps growing, Curr. Opin. Struct. Biol, 1999, 9, 732-737. 22. STUHLFELDER, C, MUELLER, M.J., WARZECHA, H., Cloning and expression of a tomato cDNA encoding a methyl jasmonate cleaving esterase, Eur. J. Biochem., 2004, 271, 2976-2983. 23. DOGRU, E., WARZECHA, H., SEIBEL, F., HAEBEL, S., LOTTSPEICH, F., STOCKIGT, J., The gene encoding polyneuridine aldehyde esterase of monoterpenoid indole alkaloid biosynthesis in plants is an ortholog of the alpha/beta hydrolase super family, Eur. J. Biochem., 2000, 267, 1397-1406. 24. FOROUHAR, F., YANG, Y., KUMAR, D., CHEN, Y., FRIDMAN, E., PARK, S.W., CHIANG, Y., ACTON, T.A., MONTELIONE, G.T., PICHERSKY, E., KLESSIG, D.F., TONG, L., Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity, Proc. Natl. Acad. Sci. U S ^,2005,102,1773-1778. 25. WAGNER, U.G, SCHALL, M., HASSLACHER, M, HAYN, M, GRIENGL, H, SCHWAB, H., KRATKY, C, Crystallization and preliminary X-ray diffraction studies of a hydroxynitrile lyase from Hevea brasiliensis, Acta. Crystallogr. D. Biol. Crystallogr., 1996, 52, 591-593.

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26. VERDONK, J.C., HARING, M.A., VAN TUNEN, A.J., SCHUURINK, R.C., ODORANT1 regulates fragrance biosynthesis in petunia flowers, Plant Cell, 2005, 17, 1612-1624. 27. ALBAN, C, BALDET, P., DOUCE, R. , Localization and characterization of two structurally different forms of acetyl-CoA carboxylase in young pea leaves, of which one is sensitive to aryloxyphenoxypropionate herbicides, Biochem. J., 1994, 300 ( Pt 2), 557-565. 28. TANG, Y., LEE, T.S., KOBAYASHI, S., KHOSLA, C, Ketosynthases in the initiation and elongation modules of aromatic polyketide synthases have orthogonal acyl carrier protein specificity, Biochemistry, 2003, 42, 6588-6595. 29. ROUGHAN, P.G., Stromal concentrations of coenzyme A and its esters are insufficient to account for rates of chloroplast fatty acid synthesis: evidence for substrate channeling within the chloroplast fatty acid synthase, Biochem. J., 1997, 327 ( Pt 1), 267-273. 30. DEVICENTE, M.C., TANKSLEY, S.D., QTL analysis of transgressive segregation in an interspecific tomato cross, Genetics, 1993,134, 585-596.

Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. Ltd. All All rights reserved. reserved.

Chapter Six

ANACARDIC ACID BIOSYNTHESIS AND BIOACTIVITY David J. Schultz,1'2* Nalinie S. Wickramasinghe,3 and Carolyn M. Klinge2'3 1

Biology Department University of Louisville Louisville, KY40292 Center for Genetics and Molecular Medicine University of Louisville School of Medicine Louisville, KY 40202 3

Department of Biochemistry & Molecular Biology University of Louisville School of Medicine Louisville, KY 40202 *Author for correspondence, email: david.schultzfgUouisville.edu

Introduction Biosynthesis of Anacardic Acids A Combination of Primary Lipid Metabolism and Polyketide Synthesis?.. Fatty Acid Profiles Anacardic Acid Biosynthesis in Geranium Trichomes Anacardic Acid Biosynthesis and Type III Polyketide Synthases Bioactivity of Anacardic Acids Antimicrobial Bioactivity Potential of Anacardic Acids for Bioengineered Pest Resistance Anacardic Acids in Cancer Cell Inhibition Summary and Future Directions

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132 135 135 136 136 141 144 144 146 148 151

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INTRODUCTION Anacardic acids (2-hydroxy-6-alkybenzoic acids) differ in the alky chain length, the number and position of double bonds, and are structurally similar to aspirin and salicylic acid (Fig. 6.1). Anacardic acids are found in a limited number of plant families including Anacardiaceae, Geraniaceae, Ginkgoaceae, Myristicaceae, and others (as reviewed by Tyman).1 With the exception of the Anacardiaceae family, where they have been found in cashew {Anacardium occidentale),2 pistachio (Pistacia vera),3 Indian marking nut (Semicarpus anacardium),4 and Zimbabwean medicine plant (Ozoroa mucronata and O. insignis),5'6 only a limited number of plants within each family contain anacardic acids. Anacardic acids QH

Salicylic acid OH

COOH

Aspirin Q

Q-CCH, COOH

R = typically a 15 or 17 carbon alkyl group with 0-3 double bonds, differing in position relative to the methyl group Figure 6.1: Structures of anacardic acids, salicylic acid and aspirin. Anacardic acids represent of group of related molecules that most commonly have alkyl side chains (R) with 15 or 17 carbons and are saturated or have 1 to 3 double bonds.

Although anacardic acids have been studied for at least four decades, relatively little is known about the physiological function of these phytochemicals within their native plants. In only one case, the common garden geranium (Pelargonium x hortorum in the Geraniaceae family), has a physiological function been determined. In geranium, anacardic acids are synthesized and secreted from tall glandular trichomes that cover most aerial surfaces. The glandular secretion of anacardic acids imparts an effective resistance to the geranium against small pests such as aphid, whitefly, and spider mites. It is critical to note here that pestsusceptible phenotypes of geranium also secrete anacardic acids, and that pest resistance is not only dependent on the presence of anacardic acids, but also on the

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physical nature of the anacardic acids.7 Specifically, pest resistance is mediated by the alkyl group desaturation. Geranium that are resistant to small pests have high concentrations of monounsaturated 22 and 24 carbon anacardic acids (predominantly 22: l m5 AnAc and 24:lffl5 AnAc anacardic acids), whereas geranium that are susceptible to pest infestations contain no 22: lm5 AnAc and 24:l m5 AnAc and a relatively higher proportion of saturated anacardic acids (22:0 AnAc and 24:0 AnAc).8 Note: we are using a modification of the nomenclature used to indicate double bond position of fatty acids described by Cahoon et al. in which cox indicates double bond position relative to the methyl group and X:Y indicates total number of carbons (X) and number of double bonds (Y).9 AnAc designates an anacardic acid rather than a fatty acid. The difference in the presence or absence of the ©5 anacardic acids (22:lffl5 AnAc and 24:l
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the potential role(s) that they may play in human health. Anacardic acids are most often found as one component of a complex mixture of structurally related phenolic lipids including cardols (alkyl resorcinols), cardanols, and/or urushiols (Fig. 6.2). Anacardic acids are proposed to be synthesized from fatty acid CoA esters and malonyl-CoA by action of a type III polyketide synthase.11 It is more than likely that the complex mixtures of anacardic acids, cardols, cardanols, and urushiols are produced by as yet unidentified type III polyketide synthase(s). The presence of these phenolic lipid mixtures in multiple plants of the Anacardiaceae family as well as in the leaves and fruits of the ginkgo tree indicates a potential ancient evolution of these phytochemicals and/or that they have evolved independently, in distinct plant families (as has been proposed for the stilbene synthase type III PKS).12

Anacardic acids OH

Cardols OH

COOH

Cardanols OH

Urushiols OH

R = typically a 15 or 17 carbon alkyl group with 0-3 double bonds, differing in position relative to the methyl group Figure 6.2: Anacardic acids are commonly found as one component of a mixture of related phenolic lipids. The phenolic lipids include anacardic acids, cardols, cardanols, and/or urushiols.

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The garden geranium is not only unique for having a defined physiological function for anacardic acids, but is also unique in that it is likely one of the only plants producing anacardic acid as a single class of compounds rather than as a complex mixture of phenolic lipids. This, coupled with the fact that anacardic acids are synthesized and secreted from a defined tissue (glandular trichomes), makes geranium an ideal system in which to study the biosynthesis and bioactivity of this unusual phytochemical. In this chapter, we review the biosynthesis of anacardic acid with an emphasis on the type III polyketide synthase family as well as our latest work understanding lipid metabolism in geranium glandular trichomes. Further, we review the bioactivity of anacardic acid with an emphasis on utilization of anacardic acids for agricultural and medicinal applications. BIOSYNTHESIS OF ANACARDIC ACIDS A Combination of Primary Lipid metabolism and Polyketide Synthesis? The first studies on biosynthesis of anacardic acid were carried out by Gellerman et a/.13'14 using leaf and seed from Ginkgo biloba. Using [1- or 2-14C] acetate labeling of anacardic acids coupled with chemical degradation to locate [14C] labeled components within the structure, Gellerman et al. concluded two distinct biochemical pathways contribute to the synthesis of anacardic acid.13 These authors were the first to propose, based on experimental data, that the ring structure was formed by a polyketide mechanism that utilized a fatty acid as a precursor. Additionally, they concluded that the carboxyl carbon of the precursor fatty acid was incorporated into the aromatic ring structure of anacardic acid. This labeling pattern is consistent with the activity of a type III polyketide synthase that utilizes an aldol condensation (C2 -» C7) as reviewed by Austin and Noel.11 Additional studies extended these results to show [2-14C]malonate was preferentially incorporated into the aromatic ring structure.14 Further, this study showed, somewhat paradoxically, that fatty acids (laurate and palmitoleate) were not incorporated into anacardic acid, but were actively incorporated into glycerolipids. This was somewhat surprising because, based on double bond position, palmitoleate was likely the precursor to the dominant anacardic acid in the tested tissue. These results indicate that a potential spatially or metabolically separated fatty acid pool is used for synthesis of anacardic acids. In contrast to the results obtained by Gellerman et al.,14 labeling studies utilizing [14C] fatty acid methylesters in geranium showed incorporation of specific fatty acids (saturated or unsaturated) into correspondingly saturated or unsaturated anacardic acids. It is not clear why incorporation of fatty acids into anacardic acids was not observed in the ginkgo system,1 while incorporation was observed in the geranium system.15'16 However, labeling duration (3-days for ginkgo and 7-10 days for geranium) as well as the modifications to the fatty acids that were supplied (free

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fatty acid versus fatty acid methyl esters) are at least partial explanations. Importantly, the labeling studies in the geranium provided indirect evidence that the 22:1™5 AnAc and 24:l ra5 AnAc that are important for the pest-resistance phenotype would be synthesized from 16:1 AU and 18:1A13 fatty acids. Fatty Acid Profiles With evidence that anacardic acids are derived from fatty acid precursors,14"16 the fatty acid profile of geranium glandular trichomes was analyzed and found to contain 17.3 % 16:1 AU and 6.1 % 18:1 A13 . 17 Based on double bond position (co5) of these fatty acids, 16:1A11 and 18:1A13 are likely the precursors to 22:lffl5 AnAc and 24:l m5 AnAc, respectively. Interestingly, the composition of fatty acids is not directly reflected in the composition of anacardic acids. For example, while the co5 fatty acids (16:1A11 and 18:1A13) comprise only 23% of the fatty acid profile, the corresponding co5 anacardic acids (22rl^ 5 and 24:I*05) comprise 80% of the anacardic acid profile. Thus, the enzyme(s) responsible for anacardic acid biosynthesis may have specificity for distinct fatty acids. Alternatively, fatty acids for anacardic acid synthesis may exist as separate pools (metabolically or spatially) from those in primary glycerolipid biosynthesis as was previously suggested.14 To gain more insight into potential pools of the co5 fatty acids for anacardic acid biosynthesis, trichome lipids were isolated and fractionated into lipid classes. While the ©5 fatty acids (16:1 AU and 18:1A13) were found at some level in all lipid classes, phosphoglycerolipids contained the highest composition of ©5 fatty acids (26% of phosphotidylinositol and 18% of phosphotidylcholine).18 In geranium, the inheritance of the pest-resistant phenotype is conditioned by a single Mendelian trait (reviewed in Schultz et a/.).8 Likewise, since pest resistance is directly correlated with the presence of the ©5 anacardic acids, production of ©5 anacardic acids and the eo5 fatty acid precursors are conditioned by the same single Mendelian trait. Thus, this single dominant Mendelian trait is most likely a fatty acid desaturase gene. A homology based cDNA library screen was used to identify this fatty acid desaturase gene.19 The desaturase gene was found to be part of the plant acyl-ACP desaturase gene family and was shown to encode a A9 14:0-ACP desaturase enzyme. This desaturase acts on myristic acid (14:0-ACP) to produce myristoleic acid (14:1A9). In geranium trichome tissue, this ©5 fatty acid would be elongated to the 16:1A11 and 18:1A13 co5-fatty acids that serve as precursors for production of the ©5 anacardic acids (22:l to5 and 24:1° 5 AnAc). Anacardic Acid Biosynthesis in Geranium Trichomes To date, biosynthetic studies of anacardic acids have used relatively long

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labeling times (3-10 days) or have provided only a static picture of lipid content and composition. Recently, we used the geranium trichome system to gain more insight into anacardic acid biosynthesis over shorter periods of time. Geranium flower pedicles were used for all labeling experiments, as this tissue provides the highest density of glandular trichomes. Geranium flowers were harvested, and ~1 cm segments of pedicles were cut into beakers containing ice-cold 50 mM CHES (pH 9) buffer. This served as an alkaline wash to remove anacardic acids that were already present as glandular trichome exudate prior to the labeling reactions. Pedicles were rinsed several times in 50 mM MES (pH 5). Each labeling reaction contained 50 x 1 cm pedicle sections in 1 ml labeling reaction (l.lxlO 7 dpm [l-14C]acetate-65 mCi/mmol in 50 mM MES pH 5) in glass scintillation vials. Samples were incubated at 25°C with shaking (250 rpm). Reactions were terminated by removing labeling mix, rinsing three times with 1 ml 50 mM MES (pH 5.0), then snap freezing the tissue in liquid nitrogen. Trichomes were recovered as described,17 and lipids were extracted using the Bligh and Dyer lipid extraction.20 The content of [14C] was quantified by scintillation counting, and aliquots of each sample were separated by TLC using a three-part development system. Plates were developed to 6 cm in methanol/acetonitrile/water/acetic acid (65/25/3/2; v/v/v/v), to 12 cm in chloroform/methanol/acetic acid (75/25/8; v/v/v) and to 18 cm in hexanes/diethyl ether/acetic acid (60/40/1; v/v/v). Plates were analyzed with a phosphorimager (BioRad, Personal FX). Incorporation of [14C] acetate was initially highest in the phosphoglycerol lipid fraction but by midway through the reaction, the most abundant labeled lipid was the anacardic acid fraction (Fig. 6.3). A small but significant amount of label was found in triacylglycerol. No incorporation into galactolipids was detected. These results indicate an active lipid metabolism in the geranium glandular trichomes and show anacardic acid metabolism is dominant in these cells. Production of anacardic acids has been proposed to utilize an acyl-CoA substrate with carbon elongation by acetate units derived from malonyl-CoA. To gain evidence for these substrates and to develop a more useful assay system to explore anacardic acid and lipid metabolism, we utilized the "bead-beater" trichome isolation method that allows for the isolation of metabolically active glandular trichomes.21' 22 Trichomes were isolated from 10 grams flower pedicle tissue in a 300-ml vessel with 40 ml 0.5 mm glass beads utilizing 3 pulses at 40 V (Rheostat setting) as described.21 The resulting cell suspension was passed through nylon screens with decreasing pore size (350, 105, and 40 micron screens). The cell material that did not pass through the final 40-micron screen was re-suspended in gland isolation buffer. This procedure resulted in isolation of gland cells at approximately 20,000 cells/ml buffer. This procedure did not separate gland and stalk cells (Fig. 6.4), but provided a preparation that was 90-95% free of nontrichome materials.

SCHULTZ, et ah al. SCHULTZ,

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incorpora ted

40000-

35000 30000

:eta

B

25000

03

u

20000

dpm [1

i—i

15000 100005000-

50

100

150

200

250

Time (min) Figure 6.3: [l-14C]acetate labeling of geranium trichome. After designated time points, reactions were terminated, trichome tissue was fractionated and lipids were recovered from the trichome tissue. Lipids were separated by TLC and identified based on migration of standards (TAG - triacylglycerol, AnAc - anacardic acids, PE phosphatidylethanolamine, PG - phosphatidylglycerol, PC phosphatidylcholine, and PI - phosphatidylinositol). [14C] lipids were quantified using a phosphorimager.

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Figure 6.4: Geranium trichome isolation. The "bead-beater" method was used to fractionate trichomes from pedicle tissue. Extracts were sequentially passed through nylon screens to sieve out non-trichome material. Trichome gland and head cells were recovered from the 40 urn screen. Average gland cell diameter was 44 ± 0.8 urn (SEM). rl4,

To test the metabolic ability of the isolated trichome cells, various [ C] labeled substrates were supplied to separate reactions. Since it was originally reported that small molecules such as NADPH and CoA could potentially diffuse from isolated gland cells,22 all required cofactors were included in the reaction mix. Each reaction was carried out in glass scintillation in 0.5 ml reaction volumes containing 0.5 mM MnCl2, 5 mM NADPH, 1 mM NAD+, 2 mM ATP, 3 mM ADP, 1 mM CoA, 3 mM Ascorbate, 0.5 mM DTT, 1 mM Sucrose, 10 mM KC1, 5mM MgCl2, 25 mM HEPES pH 7.3, and [14C] substrate. The following substrates were independently tested for incorporation into anacardic acid: 20 uM [l,5-14C]citrate (108 mCi/mmole, Moravek Biochemicals Inc.), 10 uM [2-14C]malonyl-CoA (54 mCi/mmole, Amersham Biosciences), 15 uM [l-14C]oleoyl-CoA (52 mCi/mmole, Moravek Biochemicals Inc), 100 uM [l-14C]acetate (65 mCi/mmole, ICN), 20 uM [1- 14C]myristic acid (54 mCi/mmole, Moravek Biochemicals Inc), and

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15 uM [l-14C]palmitic acid (53 mCi/mmole, Moravek Biochemicals Inc) provided as an ammonium salt of palmitic acid. All reactions were incubated at 21°C with shaking at 250 rpm for 4 hours. Reactions were terminated by addition of 0.3 ml 1.5% acetic acid followed by Bligh and Dyer lipid extraction.20 The [14C] content in the recovered lipid fraction was quantified by scintillation counting, and samples were analyzed by TLC. Samples were separated in a three-part development system. Plates were developed to ~ 6 cm in chloroform/methanol/50% aqueous NH4OH (75/25/2, v/v/v), then developed to -12 cm in chloroform/methanol/acetic acid (75/25/8, v/v/v), and finally to -18 cm in hexanes/diethyl ether/acetic acid (60/40/1, v/v/v). Plates were dried completely between developments then analyzed using a phosphorimager to detect and quantify radiolabeled products. I2 staining was used to detect non-labeled standards that were included on all TLC plates. Individual lipids were identified based on comparison to migration of lipid standards. No incorporation of palmitic acid (supplied as an ammonium salt) or myristic acid (supplied as a free fatty acid) was detected in anacardic acids but was detected in triacylglycerol and phosphoglycerol fractions. [14C] from citrate was also not detected in anacardic acid, but was found in the phosphoglycerol fraction. In contrast, acetate, malonyl-CoA, and oleoyl-CoA all showed [14C] incorporation into anacardic acids (Fig. 6.5). Incorporation of [14C] from malonyl-CoA was determined to be at a rate of 48 pmol/20,0000 gland cells. As a comparison, [14C] from malonyl-CoA was incorporated into phosphoglycerolipids at a rate of 166 pmol/20,000 gland cells. Incorporation off1 C] from acetate was similar to malonylCoA with a rate of 55 pmol f1 C] incorporated/20,000 gland cells with nearly equal incorporation into phosphoglycerolipid fractions. Oleoyl-CoA provided the highest rate of [14C] incorporation into anacardic acids at 1189 pmol/20,000 cells. In addition, [14C] from oleoyl-CoA was incorporated into phosphoglycerolipids (146 pmol/20,000 cells) and was incorporated into triacylglycerol fraction (1063 pmol/20,000 gland cells). It was somewhat surprising that citrate was not an effective source of carbon for anacardic acid biosynthesis. By activity of ATP citrate lyase and acetyl CoA carboxylase, citrate would provide cytosolic malonyl-CoA that would presumably be used for anacardic acid biosynthesis. However, the rate of [14C] incorporation from malonyl-CoA directly supplied in these reactions was very low, and thus it is possible that conversion to malonyl-CoA with subsequent incorporation into anacardic acids was below our detection limits in this experiment. A somewhat surprising result from this labeling study is the relatively high incorporation of all fatty acid substrates into triacylglycerol, indicating a high level of activity in forming storage lipids. The relationship between triacylglycerol lipid biosynthesis and anacardic acid biosynthesis in the geranium glandular trichomes will be the target of future studies. These results confirm that malonyl-CoA and fatty acid CoA esters can serve as substrates for synthesis of anacardic acid.

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1200 u o o u

1000

2

800

X

600 O Q.

O O

400

O "3

200

0 malonyl-CoA

acetate

oleoyl-CoA

[14C] substrates for anacardic acid biosynthesis

Figure 6.5: [ C] substrate incorporation into anacardic acids. Isolated geranium glandular trichome cells were incubated with [14C] labeled substrates for 4 hours. After reactions were terminated, lipids were extracted and separated by TLC and quantified using a phosphorimager. Lipids were identified based on migration patterns of standards.

Anacardic Acid Biosynthesis and Type III Polyketide Synthases Anacardic acids are likely synthesized by an enzyme that is part of the type III polyketide synthase superfamily.11 The type III polyketide synthases characteristically differ with respect to the starter molecule (although all use a CoA ester), the number of acetate units that are added, and in the type of cyclization utilized (none, lactone formation or Claisen or aldol cyclization followed by aromatization). Austin and Noel1' recently wrote an excellent review on this subject and thus, we will focus only on the polyketide synthase III family, as it may relate to anacardic acid (and other phenolic lipids) biosynthesis.

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Anacardic acids are most commonly found in plants within the Anacardiaceae family. However, anacardic acids are often not the only phenolic lipid found in these plants, but rather exists as a complex mixture of related phenolic lipids (Fig. 6.2). This is also the case for some plants not in the Anacardiaceae family, e.g., Ginkgo. One of the only plants that produce anacardic acids as a single phenolic lipid component is geranium in glandular trichomes. Of additional importance is the fact that alkyl resorcinols are found in the grain crops,23 but these plants seem to lack anacardic acids. The biosynthesis of anacardic acids, alkyl resorcinols (cardols), cardanols, and urishiol are biosynthetically similar (Fig. 6.6). In all cases, the starter molecule is a fatty acid CoA ester and is extended by addition of 3 acetate units (derived from malonyl-CoA). Each requires cyclization by aldol condensation (similar to that found in stilbene and stilbenecarboxylate synthesis) followed by aromatization. The production of anacardic acid requires action of a polyketide synthase and a reductase similar to that required to produce deoxychalcone. Cardanol also requires a reductase, but loses the carboxylate group during aldol condensation. Recently, an aldol switch was identified in the stilbene synthase that accounts for this decarboxylation event.24 Cardol requires action of only a polyketide synthase with no prior reduction and also requires decarboxylation. The production of urishiols requires a hydroxylation event, perhaps utilizing cardanol or anacardic acid (after a decarboxylation event). Based on the chain length and double bond placement, it is evident that the enzyme(s) that produces these related phenolic lipids in a single tissue utilizes the same starting fatty acids. However, it remains unclear as to the number of distinct polyketide synthases that may exist and contribute to production of these lipids in a single tissue. Production of cardol versus cardanol or anacardic acids may be explained in part through regulation of access to the intermediate required by the reductase. Recently, protein structure studies were used to propose the cyclized (nonaromatized) intermediate that is most likely the substrate for chalcone reductase and also that the ratio of chalcone and deoxychalcone can be controlled by substrate access by the reductase.25 The presence of the carboxylate group in anacardic acids may be more difficult to explain. It was recently proposed that the biosynthesis of stilbenes and stilbenecarboxylates may not be biosynthetically related. Using stereochemical consideration and structure-based prediction, the stilbenes have been proposed to be decarboxylated post-cyclization followed by concerted or stepwise decarboxylation and dehydration.24 Furthermore, these authors proposed that stilbenecarboxylates may not be produced by an enzyme-catalyzed aldol condensation, but rather, the stilbenecarboxylate is off-loaded as a lactone that in solution spontaneously undergoes an aldol condensation and facilitates the retention of the carboxylate group.24 Thus, it may be possible that a distinct polyketide synthase is required for production of anacardic acids rather than cardol and cardanol.

ANACARDIC ACID BIOSYNTHESIS BIOSYNTHESIS AND ANACARDIC ANDBIOACTIVITY BIOACTIVITY

S-CoA 3 x malonyl-CoA Type IIIPKS -3xCO

O O O O

S-CoA

Type IIIPKS

Type IIIPKS Reductase

Type IIIPKS Reductase

Cyclization

Cyclization Reduction Aromatization Decarboxlation

Cyclization Reduction Aromatization

Aromatization Decarboxlation

Type IIIPKS Reductase CHydroxylaseO Cyclization Reduction Aromatization Decarboxylation Hydroxylation

OH HOOC OH Cardol

R

R Cardanol

Anacardic acid

Urushiol

Figure 6.6: Biosynthesis of phenolic lipids. As proposed, cardols, cardanols, anacardic acids, and urushiols could be synthesized from a common intermediate such as palmitoyl-CoA. Proposed enzyme functions are given in italics and associated chemical reactions are underlined. The proposed associated chemical reactions are not intended to suggest sequence of reactions, but serve only as a point of comparison between phenolic lipids. R=15 carbon alkyl group.

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Although it is interesting to speculate on the potential reaction mechanism for synthesis of phenolic lipids based on analogy to the chalcone, stilbene, and stilbenecarboxylate synthesis, the reaction mechanism(s) must be confirmed for the polyketide synthases that produce the phenolic lipids. As a first step in this process, we have begun an EST (expressed sequence tag) based approach to isolating anacardic acid biosynthesis related clones from a trichome-specific cDNA library. These clones will provide the basis for future work on understanding the chemical mechanism used in cyclization and in the retention of the carboxylate group in comparison to that proposed for the stilbenecarboxylates. Further, by comparing the anacardic acid synthesis enzymes of geranium to the enzyme(s) producing anacardic acids and related molecules in evolutionarily distinct plants such as ginkgo and cashew, we will gain a better understanding of the evolution of the type III polyketide synthase family and provide new evolutionary insight into polyketiderelated reductases.

BIO ACTIVITY OF ANACARDIC ACIDS Bioactivity of anacardic acids has been studied largely by utilizing purified compounds extracted from cashew or ginkgo. More recently, traditional medicinal plants containing anacardic acids have been used to assess potential bioactivity.6' 26' 7 Bioactivity can be divided into broad areas of bacterial, fungal pathogens, insect, cell proliferation (anticancer activity), and enzyme inhibition. These bioactivity studies provide rationale for extraction and purification of anacardic acids for use in medical and industrial applications. Further, our studies provide the rationale for future work to bioengineer production of anacardic acids into crop plants with the intent to provide an endogenous system for plants to resist bacterial, fungal, and insect attack as well as to provide foods that may have chemotherapeutic and/or chemopreventative applications. Antimicrobial Bioactivity One of the earliest studies on bioactivity of anacardic acids (purified from cashew shell oil) provided evidence that these compounds could inhibit both gram negative and gram positive bacterial growth.28 However, gram positive bacteria were much more sensitive than were gram negative bacteria. Interestingly, this study also indicated that the number of double bonds in the alkyl portion of anacardic acid was positively correlated with increased effectiveness. This result has been confirmed in additional studies.29'34 In general, anacardic acids are effective against gram positive bacteria and are much less effective against gram negative bacteria. Also consistent with Gellerman's report,28 these studies indicated that the effectiveness of anacardic acids was influenced by the alkyl side group. Most studies have used a series of 15 carbon alkyl chain with zero to three double bonds.

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Anacardic acids with 1-3 double bonds were effective against gram positive bacteria at a range of 1 - 6 ug/ml. However, the saturated 15-carbon alkyl side group was equally effective only at much higher concentrations (10-1000 fold higher relative to the trienoic alkyl group). 29 ' 30 ' 32> 33 More recently, it has been suggested that the presence of the double bond is not the most important determinant for activity. Anacardic acids with shorter alkyl chains (10 and 12 carbons) have been found to be equally effective as the 15 carbon trienoic alkyl group.29' 34 Thus, it has been suggested that a biophysical, perhaps surfactant, nature of the side group is most important for bioactivity.34 However, it should also be noted, that the alkyl side group seems to be necessary for higher levels of activity. When compared to salicylic acid, anacardic acids regardless of alkyl chain length or saturation state, have higher bioactivity. 32 ' 35 The antibacterial activity of anacardic acids has also been compared with other phenolic lipids (cardol and cardanol) found in cashew and ginkgo tissues. Anacardic acids were reported to have greater antibacterial activity when comparing these three related phenolic lipids, thus indicating an essential role for the carboxyl group of anacardic acid.33 Anacardic acids have been tested and found to be potentially useful against medically important bacteria that are involved in tooth decay (Streptococcus mutans), acne (Propionibacterium acnes), ulcers (Helicobacter pylori), and infection (Staphylococcus aureus). ' ' Importantly, anacardic acid has synergistic effects with methicillin against methicillin-resistant Staphylococcus aureus (MRSA).34' 36 Furthermore, it has been shown that anacardic acids can function to inhibit (3lactamase, thereby suggesting a mode of action for the synergism that was observed.37 One question arising from these observations might be whether humans naturally ingest anacardic acids at concentrations commensurate with the observed antibacterial activity. Importantly, anacardic acids are found in food products such as Caju juice (17.9 mg anacardic acids/g) made from the cashew apple, 8 and which is a common beverage in Brazil. It is possible that consumption of these products may prove therapeutic and/or may prevent associated diseases. Although the bioactivity of anacardic acids against medically important bacteria is now well-known, there exists no thorough study assessing bioactivity against plant bacterial diseases. As many plant pathogenic bacteria are gram negative, it would be interesting to determine whether or not the presence of anacardic acids in plant tissues might provide some basis for defense against bacterial disease. Anacardic acids have also been tested for activity against other pathogens. Anacardic acids from cashew nut shell liquid were found to have limited effects on inhibiting the growth of protozoa (Gyrodinium cohnii and Euglena gracillis), fungi (Neurospora crassa and Penicillium griseofulvum), and yeast (Saccharomyces cerevisiae and Candida utilis)}% In other studies, anacardic acids were not effective against many fungi and yeast, although more highly unsaturated anacardic acids displayed limited bioactivity against these species. °' 33 In contrast, a study of spore germination indicated anacardic acids isolated from Rhus semialata were effective

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against Colletotrichum capsici, Fusarium oxysporum, Alternaria brassicae, Alternaria alternata, Alternaria carthami, and Curvularia lunata spore germination but were not effective against Fusarium udum spore germination.39 The potential of anacardic acid as an antifungal agent needs additional study. However, from the limited work conducted thus far, anacardic acids do not appear to be widely effective against fungal growth, but may play an important role in inhibiting spore germination. Thus, plants producing anacardic acid may be more resistant to initial challenge by fungal pathogens. Potential of Anacardic Acids for Bioengineered Pest Resistance In the geranium, production and secretion of anacardic acids from glandular trichomes provide an effective resistance against small pests. The secreted anacardic acid exudate has two modes of action, the first being a physical entrapment and the second being a toxic effect that increases mortality and reduces fecundity of the pests.7 The physical nature of the anacardic acids is a critical aspect to entrapping pests and for effective application of the toxin to escaping pests. To date, it is not known whether anacardic acids are effective at controlling larger pests or whether anacardic acids are effective when consumed rather than topically applied. To test the pesticide properties of anacardic acids when consumed, we used purified anacardic acids supplied to Colorado potato beetle (Liptinotarsa decemlineata) larvae throughout development. Anacardic acids were extracted from flower pedicles in a minimum volume of chloroform. Extracts were concentrated in a rotary evaporator then applied to a silica gel column. Samples were eluted from the column in a 5 solvent system (I hexane, II - 1% diethyl ether/1% HOAc in hexane, III - 10% diethyl ether/1% HO Ac in hexane, IV - 50% diethyl ether/1% HOAc in hexane, V - 25% methanol in diethyl ether) as described by Hesk et al.40 Fractions containing anacardic acids were identified by TLC with detection under short wave uv light. Fractions containing anacardic acids were pooled, concentrated, and purified further by HPLC (C18, 25 cm x 4.6 mm, 5 \aa, flow rate of 2.5 ml • min"1 CNCH3/dH2O/HOAc; 100/20/1; v/v/v). Bioactivity assays were conducted using newly emerged Colorado potato beetle larva reared on eggplant leaves. Each treatment consisted of 4 larva and one leaf disk (30 mm diameter circles) placed on a piece of 3 MM Whatman chromatography paper dampened with dH2O in Petri dishes (35 mm diameter). Leaf disks were replaced daily and weights were recorded as an average weight per larva during development. After 5-6 days development, each larva was placed on a single leaf disk. Leaf disks were treated with 50 mM CHES buffer (pH 9) solutions containing anacardic acids. Controls consisted of 50 mM CHES buffer (pH 9). A mixture of 22:l m5 AnAc and 24:lco5 AnAc, at proportions typically found in

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glandular trichomes of geranium (40% and 60%, respectively) (Fig. 6.7A), or purified 24:1™5 AnAc (Fig. 6.7B) were tested. The mixture of anacardic acids at lx (lx = 1 mg/ml) reduced average larval weight by day 7 of the assay. In contrast, 0.1, 0.5, and 1.0 X purified 24:1®5 AnAc showed no difference relative to the control over the duration of the assay. At the highest concentration tested (2.5 X) purified 24:1™5 AnAc was effective at reducing average larvae weight gain by day 5 of the assay.

3 4 Time (days)

1

2

3 4 Time (days)

5

6

7

Figure 6.7: Influence of anacardic acids on Colorado potato beetle larvae development. Newly emerged Colorado potato beetles were fed a diet treated with a mixture of 40% 22:1m5 AnAc and 60% 24: lm5 AnAc (A) or a diet treated with 100% 24: T 5 anacardic acids (B). Larvae weight was recorded for a duration of 7 days. Results in (A) represent the average of two independent trials (four larvae per trial) and results in (B) represent the average of four insects in one trial. Concentration of lx = 1 mg/ml.

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These data indicate that anacardic acids may inhibit larvae growth and development. Thus, anacardic acids may provide resistance to pests such as the Colorado potato beetle. It remains to be determined what effect consumption has on larvae development over a longer time period (including larvae death) and the influence of anacardic acids on pupation. Furthermore, it will be important to determine if the presence of anacardic acids impacts food preference of larvae and adult beetles. The mechanism of action for anacardic acids in pest resistance has not been determined; however, based on inhibition of prostaglandin synthase and inhibition of lipoxygenase, it has been proposed that anacardic acids can influence reproduction.41 Additionally, anacardic acids have been determined to inhibit the activity of tyrosinase, an important molting enzyme.42 It is also possible that anacardic acids are simply acting as a detergent and disrupt feeding either directly by making the tissue non-nutritive or indirectly by making the food non-palatable.

Anacardic Acids in Cancer Cell Inhibition Anacardic acids (whether purified or supplied within an extract as a traditional medicine) display anti-cancer activity. The anti-cancer activity of anacardic acids was first shown against sarcoma 180 ascites tumor growth in mice.43 Subsequently, anacardic acids from cashew apple juice were reported to inhibit proliferation of cultured human breast (BT-20) and cervix carcinoma (HeLa) tumor cells.44 More recently, Lee et al., evaluated and compared the activity of anacardic acids, cardanols, and cardols against a broader range of cancer types.45 When comparing the potential of anacardic acids to differentially effect growth of cancer cell types, anacardic acids were most effective against breast cell cancer lines. Additionally, comparing 22:1007 AnAc treatment in normal colon cells (CCD-18-Co) versus the corresponding colon cancer cells (HCT-15), the cytotoxic effective on the non-cancerous cells was nearly 8-fold lower, indicating selectivity toward cancer cell growth inhibition.45 A general conclusion that can be drawn from the studies by Itokawa et ah, Kubo et al., and Lee et a/.,43"45 is that anacardic acids, cardols, and cardanols display varying levels of anticancer activity, but differ in effectiveness against specific cell lines. Studies of traditional medicinal plants provide further evidence of the potential anticancer activity of anacardic acids. A methanol bark extract from Ozoroa insignis, which contains anacardic acids, inhibited the proliferation of a number of cancer cell lines, including HepG2 liver cancer and MDA-MB-231 breast cancer cells, in vitro.6 Semecarpus anacardium contains anacardic acids,4 and a chloroform extract of the Semecarpus anacardium nut has shown anti-tumor activity against various experimental cancer cells, e.g., B16 melanoma and leukemia L-1210 cells.26 In a more recent study, Sowmyalakshmi et al?1 found that hexane and chloroform fraction extracts of the Siddha medicine Semecarpus Lehyam (SL) were

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effective at reducing viability and increasing apoptosis of MCF-7 and MDA-MB231 breast cancer cell lines. Semecarpus anacardium is a component of SL. Furthermore, anacardic acids would be expected to be recovered in hexane and/or chloroform fractions and, thus, may be contributing factors to the observed bioactivity. The mechanism for the observed anticancer activity of anacardic acids at the molecular level has been the focus of a limited number of studies.45"47 The importance of prostaglandins in cell proliferation and involvement in cancer cell growth has become an increasingly important topic. Anacardic acids from Ozoroa mucronata and geranium have been shown to inhibit prostaglandin synthase (cyclooxygenase - COX).5'41 Anacardic acids (as sodium salts) were administered to Wistar albino rats and found to decrease production of prostaglandins in brain tissue.48 It is worth noting that these studies were conducted before the discovery of COX isoforms. COX (prostaglandin endoperoxidase synthase) is the rate-limiting enzyme for arachidonic acid metabolism to eicosanoids. The two COX isoforms (COX-1 and COX-2) have significant roles in carcinogenesis,49 and studies have reported an association of COX-2 expression with aggressive breast cancer.50 Furthermore, a recent study showed COX-2 was also over-expressed in NSCLC compared to normal lung epithelium or SCLC.51 Prolonged use of the COX inhibitor aspirin, e.g., > 6 months, was shown to have a significant inverse correlation with breast cancer risk in women.52 Anacardic acids are alkylated salicylic acids, whereas aspirin is acetylsalicylic acid (Fig. 6.1). The alkyl group of anacardic acids has been shown to influence cytotoxicity.44'45 Furthermore, in direct comparisons, salicylic acid was found to be less effective than anacardic acids at inhibiting breast cancer and cervix carcinoma cells.44 Ultimately, the relative inhibitory activity of anacardic acids toward COX-1 and COX-2 needs to be investigated. The herbal remedy BHUx contains anacardic acids as one of the principal components of Semecarpus anacardium. Interestingly, BHUx displays a more than 2-fold lower IC50 value for COX-2 compared with COX-1.53 Though this is not direct evidence for increased specificity of anacardic acids towards inhibition of COX-2, it clearly is an area in need of greater study. Anacardic acids also may have a role in genomic alterations that affect cell proliferation. When supplied to chick embryo neurons, anacardic acids cause chromosomal condensation.54 One mechanism of controlling chromosomal condensation is by regulation of histone acetylation. Anacardic acids act as a noncompetitive inhibitor of the histone acetyltransferase (HAT) activity of the transcriptional coactivators p300 and PCAF.4 ' 55 In the studies that show anacardic acids have anticancer activity, it is apparent that they are effective against breast cancer cells lines. Thus, we have initiated research to investigate the specific molecular mechanism(s) by which they limit breast cancer and lung adenocarcinoma cell proliferation. Breast cancer is the most commonly diagnosed cancer in women in the USA and a major risk factor in

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developing this disease is lifetime exposure to estrogens.56 Estrogens promote cell replication through activation of estrogen receptors (ER). Estrogen receptors occur as two isoforms, ERa and ER(3 that are ligand-dependent transcription factors.57 These receptors act in concert to control cell proliferation with ERa acting as a proliferative agent and ERP acting as an antiproliferative agent.58

COX-1 XCOX-2 OX-2^

Death Cell Death (?)

Arachidonic acid Prostaglandins PGE2

X

Apoptosis Proliferation Cell Proliferation

cAMP CBP/p300 CBP/p300 Genes for cell proliferation

Testosterone CREB Aromatase HAT

HAT

Cyclin D1 D1 B1 Cyclin B1 VEGF

ER E2

Figure 6.8: Model of the molecular mechanisms of anacardic acid mediated inhibition of breast cancer cell growth. The model suggests at least four points at which anacardic acids may prevent breast cancer cell proliferation: 1) Inhibition of cyclooxygenase would affect cell proliferation and estrogen production; 2) Interaction with estrogen receptors could alter cell proliferation; 3) Inhibition of transcription coactivators such as CBP/p300 would limit estrogen responsive cell proliferation; and 4) Anacardic acids may limit cell proliferation by directly or indirectly promoting cell apoptosis.

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Breast cancer may be prevented or treated by consumption of foodstuffs containing antiestrogenic activity59' 60 or weaker estrogenic activity61 than E2, the endogenous estrogen with highest ER binding affinity. The mechanism by which these phytochemicals may work is by altering or preventing action of the estrogen receptors. Based upon the known action of anacardic acids against breast cancer and its known bioactivities against such molecular targets as cyclooxygenase, p300/CBP, and its untested potential to interact with the ER receptors, we have developed a model for testing the ability of anacardic acids to inhibit breast (and other estrogen responsive) cancer cell lines at the molecular level (Fig. 6.8). We have predicted at least four mechanisms by which anacardic acids could limit cell proliferation in these tissues. Our initial results indicate anacardic acids may act as a type II antiestrogen against ERoc and may also influence the activity of ERp. We are currently pursuing the potential of anacardic acids to selectively inhibit COX-2. Furthermore, we are now assessing the ability of anacardic acids to induce apoptosis. Induction of apoptosis may be due to an indirect affect, for example inhibition of COX-2 would lead to a reduction in prostaglandin production thereby allowing apoptosis to proceed. Alternatively, anacardic acids may act as direct inducers of the apoptotic pathway.

SUMMARY AND FUTURE DIRECTIONS Anacardic acids are found in a number of plants, most commonly within the Anacardiaceae family. Based on results of labeling studies, anacardic acids have been proposed to be synthesized from fatty acids by action of a type III polyketide synthase adding acetate units from malonyl-CoA. Utilizing isolated geranium glandular trichome cells, a fatty acid CoA ester, as well as malonyl-CoA and acetate, have been shown to be incorporated into anacardic acids, verifying the proposed polyketide synthesis pathway (Fig. 6.6). Although anacardic acids are known to occur in a number of plants, often as a mixture of related phenolic lipids, in only one plant, the geranium, has a physiological function of anacardic acids been determined. In geranium, anacardic acids function as both a physical trap and as a toxin to impart resistance to small pests. Anacardic acids supplied as part of a diet fed to Colorado potato beetle larvae impact larval weight gain. Thus, bioengineering anacardic acid production into crop plants may provide an endogenous pest-resistance to crop plants. Although anacardic acids do not have a known physiological role in most plants, this phytochemical displays a wide range of bioactivity. One promising area is the role of anacardic acids in inhibiting breast and other cancer cell growth where anacardic acids may have an effect similar to that found for aspirin. Dietary consumption of foodstuffs containing anacardic acids may have chemotherapeutic or chemopreventative actions. Anacardic acids are now consumed in only a limited number of diets including Caju juice made from Cashew apples in Brazil.

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Bioengineered production of anacardic acids into food as chemotherapeutic or chemopreventative (dietary supplement) products may ultimately provide a more readily available consumable source of this phytochemical.

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27. SOWMYALAKSHMI, S., NUR-E-ALAM, M., AKBARSHA, M. A., THIRUGNANAM, S., ROHR, J., CHENDIL, D., Investigation on Semecarpus Lehyam - a Siddha medicine for breast cancer, Planta, 2005, 220, 910-918. 28. GELLERMAN, J. L., WALSH, N. J., WERNER, N. K., SCHLENK, H., Antimicrobial effects of anacardic acids, Can. J. Microbiol, 1969,15, 1219-1223. 29. MUROI, H., KUBO, I., Bactericidal activity of Anacardic Acids against Streptococcus mutatis and their potentiation, J. Agric. Food Chem., 1993, 41, 17801783. 30. KUBO, I., MUROI, H., HIMEJIMA, M., Structure-antibacterial activity relationships of anacardic acids, J. Agric. Food Chem., 1993, 41, 1016-1019. 31. MUROI, H., KUBO, I., Bactericidal effects of anacardic acid and totarol on methicillin-resistant Staphylococcus aureus (MRSA), Biosci. Biotech. Biochem., 1994, 58, 1925-1926. 32. KUBO, J., LEE, J. R., KUBO, I., Anti-Helicobacter pylori agents from the cashew apple, J. Agric. Food Chem., 1999, 47, 533-537. 33. HIMEJIMA, M., KUBO, I., Antibacterial agents from the cashew Anacardium occidentale (Anacardiaceae) nut shell oil, J. Agric. Food Chem., 1991, 39, 418-421. 34. KUBO, I., NIHEI, K. I., TSUJIMOTO, K., Antibacterial action of anacardic acids against methicillin resistant Staphylococcus aureus (MRSA), J. Agric. Food Chem., 2003, 51, 7624-7628. 35. KUBO, I., MUROI, H., KUBO, A., Naturally occurring antiacne agents, J. Nat. Products, 1994,57,9-17. 36. MUROI, H., NIHEI, K., TSUJIMOTO, K., KUBO, I., Synergistic effects of anacardic acids and methicillin against methicillin resistant Staphylococcus aureus, Bioorg. Med. Chem., 2004,12, 583-587. 37. BOUTTIER, S., FOURNIAT, J., GAROFALO, C , GLEYE, C , LAURENS, A., HOCQUEMILLER, R., Beta-lactamase inhibitors from Anacardium occidentale, Pharm. Biol, 2002, 40, 231-234. 38. CAVALCANTE, A. A. M., RUBENSAM, G., PICADA, J. N., DA SILVA, E. G., MOREIRA, J. C. F., HENRIQUES, J. A. P., Mutagenicity, antioxiclant potential, and antimutagenic activity against hydrogen peroxide of cashew {Anacardium occidentale) apple juice and cajuina, Environ. Mol. Mutagen., 2003, 41, 360-369. 39. PRITHIVIRAJ, B., MANICKAM, M., SINGH, U. P., RAY, A. B., Antifungal activity of anacardic acid, a naturally occurring derivative of salicylic acid, Can. J. Bot., 1997,75,207-211. 40. HESK, D., COLLINS, L., CRAIG, R., MUMMA, R. O., Arthropod-resistant and susceptible geraniums, In: Naturally Occurring Pest Bioregulators (P. A. Hedin, Ed.), American Chemical Society, Washington, D.C. 1991, pp. 224-250. 41. GRAZZINI, R., HESK, D., HEININGER, E., HILDENBRANDT, G., REDDY, C. C , COX-FOSTER, D., MEDFORD, J., CRAIG, R., MUMMA, R. O., Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids, Biochem. Biophys. Res. Comm., 1991,176, 775-780. 42. KUBO, I., KINSTHORI, I., YOKOKAWA, Y., Tyrosinase inhibitors from Anacardium occidentale fruits, J. Nat. Products, 1994, 57, 545-551.

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43. ITOKAWA, H., TOTSUKA, N., NAKAHARA, K., TAKEYA, K., LEPOITTEVIN, J.-P., ASAKAWA, Y., Antitumor principles from Ginkgo biloba L., Chem. Pharm. Bull, 1987,35,3016-3020. 44. KUBO, I., OCHI, M., VIEIRA, P. C, KOMATSU, S., Antitumor agents form the cashew {Anacardium occidentale) apple juice, J. Agric. Food Chem., 1993, 41, 1012-1015. 45. LEE, J. S., CHO, Y. S., PARK, F. J., KIM, J., OH, W. K., LEE, H. S., AHN, J. S., Phospholipase Cyl inhibitory principles from the sarcotestas of Ginkgo biloba, J. Nat. Products, 1998, 61, 867-871. 46. VARIER, R. A., SWAMINATHAN, V., BALASUBRAMANYAM, K., KUNDU, T. K., Implications of small molecule activators and inhibitors of histone acetyltransferases in chromatin therapy, Biochem. Pharmacol., 2004, 68, 12151220. 47. BALASUBRAMANYAM, K., SWAMINATHAN, V., RANGANATHAN, A., KUNDU, T. K., Small molecule modulators of histone acetyltransferase p300, J. Biol. Chem., 2003,278, 19134-19140. 48. BHATTACHARYA, S. K., MUKHOPADHYAY, M., MOHAN RAO, P. J. R., BAGCHI, A., RAY, A. B., Pharmacological investigation on sodium salt and acetyl derivative of anacardic acid, Phytother. Res., 1987,1, 127-134. 49. LANGENBACH, R., LOFTIN, C. D., LEE, C , TIANO, H., Cyclooxygenasedeficient mice - A summary of their characteristics and susceptibilities to inflammation and carcinogenesis, In: Cancer Prevention: Novel Nutrient and Pharmaceutical Developments (H.L. Bradlow, J. Fishman, M.P. Osborne, Eds.), New York Academy of Sciences, New York, 1999, pp. 52-61. 50. ARUN, B., GOSS, P., The role of COX-2 inhibition in breast cancer treatment and prevention, Semin. Oncol, 2004, 31, 22-29. 51. PETKOVA, D. K., CLELLAND, C, RONAN, J., PANG, L., COULSON, J. M., LEWIS, S., KNOX, A. J., Overexpression of cyclooxygenase-2 in non-small cell lung cancer, Respir. Med, 2004, 98, 164-172. 52. TERRY, M. B., GAMMON, M. D., ZHANG, F. F., TAWFIK, H., TEITELBAUM, S. L., BRITTON, J. A., SUBBARAMAIAH, K., DANNENBERG, A. J., NEUGUT, A. I., Association of frequency and duration of aspirin use and hormone receptor status with breast cancer risk, J. Am. Med. Assoc, 2004, 291, 2433-2440. 53. TRIPATHI, Y. B., REDDY, M. M., PANDEY, R. S., SUBHASHINI, J., TIWARI, O. P., SINGH, B. K., REDDANNA, P., Anti-inflammatory properties of BHUx, a polyherbal hormulation to prevent atherosclerosis, Inflammopharmacol, 2004, 12, 131-152. 54. AHLEMEYER, B., SELKE, D., SCHAPER, C, KLUMPP, S., KRIEGLSTEIN, J., Ginkgolic acids induce neuronal death and activate protein phosphatase type-2C, Eur. J. Pharmacol, 2001, 430, 1-7. 55. DAVIDSON, S. M., TOWNSEND, P. A., CARROLL, C , YUREK-GEORGE, A., BALASUBRAMANYAM, K., KUNDU, T. K., STEPHANOU, A., PACKHAM, G., GANESAN, A., LATCHMAN, D. S., The transcriptional coactivator p300 plays a critical role in the hypertrophic and protective pathways induced by phenylephrine

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57. 58.

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60. 61.

SCHULTZ, SCHULTZ, et ah al. in cardiac cells but is specific to the hypertrophic effect of urocortin, Chembiochem., 2005,6,162-170. HILAKIVI-CLARKE, L., CABANES, A., OLIVO, S., KERR, L., BOUKER, K. B., CLARKE, R., Do estrogens always increase breast cancer risk?, J. Steroid Biochem. Mol. Biol, 2002, 80, 163-174. KLINGE, C. M., Estrogen receptor interaction with co-activators and co-repressors, Steroids, 2000, 65, 227-251. ZHANG, W. H., MAKELA, S., ANDERSSON, L. C , SALMI, S., SAJL S., WEBSTER, J. I., JENSEN, E. V., NILSSON, S., WARNER, M., GUSTAFSSON, J. A., A role for estrogen receptors in the regulation of growth of the ventral prostate, Proc. Natl. Acad Sci. USA, 2001, 98, 6330-6335. GOTTARDIS, M. M., BISCHOFF, E. D., SHIRLEY, M. A., WAGONER, M. A., LAMPH, W. W., HEYMAN, R. A., Chemoprevention of mammary carcinoma by LGD1069 (Targretin): An RXR-selective ligand, Cancer Res., 1996, 56, 5566-5570. MOON, R. C , MEHTA, R. G., Chemoprevention of mammary cancer by retinoids, Basic Life Science, 1990, 5, 213-224. KNIGHT, D. C , EDEN, J. A., A review of the clinical effects of phytoestrogens, Obstet. Gynecol, 1996, 87, 897-904.

Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Seven

MOLECULAR AND BIOCHEMICAL INVESTIGATIONS OF SORGOLEONE BIOSYNTHESIS Daniel Cook, Franck E. Dayan, Agnes M. Rimando, Zhiqiang Pan, Stephen O. Duke,* and Scott R. Baerson Natural Product Utilization Research Unit Agricultural Research Unit United States Dept. ofAgriculture P. O. Box 8048 University, MS 38677 USA * Author for correspondence, email: sduke(g),olemiss.edu Introduction Sorghum Root Hair EST Analysis and Characterization of O-Methyltransferases Fatty Acid Desaturases Polyketide Synthases Factors Modulating Sorgoleone Production Summary and Future Directions

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INTRODUCTION Sorghum [Sorghum bicolor (L.) Moench] is grown throughout the world as a cereal grain crop, and is also planted in the United States as a green manure or cover crop.1 Injury to some plants grown in rotation with sorghum first suggested that this species was allelopathic.2 Allelopathy, the ability of a plant to suppress the growth of other plants in its vicinity by producing phytotoxins, is in the case of sorghum associated with the production of an exudate from its root hairs (Fig. 7.1). The major component (80 to 90%) of this exudate is the lipid benzoquinone sorgoleone (2-hydroxy-5-methoxy-3-[(8'Z, ll'Z)-8', 11', 14'pentadecatriene]-pbenzoquinone).3 The remaining 10 to 20% consists of several congeners of sorgoleone, differing in their aromatic ring substitutions, and/or in the number of carbons and the level of unsaturation in the tail.4"7 From a chemical ecology standpoint, all of these congeners appear to contribute to the overall allelopathic potential of sorghum since they are all phytotoxic.6'7 Sorgoleone has been found in all genotypes of S. bicolor, as well as all other Sorghum species that have been tested.8'9 This allelochemical is phytotoxic to a wide variety of plant species, with a GR50 as low as 10 uM for Digitaria sanguinalis} Soil amended with sorgoleone is phytotoxic to plants.10 Sorgoleone is generally more toxic to small-seeded plants than to larger seeded species. Ideal soil herbicides strongly adsorb to soil particles so that they do not leach from the root zone. A good soil-applied herbicide must remain in the soil for sufficient time to have the desired effect on weeds, without lingering long enough to be a problem as an environmental pollutant. For synthetic herbicides, a half-life of three to six weeks is desirable, but for an allelochemical that is continuously replenished by the producing plant, a shorter half-life may be adequate for effective weed suppression. Demuner et al.n found the half-life of sorgoleone to be ten days in a red-yellow latosol soil, and neither sorgoleone nor its metabolites could be detected after 60 days. They found sorgoleone to strongly adsorb to soil particles. Another study detected sorgoleone in soil 49 days after application.10 Einhellig and Souza reported that sorgoleone was recovered from soil where sorghum was grown the year before. Simulated root uptake of sorgoleone released to the soil rhizosphere by sorghum was found to be substantial.13 Thus, sorgoleone appears to have the desirable properties for a soil-active herbicide, making it an ideal candidate for manipulation in an annual crop in order to enhance its allelopathic properties. Sorgoleone inhibits several important plant molecular processes, including photosynthetic and mitochondrial electron transport,5' 14 " 7 the enzyme phydroxyphenylpyruvate dioxygenase (HPPD) activity,18 and root H+-ATPase activity and water uptake.19 The inhibitory activity of sorgoleone on photosystem II and HPPD in vitro is greater than that of most synthetic herbicides that target these sites of action. While the primary mechanism of action of sorgoleone as a phytotoxin on

SORGOLEONE BIOSYNTHESIS BIOSYNTHESIS SORGOLEONE

159 159

plants in the field has not yet been determined with certainty, the ability of this natural herbicide to inhibit more than one target site suggests that plant species sensitive to sorgoleone are unlikely to evolve resistance at the molecular target sites. Investigations of the biosynthetic pathway of sorgoleone using retrobiosynthetic NMR analysis of 13C-labeled exudate indicated that it is the result of the convergence of two main pathways. 4 ' 20 The lipophilic 'tail' of sorgoleone is derived from the fatty acid biosynthetic pathway (involving both fatty acid synthase and fatty acid desaturases), and the ring of sorgoleone is the result of the action of a type III polyketide synthase. The resulting lipid resorcinol intermediate is subsequently methylated by a SAM-dependent O-methyltransferase and a P450 monooxygenase to produce the reduced (hydroquinone) form of sorgoleone (Fig. 7.2).20 The release of sorgoleone into the soil appears to occur solely at the tip of the root hairs. Ultrastructural studies have also shown that these specialized cells are highly physiologically active and contain numerous mitochondria and an extensive endomembrane system.21 Therefore, it has been postulated that the biosynthesis of sorgoleone is compartmentalized within these modified epidermal cells. 21 ' 22 Our group has been engaged in identification of putative genes responsible for sorgoleone biosynthesis in Sorghum spp. root hairs, as well as the biochemical characterization of the enzymes encoded by them. Toward this end, an annotated sorghum expressed sequence tag (EST) data set containing approximately 5,500 sequences generated from a root hair-specific cDNA library was analyzed, and highly expressed candidate sequences were found representing all of the enzymes expected to be involved in the final steps of sorgoleone biosynthesis. This review discusses some of the results of these efforts, including the functional characterization of O-methyltransferase, fatty acid desaturase, and polyketide synthase enzymes proposed to be involved in this unique pathway.

SORGHUM ROOT HAIR EST ANALYSIS AND CHARACTERIZATION OF O-METHYTRANSFERASES As mentioned, our current understanding of the sorgoleone biosynthetic pathway20 suggests the participation of three different enzyme classes for biosynthesis starting with an acyl-CoA starter molecule and malonyl-CoA (Fig. 7.2). In addition, novel fatty acid desaturases would be required to generate the A912'15 double bond configuration of the proposed C16:3-CoA precursor (Fig. 7.2). We have, therefore, targeted fatty acid desaturases, polyketide synthases, Omethyltransferases, and cytochrome P450s from Sorghum bicolor for obtaining candidate sequences for subsequent biochemical studies.

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Figure 7.1: Photomicrograph of Sorghum bicolor roots showing A) sorgoleone-rich oily exudate secreted from the root hairs (Bar = 80 um) and B) closer view of a root hair with sorgoleone exuding at the tip (Bar = 15 (am) From Dayan.37

The S. bicolor cultivar BTX623 is represented by the majority of the over 208,000 public S. bicolor ESTs (http://www.tigr.org), and was chosen as the experimental system for these studies. Large-scale preparations of root hair tissue were obtained using seedlings grown on a capillary mat system developed by Czamota and co-workers,23 and root hairs were isolated using the method of Bucher et al24 (Fig. 7.3). RNA extracted from this material was used for the construction of a directional cDNA library, from which an EST database was generated. Library clones (6,624) were sequenced at random, yielding 5,469 ESTs of sufficient quality. The average EST length is 451 bp, using a moving window with a Phred quality score of 16 (corresponding to approximately 97.5% accuracy).

SORGOLEONE BIOSYNTHESIS BIOSYNTHESIS SORGOLEONE O

O

CoA

O

S

O

O

O O

O

O

O

O

O

O O O

O

O

O

O

Δ-9,12,15-C16:3-CoA

161 161

O

S-Enzyme O

O

O

O

PKS

O O

O

O

O

O

O

O

O

O

C16:3 HO

11 O

CoA S

O

O

MGD

O

O

O

O

O

O

O

O

CoA S

OH

O

O

O

O

O

O

O

CoA S

.OH OH

O f*

4

m

O "^f O

O

O

O

O

O

O

5-pentadecatrienyl resorcinol-3-methylether resorcinol-3-methylether 5-pentadecatrienyl

O

OH O0" f^V O O

HO O Her*-*

acetate acetate

P450

OH

^

O O O O O O •xO
O

H O H OH

\ ^ S - aS-adenosyl denosyl homocysteii lOCysteine homocysteine

'

' Oo-O OVO

0H OH

O

H

O

O

O

HO

OMT OMT

O

OH

O

acetyl-CoA acetyl-CoA

O HO HCT^ H OH

O

O

O

O

palmitoyl-ACP palmitoyl-ACP

HO

O

O

SAM

O O

O

NH,2 NH

OH

FAS

O

O

resorcinol 5-pentadecatrienyl resorcinol

S+

O

O

O

adenosyl adenosyl

CoA S O

O

malonyl-CoA malonyl-CoA

O

C16:0

ACP-S

O

O

O

XX

O

O

CO2

OH

O

FAD FAD

O

PKS

3x MGD

O

O

O

dihydrosorgoleone dihydrosorgoleone

OH

autooxidation autooxidation

H O

D-glucose D-glucose O O

O

O

O

I O O

O O

OH O

O

O

O

O

O

O

sorgoleone

Figure 7.2: Biosynthetic pathway of sorgoleone showing the incorporation pattern obtained with 13C-labeled substrates. FAS = fatty acid synthase; FAD = fatty acid desaturase; PKS = polyketide synthase; OMT = SAMdependent Omethyltransferase; P450 = P450 monooxygenase. Green = 213 C-D-glucose; Blue = 2-13C-acetate; Red = 13C-methyl-L-methionine (Adapted from Dayan et a/.20). (See Cover).

COOK, et al. COOK,etal.

162 162

.-

-^.

Figure 7.3: Representative root hair cell preparation. Brightfield light micrograph of S. bicolor root hair cell preparation is shown, obtained by treating whole root systems with liquid nitrogen, followed by filtration through a 250 uM steel mesh, as described by Bucher et al24

The EST data for candidate fatty acid desaturase, polyketide synthase, Omethyltransferase, and cytochrome P450-like sequences was mined by using both the Magic Gene Discovery software,25 and BLAST searches with functionallycharacterized protein sequences as queries against the EST dataset conceptually translated in all possible reading frames. From these analyses, 47 fatty acid desaturase, 9 polyketide synthase, 94 methyltransferase, and 21 P450-like ESTs were identified (Table 7.1). Assembly of the EST data into contigs suggested the representation of up to 15 different fatty acid desaturase-like, 5 polyketide synthaselike, 35 Omethyltransferase-like, and 33 P450-like sequences within the dataset (Table 7.1).

SORGOLEONE BIOSYNTHESIS BIOSYNTHESIS SORGOLEONE

163 163

Table 7.1: Sequences identified in root hair EST database Family

Clones

Root hairspecific Desaturase 11 47 4 0.859 3 Polyketide synthase 9 5 3 0.165 3 OMethyltransferase 94 23 12 1.72 >3fl P450 21 18 15 0.384 4 "total number of root hair-specific methyltransferases represented in the data may be greater than three. Contigs

Singletons

%,Total

Given that the sorgoleone biosynthetic pathway may be exclusively localized to root hair cells,21 it is reasonable to speculate that the genes encoding the biosynthetic enzymes for this pathway are specifically or preferentially expressed in this cell type. A secondary screen that used real-time PCR was employed to prioritize sequences for further biochemical characterization. Real time PCR determined expression levels of the candidates in the different tissue types from sorghum including mature leaves, immature leaves, panicles, apices, mature stems, roots, and root hairs. Gene-specific Sybr Green I assays were developed for the fatty acid desaturase, polyketide synthase, O-methyltransferase, and P450-like contigs. Remarkably, we were able to identify 3-4 candidate gene sequences from each enzyme family that were preferentially expressed in root hairs. To date, full-length open reading frames for most of these sequences have been generated and subcloned into E. coli expression vectors, or for cytochrome P450 and fatty acid desaturase-like sequences, vectors engineered for heterologous expression in Saccharomyces cerevisiae. For three O-methyltransferase-like candidate sequences that exhibited root hair-preferential expression patterns, recombinant enzymes were tested for activity with various benzene-derived substrates, including a series of 5-substituted alkylresorcinols with alkyl chain lengths ranging from 1-15 carbons (Fig. 7.4). Significantly, one of the three O-methyltransferase clones (designated SbOmt3), preferentially utilized 5-substituted alkyl-resorcinols among all of the substrates analyzed. 5-Pentadecatrienyl resorcinol (Fig. 7.2), the proposed in vivo substrate for the O-methyltransferase involved in sorgoleone biosynthesis, is closely related to these compounds. Thus, SbOMT3 could represent an O-methyltransferase participating in this pathway. Of significance, among all previously characterized plant enzymes, SbOmt3 is most closely related to an orcinol-specific (5-methylresorcinol-specific) <9-methyltransferase identified from Rosa hybrida.26

COOK, et al. COOK,etal.

164 164 /M-dihydroxy

o-methoxy-hydroxy

OH

resorcinol OH

orcinol

guaiacol

ferulic acid OH

HOOC

/M-methoxy-hydroxy

OH eugenol

[|

resorcinol monomethyl ether OH alkyl-resorcinols of increasing chain length

orcinol monomethyl ether OH

5-propyl-resorcinol OH

p-hydroxy OH

p-coumaric acid

[j

5-pentyl-resorcinol (olivetol) OH

HOO< 4-methoxyphenol 5-heptyl-resorcinol

H3CO

o-dihydroxy

catechol

caffeic acid

[j

OH OH

HOOC

Figure 7.4: Determination of substrate specificity for recombinant S. bicolor O-methyltransferases. Structures are shown for the various benzene-derived compounds tested in O-methyltransferase recombinant enzymatic assays.

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FATTY ACID DESATURASES One of the characteristic properties of sorgoleone is the unsaturated aliphatic side chain with a terminal double bond (Fig. 7.5). The polyunsaturated C15 chain of the molecule is synthesized by the modification of a saturated fatty acid precursor, a product of fatty acid synthesis. The introduction of the first double bond between carbons 9 and 10 in the Ci6 fatty acid precursor requires a soluble acyl carrier protein (ACP) desaturase, most likely a tissue-specific 16:0-ACP A9-desaturase, an enzyme yet to be characterized in sorghum. The subsequent conversion of the unsaturated fatty acid 16:lA 9 to 16:1A912'15 likely takes place sequentially by the reactions of ERlocalized membrane-bound desaturases.

B

D

H3C0

Figure 7.5: Chemical structures of various phenolic lipids (A) urushiol, (B) anacardic acid, (C) heptadecenyl resorcinol, and (D) sorgoleone.

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Two cDNA clones from sorghum root hairs, designated as SbDes2 and SbDes3, were identified from the root hair EST database as as being highly expressed in root hairs, and these were functionally characterized. Analysis of the deduced protein sequences by using the NCBI PSI-Blast program revealed that SbDes2 was significantly similar to the known plant fatty acid desaturase (or FAD2) sequences, and SbDes3 displayed high similarity to plant FAD3 sequences. Based on comparisons to known FADs, the predicted protein sequences exhibited all of the main structural characteristics possessed by FADs from other systems. Among the conserved amino acids in the two sorghum desaturase sequences were eight histidines that have been shown to be essential for desaturase activity (Fig. 7.6).27 These conserved histidine-rich motifs are most likely involved in the coordination of the diiron center of the active site.28 Like all plant desaturases, neither SbDes2 nor SbDes3 contains a fused cytochrome Z»5 domain, the electron donor required for desaturation by microsomal enzymes. Thus, it is assumed that they interact with a separate cytochrome b5. In addition, analysis of SbDes2 and SbDes3 sequences using the method of Kyte and Doolittle,29 and the program SOSUI for transmembrane helix prediction,30 revealed that all these proteins contain the four potential hydrophobic helices corresponding to membrane spanning domains predicted in the topological model developed for the membrane-bound desaturases.27 In addition to the four hydrophobic domains, the SbDes2 sequence exhibited two additional hydrophobic segments (Fig. 7.6). The existence of six transmembrane domains in SbDes2 could potentially impart the substrate/product relationships and the membrane topology of the enzyme, as well as the substrate specificity of the enzyme. Functional analyses of these clones in a yeast system showed that both SbDes2 and SbDes3 were capable of desaturating fatty acyl chains. Expression of SbDes2 in yeast resulted in the desaturation of palmitoleic acid (16:1 A9) to hexadecadienoic acid (16:2A9'12). Unlike other plant membrane-bound FAD2-type desaturases, most of which possess bifunctional or multifunctional activities, this sorghum A12-desaturase acts only on palmitoleic acid. On the other hand, expression of SbDes3 alone in yeast did not yield any new product. However, co-expression of SbDes2 and SbDes3 in yeast cells resulted in the presence of a new fatty acid which was identified as 9,12,15(9Z, 12Z)-hexadecatrienoic acid (16:1A9'12'15) by gas chromatography - mass spectrometry (GC-MS), and it was further confirmed by nuclear magnetic resonance (NMR) spectroscopy. The result of the co-expression demonstrated that the sorghum SbDes3 uniquely catalyzes the conversion of 16:2A912 to 16:3A912'15, which was proposed to serve as the starter molecule for PKS in the biosynthesis of the allelochemical sorgoleone (Fig. 7.2).

SORGOLEONE BIOSYNTHESIS SORGOLEONE BIOSYNTHESIS 110

I

146

167 167 321

I

385

SbDes2 103

306

139 |

389

SbDes3

ISP

Box1 (HXXXH)

Box 2 (HXXHH)

Box 3 (HXXHH)

Figure 7.6: Hydrophobic structure and the location of His-containing regions of sorghum fatty acid desaturases. Large boxes represent hydrophobic domains containing 23 amino acids. The locations of His motifs are indicated by small boxes and their locations are indicated by amino acid numbers.

POLYKETIDE SYNTHASES The different types of polyketide synthases catalyze the sequential condensation of two-carbon acetate units derived from a malonate thioester into a growing polyketide chain. Polyketide synthases are responsible for the synthesis of an array of natural products, including antibiotics such as tetracycline in bacteria, and mycotoxins such as aflatoxin in fungi. Furthermore, in plants these enzymes are part of the biosynthetic machinery of anthocyanins, antimicrobial phytoalexins, and phenolic lipids. Detailed reviews on polyketide synthases have recently been written.31"33 Type III polyketide synthases catalyze the iterative condensation of acetyl units derived from malonyl-CoA to a CoA-linked starter molecule. This extension is usually followed by the cyclization of the linear polyketide. A number of bacterial and plant type III polyketide synthases have been characterized that differ in the CoA linked starter molecule, the number of extensions, and the intramolecular cyclization mechanisms.33 Figure 7.7 shows a comparison of the reactions and products of a number of characterized divergent polyketide synthases. From this figure it can be observed that type III polyketide synthases are responsible for producing a large diversity of resulting molecules. A novel type III polyketide synthase catalyzes the crucial step in the formation of the pentadecatriene resorcinol intermediate in sorgoleone biosynthesis. Significantly, no polyketide synthases to date have been described that can use a

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long chain (greater than octanoyl) acyl-CoA starter to form a phlorglucinol or stilbene product. As described above, nine polyketide synthase-like ESTs were identified in the root hair EST data set (Table 7.1). To identify candidate polyketide synthases whose expression levels correlated with the accumulation of sorgoleone, the secondary screen that used real-time PCR decribed above was employed. From these analyses, three candidate polyketide synthase genes were identified showing their highest expression levels in root hairs (Table 7.1). Full-length coding sequences of the three candidates were generated by RACE (Rapid Amplification of cDNA Ends) and subcloned into E. coli expression vectors. Full-length open reading frames for all three candidates were overexpressed in E. coli, and recombinant protein was purified by using an activated Ni-column. Acyl-CoAs varying in chain length and saturation were tested in enzyme assays with all three recombinant proteins to determine their substrate specificity. In addition, various other substituted CoAs were tested as potential substrates. Most type III plant polyketide synthases characterized to date utilize substrates such as short chain acyl-CoAs that may be branched, and CoAs linked to different phenylpropanoid derivatives such as coumaroyl-CoA and benzoyl-CoA.33 Significantly, two of the polyketide synthase candidates identified from sorghum root hairs preferentially utilized long chain acyl-CoA substrates to make alkylresorcinols. Furthermore, A9',12',15'-hexadecatrienoyl-CoA, the major in vivo substrate used by the polyketide synthase involved in sorgoleone biosynthesis, is also accepted by these same two enzymes to form pentadecatriene resorcinol. It is notable that these two polyketide synthases do not utilize the smaller substrates, suggesting that these enzymes belong to a new class of polyketide synthases dedicated to the synthesis of alkylresorcinols. The third candidate polyketide synthase showed no activity towards any of the substrates tested. A phylogenetic tree was constructed by using a number of characterized and uncharacterized type III polyketide synthases (Fig. 7.8). The predicted protein sequences of the two active polyketide synthases cluster with a small family of putative polyketide synthases from rice whose function has yet to be established. Interestingly, rice is also known to synthesize alkylresorcinols.34 Thus, this small family of polyketide synthases may be responsible for their biosynthesis. Furthermore, identification and characterization of this novel polyketide synthase may help in identifying the polyketide synthases that are important in the biosynthesis of a small group of natural products with varied bioactivities and uses, the phenolic lipids.35 Examples of phenolic lipids are shown in Figure 7.5 and include urushiol, an allergen from poison ivy, anacardic acid, an anti-feedant, alkylresorcinols from various grasses possessing antifungal activity, and sorgoleone. In addition, phenolic lipids are important for the synthesis of formaldehyde-based polymers used in the automobile industry and are important in some countries in the lacquering process.

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169 169

- 2x O Acetyl-CoA

0 0 Malonyl-CoA

CoAS

p-Coumaryl-CoA OH

CoAS.

If OH O 2,3',4,6-Tetrahydroxybenzophenone

3-Hydroxybenzoyl-CoA CoA: O I Isovaleroyl-CoA

"""

O O Malonyl-CoA

OH p-Coumaryl-CoA

O

Naringenin chalcone

YY"

0 0 Malonyl-CoA

*

Tl..

T I

PCS C6—>C1, Claisen OH

O

5,7-Dihyroxy-2-methylchromone

• 6x O Acetyl-CoA

0 0 Malonyl-CoA

8x Malonyl-CoA

Figure 7.7: Comparison of the reactions and products from a number of plant type 111 polyketide synthases.

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COOK, et al. COOK,etal. Pinus sylvestris CHS (P30079) — Pinusstrobus(CAA05214) i Pinus sylvestris STS1 (Q02323) 1 PinusstrobusSTS(CAA87012) Psilotum nudum (BAA87922) Equisetum arvense CHS (Q9MBB1) — Medicago sativa CHS2 (P30074) — Glycine max (JQ2250) — Anthurium andraeanum (AAP20864) — Rubus idaeus (AAM90650) •— Camellia sinensis CHS2 (P48387) — Hydrangea macrophylla CHS (AAN76184) - Hypericum androaemum CHS (AAG30295) - Petunia hybrida (AAB36038) • Petunia hybrida (CAA32737) Ipomoea nil CHSD (022045) - Arabidopsisthaliana(AAM65314) Ruta graveolens CHS3 (Q9FSB7) - Lilium hybrida CHS (AAD49354) - Gerbera hybrida CHS (CAA86218) Triticum aestivum (AAQ19318) Sorghum bicolor CHS2 (AAD41874) — Sorghum bicolor STS (AAL49965) Bromheadia finlaysoniana (AAB62874) Rheum palmatum BAS (AAK82824) Hydrangea macrophylla STCS (AAN76182) Humulus lupulus VPS (BAB12102) VitisviniferaSTSI (P28343) - Ruta graveolens ACS (CAC14058) — Gerbera hybrida 2 PS (CAA86219) i— Sorghum bicolor PKS2 ' — Sorghum bicolor PKS3 Oryza sativa PKS3 r Oryza sativa PKS1 L Oryza sativa PKS2 Sorghum bicolor PKS1 Psilotum nudum STS (BAA87924) Psilotum nudum VPS (Q9SLX9) Bromheadia finlaysoniana BBS (CAA10514 Hypericum androsaemum BPS (AAL79808)

Figure 7.8: Phylogenetic tree showing the potential relationships between type III plant polyketide synthases.

SORGOLEONE BIOSYNTHESIS BIOSYNTHESIS SORGOLEONE

171

FACTORS MODULATING SORGOLEONE PRODUCTION Sorgoleone has been found in various amounts in all sorghum cultivars tested to date. In one study, the levels varied considerably, ranging from 0.67 to 17.8 mg sorgoleone per g root fresh weight. However, other studies have shown that there is very little variation in sorgoleone amount across germplasms.9'36 Although present ubiquitously in Sorghum spp., the factors modulating its biosynthesis are not well known. Production of sorgoleone is constitutive in the root hairs of developing plants in their early stages of development.37 Sorgoleone production is optimal at temperatures ranging from 25 to 35°C and decreases markedly outside this range (Fig. 7.9), suggesting that the allelopathic potential of this cover crop may be compromised in the field at temperatures outside the 25 to 35 °C range. Although light did not greatly affect root formation, the level of sorgoleone produced was reduced by nearly 50% upon exposure to blue light (470 nm) and by 23 % with red light (670 nm). Far-red light had a negligible effect. It is also noteworthy that the application of mechanical pressure over developing seedlings stimulated root formation, but this did not result in higher levels of this lipid benzoquinone in the root exudate. Sorgoleone production is stimulated in seedlings exposed to a crude watersoluble velvetleaf (Abutilon theophrasti Medik.) root extract (Fig. 7.10). This stimulation was not associated with increased osmotic stress, since decreases in water potential by increasing solute concentrations with sorbitol reduces sorgoleone production. Therefore, sorghum seedlings may respond to the presence of other plants by releasing more of this allelochemical.37 A similar phenomenon was reported with the stimulation of production of allelochemicals in rice seedlings grown in the presence of barnyardgrass.38 Although the concentrations of velvetleaf root extract required to elicit sorgoleone synthesis are relatively high, the more complex plant-plant interactions that occur when the rhizospheres of these two species interact in a natural environment may result in a similar elicitation of sorgoleone production at much lower concentrations of the velevetleaf elicitor(s).

172 172

COOK, al. COOK,etetal. Oft

25

OU

c*

70 -

E, D)

:oot dry

1

^_^

C _

T3

c

60 /

- 20

)

50 -

_

- 15

A

i

40 30 20 -

\ \

10 -

b

- 10

c

\

1 1

Q:

d

r :

—•— Root

\ \

Sorgoleone ,— ,

3

20

25

30

O O i_ D)

E

1 0

c -5

"o

CD

o

CO

— • —

0 -

15

\

I

H-.

0

35

40

Temperature (C) Figure 7.9: Effect of temperature on root formation and the production of sorgoleone in Sorghum bicolor cv. SX17. Sorgoleone was extracted after 7 days of growth in the dark. Means with the same letter above (root) or below (sorgoleone) the symbols are not different at P < 0.05 according to Tukey's test. From Dayan.37

SORGOLEONE SORGOLEONE BIOSYNTHESIS BIOSYNTHESIS

173 173

22 60 -

- 21

ab

o o

b

- 20

b

- 18

20 -ab >ab

Root Sorgoleone

1 eone (

- 19

o E> o

- 17

CO

16 0

2

4

6

8

10

Velvetleaf extract (mg/ml) Figure 7.10: Effect of velvetleaf root extract on root formation and the production of sorgoleone in Sorghum bicolor cv. SX17. Sorgoleone was extracted after 7 days of growth in the dark at 30°C. Means with the same letter above (root) or below (sorgoleone) the symbols are not different at P < 0.05 according to Tukey's test. From Dayan.37

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SUMMARY AND FUTURE DIRECTIONS We have pursued a strategy based on the analysis of expressed sequence tags to identify genes involved in the biosynthesis of the allelochemical sorgoleone. This approach, coupled with high-throughput gene expression analysis that uses quantitative real-time RT-PCR, has provided a highly efficient means for identifying candidate fatty acid desaturase, polyketide synthase, O-methyltransferase, and P450like sequences preferentially expressed in S. bicolor root hair cells. This has led to significant progress in the characterization of O-methyltransferase, fatty acid desaturase, and polyketide synthase enzymes potentially involved in sorgoleone biosynthesis. Recent progress has also been made in determining the environmental and developmental factors that affect sorgoleone accumulation. In addition, the annotated dataset comprised of 5,469 5' sorghum root hair EST sequences that we have generated will directly complement the existing approximately 208,000 public sorghum EST sequences, and expand our understanding of the transcriptome of a highly specialized and unique cell type. The identification of the putative biosynthetic genes involved in sorgoleone production now provides us with the necessary tools to initiate experiments designed to manipulate this pathway in planta. In addition, through the use of promoter/reporter gene fusion constructs, we will obtain basic information on the normal cell-specific expression patterns, as well as confirm the physiological roles of the fatty acid desaturase, polyketide synthase, and 0-methyltransferase sequences we have isolated and characterized. Sorghum bicolor has been one of the more recalcitrant crop species with respect to ease of regeneration in vitro from various explant sources. However, in recent years significant strides have been made toward the development of more efficient transformation protocols (e.g., Gao et al.39). In collaboration with other research groups, experiments are currently underway to generate sorghum transformants with an emphasis on the production of overexpression lines, RNA interference (RNAi) lines, and lines harboring promoter: :reporter gene fusions for the polyketide synthase genes and the desaturase gene involved in the production of the pentadecatrienyl resorcinol intermediate and C16:3 A9,12,15 fatty acid precursors, respectively. These experiments will prove or disprove the role of the putative genes of the sorgoleone pathway that we have discovered. This could ultimately pave the way for the development of highly allelopathic elite sorghum varieties through the use of genetic engineering. REFERENCES 1. WESTON, L.A., Utilization of allelopathy for weed management in agroecosystems. Agron. J., 1996, 88, 860-866. 2. BREAZEALE, J.F., The injurious after-effects of sorghum, J. Am. Soc. Agron., 1924, 16, 689-700.

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NETZLY, D.H., BUTLER, L.G., Roots of sorghum exude hydrophobic droplets containing biologically active components. Crop Sci., 1986, 26, 775-778. 4. FATE, G.D., LYNN, D.G., Xenognosin methylation is critical in defining the chemical potential gradient that regulates the spatial distribution in Striga pathogenesis. J. Am. Chem. Soc, 1996, 118, 11369-11376. 5. RIMANDO, A.M., DAY AN, F.E., CZARNOTA, M.A., WESTON, L.A., DUKE, S.O., A new photosystem II electron transfer inhibitor from Sorghum bicolor. J. Nat. Prod., 1998, 61, 972-930. 6. RIMANDO, A.M., DAYAN, F.E., STREIBIG, J.C., PSII inhibitory activity of resorcinolic lipids from Sorghum bicolor. J. Nat. Prod., 2003, 66, 42-45. 7. KAGAN, I.A., RIMANDO, A.M., DAYAN, F.E., Chromatographic separation and in vitro activity of sorgoleone congeners from the roots of Sorghum bicolor. J. Agric. Food Chem., 51, 7589-7595. 8. NIMBAL, C.I., PEDERSEN, J.F., YERKES, C.N., WESTON, L.A., WELLER, S.C., Phytotoxicity and distribution of sorgoleone in grain sorghum germplasm. J. Agric. Food Chem., 1996,44, 1343-1347. 9. CZARNOTA, M.A., RIMANDO, A.M., WESTON, L.A., Evaluation of root exudates of seven sorghum accessions. J. Chem. Ecol. 2003, 29, 2073-2083. 10. WESTON, L.A., CZARNOTA, M.A., Activity and persistence of sorgoleone, a longchain hydroquinone produced by Sorghum bocolor. J. Crop Product. 2001, 4, 363-377. 11. DEMUNER, A.J, BARBOSA, L.C.A., CHINELATTO, L.S., Jr., REIS, C, SILVA, A.A., Sorption and persistence of sorgoleone in Red-Yellow Latosol. Quimica Nova, 2005,28,451-455. 12. EINHELLIG, F.A, SOUZA, F. Phytotoxicity of sorgoleone found in grain sorghum root exudates. J. Chem. Ecol, 1992,18, 1-11. 13. WEIDENHAMER, J.D., Biomimetic measurement of allelochemical dynamics in the rhizosphere. J. Chem. Ecol. 2005, 31, 221-236. 14. NIMBAL, C.I., YERKES, C.N., WESTON, L.A., WELLER, S.C., Herbicidal activity and site of action of the natural product sorgoleone. Pestic. Biohchem. Physiol. 1996, 54,73-83. 15. RASMUSSEN, J.A., HEJL, A.M., EINHELLIG, F.A., THOMAS, J.A., Sorgoleone from root exudates inhibits mitochondrial functions. J. Chem. Ecol, 1992,18, 197-207. 16. EINHELLIG, F.A., RASMUSSEN, J.A., HEJL, A.M., SOUZA, I.F., Effects of root exudate sorgoleone on photosynthesis. J. Chem. Ecol, 1993,19, 369-375. 17. GONZALEZ, V.M., KAZIMIR, J., NIMBAL, C, WESTON, L.A., CHENIAE, G.M., Inhibition of a photosystem II electron transfer reaction by the natural product sorgoleone. J. Agric. FoodChem., 1997, 45, 1415-1421. 18. MEAZZA, G., SCHEFFLER, B.E., TELLEZ, M.R., RIMANDO, A.M., NANAYAKKARA, N.P.D., KHAN, I.A., ABOURASHED, E.A., ROMAGNI, J.G., DUKE, S.O., DAYAN, F.E., The inhibitory activity of natural products on plant phydroxyphenylpyruvate dioxygenase. Phytochemistry, 2002, 59, 281-288. 19. HEJL, A.M., KOSTER, K.L., The allelochemical sorgoleone inhibits root H+-ATPase and water uptake. J. Chem. Ecol, 2004, 30, 2181-2191.

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20. DAY AN, F.E., KAGAN, I. A., RIMANDO, A. M., Elucidation of the biosynthetic pathway of the allelochemical sorgoleone using retrobiosynthetic NMR analysis. J. Biol. Chem., 2003, 278, 28607-28611. 21. CZARNOTA, M.A., PAUL, R.N., WESTON, L.A., DUKE, S.O., Anatomy of sorgoleone-secreting root hairs of Sorghum species. Internat. J. Plant. Set, 2003, 164, 861-866. 22. YANG, X., OWENS, T.G., SCHEFFLER, B.E., WESTON, L.A., Manipulation of root hair development and sorgoleone production in sorghum seedlings. J. Chem. Ecol, 2004,30, 199-213. 23. CZARNOTA, M.A., PAUL, R.N., DAYAN, F.E., NIMBAL, C.I., WESTON, L.E. Mode of action, localization of production, chemical nature, and activity of sorgoleone: A potent PSII inhibitor in Sorghum spp. root exudates. Weed Technol, 2001, 15, 813825. 24. BUCHER, M., SCHROEER, B., WILLMITZER, L., RIESMEIER, J.W., Two genes encoding extension-like proteins are predominantly expressed in tomato root hair cells. Plant Mol. Biol. 1997, 35,497-508. 25. CORDONNIER-PRATT, M-M., LIANG, C, WANG, H., KOYCHEV, D.S., SUN, F., FREEMAN, R., SULLIVAN, R., PRATT, L.H., MAGIC Database and interfaces: an integrated package for gene discovery and expression. Comp. Funct. Genom. 2004, 5, 268-275. 26. LAVID, N., WANG, J., SHALIT, M., GUTERMAN, I., BAR, E.; BEUERLE, T., MENDA, N., SHAFIR, S., ZAMIR, D., ADAM, Z., VAINSTEIN, A., WEISS, D., PICHERSKY, E., LEWINSOHN, E., O-Methyltransferases involved in the biosynthesis of volatile phenolic derivatives in rose petals. Plant Physiol. 2002, 129, 1899-1907. 27. SHANKLIN, J., WHITTLE, E., FOX, B.G., Eight histidine residues are catalytically essential in a membrane-associated iron enzyme, stearoyl-CoA desaturase, and are conserved in alkane hydroxylase and xylene monooxygenaset. Biochemistry, 1994, 33, 12787-12794. 28. SHANKLIN, J., CAHOON, E.B., Desaturation and related modifications of fatty acids. Annu. Rev. Plant Physiol. Plant Mol. Biol, 1998, 49, 611-641. 29. KYTE, J., DOOLITTLE, R.F., A simple method for displaying the hydropathic character of a protein. J. Mol. Biol, 1982,157, 105-132. 30. HIROKAWA, T., BOON-CHIENG, S., MITAKU, S., SOSUI: classification and secondary structure prediction system for membrane proteins. Bioinformatics, 1998, 14, 378-379. 31. RAWLINGS, B.J., Type I poyketide biosynthesis in bacteria (part A-erythromycin biosynthesis). Nat. Prod. Rep. 2001,18, 190-227. 32. RAWLINGS, B J., Biosynthesis of polyketides (other than actinomycete macrolides). Nat. Prod Rep. 1999,16, 425-484. 33. AUSTIN, M.B., NOEL J.P., The chalcone synthase superfamily of type III polyketide synthases. Nat. Prod. Rep. 2003, 20, 79-110 34. BOUILLANT, M.L., JACOUD, C, ZANELLA, I, FAVRE-BONVIN, J. BALLY, R., Identification of 5-(12-Heptadecenyl)-resorcinol in rice root exudates. Phytochemistry 1994,35,769-771.

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35. KOZUBEK, A., TYMAN, J.H.P., Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem. Rev. 1999, 99, 1 36. HESS, D.E., EJETA, G., BUTTLER, L.G., Selection of sorghum genotypes expressing a quantitative biosynthetic trait that confers resistance to Striga. Phytochemistry 1992, 31, 493-497. 37. DAYAN, F.E., Factors modulating the levels of the allelochemical sorgoleone in Sorghum bicolor, Planta, 2006, in press. 38. KONG, C, XU, X., ZHOU, B., HU, F., ZHANG, C, ZHANG, M., Two compounds from allelopathic rice accession and their inhibitory activity on weeds and fungal pathogens. Phytochemistry 2004, 65, 1123-1128. 39. GAO, Z., JAYARAJ, J., MUTHUKRISNNAN, S., CLAFLIN, L., LIANG, G.H. Efficient genetic transformation of Sorghum using a visual screening marker. Genome 2005,48,321-333.

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Recent Recent Advances Advances in in Phytochemistry, Phytochemistry, vol. vol. 40 40 John T. Romeo (Editor) © © 2006 2006 Elsevier Elsevier Ltd. Ltd. All All rights rights reserved. reserved.

Chapter Eight

BIOSYNTHESIS OF TERPENOPHENOLIC METABOLITES IN HOP AND CANNABIS Jonathan E. Page* and Jana Nagel Plant Biotechnology Institute National Research Council of Canada 110 Gymnasium Place Saskatoon, SK, Canada S7N0W9 *Author for correspondence, email: ionathan.page(5>,nrc-cnrc. gc.ca

Introduction Phytochemistry of Cannabis sativa Phytochemistry of Humulus lupulus Biological Activities of Terpenophenolics from Cannabaceae Cannabinoids Hop Terpenophenolics Biosynthesis of Terpenophenolics Cannabinoid Biosynthesis Bitter Acid Biosynthesis Prenylflavonoid Biosynthesis Terpenophenolic Biosynthesis in Glandular Trichomes EST Genomics of Terpenophenolic Biosynthesis in Hop Trichomes Summary and Future Directions

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INTRODUCTION Plants in the family Cannabaceae (or Cannabinaceae) are well known for their content of biologically-active terpenophenolic metabolites. Examples of important terpenophenolics from this family include humulone in the hop plant (Humulus lupulus), which contributes to the bitter flavor of beer, and A9tetrahydrocannabinol, the main psychoactive drug in cannabis (Cannabis sativa; hemp, marijuana). Terpenophenolics have a mixed biosynthetic origin and are composed of a polyketide-derived phenolic core structure modified with isoprenoidderived prenyl side-chains. While phytochemical investigations over the last 50 years have revealed the structural diversity of terpenophenolics in Cannabaceae, the biosynthetic pathways leading to these metabolites have only begun to be understood at the biochemical and genetic level. This review focuses on recent advances in understanding how cannabis and hop synthesize terpenophenolics, and the biochemical and genomic techniques being used to identify genes and proteins involved in their biosynthesis. Terpenophenolic natural products are not unique to the Cannabaceae, although they do appear to have a limited occurrence in plants. Barron and Ibrahim reviewed the distribution of prenylated flavonoids in plants and found more than 700 different compounds to be present.1 Many of these were described from the Leguminosae and Moraceae. The pharmacological activities of prenylated flavonoids have been the subject of a recent review.2 Flavonoids are not the only phenolic molecules prenylated by plants, and other species prenylate simple polyketides such as phloroglucinol derivatives. Members of the Guttiferae (Clusiaceae) are particularly rich in such compounds with notable examples being the presence of hyperforin and hypercalin in Hypericum species and prenylated phloroglucinols and xanthones in the tropical genus Garcinia. Cannabis and Humulus are the only two genera of Cannabaceae and their close evolutionary relationship is demonstrated by the viability of intergeneric grafts. Taxonomic divisions of the genus Cannabis have proved controversial with some authors describing a single species, Cannabis sativa, divided into two subspecies, C. sativa subsp. sativa and subsp. indica, while others have split the genus into three species, C. sativa, C. indica, and C. ruderalis. ' Since most phytochemical investigations have been conducted on plant material that would be classified as C. sativa, for the purpose of this review, we use the term cannabis to denote C. sativa. Humulus is also divided into three species: Humulus lupulus (cultivated hop), H. japonicus, and H. yunnanensis. The latter two species are not used for brewing, and our review, therefore, restricts itself to terpenophenolic metabolism in H. lupulus or hop. We use hops to refer to the female inflorescences (strobiles or cones) used for brewing. Two excellent reference volumes on the biology and cultivation of hop and cannabis have been published.9'10

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PHYTOCHEMISTRY OF CANNABIS SATIVA Cannabis sativa is a dioecious annual plant that can attain heights of up to 3 m in one season. Although its center of origin is presumed to be Central Asia, today cannabis is cultivated worldwide for industrial purposes, yielding fiber, seeds, and seed oil, and as a drug and medicinal plant. The latter uses are due to its content of psychoactive terpenophenolics collectively termed cannabinoids. The principal pharmacologically active cannabinoid is A9-tetrahydrocannabinol (THC).11 THC, cannabis and its derivatives such as hashish are prohibited substances in most parts of the world. High-THC cannabis varieties grown for drug or medicinal use are termed marijuana while low-THC varieties cultivated for fiber and oil production are termed hemp. Many countries in which hemp is grown set a level of 0.3% by dry weight (bdw) as the maximum allowable amount of THC. Some monoecious varieties developed in France reportedly contain less than 0.05% bdw THC, but traditional breeding approaches have not completely removed THC from hemp, and other non-psychoactive cannabinoids remain present.12 Hemp seeds contain 20% protein and about 30% oil by weight; the latter is composed of up to 6% of the desirable polyunsaturated fatty acid, y-linolenic acid.13 Several extensive reviews on the phytochemistry of cannabis have been published, with the latest reporting the occurrence of 483 compounds.14"16 The main metabolite classes found in cannabis are polyphenolics (flavonoids and stilbenes), terpenoids, simple polyketides, and terpenophenolics. More than 20 flavonoids, mostly flavonol O- and C-glycosides, have been isolated from this plant, and several spiro-indan and dihydrostilbenes are present.17"19 Phloroglucinol glucoside has also been detected in cannabis tissue.20 Volatile terpenoids in cannabis essential oil are responsible for the characteristic odor of the plant. Ross and Elsohly analyzed the essential oil composition of fresh marijuana and found it to be composed of about 92% monoterpenes and 7% sesquiterpenes, with about 120 different terpenoids identified in total.21 Although the exact terpenoid mixture varies among different varieties, myrcene, a-terpinolene, and a-pinene tend to be the dominant monoterpenes, and caryophyllene is the major sesquiterpene.22 Owing to their pharmacological properties, the C21 terpenophenolics of the cannabinoid class have received the most attention from chemists. It is worth noting that the definition of cannabinoid has expanded from its use to denote plant-derived compounds (so called phytocannabinoids)23 to include synthetic cannabinoids {i.e., CP-55,940) and endogenous cannabinoids (endocannabinoids) such as anandamide. About 70 cannabinoids have been isolated from cannabis.14 These are grouped into eleven different types based on their core structures: cannabigerol (CBG)-type, cannabichromene (CBC)-type, cannabidiol (CBD)-type, A9-tetrahydrocannabinol (THC)-type, A8-tetrahydrocannabinol (A8-THC)-type), cannabicyclol-type,

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cannabielsoin-type, cannabinol-type, cannabinodiol-type, cannabitriol-type, and miscellaneous cannabinoids. Representative metabolites in of each of these types are shown in Figure 8.1. The cannabinoids are not the only terpenophenolics in cannabis. Cannflavin A and cannflavin B are prenylated flavones that possess C5 and C10 prenyl side-chains, respectively.

PHYTOCHEMISTRY OF HUMULUS LUPULUS Hop is a dioecious perennial vine that grows wild in the Northern hemisphere and is cultivated in temperate regions of Western Europe, the US Pacific Northwest (Washington and Oregon), and Asia. Female plants produce inflorescences that mature to form a strobile, commonly termed cone, that is composed of bracts and bracteoles attached to a central stalk. Hop cones are picked in late summer when their essential oil and terpenophenolic content is highest, then dried and processed for use in beer brewing. The major known phytochemical constituents from hop consist of terpenoids, polyphenolics (flavonoids, proanthocyanidins, stilbenes), simple polyketides, and terpenophenolics. Hop cones contain 0.5-2% of a terpene rich essential oil, which contributes the typical "hops aroma" of beer.25 Its composition differs among hop varieties, and essential oil analysis has been used as a means for varietal authentication.26 A recent paper reported that 440 compounds have been identified from hop essential oil, with the major terpenoid components consisting of the monoterpene P-myrcene, and the sesquiterpenes humulone, caryophyllene, (3farnesene, and caryophyllene oxide.26'27 Some volatile esters such as 2-methylbutyl isobutyrate are also present. The polyphenolic metabolites of hop have also received attention by phytochemists. Hop contains flavonol glycosides (reviewed by Stevens et a/.28) and proanthocyanidins (condensed tannins). McMurrough detected 16 different flavonol glycosides in hops, which consisted of mono- and diglycosides of kaempferol and quercetin.30 The proanthocyanidins contribute to beer haze through complexation with proteins, and are potent antioxidants, and thus have been analyzed more extensively than flavonoids. The proanthocyanidins of Willamette hops consist of catechin and epicatechin monomers, and polymers built mainly from catechin termini extended by epicatechin or epigallocatechin units.29 Analyses of flavonoids and proanthocyanidins in H. lupulus appear to have been undertaken only on whole hop cones, and no information exists on the relative distribution of polyphenolics in green cone parts (bracts and bracteoles) or lupulin glands. The stilbenes resveratrol and piceid are found in hops, albeit at the low amounts of 0.5 (j.g/g and 2 |j.g/g dried hops pellets, respectively.32 Several

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OH

Cannabigerol (CBG), R=H Cannabigerolic Acid (CBGA), R=COOH

A9-Tetrahydrocannabinol (THC), R=H A -Tetrahydrocannabinolic Acid (THCA), R=COOH 9

OH

Cannabidiol (CBD), R=H Cannabidiolic Acid (CBDA), R=COOH

OH

Cannabichromene (CBC), R=H Cannabichromenic Acid (CBCA), R=COOH

OH

OH

A-Tetrahydrocannabinol

Cannabicyclol

OH OH

Cannabielsoin

Cannabinol

OH

Cannabinodiol

Figure 8.1: Structures of representative members of the major classes of cannabinoids.

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acylphloroglucinol glucosides have been isolated from hops and shown to have antiinflammatory activity.33 These compounds are derived from polyketide intermediates involved in bitter acid biosynthesis. As is the case for cannabis, the terpenophenolics are the most economically important secondary metabolites in hop, and thus have dominated phytochemical investigations. The terpenophenolic metabolites of hop, collectively called "hop resins" by brewers, are divided into two fractions: the soft resins, which are hexane soluble, and the hard resins, which are insoluble in hexane but soluble in ether.9 Bitter acids are the major phytochemicals in the soft resin fraction while the hard resin fraction is composed mainly of prenylfiavonoids. Bitter acids are classified into the a-acids, consisting of humulone, cohumulone, adhumulone, prehumulone, and posthumulone, and the P-acids, consisting of lupulone, colupulone, adlupulone, prelupulone, and postlupulone (Fig. 8.2). The a-acids are the primary source of bitterness in beer, although they themselves have no bitter taste until they isomerize to form iso-a-acids (Fig. 8.3).

H

R

Humulone

—CH2CH(CH3)2

Lupulone

—CH2CH(CH3)2

Cohumulone

— CH(CH3)2

Colupulone

—CH(CH3)2

Adhumulone

— CH(CH3)CH2CH3

Adlupulone

—CH(CH3)CH2CH3

Prehumulone

—CH2CH2CH(CH3)2

Prelupulone

—CH2CH2CH(CH3)2

Posthumulone

—CH2CH3

Postlupulone

—CH2CH3

Adprehumulone —CH2CH(CH3)CH2CH3

Adprelupulone —CH2CH(CH3)CH2CH3

Figure 8.2: Structures of the major bitter acids from hop.

TERPENOPHENOLIC IN HOP AND CANNABIS TERPENOPHENOLIC METABOLITES METABOLITES IN CANNABIS HO.

HO

OH 0 Xanthohumol

CH 3 O

OH

O

Desmethy lxanthohumol

CH3O

OH O

OH O

Xanthogalenol

4'-0-Methylxanthohumol

OH O

OH 0 5'-Prenylxanthohumol

Xanthohumol B

HO HO.

OH O Xanthohumol D

OH

O

8-Prenylnaringenin

CH3O

O

lsoxanthohumol

Figure 8.3: Structures of representative members of prenylflavonoids from hop. 8-Prenylnaringenin and isoxanthohumol are flavanones formed by isomerization of desmethylxanthohumol and xanthohumol, respectively.

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This conversion occurs during wort boiling, in which malted barley and hops are extracted in boiling water prior to fermentation. P-acids are not as soluble as a-acids, and do not isomerize to give bitter five-membered ring type compounds, so do not contribute to the bitterness of beer. Because of the critical role for a-acids in flavoring beer, hop varieties are often classified into low a-acid varieties, which contain 3-5% bdw a-acids, and high a-acid acid varieties used in bittering with 1012% a-acids. "Super-a-hops" may contain as much as 19% a-acids bdw.34 Breeding for increased a-acid content seems to have occurred without parallel increases in Bacid levels, perhaps because of competition for metabolite precursors.9 The hard resin fraction of hop is composed of prenylflavonoids, which occur at concentrations of 0.1 to 1.5% bdw in mature hop cones. There are 16 prenylflavonoids known from hop, of which 13 are prenylated chalcones and three are prenylated flavanones.35'36 Representative prenylflavonoids are illustrated in Figure 8.4. Chadwick et al. have proposed a new nomenclature for prenylated chalcones from hop that takes into account known compounds and those predicted to be present.35 The two major prenylflavonoids present are the chalcones xanthohumol and its biosynthetic precursor desmethylxanthohumol. Desmethylxanthomol, xanthohumol, and related chalcones are unstable, and readily convert to their flavanone isomers. Isomerization of desmethylxanthohumol gives a mixture of 6and 8-prenylnaringenin (hopein), 5 and xanthohumol yields isoxanthohumol (Fig. 8.5). This process may occur during drying and storage of hops but, as is the case for the conversion of a-acids to iso-a-acids, the isomerization of prenylated chalcones to their corresponding flavanones is aided by the thermal conditions of wort boiling. For this reason beer contains little or no xanthohumol or desmethylxanthohumol but contains detectable amounts of isoxanthohumol, 6-prenylnaringenin, and 8prenylnaringenin.37 The presence of xanthohumol in beer indicates that hops were added late in the brewing process (i.e., dry hopping).38

BIOLOGICAL ACTIVITIES OF TERPENOPHENOLICS FROM CANNABACEAE Cannabinoids The psychoactivity of cannabis preparations such as marijuana or hashish is manifested by changes in mood and perception, and in physical effects such as increased appetite and tachycardia.39 In 1964, the compound responsible for this activity was identified as the terpenophenolic THC.11 In the 1990s, THC was shown to exert its pharmacological effects through interaction with the CBi receptor in the central nervous system and the CB2 receptor found in the immune system and, more recently, in the brainstem.40"42 In addition to its potent psychoactivity, THC has also been shown to act as an analgesic, appetite stimulant, and antiemetic, and may have

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utility in treating multiple sclerosis and other diseases.39 Other non-psychoactive cannabinoids also possess notable pharmacological activity. For example, cannabidiol (CBD), a cannabinoid found in high amounts in low-THC hemp varieties, has been shown to have anticonvulsive, anti-inflammatory, anti-anxiety, and antinausea properties.43 A9-Tetrahydrocannabivarin (THCV), a cannabinoid present in some drug varieties, is an antagonist at CBi and CB2 receptors. THC and CBD are both potent antioxidants.44 Hop Terpenophenolics The hop terpenophenolics also exhibit a range of interesting, and potentially useful, biological activities. Hops have long been known to serve the dual purpose of flavoring beer and preserving it from spoilage, and the bitter acids and iso-a-acids have been shown to function as antimicrobials.45 These compounds also exhibit health-related activities such as inhibition of bone resorption and angiogenesis, and suppression of cyclooxygenase-2 gene transcription.46"48 Recently, iso-a-acids have been shown to activate peroxisome proliferator-activated receptor (PPAR) a and y, and thus may be useful in treating diabetes.49 Hop prenylflavonoids possess diverse biological activities (reviewed by Stevens and Page ). The most notable of these are the phase 2 protein inducing activity of xanthohumol, which may be important for chemoprevention of cancer and other diseases, and the estrogenic activities of 8prenylnaringenin.50'51 Hop prenylflavonoids have also been shown to have direct antioxidant properties in addition to their "indirect" antioxidant effects mediated by phase 2 proteins.52

BIOSYNTHESIS OF TERPENOPHENOLICS The biosynthetic pathways leading to the terpenophenolics in the Cannabaceae follow a common catalytic pattern consisting of three phases: polyketide formation, aromatic prenylation, and cyclization/decoration. The structural diversity of terpenophenolics in cannabis and hop arises through variations on this common pattern. The proposed biosynthetic pathways leading to the major cannabinoids, bitter acids and xanthohumol are shown in Figures 8.4, 8.5, and 8.6, respectively. Cannabinoid Biosynthesis The cannabinoid biosynthetic pathway has been the source of some debate, and several revised metabolic routes have been proposed over the decades since THC was identified (reviewed in Turner et al. 6 ). In early work, Mechoulam accounted for the presence of neutral (i.e., THC) and acidic (i.e., THC A) cannabinoids by proposing that parallel biosynthetic pathways may be active.53

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5x Olivetolic acid

CoA-S' Hexanoyl-CoA

synthase

Aromatic Prenyltransferase "OPP Geranyldiphosphate

COOH

A9-Tetrahydrocannabinolic acid

Cannabigerolic acid

Cannabidiolic acid

Nonenzymatic conversion

Nonenzymatic conversion

OH

OH

Cannabichromenic acid

Nonenzymatic conversion

OH

HO A9-Tetrahydrocannabinol

Cannabidiol

Cannabichromene

Figure 8.4: Proposed biosynthetic pathway leading to the cannabinoids, A9tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA).

TERPENOPHENOLIC METABOLITES IN IN HOP AND CANNABIS TERPENOPHENOLIC METABOLITES CANNABIS O

189

O

3x Malonyl CoA Valerophenone Synthase Isovaleryl CoA

Phlorisovalerophenone

Aromatic Prenyltransferase

OPP 2x|

>=/

DMAPP OH O

OH

Diprenylphlorisovalerophenone (Deoxyhumulone)

Aromatic Prenyltransferase

Cytochrome P450?y

OPP OH O

OH O

DMAPP

Humulone

Lupulone

Figure 8.5: Proposed biosynthetic pathway leading to the major bitter acids, humulone and lupulone, in hop.

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Neutral cannabinoids could form through the condensation of geranyl diphosphate with olivetol to form CBG, while geranyl diphosphate condensed with olivetolic acid would form CBG acid (CBGA). Subsequent studies showed that cannabinoids are first formed as acids and decarboxylate upon drying or heating to form their neutral derivatives.54'55 Another point of contention was whether cannabidiolic acid (CBDA)/CBD, formed from oxidation of CBGA/CBG, served as a precursor for THCA/THC. Biochemical experiments, which are discussed in more detail below, showed that both CBDA and THCA are derived directly from CBGA via oxidation (Fig. 8.4). The first step in cannabinoid biosynthesis is the formation of olivetolic acid by a putative polyketide synthase enzyme, termed olivetolic acid synthase. Olivetolic acid may be synthesized via the condensation of a molecule of n-hexanoyl coenzyme A (CoA) with three molecules of malonyl CoA to yield a tetraketide that could form olivetolic acid via a Claisen condensation with retention of the carboxyl group.5 Alternatively, olivetolic acid could be formed through condensation of acetyl CoA with five molecules of malonyl CoA to form olivetolic acid.55 The latter route seems to be supported by a NMR recent study, although it is unclear if a retrobiosynthetic approach can distinguish conclusively between the two possibilities. The reaction performed by olivetolic acid synthase is similar to those catalyzed by plant type III polyketide synthases of the chalcone synthase (CHS) superfamily.58'59 Members of this group include CHS, stilbene synthase, and enzymes of more limited distribution such as acridone synthase, 2-pyrone synthase from Gerbera hybrida, /7-tricoumaroylacetic acid synthase from Hydrangea macrophylla var. thunbergii, and aleosone synthase from Rheum palmatum. These enzymes share a common reaction mechanism, catalyzing sequential condensations of a starter CoA ester with malonyl CoA to form polyketide intermediates that cyclize to yield aromatic ring systems. It is noteworthy that some cannabinoids, such as the propyl side-chain variant THCV acid (THCVA), lack the pentyl side-chain of THCA. Methyl side-chain cannabinoid derivatives are also present in some cannabis varieties.65 These variants could be formed through the use of different aliphatic CoA as starter molecules for polyketide formation or through the condensation of acetyl CoA with three (to give a methyl side chain), four (to give a propyl side-chain), or five molecules (yielding THC and other pentyl side-chain cannabinoids) of malonyl CoA. At least two groups have attempted to discover the putative polyketide synthase catalyzing olivetolic acid formation. Raharjo et al. used a homology-based cloning approach to identify a CHS homolog from cannabis.66 This enzyme does not form olivetolic acid, and instead appears to be a CHS with broad substrate specificity. Our own work, using both homology-based cloning and EST analysis of glandular trichome-specific cDNA libraries, identified several cDNAs encoding type III polyketide synthases. Heterologous expression and assay of these proteins with nhexanoyl CoA and malonyl CoA did not yield olivetolic acid and instead gave

TERPENOPHENOLIC TERPENOPHENOLICMETABOLITES METABOLITES IN INHOP HOPAND ANDCANNABIS CANNABIS 191 191 pyrone derailment products (J.E. Page, unpublished results). The identity of the enzyme catalyzing the first step in cannabinoid biosynthesis, and indeed the nature of the polyketide condensation, remains to be elucidated. To our knowledge, olivetolic acid has not been detected in cannabis. The impressive variety of polyketide-derived metabolites in cannabis, which include the cannabinoids, phloroglucinol, stilbenes and spiro-indans, and flavonoids, suggests that type III polyketide synthases in this plant have undergone much catalytic diversification. The second step in cannabinoid biosynthesis is the prenylation of olivetolic acid to form cannabigerolic acid (CBGA) (Fig. 8.4). Fellermeier et al. detected an enzyme that catalyzed the prenylation of olivetolic acid with geranyl diphosphate.67 The crude enzyme did not accept olivetol but used neryl diphosphate as a prenyl donor, albeit to a lesser extent than geranyl diphosphate. It is noteworthy that this aromatic prenyltransferase appears to be soluble, which suggests it is more similar to short-chain prenyltransferases {e.g., geranyl diphosphate synthase68) than other prenyltransferases involved in plant secondary metabolism that are membrane bound {e.g., the geranyltransferase of shikonin biosynthesis69). CBGA is a central branch-point intermediate for the biosynthesis of the different major classes of cannabinoids. Alternative cyclization of the prenyl sidechain of CBGA yields THCA or its isomers CBDA or cannabichromenic acid (CBCA) (Fig. 8.4). Pioneering work by the Shoyama group led to the identification and purification of the three enzymes responsible for these cyclizations.70"72 Subsequent cloning of THCA synthase showed it to be an oxidoreductase that catalyzes the oxidative cyclization of CBGA to form THCA.73 Based on homology and site directed mutagenesis, THCA synthase was suggested to contain a covalently linked FAD residue. The recent report of THCA synthase crystal suitable for X-ray crystallographic studies suggests that further information about this enzyme and its structural properties will be soon available.74 A cDNA encoding CBDA synthase has been patented. The putative CBDA synthase is an oxidoreductase with 89% nucleotide sequence identity to THCA synthase. CBCA synthase most likely yet another member of this family of oxidocyclases, but confirmation of this awaits its cloning. A genetic analysis of cannabinoid biosynthesis found that the amount of THC versus CBD is likely governed by one locus with two codominant alleles.76 One possible explanation for these results is that the two alleles encode either THCA or CBDA synthase so that homozygous plants would contain either THCA or CBDA as the major cannabinoid, and heterozygotes would have an approximately equal mixture of the two. Another possibility is that THCA and CBDA synthases are closely linked genes, perhaps produced as a result of a gene duplication event. A recent study that analyzed the THCA synthase sequences from drug (high-THC) and fiber (low-THC) varieties found that the amino acid sequence of THCA synthase from high-THC varieties differed by 37 major substitutions compared to low-THC varieties.77

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Other cannabinoids most likely arise from the degradation or decoration of CBGA, THCA, CBDA or CBCA, or their neutral decarboxylation products. For example, cannabinol is an artifact that forms as an oxidation product of THC.78 The formation of CBGA monomethylether79 likely occurs through the action of a methyltransferase enzyme similar to the one discussed below that is involved in xanthohumol biosynthesis. Bitter Acid Biosynthesis Bitter acid biosynthesis in hop follows the pattern of that established for cannabinoid biosynthesis, although cyclization of the prenylated polyketides does not occur (Fig. 8.5). In the case of hop bitter acids, the polyketide synthase responsible for the formation of the acylphloroglucinol core of these compounds has been identified. Paniego et al. purified and cloned phlorisovalerophenone synthase (also called valerophenone synthase, VPS) from hop. The enzyme, which showed similarity to other type III polyketide synthases from plants, utilized isovaleryl CoA or isobutyryl CoA as primers for polyketide formation. VPS gave phlorisovalerophenone (PIVP), which is the precursor for humulone and lupulone, when supplied with isovaleryl CoA and malonyl CoA. Similarly, VPS catalyzed the condensation of isobutyryl CoA and malonyl CoA to give phlorisobutyrophenone (PIBP), the precursor for cohumulone and colupulone. Okada and coworkers reported that recombinant VPS also functions as a CHS, synthesizing chalconaringenin (naringenin chalcone) from /?-coumaroyl CoA and malonyl CoA, albeit with lower activity compared to the formation of PIVP.81'82 VPS is strongly expressed in hop glandular trichomes and the VPS promoter directs reporter gene expression to glands in transformed hop plants.81'83 The second phase of bitter acid biosynthesis involves prenylation of PIVP and PIBP. Prenylation of PIVP with two dimethylallyl diphosphate (DMAPP) molecules yields diprenyl phlorisovalerophenone (deoxyhumulone), while prenylation of PIBP with three DMAPP molecules yields lupulone (Fig. 8.5). A stable isotope feeding study has shown that the prenyl groups of humulone are formed from DMAPP derived from the methylerythritol phosphate (MEP) pathway. The aromatic prenyltransferases that carry out these reactions have not been identified and it is not clear if the same enzyme is responsible for multiple prenylations. Zuurbier and co-workers showed that protein extracts from hop cones were capable of forming 4-prenyl-PIVP, 4-prenyl-PIBP, deoxyhumulone and deoxycohumulone from DMAPP and PIVP or PIBP.85 Partial characterization indicated that the enzymatic activity was soluble, as was found for the olivetolic acid:geranyl transferase in cannabis. P-acids such as lupulone are not modified after prenylation is complete but the a-acid branch of the pathway requires an oxidation step to yield humulone and its congeners. The structural relationship between a- and P-acids led to speculation

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that a-acids could be synthesized via the oxidation of P-acids. However, assays using crude protein extracts from hop cones provided evidence that deoxyhumulone serves as a precursor to humulone while lupulone did not.86 The oxidase involved is not known, but the involvement of a cytochrome P450 enzyme has been suggested84 (Fig. 8.5). Prenylflavonoid Biosynthesis The biosynthetic pathway leading to the prenyl chalcone xanthohumol (Fig. 8.6) begins as does the ubiquitous flavonoid pathway in plants, with the CHS catalyzed condensation of p-coumaroyl CoA with three molecules of malonyl CoA to form chalconaringenin. In this way, xanthohumol biosynthesis is bitter acid biosynthesis with a twist - instead of an aliphatic primer for polyketide formation, a phenylpropanoid primer (p-coumaroyl CoA) is used. A chalcone synthase gene, chsJil, was cloned from hop and found to be part of a multigene family consisting of six or more members.87' Known gene family members encode VPS, C H S H 1 , CHS2, CHS3, and CHS4.81'87 Chshl is highly expressed in glandular trichomes, leading Matousek et al. to conclude that this is the "true" chalcone synthase involved in xanthohumol biosynthesis. However, the aforementioned results, showing that VPS also possesses CHS activity and also those indicating that C H S H 1 can form PIVP when supplied isovaleryl CoA and malonyl CoA, suggest that there may be some overlap in the functions of these polyketide synthases in vivo. The catalytic functions of the other polyketide synthases from hop are not known. CHS2, which is poorly expressed in leaf and gland tissue, gave pyrone derailment products when incubated with isovaleryl CoA or />-coumaroyl CoA and malonyl CoA.81 CHS4, which is expressed in glands, produced a pyrone only from isovaleryl CoA.81 No expression of CHS3 was detected using RT-PCR and no activity was detected with this enzyme. Flavonoid biosynthesis generally proceeds via the enzymatic conversion of chalconaringenin to a flavanone, (2S)-naringenin, through the action of chalcone isomerase.89 This conversion also occurs non-enzymatically to give a mixture of (2R)- and (2.S)-naringenin. In the case of xanthohumol, however, flavanone formation is inhibited by prenylation and methylation of chalconaringenin. Prenylation of the A ring with DMAPP yields desmethylxanthohumol, which is subsequently methylated at the 6'-hydroxyl to form xanthohumol. Although the order of these two reactions is not clear, the presence of desmethylxanthohumol in hops,90 and the apparent absence of 6'-O-methylchalconaringenin, suggests that prenylation occurs before methylation. On the other hand, methylation slows down the rate of isomerization to the flavanone (due to interaction of the remaining free hydroxyl with the nearby keto group), so perhaps this reaction occurs before prenylation. Desmethylxanthohumol readily isomerizes to 6- and 8-prenylnaringenin during brewing, but this conversion seems to be inhibited inplanta.

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PAGE NAGEL PAGE and NAGEL

CoAS

p-Coumaroyl CoA

O O CoAS'^ s ^OH Malonyl CoA

3x

Chalcone Synthase

Chalconaringenin ,OH

^ s s . ^OH

OH O

Aromatic Prenyltransferase

HO

OH O Desmethylxanthohumol Chalcone O-Methyltransferase

OH O

Xanthohumoi Figure 8.6: Proposed biosynthetic pathway leading to xanthohumoi.

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The aromatic prenyltransferase, be it a desmethylxanthohumol synthase or a xanthohumol synthase, involved in xanthohumol formation has not been identified or even detected by enzyme assay. Based on the MEP pathway origin of the prenyl groups in humulone,8 we speculate that the prenyl side-chain of xanthohumol also has its origin in the plastidic isoprenoid pathway. The O-methylation step, whether it utilizes desmethylxanthohumol or chalconaringenin, is likely catalyzed by an Sadenosyl L-methionine (SAM) dependent O-methyltransferase similar to the chalcone O-methyltransferase (ChOMT) from Medicago sativa.91 ChOMT methylates the 2'-OH of the isoliquiritigen, using SAM as a methyl donor, to produce 4,4'-dihydroxy-2'-methoxy chalcone. Prenylflavonoids such as xanthohumol C (Fig. 8.3) may be formed in an analogous manner to the oxidative cyclization of CBGA to THCA. Thus, there may exist in hop THCA synthase-like enzymes that form a pyran ring system from the DMAPP-derived side-chains of xanthohumol. The formation of prenylflavonoids in which the prenyl side chain bears a hydroxyl group (e.g., xanthohumol D) could be catalyzed by cytochrome P450 enzymes. No experimental evidence for biosynthetic origins of these minor prenylflavonoids is available.

TERPENOPHENOLIC BIOSYNTHESIS IN GLANDULAR TRICHOMES The major sites for the biosynthesis and accumulation of terpenophenolics in Cannabaceae are glandular trichomes. In both hop and cannabis, glandular trichomes occur on vegetative tissues such as leaves, but are found at their highest densities on female inflorescences. Glandular trichomes of many plant species are specialized for synthesis and secretion of secondary metabolites that likely function in defense against herbivores and pathogens. Examples of trichome localized metabolites are nicotine in Nicotiana spp., monoterpenes in mint (Mentha spp.), camptothecin in Camptotheca acuminata, artemisinin in Artemisia annua, and phenylpropenes in basil (Ocimum basilicum).92'96 Secretory tissues such as glandular trichomes have been suggested to be possible sites for metabolic engineering of plant metabolism.97 The three main types of Cannabis glandular trichomes are bulbous, capitate sessile, and capitate stalked. Non-glandular cystolith hairs are also present. Capitate stalked trichomes develop at high density on the bracts of female inflorescences (Fig. 8.7a) and to a limited extent on anthers on male plants. Such trichomes have a multicellular stalk that elevates the trichome secretory disk above the epidermal surface. Capitate sessile glands have a much reduced stalk structure and lie close to the epidermal surface. The development of glandular trichomes on floral tissues has led to the selective harvesting of this trichome rich material for both illicit drug and medicinal use, and the apparent selection of varieties with increased trichome production.

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Glandular trichomes of hop consist of both bulbous and peltate types." Peltate glandular trichomes are conspicuous as a yellow "powder" at the base of bracteoles of hop cones (Fig. 8.7b). These structures have been termed lupulin glands due to their content of bitter acids (lupulin is an early name for the essence of hop). Their appearance differs from those of cannabis trichomes in that they are initially concave, resembling small cups, and take on a pyramidal appearance upon metabolite filling during ripening.100 Most lupulin glands develop on hop bracteoles and few are found on the bracts that subtend them. Glandular trichomes are also found on the underside of hop leaves and on male flowers, albeit at lower densities than on female flowers. Cystolith hairs, which give hop a rough raspy texture, are also present on leaves, petioles, and stems. In hop and cannabis, as well as many other plants that accumulate resinous substances in trichomes, secreted metabolites are synthesized in the cells of a secretory disk and secreted into a subcuticular cavity that overlies the disk cells. As metabolite secretion progresses, the secretory cavity expands to form a balloon-like sac. Kim and Mahlberg have shown that the secretory cavity is enclosed by a sheath formed of cuticle and a subcuticular wall.101 Attempts to visualize bitter acid biosynthesis in hop glandular trichomes by using the VPS promoter coupled to a reporter gene (GUS) showed that, although GUS activity was detected in glands, the VPS-expressing cells within the gland were difficult to discern either because the GUS substrate did not completely penetrate the hydrophobic glands or because the stained glands were immature.83 A point of GUS staining was observed at the center of the cup-shaped immature glands. A recent paper examining the localization of THCA synthase concluded that this enzyme is secreted into the secretory cavity where it performs its biosynthetic function. Although this seems possible, it may be that the poorly understood process by which metabolites are secreted into this compartment may also lead to leakage of the some of the enzymes that synthesize them.

EST GENOMICS OF TERPENOPHENOLIC BIOSYNTHESIS Although some of the genes involved in terpenophenolic biosynthesis in cannabis and hop are known (e.g., VPS in hop, and THCA synthase in cannabis), much remains to be done to fully elucidate terpenophenolic pathways at the molecular and biochemical levels. Our lab has been applying an EST (expressed sequence tag) genomics approach that involves the random sequencing of cDNAs from a hope glandular trichome-specific cDNA library. The goal of this research is to identify genes encoding enzymes that function in the biosynthetic pathways leading to terpenes and terpenophenolics. The use of trichome-targeted EST genomics for dissecting metabolic pathways has been validated in numerous previous studies. The enormous metabolite output of secretory structures such as

TERPENOPHENOLIC METABOLITES IN IN HOP AND CANNABIS TERPENOPHENOLIC METABOLITES CANNABIS

Figure 8.7: Capitate stalked and peltate glandular trichomes occurring on female floral tissues of cannabis (A) and hop (B), respectively. The hop lupulin glands have a pyramidal shape due to metabolite filling of the central cavity.

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trichomes, leading to the high expression of biosynthetic genes, makes them ideal tissues for EST-based genomics. Metabolism in glandular trichomes has been studied using EST analysis in peppermint (Mentha x piperita), basil (Ocimum basilicum), and tomato (Lycopersicon hirsutum) among other species. ' Monoterpenes accumulate in the glandular trichomes of peppermint. In one of the first studies of the trichome transcriptome, a cDNA library was prepared from secretory cell disks isolated from peppermint trichomes by an innovative tissue disruption (Beadbeater) method.104 Sequencing of 1316 ESTs found many were involved in monoterpene biosynthesis, with terpene synthases amounting for 3.9 % of the 1316, or the MEP pathway that supplies the isoprenoid precursors from monoterpenes.102 Basil trichomes accumulates volatile phenylpropenes, which are responsible for the characteristic smell of this culinary herb. EST analysis of a peltate trichome-specific cDNA library from basil demonstrated that these glands possess the enzymatic machinery necessary for phenylpropene biosynthesis. Analysis of the basil EST dataset showed that approximately two-thirds of the known enzymes of the pathways leading from sucrose to phenypropenes and terpenes could be detected.96 In a new variation on trichome EST analysis, a comparative EST approach was employed by Fridman et al. to take advantage of the differences in the trichome-specific accumulation of methylketones in two tomato accessions.103 Transcripts involved in the fatty acid biosynthetic pathway were more numerous in the library of the methylketone containing glands, which implied the de novo synthesis of methylketones through fatty acid biosynthesis. An abundant cDNA with similarity to plant esterases was identified and characterized as methylketone synthase. We have conducted a preliminary analysis of the transcriptome of cannabis glandular trichomes (data not shown) and are currently using EST genomics to investigate the biosynthesis of bitter acids, prenylflavonoids and terpenoids in hop. We constructed a cDNA library from the trichomes of the hop variety 'Hallertauer Taurus', which is high in a-acids (12-16% bdw) and also accumulates significant amounts of xanthohumol (typically 0.9% bdw). Trichomes were isolated by mechanically shearing them off dissected cones frozen in liquid nitrogen. Microscopic examination confirmed that the resulting yellow material consisted of a high proportion of glandular trichomes and cystolith hairs with very little contamination by other cone tissue. Although RNA isolation from the metabolite rich trichomes was problematic, we succeeded in constructing a high-quality cDNA library by using a commercial method (SMART-cDNA library construction, BD Biosciences). A total of 1990 cDNAs were sequenced yielding 1915 good quality ESTs. After clustering, these were compared to the NCBI non-redundant database using BLASTX. 105 Of the 1915 ESTs, 1162 (60.7%) formed 218 contigs and 753 (39.3%) were singletons resulting in 971 (50.7%) unique sequences. The nine most abundant ESTs, as determined by clustering, are shown in Table 8.1. The largest contig in the library (147 sequences) was composed of

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transcripts showing high similarity to an MTN19-like protein from Pisum sativum. MTN19 is a protein of unknown function that is expressed in root nodules during the early stages of symbiosis between Medicago truncatula and Rhizobium bacteria.106 In a recent study, the expression of an MTN19-like transcript was found to be altered in pea pods treated with an insect elicitor. MTN19-like proteins are also expressed in response to stresses such as drought and high light and during senescence. A putative MTN19-like transcript also belonged to one of the 20 most abundant cDNAs expressed in an alfalfa glandular trichome cDNA library. 108

Table 8.1: Most Abundant ESTs in the Hop Trichome Dataset. Most similar sequence in database MtN19-like protein {Pisum sativum) Short-chain dehydrogenase/reductase (SDR) family protein {Arabidopsis thaliand) Putative chalcone isomerase (Lycopersicon esculentum) Major allergen Pru avl (Prunus avium) Cell wall protein (Nicotiana tabacum) Glycine-rich protein {Citrus unshiu) Chalcone-flavanone isomerase family protein (A. thaliana) Cystatin-like protein {Citrus x parodist)

Accession No.

No. ESTs in dataset

AAU14999

147

NP_567300

56

AAQ55182

38

024248 CAB67122 BAA92155

29 25 23

NP_568154

23

AAG38521

19

Genes that showed similarity to metallothionein-encoding genes were also strongly expressed, accounting for 56 sequences. Metallothioneins are small, cysteine rich proteins that are known from both plants and animals. They function in metabolizing heavy metal ions and by this means protect organisms from free radical and toxic metal damage. Our finding is consistent with the hypothesis that trichomes function in detoxification processes in plants. A high expression of metallothioneins was found to occur on trichomes of Arabidopsis and Vicia. The importance of detoxification for trichomes is further supported by the relatively high abundance of enzymes of the glutathione metabolism {i.e., 20 glutathione transferases and peroxidases) in our EST dataset.

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Several of the known enzymes from the pathways leading to bitter acids and prenylflavonoids were detected in the EST dataset (Table 8.2). Ten ESTs representing vps were found in the library, and six sequences corresponding to chshl were detected. Surprisingly, chs2 was the most highly represented type III polyketide synthase in the EST dataset, with 18 copies, while only one transcript of chs4 was found. This finding is noteworthy because chs4 was shown to be specifically expressed in hop glandular tissue, while chs2 is also expressed in leaves. 7 No cDNA corresponding to chs3 homolog was found in the library, a fact that is in accordance with the finding that chsS seems to be disrupted in most of the tested hop cultivars and that it probably is a pseudogene.

Table 8.2: ESTs with Known or Possible Function in Hop Secondary Metabolism. Most similar sequence in NCBI (nr) database Phloroisovalerophenone synthase (VPS) {H. lupulus) Chalcone synthase (chs_hl) {H. lupulus) Chalcone synthase (chs2) {H. lupulus) CHS-like protein (chs4) {H. lupulus) SAM-dependent Omethyltransferases (+)-Delta-cadinene synthase isozyme A {Gossypium arboreum) Putative chloroplast terpene synthase {Quercus ilex) Lupeol synthase {Taraxacum officinale) Flavanone 3 beta-hydroxylase {Petunia x hybrida) Putative flavonoid 3'-hydroxylase {Callistephus chinensis)

Accession No.

No. ESTs in dataset

080400

10

CAC19808 BAB47194 CAD23044 various

6 18 1 18

Q43714

1

CAC41012

2

BAA86932 A42110

1 1

AAG49298

1

An intriguing result was the high number of putative chalcone isomerases (CHIs), which accounted for 3.2% of all ESTs in the library (Table 8.1). The CHIlike sequences clustered into 3 contigs and one singleton. Two large contigs are most similar to a putative CHI from tomato, whose expression was shown to be upregulated in a mutant of the anthocyanin biosynthesis pathway.110 The predicted hop CHI-like proteins do not possess the conserved residues important for catalysis and substrate binding in CHI.89 Considering that the prenylflavonoids such as xanthohumol are chalcones, and, therefore, do not require CHI for enzymatic

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isomerization to flavanones, the function of these hop proteins in hop is not easily discernible. A non-catalytic role for CHI-like proteins has been hypothesized.111 Almost all of the enzymes of the MEP pathway, which leads to the formation of IPP and DMAPP, were present in the EST library. The exception to this was the cDNA encoding CDP-ME synthetase (IspD).112 The MEP pathway also supplies building blocks for monoterpene biosynthesis, and these enzymes participate in essential oil biosynthesis in hop. Several terpene synthase like sequences were detected. Despite the fact that there are several steps in the biosynthesis of hop terpenophenolics that require the transfer of one or more molecules of DMAPP, only one cDNA with similarity to a prenyltransferase was identified in the library. The protein sequence was predicted to contain transmembrane regions by topology prediction programs. This contradicts experiments with hop cone protein extracts, which detected activity in the soluble protein fraction.85 This cDNA is therefore an unlikely candidate for the aromatic prenyltransferase involved in bitter acid and prenylflavonoid biosynthesis. To date, no enzyme catalyzing the methylation step in the biosynthesis of xanthohumol has been identified. We consider methylation of the xanthohumol precursor, which contributes to the stabilization of the chalcone, a key step in this pathway. We found 18 ESTs in the hop trichome EST dataset that were annotated as O-methyltransferases, which clustered into four contigs and one singleton. None of the hop enzymes was particularly close in sequence to known chalcone Omethyltransferases of legumes.91'1 Further experiments to identify the enzyme assay reaction products and to biochemically characterize these O-methyltransferases from hops are underway. These results, and a complete analysis of the hop trichome EST dataset, will be published elsewhere. Transcription factors accounted for a significant number of the ESTs. However, the MYB transcription factor that has been suggested to be involved in hop cone development and the regulation of terpenophenolic biosynthesis was not present.114

SUMMARY AND FUTURE DIRECTIONS Cannabis and Humulus are closely related genera of Cannabaceae that share many unusual biological and phytochemical characteristics. These include their dioecious nature, the presence of glandular trichomes on inflorescences, and the biosynthesis of terpenophenolic secondary metabolites in these trichomes. As this review make clear, while the structures of the terpenophenolics found in hop and cannabis may vary, the biosynthetic pathways by which they are formed display a common pattern of polyketide formation, prenylation, and cyclization/decoration. Several of the genes involved in terpenophenolic pathways have now been identified, including those encoding the polyketide synthases that catalyze the formation of the polyketide core of the bitter acids and prenylflavonoids in hop, and the oxidocyclases

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that catalyze the last steps of THCA and CBDA biosynthesis. Our genomics approach of using trichome-targeted EST analysis is aimed at unraveling some of the remaining steps in hop terpenophenolic biosynthesis. The discovery of the polyketide synthases, prenyltransferases, and other enzymes responsible for terpenophenolic synthesis in cannabis and hop will open up new opportunities for metabolic engineering of these economically important crops. Applications could include the production of zero-cannabinoid cannabis varieties for use in food and fiber production and varieties with tailored cannabinoid profiles for medicinal use. Although genetic modification of hop is unlikely to be accepted by consumers, the creation of hop varieties with modified terpenophenolic contents through molecular breeding efforts could be envisioned. Considering the significant interest in the potent estrogenic properties of 8-prenylnaringenin, one route to producing larger amounts of this molecule would be to block the methylation step in xanthohumol biosynthesis, possibly leading to the accumulation of the 8prenylnaringenin precursor, desmethylxanthohumol. The diverse biological activities of cannabinoids and hop terpenophenolics demonstrate the importance of prenylation as a modification for imparting biological activities to phytochemicals. The aromatic prenyltransferase enzymes from these plants may, therefore, be useful for as biocatalysts for introducing prenyl groups into a variety of low-molecular weight phenolic substrates. As we note above, glandular trichomes have been suggested to be possible sites for the heterologous production of valuable metabolites in plants. Perhaps understanding terpenophenolic biosynthesis in hop and cannabis trichomes will allow these plants to be used as platforms for industrial production of other molecules that are of limited supply in nature? Humankind's long history of growing cannabis and hop as sources of terpenophenolic metabolites, and our increasing understanding of how these molecules are synthesized in planta, suggests that new agricultural, medical and biotechnological uses for these plants will be found in the future.

ACKNOWLEDGEMENTS We are grateful for the support of the hop EST genomics project by S.S. Steiner (Hopsteiner). Our EST analysis was assisted by Jacek Nowak (Bioinformatics Unit, NRC-PBI). We also acknowledge the contributions of Dr. Fred Stevens (Oregon State University), Dr. Paul Matthews (S.S. Steiner), Dr. Martin Biendl (Hopsteiner) and the staff at the Institute of Plant Biochemistry (Halle, Germany). REFERENCES 1.

BARRON, D., IBRAHIM, R.K., Isoprenylated flavonoids Phytochemistry, 1996, 43, 921-982.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Nine

ENGINEERING PATHWAY ENZYMES TO UNDERSTAND THE FUNCTION AND EVOLUTION OF STEROL STRUCTURE AND ACTIVITY Pruthvi Jayasimha, C. Bryson Bowman, Julia M. Pedroza, and W. David Nes* Department of Chemistry and Biochemistry Texas Tech University Lubbock, TX 79409-1061

*Authorfor correspondence, email: [email protected]

Introduction Natural Occurrence and Limits to Structural Variation Phytosterol Biosynthesis Structural Determinants for Activity Sterol Methyltransferases: Steric-Electric Plug Model SMT: Substrate Specificities and Functional Domains Engineering Product Diversity Summary and Future Directions

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INTRODUCTION The relationship between chemical structure and biological function, a problem of molecular recognition, can be seen as one of the central issues that determine the production and processing of phytosterols. For a given substrate, modifications that are allowed or forbidden, define sterol specificity toward enzymes, while knowledge of the specificity for sterol in membranous interactions define the performance of the lipid leaflet. At the molecular level, the general principles of sterol recognition are not easily formulated, and structural features specific to catalytic competence may not be the same features required of lipids to affect fluidity of the membrane. An evolutionary analysis of these differences can be used to uncover the problem of structure and its relationship to function. Pathway evolution, which has a cumulative effect on the design of sterol structure, requires two key elements: pathway data {e.g., compounds, enzymes, their amounts and interactions) and phylogenetic data (i.e., knowledge of the genes encoding sterolbased enzymes and their evolutionary relationships). This information is fragmented, but sufficient data is available from a variety of sources to rationalize why plant enzymes that act on sterols favor synthesizing products with a specific side chain and stereochemistry. We surmise that a purpose-driven synthesis of phytosterols evolved from enzymes of low substrate specificity that can produce product sets of mixed stereochemistry to enzymes that possessed strict requirements for substrate yielding products of specific stereochemistry. In our analysis of sterol evolution, we survey first the structures of sterols to establish the limits to diversity, followed by a brief review of sterol biosynthesis and structure-activity studies to provide background information on factors that can impact sterol phylogenesis. We then move on to outline parallel strategies performed with a critical enzyme of phytosterol synthesis, the sterol methyltransferase (SMT) to show that mutations engineered in the enzyme structure can lead to a change in the sterol structure thereby affecting cell growth, maturation and evolution.

NATURAL OCCURRENCE AND LIMITS TO STRUCTRAL VARIATION Plant sterols differ from sterols of other origins in their diversity. In the plant kingdom, about 250 sterols have been reported and as many as 60 sterols have been detected from a single plant.'' 2 Many of these compounds comprise intermediates in the various biosynthetic pathways to A5-sterols. Table 9.1 summarizes the major differences that are known to arise in sterol structures. Whereas the major sterol of animals is almost always cholesterol, in plants, fungi, and protozoa the end products contain a cholesterol molecule modified by the insertion of double bonds at various positions in rings B and C of the nucleus and in the side chain, and by the addition of

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one or two supernumerary carbon atoms at C-24. Amidst all this diversity, what uniformity exists in the sterol structure? The sterol molecule can be divided into four domains based on chemical features: A, equatorial 3-hydroxyl group; B, planar tetracyclic ring system; C, "righthanded" C-20R configuration; and D, C& to Cio- side chain (Table 9.1). In naturally occurring sterols, domains A and C are fixed in the molecule. These domains contain chiral carbons at key positions in the nucleus that affect the overall polarity and shape established by the alternating all trans-anti stereochemistry of the ring system. In the B-domain, variations occur from metabolism of a 4,4-dimethyl sterol to a 4-desmethyl sterol. The change in the structure of the nucleus can affect the tilt of the 3-hydroxyl group and tilt of the 17(20)-bond. In some specialized cases, such as in marine invertebrates, modifications in the A-ring occur. In the D-domain, variations in the size and direction of the 24-alkyl group and double bonds singly or in combination can be found introduced to the side chain. The geometry of double bonds in the side chain is usually trans-oriented, but in the case of sterols with a double bond associated with the 24(28)-ethylidene group the geometry can be either Z or E. In some specialized cases, again in marine invertebrates, methylation can occur at C-23 and C-25, and the side chain can be shortened or lengthened by one carbon atom.4

PHYTOSTEROL BIOSYNTHESIS The sterol pathway is fundamentally the same in all organisms yet the enzymatic reactions involved in the biosynthesis of A -sterols do not necessarily occur in a set order.5 Modifications occur in the A ring as a result of different reactions that involve C4 demethylations and double bond rearrangement of the A8to A5-bond. Modifications in the B, C, and D rings can occur before or after modifications in the side chain. The number of enzymes required to synthesize animal cholesterol is less than the number of enzymes required to synthesize phytosterols (24-alkyl sterols). Three enzymes that are not synthesized by animals are the SMT, the 9p\19-cyclosterol to A -isomerase, and the A22-desaturase. The several reactions that occur in the post-squalene part of sterol biosynthesis are summarized in Figure 9.1 for the conversion of cycloartenol to sitosterol and lanosterol to ergosterol. The organic approach involving natural product isolation and characterization as well isotope labeling studies is used to establish the broad outlines of the ordering of intermediates and provides the first indication about the substrate specificity of enzymic transformation. The diversity in sterol side chain structure, controlled by the SMT, can appear as the first enzymatic step in the cycloartenol pathway or can occur as an early or late enzymatic step in the lanosterol pathway. The C-methylation reaction is a physiologically irreversible step subject to

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Table 9.1: Domains of Sterol with Natural Variants

D

Domain A Domain B

Domain C Domain D

Constant 3/? OH 1) Double bonds at A0, A5, A7, A8, A8(14), A14, A9(11) and combinations 2)CH 3 (S)atC-4andC-14 3) 19-nor 4) A-nor 5) 9/?, 19-cyclopropane constant 20/? 1) Double bonds at A22, A23, A7, A24(28), A25 and combinations 2) Shortening or lengthening by one C-atom 3) Broadening with d - C 3 (cyclopropane) at C-22,C-23, C-24 and/or C-25 4) Addition of methyl group(s) to C-23, C-24, C-25

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strict regulatory control by the 24-alkyl sterol end product or allosteric effectors such as ATP.6'7 Excellent summaries of these reactions in plants and in nature generally have been recorded in review articles and repetition of details is unnecessary here.8"10 The start of the sterol pathway that ultimately produces membrane inserts, ergosterol in fungi and sitosterol in plants, begins with the formation of the tetracycles, lanosterol in fungi, protozoa, and animals or cycloartenol in plants. ' Alternatively, prokaryotes produce pentacylic hopanoids as the membrane component. 3 In sterol biosynthesis, squalene is oxidized to squalene- 2,3 (S)epoxide with the participation of molecular oxygen. The key enzyme involved in the cyclization of squalene-2,3(S)-epoxide to the first cyclic sterol precursor in animals is 2,3 oxdiosqualene:lanosterol synthase (cyclase). The asymmetry in lanosterol and cycloartenol is furnished by a stereospecific cyclization reaction defined by the stereochemistry of the olefin substrate. In the case of (-)-sterols, these compounds were synthesized later in evolution than hopanoids after the appearance of oxygen in the atmosphere.14 The hopanoids are considered together with sterols in this section because an evolutionary transition is thought to have taken place in isoprenoid biochemistry to switch from making hopanoids to cholesterol and then to 24-alkyl sterols. ' 5 The genes for cholesterol synthesis have been detected in prokaryotes, including genes that have been cloned for the lanosterol and cycloartenol synthases and the 14a-demethylase enzymes.16"18 The biosynthesis of tetrahymanol proceeds via the direct cyclization of squalene (Fig. 9.2) and is, therefore, an oxygen-independent process. The first step in the pathway by which squalene is transformed anaerobically to hopanoids is the lefthanded coiling of the substrate to produce (+)-tetrahymanol.19 The ability of cyclases to distinguish between either ends of the coiled substrate establishes the absolute stereospecificity of enzyme catalysis, a consequence of substrate binding to a chiral right-handed enzyme active site. The squalene-2,3(S)- epoxide to sterol synthase (= 2,3-oxidosqualene cyclase) also recognizes a left-handed coil of the substrate olefin but the squalene synthase-imposed substrate conformation is notably different from the squalene-2,3(S)-epoxide synthase-imposed substrate conformation, suggesting different positioning effects related to binding in the active sites of these enzymes. Enzymes (binding proteins, etc.) that act on sterols are highly asymmetric agents and are expected to react quite differently with the two mirror image forms of sterol compounds that contain an optically active atom(s). The precise interactive fit between, for example, a SMT enzyme or receptor protein involved with the signaling process, and its substrate, which is necessary for function, occurs with just one mirror-image, while the other is expected not to be accommodated. The stereochemistry of sterols can undergo an exact opposite change in the optical activity (-) to (+) of the molecule due to the cyclization reaction to produce antipodes or result from metabolism following the production of the first tetracycle. Alternatively, a single change in a stereocenter, such as the inversion of

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configuration at C20 in cholesterol, can be introduced into the molecule, and the altered stereochemistry is recognized by a negative shift in the optical activity on [a]o from -39.6 to -56.2 0 . 20 ' 21 Introduction of the systematic nomenclature used to identify the stereocenters in the cholesterol molecule at 3R, 8S, 9S, 10R, 13R, 14S, 17R, and 21R can be confusing. Thus, it appears that hydrogen atoms at C8S and C9S are not opposite to each other spatially, yet the molecule possesses an alternating all trans-anti stereochemistry in the ring to produce a pseudo-planar shape. Because of these complications, we introduced the convention that natural sterols, like sugars, are of the D-series.22 The absolute stereochemistry for a typical sterol, sitosterol, is shown in Figure 9.3. The X-ray structure of sitosterol yields the relative stereochemistry (Fig. 9.3, B) and provides the molecular dimensions of length, width, and height of the molecule. For sitosterol, there are 512 possible stereoisomers, yet only 2 stereoisomers exist as membrane inserts, sitosterol and 24episitosterol (clionasterol). For cholesterol, 256 possible stereoisomers exist, and only one is found to occur as a membrane insert. Similarly, there is one stereoisomer in the hopanoid family of compounds known to exist biologically. Primitive cyclase enzymes catalyze a mechanistically and thermodynamically simpler process than their eukaryote counterparts. The membranes of these organisms may have evolved in a stereochemical background, and if so, catalytic control to produce an asymmetric product [either D- or L] may be an evolutionary determinant for advancement. For stereospecific catalysis at time zero, the activated complex of a primitive enzyme {i.e., an enzyme that can act on more than one substrate and/or produce multiple products) conceivably can generate pairs of enantiomers [multiple products] such that with random mutation the specificity constant changed to limit catalysis to a single chiral product. Alternatively, the enzyme may originate to recognize a substrate conformation that corresponds to a binding site that conforms in a "lock and key" sense to a specific coiled structure of squalene or squalene-oxide. The cyclization of squalene to hopanoids proceeds basically in one step to generate the end product for physiological utilization. In the phytosterol pathway, the cyclization products of squalene 2,3(S)-epoxide, lanosterol or cycloartenol, are converted in several steps to the compound utilized as a membrane insert such that several opportunities arise during conversion of the intermediate to change the final global stereochemistry of the product, which apparently does not happen. Although the global stereochemistry of the molecule remains fairly constant in the sterol structure during pathway conversion, the stereochemistry at C24 of phytosterols can vary with the less-advanced organisms generating primarily 24Pmethyl sterols and the more advanced organisms generating 24a-ethyl sterols. The C24-alkylation pathway can produce four olefins that differ stereochemically and regiospecifically during the course of the C-methylation reaction: A24(25) - A 4(28) A2 (24) -and A25(27) -sterols, and each intermediate can be processed to a A5-

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Cycloartenol [D-Sterol]

[Left-handed coif] Ifor cyclization J

Prokaryotes

Tetrahymanol

Lanosterol [D-Sterol]

|[Left-handed coil for cyclization] Protozoa/Fungi/Animals

3(S)-Squalene-2,3-epoxide

I

A-Sterols Figure 9.2: Cyclization routes to hopanoids and sterols.

to

24aC 2 H 5 = Sitosterol

24aCH 3 = Campesterol

24H = Cholesterol

ENGINEERING PATHWAY ENZYMES – PHYTOSTEROLS

OH

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B

OH

Figure 9.3: Stereochemistry of sitosterol. The effect on structure by changing one or all of the stereocenters in the molecule is illustrated

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phytosterol.1'23 Assuming that there exists a membrane asymmetry to the lipid leaflet, then to accommodate different mixtures of phytosterols in membranes of fungi versus plants, the SMT enzyme may have moved to recognize structurally distinct substrates (flat or bent) and to catalyze stereochemically opposite Cmethylation pathways that utilize a sr-face (P) (fungi) or a re-face (a)-methyl (plants) attack mechanism during the evolution of eukaryotes, as some investigators have proposed.24'25 To address these fundamental issues, we begin with a detailed analysis of the structural requirements of sterol to function in multiple roles and later compare this data with studies directed at model membrane systems that are discussed next.

STRUCTURAL DETERMINANTS FOR ACTIVITY There are two major approaches used to investigate the importance of sterol structure to biological function: the biological and the physical biochemistry approaches. In the biological approach, an organism is studied that is auxotrophic for sterols to grow or reproduce. Typical organisms tested are (i) insects and pythiaceous fungi (Phytothora cactorum) that possess a natural block in the isoprenoid section of the sterol pathway,26'27 (ii) yeast cultured anaerobically that possess a block at the squalene to squalene oxide stage of synthesis,28'29 (iii) mutants in sterol biosynthesis with a specific block in the post-squalene segment, such as the yeast strain GL7 with an impaired squalene-oxide to lanosterol cyclase,30"32 or (iv) cultures treated with sterol biosynthesis inhibitors designed to block a specific step in the pathway, such as, with 2,3-epiminosqualene to block squalene-oxide cyclization to lanosterol.33'34 There is remarkable agreement in these studies for the sterol requirements in growth support. The physical biochemistry approach involves different instrumentations used to measure the change in viscosity or permeability as a function of sterol supplementation to artificial membranes.35'3 Sterol-induced changes in packing of the acyl chains of phospholipids in the hydrophobic phase of the lipid leaflet are related to sterol affects on the mechanical strength and regulation of membrane permeability.37 Sterol features involved with the bulk role are supported by these studies. The function of sterols has traditionally been viewed in relation to the fluidity of the membrane. However, recent studies report that cholesterol biosynthesis disturbed in animals can lead to high cholesterol levels that may be deleterious {e.g., lead to heart disease) or lead to low cholesterol levels that can engender birth defects {e.g., lead to Smith-Lemli-Opitz syndrome).38 A phenomenon similar to the developmental defects in humans that result from altered cholesterol homeostasis has been observed in the congenital deformities in insects provided a non-utilizable sterol, blockage of the ergosterol pathway at the stage of C24-methylation or via genetic engineering in plants to impair phytosterol synthesis.26'39'40 In their capacity

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to regulate embryonic development, sterols can possess a non-metabolic function by serving as signaling molecules involved with the hedgehog protein or related signaling process.41' 2 A dual non-metabolic role of sterols in these organisms is, therefore, implied based in part on quantitative differences for pairs of dietary sterol supplements; the bulk membrane role requires most of the total cellular sterol and the "sparking role" [where the sterol acts as a signal molecule] involves trace sterol levels. 30 ' 3 Usually the difference in the amounts of sterols required to play the 2 functions is approximately 99 to 1 - membrane to signal molecule as reported in the dual role for cholesterol in animal cells and dual role for phytosterols in microbes.42"45 The A5-sterols can also undergo metabolism to produce hormonal levels of an oxygenated compound such as the insect ecdysteroids, plant brassinosteroids, and fungal antheridiol and oogoniols that control growth and/or reproduction in these organisms.46'47'27 Eukaryote cells usually accumulate about 80% to 90% of the total sterol as 5 A -sterol (e.g., cholesterol), and in the case of cultured animal and plant cells undergoing active proliferation, the total sterol is about 3,000 fg/cell whereas for the smaller sized yeast the total sterol content is approximately 20 fg/cell.48 Based on the abundance of A5-sterols in cells and in cell membranes, structure-activity studies have focused primarily on variations in the 4-desmethyl sterol structure. By systematically varying the structure of cholesterol, the importance of inverting the configuration of a single chiral carbon atom was tested using a set of compounds that differed in a single feature. For example, in assays with the GL7 mutant or yeast cultured anaerobically tested with cholesterol and 3-epicholesterol or cholesterol and 20-epicholesterol, only cholesterol supported growth.29'32 Inverting C24 in the phytosterol side chain has been examined by incubating a panel of 24-alkly sterols with different sterol auxotrophs, and no sterol was superior to cholesterol as the bulk membrane component.29'32'4 The GL7 yeast used to evaluate the enantiomer pair «a/-cholesterol and entcholesterol was cultured in a medium containing 5 ug/mL sterol to support optimum growth.21' 32 The GL7 cells were conditioned to possess an up-regulated heme pathway to permit the yeast to accumulate dietary sterol such as lanosterol, which can be converted stoichiometrically to ergosterol.50 Thus, all the relevant enzymes of the post-lanosterol pathway can become operational in these cells, including the A22-desaturase that accepts cholesterol as substrate. Replacement of the dietary ergosterol supplement with a cholesterol supplement to GL7 will not prevent the cells from growing optimally, suggesting that the membrane requirements for sterol are met by the structural features of cholesterol. In cholesterol-grown cells, it is possible to show that the GL7 is leaky for ergosterol, producing hormonal levels of the compound at approximately 0.2 fg/cell or 1% of the total sterol in the cell (Fig. 9.4). When natural (nai) cholesterol (purified by HPLC to remove trace oxidation products) is fed to GL7, three sterols are detected in the cell, cholesterol, ergosterol and cholesta-5,22-dienol.34 On the other hand, when synthetically prepared

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Figure 9.4 (Prev.): Chromatographic analysis of the sterol composition of the yeast strain GL7 supplemented (5 ug/mL) with cholesterol and e«r-cholesterol (and Tween 80 to satisfy the unsaturated fatty acid requirement) in yeastpeptone-dextrose medium. The upper panels A and B are GC trace of the total sterols of GL7 fed cholesterol (A) and e«/-cholesterol (B). The main GC peaks are the added sterol and the peak eluting as a shoulder of the main peak in panel A is cholesta-5,22-dienol (mass spectrum, M+ 384 amu). Panel C is the HPLC (25-cm Ci8 Whatman column, eluted with 4% aq. MeOH at 40 °C). Fractions eluting at ca. 9 min and 11 min were collected and analyzed by GCMS (Panels D and E) and using a multiple wavelength diode array detector, the uv character corresponding to these compounds was determined (Panels F and G). In Panel D, the major compound eluting at ca. 9.4 min was determined to be cholesta-5,22-dienol and the later eluting peak at ca. 11 min was determined to be ergosterol by comparison of the mass spectra and relative retention times to cholesterol of these compounds with that of authentic specimens.

o 5

20

Time (min)

Figure 9.5: HPLC-radiocount analysis of the total sterol fraction from GL7 yeast incubated with [3-3H]cholesterol (Panel A) or [2-14C]acetate (Panel B). See reference 34 for a key to the sterol analysis and chromatographic conditions.

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enantiomeric (ent) cholesterol (provided by Dr. Scott Rychnovsky at UC Irvine) was fed to GL7, two sterols were detected in the cell, e«/-cholesterol and ergosterol (unpublished). Ergosterol synthesis continues whether cholesterol or e«^-cholesterol is supplied in the medium (Fig. 9.5), suggesting that any feedback of the exogenous cholesterol on de novo biosynthesis of ergosterol is not enantiospecific. The ability for trace levels of ergosterol to be synthesized by the cells is important to the multiple functions of sterols involved in cell growth, specifically to the so-called "sparking function" which is functionally distinct from the bulk membrane role.30"43 The results are interpreted to imply that e«?-cholesterol can replace cholesterol as a bulk membrane insert, but it cannot act as a substrate for enzymes of the ergosterol pathway nor can it act on ergosterol synthesis by an enantiospecific protein. These findings are supported by studies involving monolayer systems showing that cholesterol and ercf-cholesterol are basically the same.51 In the case of the sparking role, yeast will accept only a 24p-methyl sterol to support growth.34 This implies that there exists a stereochemical specificity for sterols in Saccharomyces cerevisisae most likely involving sterol-protein binding. The length of the sterol side chain has been evaluated with GL7 and yeast cultured anaerobically.22'43 In the case of the bulk membrane role, no significance can be attributed to C-methylation of the sterol side chain at C24 since cholesterol, ergosterol, and sitosterol support growth of the cells. Phytosterols compared to cholesterol have structures that possess more carbon atoms in the side chain, however, the position of these "added" methyl groups appears to be limited to C24. Synthetic analogs bearing straight rather than terminally branched chains of various lengths (n =2 to n = 9) were tested with yeast sterol auxotrophs and the maximal effective sterols possessed a side chain length similar to that of cholesterol.36 Thus, the native sterol side chain can be modified at C24 in terms of size and bulk to affect the flexibility of the side chain, but the side chain can not be modified to make it much longer or shorter than 6 carbons from C20, presumably in order to maintain a proper fit of the sterol in the lipid leaflet. Dreiding models of lanosterol and cyloartenol show they possess a fused ring stereochemistry that is similar to the final A5 -sterols. The major structural difference between lanosterol and cyloartenol and A5- sterols is the addition of the C4 and C14 methyl groups in the intermediates and the addition of a 24-alkyl group in the final products associated with plants and fungi. The hypothesis that cycloartenol is bent and lanosterol flat thereby providing significant structural differences to sterols that compose the phytosterol pathway in plants and fungi was found invalid.24'25'49 Both of these isomers are flat, as revealed by detailed conformational analysis work on the solution and solid state properties of cycloartenol and several of its 9p,19-cyclosterol relatives.52 The X-ray structures of lanosterol, cycloartenol, and sitosterol share similar shapes, all are flat, and the side chain orients to the "right" in the solid state.3'52'53

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The methyl group(s) at C24 are not harmful to phytosterol function in microbes, nor is the C14 methyl necessarily a deleterious group even though the back face planarity of the sterol molecule will be exposed to steric interference. However, the C4 methyl groups are deleterious to the ability of lanosterol or cycloartenol as well as hopanoids to function as eukaryote membrane inserts, as shown with GL7 and in studies with artificial membranes.32'54'55 It appears that the inability of these 4,4-dimethyl compounds to replace ergosterol as an architectural component results from steric interference of the C4 methyl groups on the hydrogen bonding interactions with the C3 hydroxyl group. Yeast sterol auxotrophs make accommodating changes in their phospholipids as a function of the sterol that is supplied in the growth media.49 These modifications in membrane composition are in response to the particular sterol added to the cell, and appear to be specific for that sterol. Thus, a compensatory adjustment in the membrane lipids to an abnormal sterol can occur, and whether such changes take place when enr-cholesterol is fed to GL7 was not studied. When care is not taken to determine the exact type and amount of sterol in the growth preparation, misconceptions about the role of a specific sterol can arisesuch as to whether the 24-alkyl group is essential in sterol-controlled yeast physiology or to support growth of cultured animal cells. For example, commercial preparations of Tween 80 and serum albumin, as well as so-called dilapidated media and agar, can possess trace levels of phytosterols and/or cholesterol that can affect structure-activity assessments. In the case of the peptone-yeast extract-dextrose compounded medium used for culturing yeast, the amount of sterol contaminant that can originate in the medium was determined to be 25 jxg/L; if the sterols in the medium were accumulated by the GL7 sterol auxotroph, the amount of 24p-methyl sterol in growth arrested cells can be as much as 0.01 fg/cell.32 The physiological requirement for the diversity of sterols synthesized by microbes and plants is unclear, although preliminary evidence suggests that the complex mixture of structures is advantageous to the cell. Support for the involvement of the different sterols in different physiological roles has been obtained with Gibberella fujikuroi treated with an inhibitor of the squalene-2,3(S)-epoxide to lanosterol synthase (2,3-epiminosqualene) where there is even delayed expression of certain pathway enzymes. The position of the SMT enzymes in the order of steps 5 to the final A -sterol is critical to the potency of an inhibitor of the C-methylation reaction. For example, when the C-methylation reaction occurs as a first step in the pathway compared to a late step, significantly less inhibitor is required to impair growth.57'58 The reason for the difference in physiological response appears to associated with the accumulation of the type of intermediates, e.g., 4,4-dimethyl sterols compared to 4-desmethyl sterols, and to the absence of the end product.

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STEROL METHYLTRANSFERASES: STERIC-ELECTRIC PLUG MODEL The search for a common biogenetic origin of the phytosterols has occupied the efforts of organic and biological chemists for the last half Century. The origin of the phytosterols is associated with the one/two carbon transfer to the sterol side chain and, therefore, is linked to the evolutionary appearance of the SMT and to its catalytic properties. The timing of when the SMT appeared in evolution is a matter of debate; some investigators believe that the SMT evolved in the prokaryotes since blue-green algae are shown to synthesize trace amounts of unusual phytosterols, while other investigators believe that the SMT evolved in the eukaryotes.12'15"17 A number of SMTs from plants, fungi, and protozoa have been purified and characterized.23-59"61 All have similar properties; native molecular mass in the 160 to 172 kDa range (as tetramers), a p / value that ranges from 5 to 8, a pH optimum within one unit of neutrality, a turnover number of approximately 0.01 s"1, and the optimal substrate occurs naturally in the organism. The SMTs of plants are thus far distinguishable from their protozoan and fungal counterparts in that plants synthesize multiple isoforms to generate the side chains of campesterol (first Ci-transfer activity) and sitosterol (second Q -transfer activity), whereas the SMT in microbes carry out multiple Ci-transfer activities to produce the monoalkylated (ergosterol) or doubly alkylated sterol side chains (clionasterol) via a successive methyl transfer to the A24-substrate.60'62'63 The C24-alkyl group of fungal ergosterol and plant sitosterol side chains are added by a SMT enzyme, however, the configuration of the 24-alkyl group is established by a A24-reductase-type enzyme.25 Alternatively, the 24-alkyl group of algal ergosterol is determined by the SMT.10 An interesting feature of the SMT from yeast is that it produces a single product by a non-stop (concerted) mechanism, and all the other SMTs examined to date are capable of producing multiple products by a step-wise ionic mechanism involving the production of cations at C24 and C25.10 The kinetic mechanisms vary among this class of catalyst, the yeast SMT operates as random bi bi mechanism, and the plant SMT from soybean operates an ordered mechanism with AdoMet binding first.61'64 The catalytic properties of the SMT have been established recently by using cloned enzymes, and they agree with the enzyme properties associated with the steric-electric plug (SEP) model, proposed by Nes (Fig. 9.6).65 This model provides a unified concept for a common structural relationship among sterols to bind to the SMT. Studies with substrate analogs to explore the active site of SMTs indicate interactions between the enzyme and sterol 3p-hydroxyl group and A24-bond show that the major binding forces and that C-methylation is catalyzed stereospecifically through a bi-substrate reaction that involves AdoMet as the methyl donor. The model also outlines the catalytic cycle whereby C-methylation of the A -sterol

\

3

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2 Activated COMPLEX

EP COMPLEX

E + S COMPLEX

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1

pseudocychc side chain conformation

AdoHcy

+ Free Enzyme

Figure 9.6: Steric-electric plug model of sterol-sterol methyltransferase interactions that occur during catalysis. Putative contacts, conformational changes, and release of products are indicated.

=I I

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substrate gives rise to the parent cations of the various major structural types. Termination of these ionic reactions by elimination of a proton generates the corresponding olefin, which typically is a A24(28) - or A25(27) -sterol. A key feature of the C-methylation reaction is the predicted methyl group attack to C24(25) from the 5/-(P)face of the substrate A24 -bond. In support of this proposal are the findings that i) the yeast SMT catalyzes a SAT2 mechanism, ii) the A25( 7) -sterols possess a 24p*methyl group, iii) mechanistic studies with 13C27-labeled substrates show the (reversible) 1,2-hydride shift of H24 to C25 occurs from the re-face of the original substrate double bond [thus, the Z-C27-methyl on zymosterol becomes the RC27methyl on fecosterol), and iv) differential inhibition studies that use 24a/(3methyl sterol pairs show only the 24p-methyl sterol can down-regulate activity.10'61'66 Although several investigators considered the plant SMT to have a different kinetic mechanism by using presumed conformationally different substrates and transition state analogs of the 24a/B-methyl configuration (observations that can bear on evolution of the enzyme24' 5 ' 49 ), the recent findings suggest that SMTs of plant and fungal origin operate a similar mechanism and bind only flat sterols productively.10 Kinetic studies ruled out an X-group-bound adduct involved with the coupled methylation-deprotonation reaction, consistent with the S#2 mechanism proposed by Arigoni.10'66 Scatchard plots showed one binding site for sterol and AdoMet in the native SMT structure.67 The observed regiochemistry and stereochemistry of the SMT catalyzed reaction critically depends on the precise folding of the sterol side chain and the positioning of the A 4-bond relative to AdoMet in the active site. Conformational constraints imposed by the configuration at C20 to orient the side chain to a "righthanded" structure, along with the parallel alignment required to posit the reacting A24-bond with its si-iaco, toward AdoMet, suggest that a small number of substrate conformations can account for the majority of known phytosterol side chain skeletal types.10 The types of enzymatic catalysis performed by SMTs relate to specificity and rate accelerations both derive from the free energy made available on binding of a specific substrate to the SMT active center. Binding energy of the substrates is the major source of free energy used by the SMT to lower the activation energy on the C-methylation reaction. The binding energy provides specificity as well as catalysis. In keeping with the Koshland model, when a "good" substrate binds to the active site, the binding forces between the enzyme and the substrate are used to drive the enzyme into an energetically less favorable but catalytically active conformation (the induced-fit model). Alternatively, the "poor" substrate lacks the necessary structural features to induce conformational change required for catalytic activity, and thus does not undergo reaction.68 It is not completely understood how the SMT actually lowers the activation energy for the chemical step of making and breaking of bonds [methylation, hydride transfer, and deprotonation], or forces the selection of a

ENGINEERING ENGINEERING PATHWAY PATHWAY ENZYMES ENZYMES -– PHYTOSTEROLS PHYTOSTEROLS

229

specific reaction channel by exacting a preferred substrate conformation and the positioning of a counter ion. Recently, we investigated the role of binding energy to catalysis, and the binding energy to make the enzyme specific for sterol acceptors by using the SMT from Saccharomyces cerevisiae (Fig. 9.7). Putative native substrates of the yeast SMT carrying a A24-bond, a dead end inhibitor, and a transition state analog of the Cmethylation reaction were tested with the enzyme. Tested compounds were zymosterol, fecosterol, lanosterol, ergosta-8,25(27)-dienol and 25-azalanosterol. Of the five compounds tested, only zymosterol was catalyzed, approximate Km of 15 uM and £cat = 0.01 s"1.67'69 The equilibrium constant (K
230

A NES, et al.

Ergosta-8,25(27)-dienol

SMT %

Fransition State Intermediate In Activated Complex-(ES*)

—^~ SMT '

l

Ad<>vlet

|Sterol/Methyl Acceptor- (S)

^olJ-J

rTV Hc/-^ Zymosterol

Lanosterol

|Methyl Producl-(P)[

Fecosterol

25-Azalanosterol

ES*

E

B

Reaction Coordinate

^ E.AdoMet ^

E.Zym.AdoMe , ^

[E.Zym.AdoMet|*

E.AdoHcy

X

E.Feco.AdoHcy

A E.Feco

E+Feco+AdoHcy

Figure 9.7: Enzymatic steps involved with Erg6p catalysis. Panel A, chemical steps involved in zymosterol conversion to fecosterol; Panel B, kinetic mechanism illustrating a random bi bi mechanism; Panel C, energy diagram showing important reaction coordinates in catalysis plotted against the freeenergy. The following notations are used to represent the key-steps in the C-methylation catalysis: ES, Enzyme-substrate complex; ES*, Enzymesubstrate in activated complex; EP, Enzyme-product complex. The apparent free energy required for the formation of these reaction coordinates are calculated from steady-state kinetic parameters, Kd, kcM and the thermodynamic parameter, £a, established over the temperature range 15 °C to 40 "C.

to CO

o

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ES, is the enzyme-substrate complex; ES* is the enzyme-substrate in the activated complex; EP, is the enzyme-product complex. The apparent free energy required for the formation of these reaction coordinates is calculated from the steady-state kinetic parameters, K& kcat and the thermodynamic parameter, Ea, taken from an Arrhenius plot of ln^ca/Km) of the SMT reaction as a function of 1/T, is measured over a temperature range 10- 40 °C. The free energy (AGES) associated with binding of the sterol to the SMT is measured to be - 7.65 kcal/mol, which reflects the net energy gained by the SMT enzyme as a result of thermodynamically favorable SMTzymosterol complex formation. Utilizing the mathematical equation [AGES* = Ea= RTln(&cat/£s)+RTln(&BT//2)], the free energy of the transition state that comprises the SMT enzyme and co-substrates in the activated complex (the free energy of activation, Ea) is measured to be 14 kcal/mol, while the data from the Arrhenius plot for the same indicated 13.9 kcal/mol, thus providing excellent experimental evaluation.69 The total energy input (AGfcat) by the SMT enzyme to reach the transition-state (Fig. 9.7, Panel C) can be directly obtained by summing AGES and Ea, which turns out be around 22 kcal/mol. Considering the steady-state kinetic parameter, the £cat value in the Eyring equation [AGicat= RT{ln(&BT//z)}-ln(&cat)] is determined to be 21 kcal/mol, which is in good agreement with the experimentally determined values for data obtained on kinetic constants derived from the pretransition stages of the reaction coordinate. As the key step in the evolution of phytosterols is the C-methylation reaction, such processes are emphasized in the following sections, particularly the evolutionary development of sterol specificity experimentally monitored by using Ea information obtained on cloned SMTs from different organisms.

SMT: SUBSTRATE SPECIFITIES AND FUNCTIONAL DOMAINS Comparison of the deduced amino acid sequences (a few representative SMT sequences are given in Figure 9.8) and knowledge of the deduced or expected substrate specificities of SMTs from these organisms (Fig. 9.9) indicates that SMTs can be classified into five subdivisions with three gene families acting as a SMT1 type, recognizing zymosterol, cycloartenol, or lanosterol, and two gene families acting as a SMT2 type, recognizing 24(28)-methylene lanosterol and 24(28)methylenelophenol (Table 9.2). The cDNA to a SMT gene cloned and sequenced first was from S. cerevisiae (ERG6), and this gene and its gene product (Erg6p) are often used as the reference specimens for comparative purposes. 70 ' 71 SMT cDNAs encode proteins of 360 to 383 aa (Fig. 9.10), in accord with observed native molecular masses of 40 to 43 kDa from SDS-PAGE analysis. There is no apparent relationship between the length of the primary structure in terms of its amino acid composition for the SMT1 and SMT2 isoforms. However, sequence analysis among SMT1 {A. thaliana, G.max, R. communis, Z. mays, N. tabacum, and O. sativa) showed that they share nearly 80% sequence identities while possessing only 40%

232

NES,etal NES, et al.

identity with SMT 2 {A. thaliana2-\ and A. thaliana2-2). In addition, when the predicted amino acid sequences of SMT1 and SMT2 enzymes are compared to one another, three regions of substantial similarity are observed: (Fig. 9.10): i) Region-I exhibiting a YEY/F/WGWGXSFHF sequence motif near the N-terminal region of the polypeptide. This motif is common only to the SMT enzyme;10'72 ii) Region-II comprises LDXGCGXGGPXRXI, a sequence motif that exhibits similarity to the glycine-rich consensus motif described in all AdoMet-dependent methyltransferases;72'73 and iii) Region-Ill that shows IEATCHAP and also appears to be unique to the SMT. When the % identity within the three conserved Regions is compared among the SMTs, the range in identity across isoforms increases to 83 to 93% relative to the identity of the yeast SMT1. The indication of a close structural relationship among SMTs using the conserved regions provides a different measure of similarity from using the substrate specificities for these enzymes (Table 9.2). Apart from these, SMT2 type of enzymes are known to possess a hydrophobic domain of approximately 25 amino acids at the N-terminal position that is absent in SMT1 type of enzymes.8'71 The presence of these identities suggests a divergent evolutionary relationship between the two classes of enzymes. The first experimental evidence to prove that the Region-I sequence motif functions as a sterol substrate recognition site was provided by the chemical-affinity labeling of yeast-SMT enzyme with the mechanism-based inactivator [3-3H]26, 27dehydrozymostrol. In addition, photoaffinity labeling of [3H3-/we//2y/]AdoMet to the wild-type Erg6p and chemical affinity labeling with the mechanism-based inactivator assayed with a Y81W mutant of Erg6p revealed that Regions-II and III contain an AdoMet recognition site and sterol recognition site, respectively [Jayasimha,P., Zhou, W., Marshall, J.A., Song, Z., P. Veeramanchanemi and Nes, W.D., unpublished work]. Further experimental evidence to support that Regions 1 and 3 serve as a sterol recognition site and Region 2 serves as an AdoMet recognition site was obtained from site-directed mutagenesis experiments on the yeast-SMT, after which we determined if activity was greatly impaired as a consequence. The properties of the mutagenized proteins were examined with particular attention to the kinetic parameters Vmiai/Km and product analyses by GC-MS or as necessary affinity information obtained from the equilibrium binding constants as sensitive indicators of altered function. Changed equilibrium and kinetic binding properties of the mutant enzymes from the yeast SMT indicated that residues required for catalytic activity are also involved in inhibitor binding. Thus, 25-azalanosterol appears to bind in the same active center as the co-substrates, zymosterol and AdoMet.6

*i \

c i

* J

•

Selected species

n

r

j

u

i

i

24(28)-Methylenelophenol

24(28)-Methylenelanosterol

Lanosterol

Cycloartenol

Zymosterol

Preferred substrate

n

-

-

T

*

i

233 -

„ .. . Functional . .r .

SMT1

ci&ssixic&xion

Crmethylation on 24, 25-double bond

SMT1

SMT1

Ci-methylation on 24, 25-double bond

SMT2

SMT2

Ci-methylation on 24(28)-double bond

Crmethylation on 24(28)-double bond

Crmethylation on 24, 25-double bond

Principal catalysis

ENGINEERING PATHWAY ENZYMES – PHYTOSTEROLS Table 2. Phytosterol diversity in relation to substrate preference . p

SMT , . „ ,. classification ,

(gene family) SMTb-fungal

Trypanosoma brucei

Saccharomyces cerevisiae

SMTe-protozoan

Gibberella zeae

Glycine max

SMTc-fungal

Pneumocystis carinii

SMTa-plant

SMTc-fungal

SMTd-plant

Arabidopsis thaliana

% Identity within _ . , „ TI Region-I, TT II & TIII , • r-

100 3

... 0/ r , % Identity .. overall i-iA/^t-o

44

sequence motif 100.0'

83.0

52

37

51

90.0

90.0

90.0

93.0

1 See text for details.' % Identity calculation referenced to S. cerevisiae SMT sequence were calculated using SIM alignment in ExPaSy server. The estimated values reflect the combined Region-1, 2 &3 identities. 3 % Overall identities were estimated with reference to S. cerevisiae using SIM alignment. % Identity means the percentage of identical amino acid residues that occupy same relative position when two or more sequences are overlapped with each other. The positions to Regions 1 to 3 are discussed in text. The gene family for SMT is as outlined in Fig 9.

1

I

2 =

1 On

I to

234

S. G. G. T. A. P.

S. G. G. T. A. P.

S. G. G. T. A. P.

cerevisiae zeae max brucei thaliana carinii

cerevisiae zeae max brucei thaliana carinii

cerevisiae zeae max brucei thaliana carinii

S. cerevisiae G. zeae G. max T. brucei A. thaliana P. carinii

s. cerevisiae G. zeae G. max T. brucei A. thaliana P. carinii

SMT1 SMT1 SMT1 SMT1 SMT2 SMT2

SMT1 SMT1 SMT1 SMT1 SMT2 SMT2

SMT1 SMT1 SMT1 SMT1 SMT2 SMT2

SMT1 SMT1 SMT1 SMT1 SMT2 SMT2

SMT1 SMT1 SMT1 SMT1 SMT2 SMT2

1 80 TDKDAEERRLEDYNEA NES, et al.MSETELRKRQAQFTRELHGDDIGKKTGLSALMSKNNSAQKEAVQKYLRNWDGR (1) (1) MVASSNTGLEREDHQRDADFNKAMHGSSAQARGGVAAMFRKGGAAKQAAVDEYFKHWDNKPAENETPEERAARQAEYATL MQKKKKNRNEWLCSAEGTGGCSRLAAMDLASNLGGKIDKAEVLSAVQKYEKYHV- -CYGGQEEERKANYTDM (1) MSAGSRGPLSLLIARERDANGVNGDVNATAGRLRDRYDGKGASASERRQDATSL (1) MDSLTLFFTGALVAVGIYWFLCVLGPAERKGKRAVDLSGGSISAEKVQDNYKQYWSF FRRPKEIETAEKVPDF (1) MSFELERIDIEKDREFSEIMHGKDAAKERGLLSSFRKDKEAQKIALDSYFGFWGDKCTSEKNDIHQQERFKFYATL (1) 81 160 (70) THSYYNWTDFYEYGWGSSFHFSRFYKGESFAASIARHEHYLAYKAGIQRGDLVLDVGCGVGGPAREIARFTGCNVIGLN (81) TRHYYNLATDLYEYGWGQSFHFCRFSQGEPFYQAIARHEHYLAHQIGIKDGMKVLDVGCGVGGPAREIAKFTGAHITGLN (72) VNKYYDLVTSFYEFGWGESFHFAPRWKGESLRESIKRHEHFLPLQLGLKPGQKVLDVGCGIGGPLREISRFSSTSITGLN (55) TNEYYDIVTDFYEYGWGQNFHFAPRYMNETFYESLARYEYFLAYHAQFKPTDTVLDVGCGIGGPARNMVRFTSCNVMGVN (74) VDTFYNLVTDIYEWGWGQSFHFSPSIPGKSHKDATRLHEEMAVDLIQVKPGQKILDVGCGVGGPMRAIASHSRANWGIT (77) TRHYYNLVTDFYEYGWSTSFHFCRFAKDESFSQAIARHEHYIALHAGIREGETVLDVGCGVGGPACQISVFTGANIVGLN 161 240 (150) NNDYQIAKAKYYAKKYNLSDQMDFVKGDFMKMDFEENTFDKVYAIEATCHAPKLEGVYSEIYKVLKPGGTFAVYEWVMTD (161) NNNYQIERATHYAFKEGLSNQLEFVKGDFMQMSFPDNSFDAVYAIEATVHAPTLKGIYSEIFRVLKPGGVFGVYEWLMTD (152) NNEYQITRGKELNRIAGVDKTCNFVKADFMKMPFPDNSFDAVYAIEATCHAPDAYGCYKEIFRVLKPGQYFAAYEWCMTD (135) NNEYQINRARQHDSRYGMSGKINYTKTDFCNMCFGDNEFDGAYAIEATCHSESKVKCYSEVFRAIKPGAYFMLYEWCLTD (154) INEYQVXJRARLHNKKAGLDALCEWCGNFLQMPFDDNSFDGAYSIEATCHAPKLEEVYAEIYRVLKPGSMYVSYEWVTTE (157) NNDYQIQRAKYYSEKKGLSDKLKFIKGDFMQMPFPENSFDKIYSIEATIHAPSLEGVYSEIYRVLKPGGLYASYEWVMLN 241 320 (230) KYDENNPEHRKIAYEIELGDGIPKMFHVDVARKALKNCGFEVLVSEDLAD NDDEIPWYYPLTGEWKYVQNLANLA (241) EYDNDNLRHREIRLGIEQGDGISNMCKVSEGIAAIHDSGFEMLHHEDLAD RPDALPWYWPLAGELRYVQTVGDFF (232) SFDPQNPEHQKIKAEIEIGDGLPDIRLTAKCLEALKQAGFEVIWEKDLA VDSPLPWYLPLDKSHFSLSSF (215) LYDPANEEHQRVRHGIELGDGLPELDTMRQWAAVKAAGFWEESFDMAERFESGEPKSVPWYEPLQGSYTSLSGL (234) KFKAEDDEHVEVIQGIERGDALPGLRAYVDIAETAKKVGFEIVKEKDLA SPPAEPWWTRL (237) KYDENDPEHQQIVYGIEIGDSIPKISKIGEAEAALIKVGFEIIHSEELSTK NSPLPWYYYLDGDLRKVRSFRDFI 321 399 TFFRTSYLGRQFTTAMVTVMEKLGLAPEGSKEVTAALENAAVGLVAGGKSKLFTPMMLFVARKPENAETPSQTSQEATQ TIVRMTTWGRTIAHGLAGLLETFKLAPAGTKKTADSLALAADCLVAGGRDKLFTPMYLMVARKPAA --RLTAVGRLFTKNMVKVLEYVGLAPKGSLRVQDFLEKAAEGLVEGGKREIFTPMYFFLARKPDLDRN - - -RATPAGRWLTSVTCRLLEAVRLAPAGTCKATEILEEGAVNLVKGGELGIFTPSFFVKARKPRLGEELSC KMGRLAYWRNHIWQ1LSAVGVAPKGTVDVHEMLFKTADCLTRGGETG1FSPMHMILCRKPESPEESS SIARMTTIGKWLISSFIGLMEFIGLLPKGSKKVNDILLVAADSLVKAGKKEIFTPMQLWVCRKPLV (305) (316) (302) (291) (294) (312)

Figure 9.8: Alignment of select sterol methyltransferase amino acid sequences (GenBank accession no.); Saccharomyces cerevisiae (NP013706), Gibberella zea ( X P 3 82959), Glycine max (T06780), Trypanosoma brucei (AAZ40214), Arabidopsis thaliana (CAA61966) and Pneumocystis carinii (AKK54439). The sequences were aligned using Align X (Informax Inc.) with defaulted parameters. The deduced substrate preference of SMT that catalyzes the first or second Q -transfer activity, SMT1 and SMT2 is reported.

to

1

ENGINEERING PHYTOSTEROLS ENGINEERING PATHWAY PATHWAY ENZYMES ENZYMES -– PHYTOSTEROLS Populus trichocarpa trichocarpa 111 1 communis Ricinus communis Gossypium arboreum 1 •Gossypium 100 Glycine max 1I Glycine 98 Medicago truncatula truncatulal1 Medicago 60 Arabidopsis Arabidopsis thaliana thatiana 1 ^ ^ ^ ^ ^ ^ ^ — ^ ^ ^ ^ ^ ^ ^ — Populus Populu: trichocarpa 1 2 •Nicotiana Nicotiana tabacum 111 1 97 Lycopersicon esculentum 11 Lycopersicon esculentum 100 Nicotiana tabacum •Nicotiana tabacum 11 22 Zea mays 111 1 62 Oryza 1 Oryza sativa sativa 111 100 Hordeum vulgare vulgare 1 57 Triticum aestivum 1I Oryza sativa sativa 112 Oryza 2 Zea mays 1 2

235

88

52

93

51 93 100

75 97 100 99 100 100

53

100 53 56

100

63 81

100 80

100

53 73

91

100

100

100 88

Saccharomyces cerevisiae cerevisiae •Saccharomyces Candida glabrata albic Candida Kluyveromyces lactis Kluyveromyces 'Eremothecium Eremothecium gossypii gossypii Clavispora lusitaniae lusitaniae Clavispora Candida albicans Candida albicans •Debaryomyces Debaryomyces hansenii Yarrowia lipolytica Yarrowia lipolytica Ustilago maydis Ustilago Cryptococcus neoformans Cryptococcus Schizosaccharomyces pombe 'Schizosaccharomyces Aspergillus nidulans Aspergittus Gibberell Gibberella zeae 1 Coccidioides posadasii Coccidioides posadasii Magnaporthe grisea grisea22 crassa Neurospora crassa MbbereUazeae zeae 22 Gibberella Magnoporthe grisea 3 •Magnoporthe Magnoporthe grisea 1I •Magnoporthe Pneumocystis carinii Thalassiosira pseudonana Thalassiosira Dictyostelium discoideum Triticum aestivum 2 1 100 Hordeum vulgare vulgare 22 22 Hordeum 56 •Oryza Oryza sativa 2 81 Zea mays 2 100 Hordeum vulgare vulgare 2 1 Triticum aestivum 2 2 •Allium cepa22 Allium cepa Lotus japonicus japonicus 2 73 Glycine max •lycine 2 63 ;sypium arboretum 2 Gossypium •Populus Populus trichocarpa trichocarpa 2 ,Medicago truncatulal2 Medicago truncatula Arabidopsis thaliana 2 2 92 62 •Arabidopsis Arabidopsis thaliana 2 1 Lycopersicon esculentum 2 2 'Lycopersicon 100 100 94 •Solanum Solanum tuberosum 2 2 •Nicotiana Nicotiana benthamiana 2 2 Lycopersicon esculentum 2 1 99 100 Solanum lolanum tuberosum 2 1 96 Nicotiana benthamiana 2 1 100 Nicotiana tabacum 2 Pinus 2 Pinus2 Chlamydomonas reinhardtii Leishmania major 100 LeishmanUi donovani Leishmania Trypanosoma brucei Trypanosoma cruzi

SMTb Fungal

SMTc Fungal

96 83

SMTa Plant

SMTd Plant

SMTe Protozoan (kinetoplastida)

• 0.05 changes changes

Figure 9.9: Rooted phylogenetic tree of eukaryotic sterol methyltransferase was created with PAUP* using the neighbor-joining method with kinetoplastida SMTs as the out group. The scale bar represents a distance of 0.05 substitutions per site. Numbers are the percentage of bootstrap values for 1,000 replicates. SMTa through SMTe designate SMT subfamilies defined by a minimum of 35% identity between members at the amino acid level. Adapted from reference 85.

NES, et al. NES,etal.

236 236

'81

S.c G.m G.z T.b A. t-2 P.c

91

YEYGWGSSFHF YEFGWGESFHF YEYGWGQSFHF YEYGWGQNFHF YEWGWGQSFHF YEYGWSTSFHF

124

135

LDVGCGVGGP

LDVGCGIGGP LDVGCGVGGP LDVGCGIGGP LDVGCGVGGP LDVGCGVGGP

192

200

IEATCHAP IEATCHAP IEATVHAP IEATCHSE IEATCHAP IEATIHAP

383 367 389 360 361 377

aa aa aa aa aa aa

Figure 9.10: Partial alignment of deduced primary sequence highlighting the conserved regions in SMT1 and SMT2 isoforms from different organisms as found in GenBank®: Shaded amino acids indicate identical residues among the SMT proteins and bold amino acids represent a different residue from the residue that appears to be conserved in that position.

By the design of the site directed mutant and the resulting modified kinetic behavior and change in the normal product distribution, it becomes possible to distinguish the location of a substrate binding site and which step(s) of the reaction sequence is altered by the mutation. Substitutions in the Erg6p Region 1 uncovered two amino acid residues at positions Glu79 and Tyr81 that can control the reaction pathway and product diversity, suggesting that amino acids of Region 1 likely interact with the side chain of the sterol.74'75 Leucine screening of conserved aromatic amino acids and histidine of the yeast and soybean SMT Region 1 indicated that histidine90 in yeast contiguous to histidine92 in soybean SMT were essential to activity (Fig. 9.11, Panel A). Additional leucine screening of 12 conserved acidic amino acid residues (Asp or Glu), and other histidine residues in the yeast-SMT (Fig. 9.11, Panel B) provided possible contact residues to the co-substrates in the three conserved regions.67 To date, no three-dimensional structure of this class of catalyst has been determined. However, the studies outlined herein, coupled with homology modeling using other AdoMet-dependent methyltransferases of known crystal structure, led to a prediction of the secondary structure with the sterol and AdoMet complexed to the yeast-SMT shown in Figure 9.12.67 The secondary structure of the yeast and soybean SMTs determined experimentally by using Circular Dichroism, and the homology modeling predicts that the active sites of SMT from fungi and plants have a common three-dimensional structure. ' It is interesting to note that the equivalent functional groups in the active center are situated on non-equivalent

ENGINEERING PATHWAY ENGINEERING PATHWAYENZYMES ENZYMES–- PHYTOSTEROLS PHYTOSTEROLS

237

^ ^ ^ Reqion-I Sequence Motif — Yeast: Y81EYGEWGSSFHF91 I I Soybean: Y83EFGEWGESFHF93

/VVVVVVVVVVVV" Figure 9.11: Histograms displaying the effect of select mutations on yeast and soybean SMT activity measured by steady-state kinetics. Panel A, the amino acid residues in Region-1 substituted with leucine are reported in the sequence underlined and the resulting activities plotted as a function of the location of the substitution. Panel B, the mutational analysis of conserved acidic amino acids and histidine residues in regions downstream to Region-1 in the yeast-SMT.

238

NES, et al.

C-term.

N-term.

NH2

Figure 9.12: Schematic representation of the methyltransferase fold of Erg6p. Spatial arrangement of the secondary structure elements in relation to sterol and AdoMet substrates is shown along with possible contact amino acids that interact with the substrates. Adapted from reference 67.

to 00

I

ENGINEERING ENGINEERING PATHWAY PATHWAY ENZYMES ENZYMES -– PHYTOSTEROLS PHYTOSTEROLS

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secondary structure elements, and they are distant with respect to location in the primary structure. This is consistent with the observation that the active site in AdoMet-dependent methyltransferases is formed usually from several different regions in the polypeptide chain.72

ENGINEERING PRODUCT DIVERSITY SMT enzymes carry out a one-carbon transfer of methyl from AdoMet to a A24(25) -sterol acceptor. The Primitive microbes from protozoa and plants appear to catalyze the simplest of all SMT catalyzed reactions in requiring only A24(25' -sterols to yield a A25(27) -sterol that cannot undergo further C-methylation (Table 9.3). SMTs from Prototheca wickerhamii (colorless algae) and Trypanosma brucei (protozoan) catalyze the formation of A25(27) -sterols, but in both instances the enzymes will generate minor amounts of the A24(28) -sterol, suggesting there is a more primitive form of the enzyme that will be discovered.23'57 As microbes evolved, the SMT in these organisms changed structurally so that the principal enzyme-generated product can be a A24(28) -sterol. Studies of kinetic isotope effects on SMT enzyme-generated product profiles using ^Yi^-methyl]AdoMet assayed with corn sprouts indicated that two SMT activities exist in these plants to produce methylated olefins containing the A23(24) and A24(28)- side chains.77 The observation of different product profiles and separable Ci -methyl transfer activities that generate different olefin-containing side chains in crude enzyme preparations from plants could be interpreted to be the result of multiple SMTs.78'79 However, recent work using cloned SMTs from vascular plants indicates that the SMT can be bifunctional to accept either a A24(25) - or a A24<28) -sterol substrate, typically elaborating a product profile altered from the product set generated by SMTs from primitive organisms.8 From this information, it is unclear what mutational pathways evolved to move a primitive SMT1 to a moreadvanced SMT1 or advance the SMT1 to a SMT2. We surmise that improving activity towards a new substrate such as to a A24(28) -sterol, the product of a Citransfer reaction utilizing a A24(25) -sterol, and tuning the product specificity via specific reaction channels to generate A24<28) - sterols rather than a A24(25' -sterols may be factors that govern the evolution of the SMT. This idea is amendable to experiment as discussed next. In vascular plants, the SMT has diverged such that isoforms of the SMT exist, SMT1 and SMT2, that recognize a A24(25) -sterol or A24(28) -sterol as the optimal substrate, respectively. For these isoforms, the function has not necessarily changed in evolution since both enzymes perform a Ci-transfer activity. However, the yeast SMT accepts only A24(25) -sterol substrates and, therefore, differs significantly from the SMT1 of plants which can accept A24(25) - or A24(28) -sterols. 80 The yeast SMT can be engineered to acquire an improved trait never required for the biological function in the wild-type enzyme by mutating a Region 1 amino acid at position-81

NES, et al. NES,etal.

240

Table 3. Product diversity in SMT catalyzed C-methylation reactions Source

High Energy Intermediate

Substrate

Product(s) Generated

TB-SMT

SC-SMT ZM-SMT1

E82L-SMT PW-SMT1

TB-SMT

Y81F-SMT AT-SMT2 GM-SMT1 PC-SMT2

SC-SMT 26,27-Dehydro zymosterol AdoMet

ZM-SMT1

N Cycloartenol

SMT

N

H

The following strains/organisms were used as a source of SMT: Saccharomyces cerevisiae (SC); Trypanosoma brucei (TB); Prototheca wickherhamii (PW); Arabidopsis thaliana (AT); Glycine max (GM); Zea mays (ZM); Pneumocystis carinii (PC). See text for key to the literature.

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from tyrosine to phenylalanine.75 This amino acid substitution alters substrate specificity to promote C2-activiy such that fecosterol, a A24(28) -sterol, can become a substrate. The substrate for C2-activity recognized by the plant SMTs was not recognized by the Y81F Erg6p mutant. Remarkably, the Y81F Erg6p mutant will catalyze fecosterol to three 24-ethyl(idine) sterol products that contain the same side chain structures as the three 24-ethyl(lidine) sterol products generated by the SMTl or SMT2 enzymes of vascular plants assayed with 24(28)-methylene lophenol or 24(28)-methylene cyclartanol.59' ' The product profiles of the three 24-ethyl(lidine) sterols [24(28)Z-ethylidene, 24(28)£-ethylidene, and 24J3-ethyl] differs significantly with the 24(28)Z-ethylidene side chain making up greater than 90% of the total product set in the SMTl from Arabidopsis, whereas in the native soybean SMTl and Y81F yeast mutant the distribution of the enzyme-generated products was approximately 4:1:2 and 2:4:1, respectively.59'61'75 Other aromatic residues in Region 1 of the yeast SMT when similarly mutated failed to perform d-activity (Fig. 9.13, Panel A). When tyrosine-83 in the soybean SMT (equivalent to the Tyr81 in Erg6p) is mutated to phenylalanine, the C2-activity increased five fold as found for the same mutation generated in the yeast SMT (Fig. 9.13, Panel B). These results suggest that the first tyrosine residue in Region-I (81 in yeast and 83 in soybean), absolutely conserved among SMT proteins in 16 different species, plays a pivotal role in sterol substrate recognition and that a single-point mutation at this position is sufficient to either enhance or diminish the catalytic competence. Enzyme variation in active site structures of SMTs improves substrate specificities and reaction channeling, thereby increasing diversity to afford a second Ci-transfer activity to produce the 24-ethyl sterol side chain. For example, vascular plants that have adapted to utilize 24-ethyl sterols as the preferred membrane insert require C2-activity to grow normally, and a change to produce high levels of cholesterol can impair growth.81'62 However, the native proclivity to perform either Ci -transfer activity seems to be present in all SMTs but masked by conformational constraints peculiar to individual active site topographies. The combination of recent work on the yeast SMT revealed that the enzyme has the relevant enzymatic machinery to produce multiple products common to plants. Thus, i) the native Erg6p treated with a mechanism-based inactivator, 26,27-dehydrozymosterol, will convert the substrate to a A23(24) -olefin, ii) the native Erg6p will convert zymosterol to a A24(28) -olefin, iii) the Y81F mutant will convert fecosterol to a set of 24-ethyl sterol products, and iv) the E82L mutant will convert zymosterol to a A25(27) -olefin (Table 9.3). It is interesting that plant sterols can replace ergosterol as a membrane insert but they cannot replace ergosterol as the "sparking" compound when the cells are cultured anaerobically.34'43 It appears that Tyrosine-81 (ERG6 nomenclature) is a critical amino acid in the SMT active site and that to change its identity can have serious consequences on substrate specificity and product distribution. The impact of changing the amino

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Figure 9.13: Histograms displaying the effect of mutations on the first and second Ci-transfer activities performed by the sterol methyltransferase from yeast and soybean. Panel A reports the activities that result from the substitution of tyrosines -81 and 83 and tryptophan85 to phenylanine in yeast-SMT. Panel B reports that activities that result from substitution of phenylalnine-82 and tyrosine-83 to isoleucine and phenylanine, respectively in soybean-SMT.

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acid at position-81 in the Erg6p on catalytic competence has been examined thermodynamically with the idea that a change in the activation energy (Ea) required to reach the transition state is related to improvement, or lack thereof, in the SMT reaction rate. It has been previously proposed that enzymes maximize rates by binding transition states strongly and substrate weakly, and that they have evolved in this way.82'83 In addition, we hypothesize that the difference in free energy profiles associated with the free energy of activation for the SMT catalyzed reaction can be a biomarker in which the higher the energy of activation required to generate a particular product set, such as A25(27) -sterols versus A24(28) -sterols, or to catalyze one set of acceptors versus another, such as A24(25) -sterols versus A24(28) -sterols, reflects the more primitive enzyme. With these considerations in mind, we assayed a set of SMT1 and SMT2 enzymes from different sources to establish the activation energies associated with this class of catalyst. The SMTs were assayed with substrates that represent the native acceptors for SMT1 and SMT2 enzymes (Fig. 9.14). The energetics involved to destabilize the SMT conformation to afford catalysis was similar in the SMTs tested, approximately 14 to 17 kcal/mol. The SMT from protozoa is considered more primitive since it requires a higher energy route to catalyze zymosterol (preferred substrate) than the other SMTs catalyzing their optimal substrates; the primitive SMT from protozoa catalyzes primarily the A25(27) - methylation pathway. The yeast SMT1 and soybean SMT1 prefer A24( 5) -substrates and ca. 2 kcal/mol difference is expended to accept the unnatural A24(28) -sterol in either the Y81F mutant or wildtype soybean SMT. Thus, for these SMTs, the evolution to accept A24(28) -sterol substrates is costly energetically and provides an explanation for the divergence of the SMT1 into a SMT2, where the utilization of the A -sterol substrate is favored by approximately 2 kcal/mol [Jayasimha, P., Nes, W.D., unpublished work]. These results also suggest that the mutational pathway to affect change in the primary structure of SMTs to improve catalysis affects the electrostatic/hydrophobic interactions of the active site, making it less rigid for contact residues to recognize the sterol substrate at binding or in the transition state. The availability of rational redesign to change the SMT active site to affect these interactions can be employed with greater precision to mimic the natural evolution of SMT, i.e., for the generation of an efficient catalyst with altered chemical mechanism that employs novel selectivity to generate unique product sets.

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Trypanosome

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Arabidopsis (2) {SMT-2}

.Ea-Zymosterol catalysis ^a-Cycloartenol catalysis Ea-C2 -catalysis (Fecosterol/24-methylenelophenol)

Yeast

Figure 9.14: Comparison of the thermodynamics of transition state formation for enzymatic reactions performed by sterol methyltransferases from different sources.

I

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SUMMARY AND FUTURE DIRECTIONS Fossil C28 - and C29- phytostearanes recovered from the environment reveal the existence of eukaryotes 500 million to 1 billion years before the extant fossil record indicates that the lineage arose.84 Clearly, the genes to make phytosterols and hence genes for the SMT arose early in evolution, at least coincidental with the appearance of the eukaryotes. As a first guess, the SMT evolved from an AdoMetdependent methyltransferase, which acquired a binding site for sterol that replaced a binding site for some other natural product. The SMT catalyzed reaction operates anaerobically and requires no cofactors; therefore, there is no restriction for this enzyme class to originate with the prokaryotes. The SMTs share a common evolutionary origin based upon their similar reaction mechanism and conserved structural characteristic, including amino acid homology and conserved sequence motifs (Regions 1 to 3). Phylogenetic analysis of 64 SMTs from plants, fungi, and protozoa allowed recognition of five SMT gene subfamilies on the basis of substrate specificity and genetic similarity; both plant and fungal SMTs contain subfamilies of SMT1 and SMT2 that catalyze preferentially either the first or second Ci -transfer reaction, respectively. The membranes of these eukaryotes may have evolved in a stereochemical background, and if so, catalytic control to produce specific product sets may affect evolutionary advancement related to the sterol structure. The ability, for example, for the yeast membrane to tolerate a range of stereochemically different compounds indicates there is no stereospecific requirement for sterols to act as membrane components. The inability for 4,4-dimethyl sterols and hopanoids to act as membrane inserts in many eukaryotes is best explained by the harmful geminal C4methyl group on activity. The addition of methyl groups to C24 can benefit the sterol function by adding increase bulk to the sterol side chain, thereby allowing the side chain to "sweep" out a larger cone in the lipid leaflet than will exist with the cholesterol side chain, and the C24-alkylation can be recognized specifically by a protein receptor evolved specifically for that sterol structure. The basic assumption is that enzymes control biosynthetic pathways and amino acid side chains in a binding site determine affinity of a bound sterol and the chemical transformation that it may undergo. Thus, for sterol evolution, the diversity in phytosterols is controlled by 3 major factors: i) the structure of enzymes that act on sterols, ii) the requirements imposed by the membrane for a sterol of particular shape, polarity, and length, and iii) recognition elements of proteins that utilize sterols as signal (regulatory, sparking etc) molecules. So far, it appears that molecular recognition of sterol is purpose-driven, and the different systems involved in molecular recognition have different sterol specificities. The mutational pathway directed at the SMT appears to have afforded improvements in catalytic efficiency in terms of specificity and product distribution; one benefit is the evolution of SMT2 enzymes with specificity toward 24(28)-methylene lophenol to regulate the

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campesterol/sitosterol ratio in plants.85 Another evolutionary benefit to these mutations is the substrate preference of the yeast SMT to accept zymosterol. This structure is the basis for the ergosterol side chain, the compound which can play multiple roles in the yeast physiology. The function, enzymology, and evolution of phytosterols is an area which has received little attention. In spite of numerous indications that the C-methylation reaction in sterol biosynthesis in plants, fungi, and protozoa are essential and that to disturb phytosterol homeostasis in these organisms can be of therapeutic value, the bulk of the evidence along these lines comes from scattered observations. A systematic and interdisciplinary approach to the area of phytosterols is likely to be fruitful, not only in expanding our understanding of this poorly known area but also in uncovering genetic opportunities to develop transgenic plants with value-added traits. An important step will be to define a phytosterol pathway completely, including uncovering and then characterizing the properties of each of the enzymes and their corresponding genes, to establish the level of the active enzymes and their transcripts during development, and to relate this information to the sterol composition of the cells. As the X-ray structures of these enzymes become available, a more sophisticated understanding of the catalytic properties can be determined, from which enzyme redesign can be employed to generate useful enzymes that can be engineered into plants and other organisms.

ACKNOWLEDGMENTS We thank the support of National Science Foundation (Grant-MCB0417436), National Institutes of Health (Grant-GM63477) and Welch Foundation (Grant-D-1276) for financial support.

REFERENCES 1.

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5. NES, W.R., MCKEAN, M.L., Biochemistry of Steroids and Other Isopentenoids, University Park Press, Baltimore. 1977, 680p. 6. NES, W. D., SONG, Z., DENNIS, A. I., ZHOU, W., NAM, J., MILLER, M. M., Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols., J. Biol. Chem., 2003, 278, 34505-34516. 7. NES, W. D., Sterol methyltransferase: Enzymology and inhibition., Biochim. Biophys. Ada., 2000, 1529, 63-88. 8. BENVENISTE, P., Biosynthesis and accumulation of sterols., Annu. Rev. Plant. Biol., 2004, 55, 429-457. 9. ROBERTS, C. W., McLEOD, R., RICE, D. W., GINGER, M., CHANCE, M. L., GOAD, L. J., Fatty acid and sterol metabolism: Potential antimicrobial targets in apicomplexan and trypanosomatid parasititc protozoa., Mole. Biochem. Parasit., 2003, 126, 129-142. 10. NES, W. D., Enzymatic mechanisms for sterol C-methylations., Phytochemistry., 2003, 64, 75-95. 11. NES, W. D., NORTON, R. A., CRUMLEY, F. G., MADIGAN, S. J., KATZ, E. R., Sterol phylogenesis and algal evolution., Proc. Natl. Acad. Sci. USA., 1990, 87, 75657569. 12. NES, W. R., NES, W. D., Lipids in Evolution. Plenum Press, New York. 1980, 244 p. 13. NES, W. R., Role of sterols in membranes., Lipids, 1974, 9, 596-612. 14. PORALLA, K., KANNENBERG, E., Hopanoids: sterol equivalents in bacteria., ACS Symp. Ser., 1987, 325, 239-251. 15. OURISSON, G., ROHMER, M , ANTON, R., From Terpenes to sterols: Macroevolution and microevolution., Rec. Adv. Phytochem., 1978, 13, 131-162. 16. BODE, H. B., ZEGGEL, B., SILAKOWSKI, B., WENZEL, S. C , REICHENBACH, H., MULLER, R., Steroid biosynthesis in prokaryotes: Identification of myxobacterial steroids and cloning of the first bacterial 2,3(S)-oxidosqualene cyclase from the myxobacterium Stigmatella aurantiaca., Mole. Microbiol., 2003, 47, 471-481. 17. PEARSON, A., BUDIN, M., BROCKS, J. J., Phylogenetic and biochemical evidence for sterol synthesis in the Gemmata obscuriglobus., Proc. Natl. Acad. Sci. USA, 2003, 100, 15352-15357. 18. BELLAMINE, A., MANGLA, A. T., NES, W. D., WATERMAN, M. R., Characterization and catalytic properties of the sterol 14a-demthylase from Mycobacterium tuberculosis., Proc. Natl. Acad. Sci. USA, 1999, 96, 8937-8942. 19. WENDT, K. U., SCHULZ, G. E., COREY, E. J., LIU, D. R., Enzyme mechanisms for polycylic triterpene formation., Agnew. Chem. Int. Ed., 2000,39, 2812-2833. 20. RYCHNOVSKY, S. D., MICKUS, D. E., Synthesis of e«?-cholesterol, the unnatural enantiomer., J. Org. Chem., 1992, 57, 2732-2736. 21. NES, W. D., VENKATRAMESH, M., Molecular asymmetry and sterol evolution., ACS Symp. Ser., 1994, 562, 57-89. 22. JOSEPH, J. M., NES, W. R., The configuration at C-20 of a natural A5-C26- sterol. Chem. Commun., 1981, 367-368. 23. ZHOU, W., LEPESHEVA., G. I., WATERMAN, M. R., NES., W. D., Mechanistic analysis of a multiple product sterol methyltransferase implicated in ergosterol biosynthesis in Trypanosoma brucei, J. Biol. Chem., in press.

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43. NES, W. R., Structure-function relationships for sterols in Saccharomyces cerevisiae., ACSSymp. Ser., 1987, 325, 252-267. 44. XU, F., RYCHONOVSKY, S. C , BELANI, J. D., HOBBS, H. H., COHEN, J. C , RAWSON, R. B., Dual roles for cholesterol in mammalian cells., Proc. Natl. Acad. Sci. USA, 2005,102, 14551-14556. 45. WHITAKER, B. D., NELSON, D. L., Growth and metabolism of phytosterols in Paramecium tetaurelia., Lipids, 1987, 22, 386-396. 46. SVOBODA, J. A., THOMPSON, M. J , Variability in steroid metabolism among phytophagous insects., ACS Sym. Ser., 1987,325, 176-186. 47. LI, J., NAGPAL, P., WITART, V., MCMORRIS, T.C., CHORY, J., A role of brassinosteroids in light-dependent development of Arabidopsis., Science, 1996, 272, 398-401 48. NES, W.D., Control of sterol biosynthesis and its importance to developmental regulation and evolution., Rec. Adv. Phytochem., 1990, 24, 283-327. 49. BLOCH, K. E., Sterol structure and membrane function., CRC Crit. Rev. Biochem., 1983,14, 47-82. 50. ZHOU, W., GUO., D., NES, W. D., Stereochemistry of hydrogen migration from C-24 to C-25 during biomethylation in ergosterol biosynthesis., Tetrahedron Letts., 1996, 37, 1339-1342. 51. WESTOVER, E. J., COVEY, D. F., BROCKMAN, H. L., BROWN, R. E., PIKE, L. J., Cholesterol depletion results in site-specific increases in epidermal growth factor receptor phosphorylation due to membrane level effects., J. Biol. Chem., 2003, 278, 51125-51133. 52. NES, W.D., KOIKE, K., JIA, Z., SAKAMOTO, Y., SATOU, T., NAKAIDO, T., GRIFFIN, J.F., 9#19-cyclosterol analysis by *H and 13C NMR, crystallographic observations, and molecular mechanics calculations., J. Am. Chem. Soc, 1998, 120, 5970-5980.14, 47-82. 53. NES, W.D., WONG, R.Y., BENSON, M., LANDREY, J.R., NES, W.R., Rotationalisomerism about the 17(20)-bond of steroids and euphoids as shown by the crystalstructures of euphol and tirucallol., Proc. Natl. Acad. Sci. USA, 1984, 81, 5896-5900. 54. BERNSDORFF, C , WINTER, R., Differential properties of the sterols cholesterol, ergosterolm P-sitosterol, fr-aw.s-7-dehydrocholesterol, stigmasterol and lanosterol on DPPC bilayer order., J. Phys. Chem., 2003,107, 10658-10664. 55. PARKS, L. W., CROWLEY, J. H., LEAK, F. W., SMITH, S. J., TOMEO, M. E., Use of sterol mutants as probes for sterol functions in the yeast, Saccharomyces cerevisiae., In: Biochemistry and Function of Sterols. (E. J. Parish and W. D. Nes, eds,), CRC, Boca Raton. 1997, pp.257-262 56. NES, W. D., LE, P. H., Evidence for separate intermediates in the biosynthesis of 24f3methylsterol end products by Gibberella^wjikuroi., Biochim. Biophys. Acta, 1990, 1042, 11-125. 57. MANGLA, A. T., NES, W. D., Sterol C-methyl transferase from Prototheca wickerhamii: Mechanism, sterol specificity, and inhibition., Bioorg. Med. Chem., 2000, 8, 925-936. 58. KANAGASABAI, R., ZHOU, W., LIU, J., NGUYEN, T. T. M., VEERAMACHANENI, P., NES, W. D., Disruption of ergosterol biosynthesis, growth,

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and the morphological transition in Candida albicans by sterol methyltransferase inhibitors containing sulfur at C-25 in the sterol side chain., Lipids, 2004, 39, 737-746. 59. ZHOU, W., NES, W. D., Sterol methyltransferase2: purification, properties and inhibition., Arch. Biochem. Biophys., 2003, 420, 18-34. 60. NES, W. D., McCOURT, B. S., ZHOU. W., MA, J., MARSHALL, J. A., PEEK, L-A., BRENNAN, M., Overexpression, purification and stereochemical studies of the recombinant (S)-adenosyl-methionine: A24(25) -to-A24(28) -sterol methyltransferase from Saccharomyces cerevisiae., Arch. Biochem. Biophys., 1998, 353, 297-311. 61. NES, W. D., SONG, Z., DENNIS, A. L., ZHOU, W., NAM, J., MILLER., M. B., Biosynthesis of phytosterols. Kinetic mechanism for the enzymatic C-methylation of sterols., J. Biol. Chem., 2003, 278, 34505-34516. 62. DIENER, A. C , LI, H., WHORISKEY, W. J., NES, W. D., FINK, G. R., Sterol methyltransferase 1 controls the level of cholesterol in plants., The Plant Cell, 2000, 12, 853-870. 63. BOUVIER-NAVE, P., HUSSELSTEIN, T., BENVENISTE, P., Two families of sterol methyltransferases are involved in the first and second methylation steps of plant sterol biosynthesis., Eur. J. Biochem., 1998, 256, 88-96. 64. VENKATRAMESH, M., GUO, D., NES, W. D., Mechanism and structural requirements for transformation of substrates by the (S)-adenosyl-L-methionine: A24(25) sterol methyl transferase from Saccharomyces cerevisiae., Biochim. Biophys. Ada, 1996,1299,313-324. 65. PARKER, S. R., NES, W. D., Regulation of sterol biosynthesis and phylogenetic implications., ACS Symp. Ser., 1994, 497, 110-145. 66. ARIGONI, D., Stereochemical studies of enzymic C-methylations., Ciba Found. Symp., 1978, 60, 243-258. 67. NES, W. D., JAYASIMHA, P., ZHOU, W., KANAGASABAI, R., JIN, C , JARADAT, T. T., SHAW, R. W., BUJNICKI, J. M., Sterol methyltransferase. Functional analysis of highly conserved residues by site-directed mutagenesis., Biochemistry, 2004, 43, 569576. 68. KOSHLAND, D., E., Jr., NEET, K. E., The catalytic and regulatory properties of enzymes., Annu. Rev. Biochem., 1968, 37, 359-410. 69. JAYASIMHA, P., SONG, Z., NES, W. D., Biochemistry, in press. 70. HARDWICK, K. G., PELHAM, H. R. B., SED6 is identical to ERG6 and encodes a putative methyltransferase required for ergosterol biosynthesis., Yeast, 1994, 10, 265269. 71. VENKATRMESH, M., GUO, D., HARMAN, J. G., NES, W. D., Sterol specificity of the Saccharomyces cerevisiae ERG6 gene product expressed in Escherichia coli., Lipids, 1996,31,373-377. 72. SCHUBERT, H. L., BLUMENTHAL, R. M., CHENG, X., Many paths to methyltransferase: a chronicle of convergence., Trend Biochem. Sci., 2003, 28, 329-336. 73. KAGAN, R.M., CLARKE, S., Widespread occurrence of three sequence motifs in diverse S-adenosyl-L-methionine-dependent methyltransferases suggests a common structure for these enzymes., Arch. Biochem. Biophys., 1994, 310, 417-427.

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74. NES, W. D., MARSHALL, J. A., JIA, Z., JARADAT, T. T., SONG, Z., JAYASIMHA, P., Active site mapping and substrate channeling in the sterol methyltransferase pathway., J. Biol. Chem., 2002, 277, 42549-42556. 75. NES, W.D., MC COURT, B. S., MARSHALL, J. A., MA, J., DENNIS, A. L., LOPEZ, M., Li, H., HE, L., Site-directed mutagenesis of the sterol methyltransferase active site from Saccharomyces cerevisiae results in formation of novel 24-ethyl sterols., J. Org. Chem., 1999,64, 1535-1542. 76. NES, W. D., SINHA, A., JAYASIMHA, P., ZHOU, W., SONG, Z., DENNIS, A. L., Probing the sterol binding site of soybean sterol methyltransferase by site-directed mutagenesis: Functional analysis of conserved aromatic amino acids in Region 1., Arch Biochem. Biophys., 2005, in press. 77. GUO, D., JIA, Z., NES, W. D., Phytosterol biosynthesis. Isotope effects associated with biomethylation formation in 24-alkene sterol isomers., Tetrahedron Letts., 1996, 37, 6823-6826. 78. FONTENEAU, P., HARTMANN, M. A., BENVEISTE, P., A 24-Methylene lophenol C-28 methyltransferase from suspension cultures of bramble cells., Plant Sci. Lett., 1977,10, 147-155. 79. MISSO, N.L.A., GOAD, L. J. The synthesis of 24-methylene cycloartanol, cyclosadol, and cyclolaudenol by a cell free preparation from Zea mays shoots., Phytochemistry, 1983,22,2473-2476. 80. NES, W. D., Enzyme redesign and interactions of substrate analogues with sterol methyltransferase to understand phytosterol diversity, reaction mechanism and the nature of the active site., Biochem. Soc. Trans., 2005, 33, 1189-1196. 81. SCHALLER, H., BOUVIER-NAVE, P., BENVENISTE, P., Overexpression of an Arabidopsis cDNA encoding a sterol-C-241-methyltransferase in tobacco modifies the ratio of 24-methyl cholesterol to sitosterol and is associated with growth reduction., Plant Physiol, 2001,118, 461-469. 82. JENCKS, W. P., Binding energy, specificity, and enzymic catalysis: the Circe effect., Adv. Enzymol, 1975, 43, 210-410. 83. ALBERY, W. J., KNOWLES, J.R., Evolution of enzyme function and the development of catalytic efficiency., Biochemistry, 1976,15, 5631-5640. 84. BROCKS, J.J., LOGAN, G.A., BUICK, R., Archean molecular fossils and the early rise ofeukaryotes.^c/ewce, 1999,285, 1033-1036. 85. ZHOU, W., NGUYEN, H.T., NES, W. D., Plant sterol methyltransferases: Phytosterolomic analysis, enzymology and bioengineering strategies. In: Advances in Plant Biochemistry and Molecular Biology (Lewis, N.G. ed.), Vol. 1, Bioengineering and Molecular Biology of Plant Pathways (Bohnert, H. J. and Nguyen, H. T., eds,), Elsevier Science, Oxford. In press.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Ten

METHYLATION AND DEMETHYLATION OF PLANT SIGNALING MOLECULES Yue Yang,1 Marina Varbanova,1 Jeannine Ross,2 Guodong Wang,1 Diego Cortes,3 Eyal Fridman,1 Vladimir Shulaev,3 Joseph P. Noel,2 Eran Pichersky 1

Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Street, Ann Arbor, MI 48109-1048 2

Jack Skirball Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, LaJolla, CA 92037 3

Virginia Bioinformatics Institute, Virginia Polytech University, Blacksberg VA * Author for correspondence, email: [email protected]

Introduction 254 Potential Substrates of Arabidopsis SABATH Methyltransferases 257 Atl g 19640 Encodes an Enzyme that Methylates Jasmonic Acid 257 At3gl 1480 Encodes an Enzyme that Methylates Salicylic & Benzoic Acids.. 257 At5g55250 Encodes an Enzyme that Methylates Indole-3-acetic Acid 257 At4g26420 and At5g56300 Encode Enzymes that Methylate Gibberellins .... 258 At3g44860 Encodes an Enzyme Capable of Methylating Farnesoic Acid 259 At5g04370 Encodes an Enzyme Capable of Methylating Nicotinic Acid 261 Possible Roles for Methylation of Signaling Molecules, and the Conversion of Methylesters Back to the Free Acids by Arabidopsis Esterase Family 261 Summary and Future Directions 265

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INTRODUCTION Many developmental and physiological processes in plants involve the transmission of chemical signals from one organ to another, or from one tissue to another, or even from one neighboring cell to another. Compounds that constitute the signal are designated as hormones, or more generally, signaling molecules. Examples of such natural chemicals in plants include ethylene, auxins, gibberellins, brassinosteroids, cytokinins, jasmonates, and salicylic acid. A signaling molecule needs to be synthesized at the source in response to some stimulus, transmitted, and finally perceived at the target site in order to elicit a response. After carrying out its function, the signal must be switched off, or the agent and physiological response will continue to be perceived and propagated after the need to respond expires. Historically, the evidence for the presence of signaling compounds has typically come from genetic experiments, through the analysis of mutations that were defective in the initiation of a specific process. In other cases, the exogenous application of an array of compounds has revealed effects that some of these chemicals have on physiological or developmental processes. In both experimental examples, the aspect that has received most attention is signal perception. Indeed, many exciting discoveries have recently been made regarding protein receptors and their interaction with signaling molecules, although for many such compounds the identity of the receptor has not yet been elucidated. For many signaling compounds we do not yet have a complete understanding of the intricacies of the pathways involved in their syntheses or their catabolic fates. In some cases, the exact chemical structure of the actual mobile compound is not clear. For example, salicylic acid (SA) is generally believed to be involved in the initiation of systemic acquired resistance (SAR), but its exact biosynthetic route in plants has not yet been completely elucidated, and there are indications that it may be derived from multiple pathways.1 Furthermore, although the exogenous application of SA leads to the initiation of a SAR response, several reports have suggested that SA itself is not the mobile signal.2 In our work on the biosynthesis of floral volatiles, we have encountered a class of enzymes that methylate various compounds at their carboxyl moiety to produce volatile methylesters that serve to attract pollinators. Interestingly, at least two such esters are the methylation products of known plant signaling molecules namely, flowers of some species emit methylsalicylate (MeSA) and methyljasmonate (MeJA) that they synthesize from SA and jasmonic acid (JA), respectively, using Sadenosyl-L-methionine (SAM) as the methyl donor (Fig. 10.1). We asked whether plants methylate these two signaling molecules, SA and JA, for purposes other than the production of floral scent, and if so, what are the physiological consequences to the plant. For example, will a methylated signaling molecule be perceived in the same way as a non-methylated one? Will it be transported in the same way? Will

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methylation prevent the further conjugation reactions that are known to occur at the carboxyl group of some signaling molecules, such as linking them to amino acids or sugars?3'4 Furthermore, what would be the metabolic fate of the resultant ester products?

JMT 0

0H

Jasmonic acid

Methyljasmonate

SAMT Salicylic acid

Methylsalicylate

Figure 10.1: Representative reactions of SABATH methyltransferases. Jasmonic acid carboxyl MT (JMT) catalyzes the formation of methyljasmonate, and salicylic acid carboxyl MT (SAMT) catalyzes the formation of methylsalicylate. Not shown here is the S-adenosyl-L-methionine (SAM) molecule that serves as the methyl donor.

With the availability of the genome sequence of Arabidopsis, we were able to determine that the Arabidopsis thaliana genome contains 24 genes encoding such methyltransferases (MTs), named the AtSABATH family5 (Fig. 10.2). We have, therefore, endeavored to determine the substrate specificity of the enzymes encoded by the Arabidopsis AtSABATH genes, and examine their role in signal transduction pathways or in other physiological processes of the host plant. The current results of this ongoing project are described below.

at PICHERSKY, et al.

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0.1

At1g68040 At1g19640 JMT At4g36470 At2g14060 At3g21950 At5g38020 At3g11480 BSAMT At5g04380 At5g66430 At5g04370 NAMT At5g55250 IAMT At5g56300 GAMT2 GAMT1 At4g26420 -At1g15125 - At5g37990 - At5g37970 - At5g38780 At5g38100 At1g67720 At1g66690 At1g66700 - At3g44840 At3g44860 FAMT - At3g44870

Figure 10.2: A phylogenetic tree of the Arabidopsis SABATH methyltransferase family. The neighbor-joining tree was constructed using the aligned protein sequences of 24 SABATH MTs. The Arabidopsis gene locus symbol is used to identify each MT. MTs whose catalytic functions have been determined are designated by names (in gray).

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POTENTIAL SUBSTRATES OF ARABIDOPSIS SABATH METHYLTRANSFERASES Arabidopsis Atlgl9640 Encodes an Enzyme that Methylates Jasmonic Acid The first AtSABATH enzyme to be characterized was shown by Seo et al. to encode an enzyme that methylates JA and was designated JMT.6'7 This group isolated a cDNA from Arabidopsis that was orthologous to a Brassica cDNA that was differentially expressed in flowers, mostly in nectaries. These two cDNAs encode proteins that are homologous to the enzyme salicylic acid carboxyl methyltransferase (SAMT) that we had previously characterized from the flowers of Clarkia breweri* The Clarkia SAMT which contributes methylsalicylate to the floral scent of this plant, constitutes one of the founding members of the SABATH methyltransferase family.5 Because of the homology to SAMT, Seo et al. assayed JMT for activity with several common plant acids and showed that it was specific for JA. This group, as well as our own, showed that the gene encoding JMT is inducible by wounding, alamethicin, and herbivory.5'7 They went on to show that plants overexpressing JMT were more resistant to fungal attacks. However, they did not test a null mutant of JMT. We have subsequently obtained a T-DNA mutant of JMT, and so far, have not observed any obvious physiological phenotype for this mutation in planta. Arabidopsis At3gll480 Encodes an Enzyme that Methylates Salicylic Acid and Benzoic Acid As mentioned, the first SABATH MT, characterized in C. breweri flowers, is a MT with strict specificity to SA. Dudareva et al. later isolated a SABATH enzyme from snapdragon flowers, which acts on benzoic acid (BA) to give methylbenzoate (MeBA). We have shown that Arabidopsis contains an expressed SABATH gene encoding an enzyme that can methylate both SA and BA with similar specificities, and this enzyme was, therefore, designated BSMT1.10 The Arabidopsis BSMT gene possesses low-level constitutive expression in flowers, and also in cells found at the base of the trichomes. Much like JMT, BSMT is induced by wounding, alamethicin, and herbivory.10 A T-DNA insertion null mutant obtained has not shown any obvious morphological or physiological phenotype under the conditions tested thus far. Arabidopsis At5g552500 Encodes an Enzyme that Methylates Indole-3-acetic Acid The AtSABATH gene At5g55250 encodes an enzyme that converts indole-3acetic acid (IAA) to IAA methyl ester (MelAA). Under steady state kinetics, IAMT

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exhibits KM values of 13 uM for IAA.11 IAMT exhibited no methyltransferase activities with other signaling molecules or chemicals with similar structure to IAA, such as tryptophan and indole-3-carboxylic acid. However, it did methylate the active auxins indole-3-butyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4D), but at much reduced rates as compared with IAA (unpublished data). Protein structure modeling using experimentally determined C. breweri SAMT crystal structure suggested that the replacement of Trp-226 of CbSAMT by Gly of AtlAMT in the active site creates a spacious pocket for the recognition and binding of the indole ring of IAA, which explains the difference in the substrate specificity of the two enzymes.11 Expression analysis of IAMT in Arabidopsis (Col) using Real-time RT-PCR and promoter-GUS fusion revealed higher expression levels in flowers and siliques, and MelAA was detected in all tissues examined by gas chromatographymass spectrometry (GC-MS) analysis (unpublished data). A plant carrying a null allele mutation in IAMT showed diminished levels of MelAA (unpublished data). Plants overexpressing IAMT displayed loss of root gravitropism and an upwardcurling leaf phenotype,12 suggesting disruption of auxin gradients.13 Arabidopsis At4g26420 and At5g56300 Encode Enzymes that Methylate Gibberellins At4g26420 and At5g56300 encode enzymes, respectively, designated GAMT1 and GAMT2, that methylate several gibberellins (GAs) in vitro. GAMT1 showed high methyltransferase activity with GA9 and GA20, followed by GA4, GA3, and GA1, while GAMT2 was optimally active with GA4. Under steady state kinetics, the KM values of GAMT1 and GAMT2 with these substrates range from 2 uM to 15 uM. Both enzymes have low or absent levels of activity with gibberellins containing a y-lactone (unpublished data). GAs have regulatory roles in seed development and germination, stem elongation, flowering, and fruit set.14'15 Arabidopsis mutants with impaired GA biosynthesis are generally dwarfed, having small dark green leaves, flowering delays, and showing various degrees of male sterility.16 Overexpression of GAMT1 and GAMT2 in Arabidopsis plants under the control of CaMV 35S promoter resulted in phenotypes that mimicked GA deficiency. GAMT1 overexpression lines are dark green dwarf plants with shortened pedicels and petioles, and are partially sterile (Fig. 10.3). The fertility of these plants could be partially restored by supplementing bioactive GA. GAMT2 overexpression lines also have a dwarf habit, however, with cauline leaves lighter in color than wildtype plants. In addition, the plants show bushy phenotype with extensive branching and auxiliary floral meristem. In contrast to GAMT1, the sterility of GAMT2 overexpression lines cannot be recovered by spraying the plants with GA4.

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Figure 10.3: Arabidopsis plants overexpressing GAMT1 display a dwarf growth habit with dark green leaves and shortened pedicels and petioles, compared to wildtype plant (shown in the middle of the picture). All plants are 1-month old.

Arabidopsis At3g44860 Encodes an Enzyme Capable of Methylating Farnesoic Acid The AtSABATH gene At3g44860 encodes a protein with high catalytic activity and specificity towards farnesoic acid (FA). Under steady state conditions, this farnesoic acid carboxyl methyltransferase (FAMT) exhibits KM values of 41 uM for FA. A three-dimensional model of FAMT constructed based upon similarity to the experimentally determined structure of C. breweri SAMT indicated a snug fit for FA recognition in the FAMT active site (Fig. 10.4). In planta, the mRNA levels of

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FAMT increased in response to the exogenous addition of several compounds, such as alamethicin, previously shown to induce plant defense responses at the transcriptional level.1018 Although methyl farnesoate (MeFA) has not yet been detected in Arabidopsis, the presence of a FA-specific carboxyl methyltransferase in Arabidopsis capable of producing MeFA, an insect juvenile hormone precursor made by some plants as a presumed defense against insect herbivory,19'20 suggests that MeFA or chemically similar compounds are likely to serve as new specialized metabolites in Arabidopsis. Alternatively, low levels of FA and/or MeFA may serve as signaling molecules in Arabidopsis.

S-adenosyJil-Thomocvs

Figure 10.4: Surface representation of the FAMT active site with the substrate, farnesoic acid (FA), and product, S-adenosyl-L-homocysteine (SAH) bound. The carboxyl group of FA is shown poised to receive a methyl group from the methyl donor, S-adenosyl-L-methionine. The FAMT protein structural model, including the active site, was constructed by using the experimentally determined structure of the C. breweri SAMT co-crystallized with SA and SAH as the template for homology modeling.

METHYLATION METHYLATION AND AND DEMETHYLATION DEMETHYLATION OF PLANT PLANT SIGNALING SIGNALING 261 Arabidopsis At5gO437O Encodes an Enzyme Capable of MethylatingNicotinic Acid Another AtSABATH enzyme for which an in vitro susbtrate has been found is the methyltransferase encoded by At5g04370. We have determined that this enzyme possesses high specific activity for nicotinic acid (NA), an important intermediate compound in the salvage biosynthetic pathway of NAD + , where NA is found in the amide form. NAD + is an essential chemical cofactor for all organisms, both as a coenzyme for oxidoreductases and as a source of ADP-ribosyl groups used in various metabolic pathways.21 The carboxyl methylation of NA, to give methyl nicotinate, precludes it from forming the amide. NA also serves as the precursor of trigonelline, which is produced in plants through the N-methylation of NA and is hypothesized to be involved in desiccation and salt tolerance as well as several other developmental processes.22 Two-week-old Arabidopsis seedlings express NAMT in all tissues examined. In mature plants, NAMT expression is detected in flowers, roots, and siliques. NAMT was induced by NaCl, ABA (another signaling molecule involved in desiccation response), trigonelline, quinolinic acid (a NA analog), and NA.

POSSIBLE ROLES FOR METHYLATION OF SIGNALING MOLECULES, AND THE CONVERSION OF METHYLESTERS BACK TO THE FREE ACIDS MEDIATED BY THE ARABIDOPSIS ESTERASE FAMILY The natural occurrence of methylated signaling molecules has seldom been reported. It is likely that these molecules inside tissues are often not detected because they are found at low levels, and also because many investigators, who are mainly concerned with measuring internal concentrations of the nonvolatile forms of signaling molecules, use methods that are incompatible with the detection of such methylesters.23 In the extreme, some of these molecules, including auxin, are actually chemically methylated as part of the analytical procedure so that they can be studied by using techniques such as GC-MS.24'25 This experimentally introduced methylation would obviously completely eliminate the ability of the experimentalist to ascertain the naturally occurring methylated forms of such compounds. The most extensively documented occurrence of a methylated signaling molecule is that of MeSA. As mentioned, MeSA has been detected in the floral scent of many species. However, there are also many reports indicating that MeSA is produced in and emitted from vegetative parts of the plant upon induction of systemic acquired resistance (SAR) and during herbivory in general.26'27 While emission of the volatile MeSA may help the plant attract parasitic wasps that prey

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upon the caterpillars inflicting tissue damage,28'29 in some cases, for example in tobacco leaves during tobacco mosaic virus (TMV) infection, the internal concentration of MeSA is quite high as well (on the order of a few (j,g/g FW),26 implying possible roles other than in predatory insect attraction. Salicylic acid (SA) has been shown to be an essential component of the signal transduction pathway in SAR.2 Recently, we showed that a tobacco protein originally designated SABP2 (for its ability to bind SA) that is induced during SAR is a methylsalicylate esterase, capable of de-esterifying MeSA back to SA.30'31 When the expression of SABP2 is suppressed, tobacco plants infected with TMV cannot mount an effective SAR response, ° indicating that MeSA is also an essential part of the signal transduction pathway in SAR. Bioinformatics analysis has revealed that the Arabidopsis genome contains 20 genes encoding proteins with sequence similarity to tobacco SABP232 (Fig. 10.5). Additional work has shown that several of them have methylsalicylate esterase activity (Yang, Kumar, Klessig and Pichersky, unpubl.). The role of MeSA in the signal transduction pathway is not yet clear. It is likely that MeSA itself is not capable of eliciting a response at the target cells, since plants that cannot convert MeSA to SA are defective in SAR. Since MeSA is produced from SA, why would plants make MeSA only to convert it back to SA? The most likely explanation is that converting SA to MeSA is part of mobilizing the signal. MeSA is more non-polar than SA and, therefore, is capable of diffusing through membranes. However, whether MeSA is a mobile signal, involved in either short-distance, cell-to-cell transport, or in long-distance transport, remains to be experimentally determined. MeJA has also been reported in floral volatiles. The importance of JA as a signaling molecule that regulates many defense responses is now well appreciated.4'33 In studying the effect of JA, many investigators typically exogeneously apply MeJA instead of JA because MeJA is much cheaper than JA and because MeJA, like MeSA, is much more non-polar than its free acid and more readily penetrates into plant tissue. It has generally been assumed that once MeJA enters the cell, it is de-esterified back to the active compound JA. However, only recently has it been shown by Stuhlfelder et al. that tomato possesses a methyljasmonate esterase (MJE).34'35 Stuhlfelder et al. applied MeJA to tomato cell culture, showing that a methylesterase activity was induced. The protein was purified and shown to be active with both MeJA and MelAA but not with MeSA.34 The protein sequences of the tomato MJE34 and a closely related MJE from potato (accession number AY684102) indicate that they are homologous to tobacco SABP2 and to the Arabidopsis esterase family (Fig. 10.5).

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At4g37140

At2g23570 At2g23550 At2g23580 At2g23560

At2g23590 At5g10300

At2g23610

MJE

At4g16690

At3g50440 At4g09900

At5g58310

At1g33990

o.i

At3g29770 At3g10870 At1g26360 At1g69240

Figure 10.5: The Arabidopsis genome has 20 genes likely to encode small-molecule esterases. Shown is an unrooted phylogenetic tree constructed with protein sequences of Nicotiana tabacum methylsalicylate esterase (SABP2), Lycopersicon esculentum methyljasmonate esterase (MJE), and with the Arabidopsis thaliana protein sequences.

As with MeSA, the function of MeJA is not yet known. Seo et al. showed that when JMT was overexpressed under the control of the 35S promoter in Arabidopsis, the concentration of MeJA in the transgenic plants increased, the concentration of JA did not change, and the plants were more resistant to fungal

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attack.7 We have examined the JMT overexpressing line constructed by Seo et al. by DNA microarray analysis and have found that a member of the Arabidopsis esterase family (encoded by gene At5g58310, Fig. 10.5) is induced 4-fold in these transgenic plants. A cDNA of At5g58310 was expressed in E. coli, and the protein was shown to have methylesterase activity with MeJA and MelAA, but not with MeSA (Yang, Fridman and Pichersky, unpubl.). The increase in disease resistance of plants overexpressing JMT observed by Seo et al. may thus be due to increased mobility of MeJA, which is then converted back to JA to exert its regulatory function in the target cells. MelAA has rarely been reported in plants,11'36'37 although it has been applied exogenously by investigators because of its higher penetrability compared with the more polar IAA.38 Based on our analysis, MelAA is present at different levels in different tissues but always at lower concentrations than free IAA, with the exception in the roots, where MelAA levels are higher than free IAA levels (unpublished data). There are more MelAA in siliques and flowers than in leaves, likely due to the higher IAMT expression in these organs compared with leaves. The MelAA levels in roots are 2-fold higher than the levels in the siliques, although IAMT mRNA levels, determined by real-time RT-PCR analysis, are more than 20-fold lower in roots than in siliques. This discrepancy might be explained by substrate availability, since roots are known to have more capacity for de novo synthesis of IAA.39 However, the possibility that MelAA synthesized elsewhere in the plant is mobilized to the roots cannot be ruled out, particularly since the levels of MelAA in the roots were reduced by more than 50% in a knockout mutant in IAMT, a gene known to be expressed at very low levels in roots compared to siliques and flowers. The function of MelAA remains unclear. It has been demonstrated that the perception of gravity involves the redistribution of auxin to generate a gradient between the lower and upper flanks of organs to cause the phenomenon known as gravitropism, in which the roots grow downward and the above-ground portion of the plant grows upward.40 Plants overexpressing IAMT, however, are partly deficient in gravitropism. One possible explanation is that IAMT inactivates IAA by converting it to MelAA, and thus IAMT overexpressing lines are deprived of active auxins, resulting in loss of gravitropism, a phenotype that is similar to that displayed by mutants with reduced endogenous auxin.41 On the other hand, by converting IAA to MelAA, IAMT might play a role in the establishment of auxin gradients through methylating and mobilizing IAA, followed by de-methylation to IAA at the target site by an esterase. To test these hypotheses, the induction patterns of IAMT and esterases capable of de-esterifying MelAA upon gravistimulation need to be examined. Our in vitro biochemical assays have determined that MelAA could be demethylated by enzymes encoded by several Arabidopsis genes with sequence similarity to tobacco SABP2 (Fig. 10.5) (Yang, Pichersky, unpublished data). These MelAA esterases are induced in different organs and under different conditions

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(information obtained from publicly available DNA-chip database, for example https://www.genevestigator.ethz.ch/ 42). Since the tomato MeJA esterase from tomato was reported to be equally active with MelAA,34 we tested these Arabidopsis SABP2 homologs with MeJA and found that most, but not all, of the esterases that can de-methylate MelAA can also de-esterify MeJA (unpublished data). The reasons for the dual specificity are not yet clear. There is little data regarding the in vivo presence and activity of other methylated signaling molecules and hormones, such as gibberellins. Since overexpression of GAMTl and GAMT2 using the 35S promoter resulted in phenotypes resembling those caused by GA deficiency, it appears that methyl gibberellins are not active or may act as true antagonists. Whether the in vivo role of GAMTl and GAMT2 is to inactivate GAs, convert them to antagonists, or to help in their mobilization remains to be determined. For the other identified substrates of AtSABATH methyltransferases, there is even less evidence for roles as signaling molecules, and it is premature to speculate on the significance of the observation that they may be methylated. Farnesoic acid and its analogues have been shown to be signaling molecules in insects, fungi and bacteria,43"45 but not yet in plants. Nicotinic acid is not known to be a signaling molecule in any organism; but trigonelline, a compound found in plants that is biosynthesized by N-methylation of NA, has been hypothesized to play a role in plant defense signaling. ' 7 It remains to be determined whether other AtSABATH enzymes methylate other plant signaling molecules, and whether plants also have esterases that can reverse such methylation reactions. SUMMARY AND FUTURE DIRECTIONS The Arabidopsis thaliana genome contains 24 genes encoding carboxyl methyltransferases belonging to the AtSABATH family. Our recent work has shown that enzymes in this MT family catalyze the formation of methylesters of salicylic acid (SA), indole-3-acetic acid (IAA), various gibberellins (GAs), farnesoic acid, nicotinic acid, as well as of jasmonic acid (JA), as previously described. JA, SA, IAA and GAs are known signaling molecules in plants, and they also play important roles in many other facets of the plant life cycle. Some of these signaling compounds, notably JA and IAA, have also been shown to be conjugated at their carboxyl end to other compounds including sugars and amino acids, and such conjugations may affect the mobility and activity of these as well as other hormones. Methylation of signaling molecules at their carboxyl end blocks further conjugation at this moiety; thus, carboxyl-directed methylation of these signaling molecules antagonizes sugar- and amino acid-directed biochemical conjugations (Fig. 10.6). Furthermore, the methylesters are more non-polar than the free acids and are, therefore, more able to diffuse through membranes. Thus, methylation may be involved in cell-to-cell transport or even long-distance transport of signaling

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molecules. We have also shown that the A. thaliana genome possesses a family of genes encoding enzymes that hydrolyze methylesters of some, and perhaps all, of these signaling molecules to give back the free acids. In the future, we plan to test whether methylation and demethylation of plant signal molecules play a role in deactivating/activating such molecules or in mobilizing them, or in both. These questions will be addressed in part by examining the sites of action of both methylatransferases and methylesterases. For example, the spatial separation of the expression of the methyltarnsferases and the esterases to the origin and destination, respectively, of the signal molecules will provide evidence for involvement in a transport mechanism. A correlation of methyltransferase expression with a diminution in hormonal activity, on the other hand, will suggest involvement in deactivation. Other experiments are designed to determine the exact substrate specificity of each of the remaining carboxyl methyltransferases and their corresponding methyl esterases.

O R-C-X

O R-C-OH

MT «

O R-6-OCH3

Esterase Figure 10.6: A schematic diagram of the interactions between methylation, de-esterification, and other conjugation reactions involving signaling molecules. Methylation by the SABATH MTs is mutually exclusive with the other known conjugation reactions carried out on the carboxyl group, and esterases convert methylesters back to free acids.

ACKNOWLEDGMENTS This work was supported by National Science Foundation Arabidopsis 2010 Project MCB-0312466 to EP, MCB-0312449 to JPN, and MCB 0312857 to VS.

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17. YANG, Y., YUAN, J.S., ROSS, J.R., NOEL, J.P., PICHERSKY, E., CHEN, F., An Arabidopsis thaliana methyltransferase capable of methylating farnesoic acid, Arch. Biochem. Biophys., 2005, 444, In Press. 18. ENGELBERTH, J., KOCH, T., SCHULER, G., BACHMANN, N, RECHTENBACH, J., BOLAND, W., Ion channel-forming alamethicin is a potent elicitor of volatile biosynthesis and tendril coiling. Cross talk between jasmonate and salicylate signaling in lima bean, Plant Physiol, 2001,125, 369-377. 19. TOONG, Y.C., SCHOOLEY, D., BAKER, F., Isolation of insect juvenile hormone III from a plant, Nature, 1988, 333,170-171. 20. BEDE, J.C., GOODMAN, W.G., TOBE, S., Developmental distribution of insect juvenile hormone III in the sedge, Cyperus iria L., Phytochemistry, 1999, 52, 12691274. 21. BIEGANOWSKI, P., BRENNER, C , Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans, Cell, 2004,117,495-502. 22. SHIMIZU, M.M., MAZZAFERA, P., A role for trigonelline during imbibition and germination of coffee seeds, Plant Biol, 2000, 2, 605-611. 23. SCHMELZ, E.A., ENGELBERTH, J., TUMLINSON, J.H., BLOCK, A., ALBORN, H.T., The use of vapor phase extraction in metabolic profiling of phytohormones and other metabolites, Plant J., 2004, 39, 790-808. 24. CHEN, K.-H., MILLER, A., PATTERSON, G., COHEN, J., A rapid and simple procedure for purification of indole-3-acetic acid prior to GC-MS-SIM analysis, Plant Physiol, 1988,86,822-825. 25. LJUNG, K., BHALERAO, R.P., SANDBERG, G., Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth, Plant J., 2001, 28, 465474. 26. SHULAEV, V., SILVERMAN, P., RASKIN, I., Airborne signalling by methyl salicylate in plant pathogen resistance, Nature, 1997, 385, 718-721. 27. VAN POECKE, R.M.P., POSTHUMUS, M.A., D1CKE, M., Herbivore-induced volatile production by Arabidopsis thaliana /eads to attraction of the parasitoid Cotesia rubecula: Chemical, behavioral, and gene-expression analysis, J. Chem. Ecol, 2001,27, 1911-1928. 28. TURLINGS, T.C.J., LOUGHRIN, J.H., MCCALL, P.J., ROSE, U.S.R., LEWIS, W.J., TUMLINSON, J.H., How caterpillar-damaged plants protect themselves by attracting parasitic wasps, Proc. Natl. Acad. Sci. U.S.A., 1995, 92,4169-4174. 29. TAKABAYASHI, J., DICKE, M., Plant-carnivore mutualism through herbivoreinduced carnivore attractants, Trends Plant Sci., 1996,1, 109-113. 30. KUMAR, D., KLESSIG, D.F., High-affinity salicylic acid-binding protein 2 is required for plant innate immunity and has salicylic acid-stimulated lipase activity, Proc. Natl. Acad. Sci. U.S.A., 2003, 100, 16101-16106. 31. FOROUHAR, F., YANG, Y., KUMAR, D., CHEN, Y., FRIDMAN, E., PARK, S.W., CHIANG, Y., ACTON, T.B., MONTELIONE, G.T., PICHERSKY, E., KLESSIG, D.F., TONG, L., Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity, Proc. Natl. Acad Sci. U.S.A., 2005,102,1773-1778.

METHYLATION AND AND DEMETHYLATION DEMETHYLATION OF PLANT PLANT SIGNALING SIGNALING 269 METHYLATION 32. FRIDMAN, E., WANG, J., IIJIMA, Y., FROEHLICH, J.E., GANG, D.R., OHLROGGE, J., PICHERSKY, E., Metabolic, genomic, and biochemical analyses of glandular trichomes from the wild tomato species Lycopersicon hirsutum identify a key enzyme in the biosynthesis of methylketones, Plant Cell, 2005,17, 1252-1267. 33. HOWE, G.A., SCHILMILLER, A.L., Oxylipin metabolism in response to stress, Curr. Opin. Plant Biol., 2002, 5,230-236. 34. STUHLFELDER, C , LOTTSPEICH, F., MUELLER, M.J., Purification and partial amino acid sequences of an esterase from tomato, Phytochemistry, 2002, 60, 233240. 35. STUHLFELDER, C, MUELLER, M.J., WARZECHA, H., Cloning and expression of a tomato cDNA encoding a methyl jasmonate cleaving esterase, Eur. J. Biochem., 2004,271,2976-2983. 36. NARASIMHAN, K., BASHEER, C , BAJIC, V.B., SWARUP, S., Enhancement of plant-microbe interactions using a rhizosphere metabolomics-driven approach and its application in the removal of polychlorinated biphenyls, Plant Physiol, 2003, 132, 146-153. 37. WOODWARD, A.W., BARTEL, B., Auxin: Regulation, action, and interaction, Ann. Bot., 2005, 95, 707-735. 38. ZIMMERMAN, P., HITCHCOCK, A.E., Comparative effectiveness of acids, esters, and salts as growth substances and methods of evaluating them, Contrib. Boyce Thompson Inst., 1937, 8,337-350. 39. KOWALCZYK, M., SANDBERG, G., Quantitative analysis of indole-3-acetic acid metabolites in Arabidopsis, Plant Physiol, 2001,127, 1845-1853. 40. MORITA, M.T., TASAKA, M., Gravity sensing and signaling, Curr. Opin. Plant Biol, 2004, 7,712-718. 41. ZHAO, Y., HULL, A.K., GUPTA, N.R., GOSS, K.A., ALONSO, J., ECKER, J.R., NORMANLY, J., CHORY, J., CELENZA, J.L., Trp-dependent auxin biosynthesis in Arabidopsis: Involvement of cytochrome P450s CYP79B2 and CYP79B3, Genes Dev., 2002,16,3100-3112. 42. ZIMMERMANN, P., HIRSCH-HOFFMANN, M., HENNIG, L., GRUISSEM, W., GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox, Plant Physiol, 2004,136,2621-2632. 43. DAVEY, K.G., The modes of action of juvenile hormones: Some questions we ought to ask, Insect Biochem. Mol Biol, 2000,30, 663-669. 44. WANG, L-H., HE, Y., GAO, Y., WU, J.E., DONG, Y-H., HE, C , WANG, S.X., WENG, L-X., XU, J-L., TAY, L., FANG, R.X., ZHANG, L-H., A bacterial cell-cell communication signal with cross-kingdom structural analogues, Mol. Microbiol, 2004,51,903-912. 45. OH, K.-B., MIYAZAWA, H., NAITO, T., MATSUOKA, H., Purification and characterization of an autoregulatory substance capable of regulating the morphological transition in Candidaalbicans, Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 4664-4668. 46. HUNT, L., LERNER, F., ZIEGLER, M., NAD - new roles in signalling and gene regulation in plants, New Phytol, 2004,163,31-44.

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47. BERGLUND, T., KALBIN, G., STRID, A., RYDSTROM, J., OHLSSON, A.B., UV-B- and oxidative stress-induced increase in nicotinamide and trigonelline and inhibition of defensive metabolism induction by poly(ADP-ribose)polymerase inhibitor in plant tissue, FEBS Lett., 1996,380, 188-193.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved. Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Eleven

RECENT ADVANCES IN AUXIN BIOSYNTHESIS AND CONJUGATION Amber Kei Bowers and Yunde Zhao* Section of Cell and Developmental Biology Division of Biological Sciences University of San Diego 9500 Gilman Drive La Mia, CA, USA 92093-0116 *Author for correspondence, email: [email protected] Introduction Auxin Biosynthesis Transgenic Plants that Overexpress the iaaM gene YUCCA Family of Flavin Monooxygenase-like Enzymes Transgenic Lines that Overexpress the Cytochrome P450 CYP79B2 The superroot2 Mutant The superrootl Mutant UGT74B1 Glucosyltransferase Other Auxin Biosynthesis Genes Auxin Conjugation The Bacterial IAA-Lysine Synthetase (iaaL) IAA-amido Synthetases in Plants IAA-glucose Synthetase IAA Carboxyl Methyltransferases Summary and Future Directions

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INTRODUCTION Auxin was the first plant hormone ever identified; yet it is probably the least understood among the plant hormones in terms of biosynthesis and metabolism. The study of the biosynthetic pathway and metabolism of auxin has been hampered in past years by genetic redundancy and complexity, but more modern tools have aided great strides in recent progress in the fields of auxin biosynthesis and conjugation. This review will focus on some of the progress made in these fields. AUXIN BIOSYNTHESIS Indole-3-acetic acid (IAA), the main auxin in plants, has a relatively simple structure and can be easily synthesized in a laboratory. However, the biosynthesis of auxin in plants appears to be incredibly complex and remains poorly defined despite many years of research. Because of the structural similarities between IAA and tryptophan, tryptophan has long been proposed as the main precursor for auxin biosynthesis (see review Bartel, 1997).1 In fact, plant pathogenic bacteria such as Agrobacterium tumefaciens and Pseudomonas syringae use tryptophan as the precursor to synthesize IAA in a two-step pathway (Fig. 1 l.l).2"5 The bacteria use a tryptophan-2-monooxygenase (iaaM) to convert tryptophan into indole-3-acetamide that is subsequently hydrolyzed by a hydrolase (iaaH) to produce IAA (Fig. 1 l.l). 2 ' 3 However, the iaaM/iaaH pathway has not yet been found in plants. Early molecular genetics studies on auxin biosynthesis were mainly focused on exploring the effects of mutations in tryptophan biosynthesis and metabolism on auxin biosynthesis.6"11 Such studies resulted in the discovery of a tryptophan-independent auxin biosynthesis pathway, but did not shed much light on how tryptophan is converted to IAA in plants.6'7'12'1 Our current understanding of the tryptophan-dependent auxin biosynthesis mainly stems from research on auxin overproduction Arabidopsis mutants. In this paper, we analyze the identified auxin overproduction mutants and discuss their roles in converting tryptophan to IAA.

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laaM CYP79B2 CYP79B3

-OH

S-alkyl-thiohydroximate

I lndole-3-acetaldehyde

lndole-3-acetonitrile

Indolic glucosinolate

indole-3-acetic acid (IAA)

Figure 11.1: Proposed pathways for the biosynthesis of indole-3-acetic acid. These pathways are all potential tryptophan-dependent sources of IAA. Genes in bold have been shown to cause overproduction of IAA in plants when overexpressed. SUR2, SUR1, and UTG74B1 are all involved in the biosynthesis of glucosinolates, and their disruption is known to cause auxin overproduction. The iaaM/iaaH pathway has been demonstrated in bacteria.

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Transgenic Plants that Overexpress the iaaM Gene The iaaM/iaaH pathway in bacteria has been well established both genetically and biochemically.2"5 It is also known that iaaM converts tryptophan to indole-3acetamide in plants that are infected by pathogenic bacteria.14"16 Overexpression of iaaM in tobacco, petunia, and Arabidopsis leads to auxin overproduction.14"16 Studies on iaaM overexpression in transgenic plants not only have provided insights on what the consequences are when a plant produces too much auxin, but also have provided characteristic phenotypes that can be used to score auxin overproduction mutants that may reveal the key auxin biosynthesis genes in plants. In Arabidopsis, overexpression of the iaaM gene leads to increased hypocotyl length in light, epinastic cotyledons, epinastic rosette leaves, and increased apical dominance.16 Very high level expression of the iaaM gene in Arabidopsis often leads to defects in vascular tissue development and formation of a pin-like inflorescence. In contrast to some other auxin overproduction mutants, Arabidopsis lines overexpressing iaaM do not display dramatic defects in root development (see below).16 YUCCA Family of Flavin Monooxygenase-like Enzymes The mutant yucca was initially identified from an activation-tagging screen for phyA-211 enhancers.17 Later, we found that yucca displayed characteristic auxin overproduction phenotypes, namely, long hypocotyls and epinastic cotyledons when grown in the light.17 In the dark, yucca has shorter hypocotyls and lacks an apical hook.17 The identical phenotypes of the iaaM overexpression lines and yucca prompted us to investigate whether yucca is an auxin overproduction mutant. Several lines of evidence unequivocally demonstrated that yucca is an auxin overproduction mutant. First, direct GC-MS analysis of yucca indicated that there was a 50% increase of free IAA levels in the weak yucca alleles.17 Second, explants of yucca mutant produced massive roots in the absence of any exogenous plant hormones. In fact, yucca explants can generate callus, and yucca plants can be regenerated from an explant without any exogenous auxin, indicating that yucca produces sufficient endogenous auxin to support callus formation and regeneration of yucca plants through tissue culture. Furthermore, auxin inducible genes are upregulated in the yucca mutant.17 YUCCA was proposed to be involved in tryptophan-dependent auxin biosynthesis on the basis of the responses of yucca to tryptophan analogs such as 5methyl tryptophan. The yucca mutant was resistant to the toxic effects of 5-methyl tryptophan through the conversion of 5-methyl tryptophan to 5-methyl IAA, an active auxin that stimulates adventitious root formation.17 The yucca phenotypes were caused by increased levels of expression of a flavin monooxygenase-like (FMO) enzyme that is capable of catalyzing the conversion of tryptamine into N-

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hydroxyl tryptamine, which can proceed to IAA through indole-3-acetaldoxime (Fig. 11.1).17 Because overexpression of the YUCCA gene conferred higher levels of auxin production in the yucca mutant, we proposed that YUCCA catalyzes a ratelimiting step in the auxin biosynthesis in Arabidopsis.11 The identification of YUCCA and elucidation of its role in auxin biosynthesis is a key step forward in determining the auxin biosynthesis mechanisms. However, there is a caveat in the yucca mutant study. The yucca mutant is a gain-of-function dominant mutant caused by overexpression of the YUCCA gene. Therefore, it is debatable whether the YUCCA gene under normal expression conditions also functions in auxin biosynthesis. Analysis of loss-of-function mutants of YUCCA should provide answers to the above argument. However, YUCCA belongs to a family with 11 members in Arabidopsis, and a subset of the family has been shown to have overlapping functions. It was not surprising that T-DNA insertion mutants of either YUCCA1 or YUCCA2 did not display obvious developmental phenotypes. There was essentially no phenotypic difference between the yuccal yucca2 double mutant and wild-type.17 Nevertheless, the identification of all the YUCCA genes provides us the opportunity to analyze systematically each YUCCA gene in Arabidopsis and their contributions to auxin biosynthesis and plant development. We believe that mutants in which the functions of several YUCCA genes in the correct combinations are compromised will tell us the differing functions of the many YUCCA gene family members. The expression patterns of the YUCCA genes will tell us how to make the multiple loss-of-function mutants that may show developmental defects. The importance of YUCCA genes in plant growth and development has been further supported by studies of a petunia mutant called floozy, which has a transposon insertion in the YUCCA ortholog in petunia.18 The petunia floozy mutant is a recessive loss-of-function mutant with defects in floral organ and leaf vascular tissues. The floozy mutant also displays a loss of apical dominance similar to that of auxin signaling mutants, but no apparent reduction in free auxin levels. Overexpression of FLOOZY causes plants to have elongated and narrow leaves and a proliferation of root hairs similar to the YUCCA overexpressing Arabidopsis mutants. 1718 Taken together, the work on YUCCA genes in both Arabidopsis and petunia provides a handle for further studying auxin biosynthesis and the roles of the YUCCA genes in plant growth and development. Transgenic Lines that Overexpress the Cytochrome P450 CYP79B2 CYP79B2 was first isolated as a tryptophan metabolism enzyme by screening yeast cells transformed with an Arabidopsis cDNA library for cDNAs that confer resistance to toxic tryptophan analogs.19 CYP79B2 was found to convert tryptophan to indole-3-acetaldoxime in vitro (Fig. 11.1).1 ' Overexpression of CYP79B2 in Arabidopsis led to resistance to toxic tryptophan analogs.19'21 More importantly,

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light grown CYP79B2 overexpression lines also displayed long hypocotyls and epinastic cotyledons, which are characteristic phenotypes of auxin overproduction plants observed in the iaaM overexpression lines and in the yucca mutants. ' ' Indeed, the CYP79B2 overexpression lines have elevated free IAA levels, and various auxin-inducible genes are induced in the overexpression lines.21 CYP79B2 has a close homolog in Arabidopsis called CYP79B3.19 The cyp79b2 cyp79b3 double loss-of-function mutant shows both measurably lower levels of free IAA than wild-type and displays phenotypes consistent with lower levels of auxin.21 These studies indicate that indole-3-acetaldoxime is an important auxin biosynthesis intermediate (Fig. 11.1), and the production of indole-3-acetaldoxime appears to be a rate-limiting step in auxin biosynthesis. The identification of CYP79B2 and CYP79B3 added more complexity to the auxin biosynthesis mechanism. It will be interesting to investigate the relative contributions of the YUCCA pathway and the P450s to auxin biosynthesis and how the two pathways are regulated and coordinated. The superroot2 Mutant The superroot2 (sur2) mutant was initially identified as a recessive mutant with massive adventitious roots originating from hypocotyls.22 In addition to the root phenotypes, light grown sur2 also has long hypocotyls and epinastic cotyledons, characteristic auxin overproduction phenotypes that are observed in the iaaM overexpression lines, yucca mutants, and the CYP79B2 overexpression lines.22 Direct free IAA analysis showed that sur2 produced more free IAA than wild-type.22 SUR2 encodes a cytochrome P450 named CYP83B1 that catalyzes the Nhydroxylation of indole-3-acetaldoxime to produce the corresponding acz-nitro compound, l-acz-nitro-2-indolyl-ethane, a key intermediate for indolic glucosinolate biosynthesis (Fig. 11.1).23'24 Loss-of-function sur2/cyp83Bl mutants cannot convert indole-3-acetaldoxime to glucosinolate intermediates; therefore, more indole-3acetaldoxime is fluxed to IAA biosynthesis to cause auxin overproduction. Although SUR2 does not participate in auxin biosynthesis directly, work on SUR2 provided additional evidence that indole-3-acetaldoxime is an important auxin biosynthesis intermediate. Interestingly, sur2 has the adventitious root phenotypes that have not been observed in iaaM overexpression lines, the CYP79B2 overexpression lines, or yucca mutants.16'17'21 It is not clear why the auxin overproduction mutants show such differences in root phenotypes. One possible explanation for the observed differences may be the result of different expression patterns of the genes that cause elevated auxin levels in different cells.

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The superrootl (rooty) Mutant The superrootl {surl) mutant was probably the first identified auxin overproduction mutant in Arabidopsis.25'26 Like sur2, surl also produces massive adventitious roots from hypocotyls. When grown in the light, surl also displays long hypocotyls and epinastic cotyledons. In the dark, surl has short hypocotyls without an apical hook, a phenotype also observed in the yucca mutant.25'2 SUR1 encodes a C-S lyase that has been shown to participate in glucosinolate biosynthesis as well (Fig. 11.1).27 SUR1 catalyzes S-alkylthiohydroximate to thiohydroximic acid, a key step in glucosinolate biosynthesis (Fig. 11.1).27 Like sur2, the auxin overproduction phenotypes of surl can also be attributed to the funneling of excess indole-3acetaldoxime into IAA biosynthesis, further supporting that indole-3-acetaldoxime is a key intermediate in auxin biosynthesis. UGT74B1 Glucosyltransferase Another protein involved in glucosinolate biosynthesis that appears to be involved in auxin homeostasis is the glucosyltransferase, UGT74B1, which catalyzes a step downstream of SUR1 in glucosinolate biosynthesis (Fig. 11.1).28 Like surl and sur2, recessive ugt74bl mutants show high levels of auxin and high auxinrelated phenotypes, such as epinastic leaves and shortened hypocotyls in dark-grown seedlings, in addition to some phenotypes apparently unrelated to the overproduction of auxin.28 Additionally, expression of UGT74B1 is induced in the cotyledons of plants upon auxin treatment, which, together with the observations that SUR2 expression is also regulated by auxin levels, suggests that auxin overproduction can be compensated for in plants by a negative feedback mechanism that will funnel excess intermediates into the production of glucosinolates and down regulation of IAA production.28 Other Auxin Biosynthesis Genes Research on dominant auxin overproduction lines and the indolic glucosinolate biosynthesis pathway has established that indole-3-acetaldoxime is a key intermediate in IAA biosynthesis. However, how indole-3-acetaldoxime is converted to IAA is not understood at present. Indole-3-acetaldoxime can be converted to indole-3-acetonitrile by eliminating one molecule of water, and indole3-acetonitrile is subsequently converted to IAA by a family of nitrilases in Arabidopsis.29'31 Indole-3-acetaldoxime may be converted to indole-3-acetaldehyde by hydrolysis, and the aldehyde can then be converted to IAA by an aldehyde oxidase or an aldehyde dehydrogenase. There is some evidence that Aldehyde Oxidase 1 (AO1) in Arabidopsis may be important for auxin biosynthesis. In the surl mutants, AO1 is up-regulated, and the enzyme shows substrate preference for

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indole-3-acetaldehyde.32 The regulation of this enzyme is unaffected by the addition of more IAA, so the effects are not due to the overproduction of IAA in surl plants. However, mutants in the biosynthetic pathway of molybdopterin, an essential cofactor for all aldehyde oxidases, did not show auxin deficient phenotypes, suggesting that the aldehyde oxidases are probably not essential for auxin biosynthesis (Zhao et al., unpublished data). Another potential player in auxin biosynthesis is an amidase that can hydrolyze indole-3-acetamide to IAA.33 As described above, the iaaM / iaaH pathway has not been found in plants. However, it was reported that plants grown in sterile conditions contain indole-3-acetamide, although it is not clear how the indole3-acetamide is produced in plants. It was recently proposed that indole-3-acetamide in plants is processed into IAA by an amidase known as AMI I.33 In summary, genetic and biochemical studies have identified several key genes in the tryptophan dependent auxin biosynthesis pathway (Fig. 11.1). We focused on the routes that are supported by genetic and biochemical studies, although tryptophan can be converted to IAA through various routes in theory (Fig. 11.1). The indole-3-pyruvate route is widely used in bacteria, but little evidence suggests that this is the case in plants. However, recently a gene encoding the pyruvate dehydrogenase El alpha homolog was identified from a genetic screen for mutants resistant to exogenous IAA-amino acid conjugates, indicating that indole-3-pyruvate may be an important intermediate in auxin biosynthesis in plants.34 AUXIN CONJUGATION The carboxyl group in IAA is necessary for the auxin activities displayed by IAA. Almost all the known synthetic auxins also need a carboxyl group for their auxin activities. Therefore, modifications of the carboxyl group can serve as an effective means to regulate IAA activity. In theory, the carboxyl group in IAA can form either an ester bond with a hydroxyl group of sugars or alcohols, or an amide bond with amino acids or primary amines. However, the IAA carboxyl group is not normally very reactive. In order to form conjugates with sugars or amino acids, the carboxyl group either needs to be activated or to react with an activated intermediate. Here, we discuss how the carboxyl group can be activated and how various auxin conjugates are formed. We also discuss the implications of forming auxin conjugates in regulating auxin activities. The Bacterial IAA-Lysine Synthetase (iaaL) Research on auxin biosynthesis in bacteria has provided valuable information for analyzing auxin biosynthesis in plants. Auxin conjugations were first identified in bacteria.3 The biochemical mechanisms governing auxin conjugation in bacteria were solved long before the plant auxin conjugate enzymes were identified. The

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bacterial auxin conjugation system has provided useful information for analyzing auxin conjugation in plants. The first enzyme responsible for converting free IAA to an IAA-amino acid conjugate was isolated from Pseudomonas syringae?5^6 The iaaL protein catalyzes the reaction between IAA and the amino acid lysine in an ATP dependant manner to form NE-indole-3-acetyl-L-lysine (Fig. 11.2).35'36 The reaction probably proceeds in two steps: i) the carboxyl group of IAA reacts with ATP to form the acyl-AMP intermediate and pyrophosphate; ii) the s-amino group nucleophilic substitution of the AMP group forms the IAA-Lys conjugate (Fig. 11.2). The reaction is similar to the activation of fatty acids, where ATP is also used to form an acyl-AMP intermediate and pyrophosphate. Our sequence analysis of iaaL indicates that iaaL contains an AMP binding domain at its N-terminal region. Such an AMP binding domain has been found in many other ATP utilizing enzymes. The discovery of iaaL and the IAA-Lys conjugate not only provided a mechanism to modify auxin activity in bacteria, it also provided a tool to regulate auxin activities in plants. When iaaL was overexpressed in plants, it caused dramatic developmental defects that are consistent with lower auxin activities in plants.37 Tobacco plants expressing the iaaL gene displayed reduced rooting, and dramatically reduced apical dominance, which presumably are caused by the reduction of free IAA levels in the transgenic plants.37 In Arabidopsis, overexpression of the iaaL gene also led to dramatic decrease of apical dominance. Since the initial work on the transgenic iaaL plants, iaaL has been widely used as an anti-auxin gene in vivo to regulate auxin activities.17'38 IAA-amido Synthetases in Plants IAA-amino acid conjugates have been isolated from plant tissues for a long time, but only recently have the enzymes responsible for making the conjugates been identified.39 Staswick and colleagues at the University of Nebraska elegantly elucidated that plant GH3 proteins belong to the firefly luciferase family of adenylate-forming enzymes.4 '41 A subset of GH3 proteins in Arabidopsis was shown to make IAA-amino acid conjugates. Although iaaL and GH3 proteins do not share primary sequence homology, they use a similar mechanism to make IAAamino acid conjugates by using ATP to activate the carboxyl group in IAA (Fig. 11.2).40'41

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A) iaaL catalyzed reactions OH ATP

Lysine

PPi

water

"AMP

IaaL

iaaL

B) lAA-amido synthases (GH3) OH Lysine

water

AMP GH3

GH3 'COOH

C) lAA-Glucose synthetase CH2OH CH,OH

CH2OH UTP

O. H

Pi

'AH OH

H

OH

IAGlu

H

OH

O-P-O-P-O-Uridine OH 6" 6"

H

OH

D) IAA carboxyl methyltransferase +

COO' H3N—CCH 2

NH2

IAA methyl transferase

H 3 C-S + H

OHOH

Figure 11.2: Proposed synthesis of IAA conjugates. A) Synthesis of the IAA-Lys conjugate catalyzed by iaaL. B) Synthesis of IAA-amino acid conjugates catalyzed by GH3 proteins. C) Synthesis of IAA-Glucose catalyzed by IAGlu. D) Synthesis of MelAA catalyzed by a methyltransferase.

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IAA-glucose Synthetase In addition to IAA-amino acid conjugates, IAA has been found to form conjugates with a mono or disaccharide by ester linkage. Unlike formation of the IAA-amido linkage where the carboxyl group is activated by adenylation, formation of IAA-sugar conjugation relies on the activation of the sugar by forming UDP-sugar intermediates (Fig. 11.2). The first plant IAA-conjugate-biosynthesis gene, IAGlu, isolated from maize, catalyzes the formation of IAA glucose ester (IAGlc) from IAA and UDP-glucose.42 The physiological roles of the IAA-glucose conjugate have been analyzed by overexpression of the IAGlu gene. Over-expression of the Arabidopsis homolog of IAGlu, UGT84BI, produced wrinkly and curling leaves.43 IAA Carboxyl Methyltransferase Small organic acid molecules such as jasmonic acid and salicylic acid have been shown to form methyl esters in plants.44 However, whether IAA can also be modified by methylation in plants was not clear until recently when an IAA-specific methyl transferase (IAMT1) was identified in Arabidopsis.45'46 IAMT1 belongs to a large family of novel carboxyl methyltransferases with at least 23 members in Arabidopsis45'46 This plant specific methyl transferase family catalyzes the formation of methyl esters from small organic acid molecules and S-adenosyl methionine (SAM). Interestingly, methylation of IAA is not an ATP dependent reaction; instead the reaction uses an active methyl donor, SAM. So far, all of the IAA-amino acid conjugation reactions use ATP to generate an activated acyl-AMP intermediate to drive the reaction, whereas the IAA ester formation does not use ATP to activate the carboxyl group. Instead, the other activated substrates (SAM or UDP-sugar) are used (Fig. 11.2). Unlike the IAA conjugates with sugar and amino acids, which are either polar or charged, methyl IAA is essentially non-polar. The non-polar nature of IAA, of course, renders it different properties, and perhaps different functions, from the IAAsugar or IAA-amino acid conjugates. Methyl IAA (MelAA) is diffusible inside the plants, whereas IAA-sugar and IAA-amino-acid need active transport. We observed that MelAA is at least 10 times more potent than IAA in root and hypocotyl elongation assays. The different potency between MelAA and IAA is probably caused by the different efficiency in uptake of the two compounds.46 The physiological significance of IAA methylation has been demonstrated by analyzing overexpression lines of IAMT1.46 When IAMTI is overexpressed in Arabidopsis, the transgenic plants lose normal gravitropic responses in both the root and the aerial parts of the plants. Furthermore, overexpression of IAMTI causes extreme curvature of rosette and cauline leaves. Overexpression of IAMTI also leads to altered responses to exogenous auxin, indicating that methylation is indeed an effective means to regulate auxin activity.46

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SUMMARY AND FUTURE DIRECTIONS Tremendous progress has been made in the auxin biosynthesis and conjugation field in the past few years. The emerging picture of auxin homeostasis is that a complicated network composed of multiple pathways and genes in auxin biosynthesis and metabolism is responsible for regulating auxin levels in response to environmental and developmental signals. Available genetic and genomic tools will enable us to further analyze the biochemical basis of auxin biosynthesis and metabolism and to investigate the underlying mechanisms on how auxin regulates various plant growth and developmental processes. Although a number of pathways for auxin biosynthesis have been proposed, it still remains obscure what role each of those pathways plays in plant growth and development. It will be important to analyze how each pathway is regulated by various hormonal and environmental signals. The current understanding of auxin homeostasis mainly originated from research that used Arabidopsis thaliana as a model system. It will be important to investigate whether different plant species have different ways of handling auxin homeostasis. It will be especially interesting to explore how the various genes and pathways interact, and the relative contributions of each to auxin homeostasis and, ultimately, to the overall development of the plant. Many auxin conjugates have been discovered in plants, but the regulation and function of each of these has yet to be fully understood. How, for example, does spatial and temporal regulation of these genes fit into the overall picture of auxin biology? This will be a critical question in the elucidation of auxin homeostasis.

ACKNOWLEDGEMENTS We thank the Zhao lab members for critical reading of this manuscript. This work is supported by NIH grant 1RO1GM68631 (to YZ). AKB was supported in part by funds from the NIH CMG Graduate Student Training Grant. REFERENCES 1. BARTEL, B., Auxin Biosynthesis. Annu. Rev. Plant Physiol. Plant Mol. Biol, 1997, 48, 51-66. 2. COMAI, L., KOSUGE, T., Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi. J. Bacteriol, 1980, 143, 950-957. 3. COMAI, L., KOSUGE, T., Cloning characterization of iaaM, a virulence determinant of Pseudomonas savastanoi. J. Bacteriol, 1982,149, 40-46. 4. KOSUGE, T., HESKETT, M.G., WILSON, E.E., Microbial synthesis and degradation of indole-3-acetic acid. I. The conversion of L-tryptophan to indole-3-acetamide by an enzyme system from Pseudomonas savastanoi. J. Biol. Chem., 1966, 241, 3738-3744.

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5. MAGIE, A.R., WILSON, E.E., KOSUGE, T., Indoleacetamide as an Intermediate in the Synthesis of Indoleacetic Acid in Pseudomonas savastanoi. Science, 1963, 141, 12811282. 6. WRIGHT, A.D., MOEHLENKAMP, C.A., PERROT, G.H., NEUFFER, M.G., CONE, K.C., The maize auxotrophic mutant orange pericarp is defective in duplicate genes for tryptophan synthase beta. Plant Cell, 1992, 4, 711-719. 7. WRIGHT, A.D., SAMPSON, M.B., NEUFFER, M.G., MICHALCZUK, L., SLOVIN, J.P., COHEN, J.D., Indole-3-acetic acid biosynthesis in the mutant maize orange pericarp, a tryptophan auxotroph. Science, 1991, 254, 998-1000. 8. BERLYN, M.B., LAST, R.L., FINK, G.R., A gene encoding the tryptophan synthase beta subunit of Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, 1989, 86, 4604-4608. 9. LAST, R.L., BISSINGER, P.H., MAHONEY, D J , RADWANSKI, E.R., FINK, G.R, Tryptophan mutants in Arabidopsis: the consequences of duplicated tryptophan synthase beta genes. Plant Cell, 1991, 3, 345-358. 10. NIYOGI, K.K., FINK, G.R., Two anthranilate synthase genes in Arabidopsis: defenserelated regulation of the tryptophan pathway. Plant Cell, 1992, 4, 721-733. 11. NIYOGI, K.K., LAST, R.L., FINK, G.R., KEITH, B., Suppressors of trpl fluorescence identify a new Arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit. Plant Cell, 1993,5, 1011-1027. 12. NORMANLY, J., COHEN, J.D., FINK, G.R., Arabidopsis thaliana auxotrophs reveal a tryptophan-independent biosynthetic pathway for indole-3-acetic acid. Proc. Natl. Acad. Sci. USA, 1993,90, 10355-10359. 13. OUYANG, J., SHAO, X., LI, J., Indole-3-glycerol phosphate, a branchpoint of indole-3acetic acid biosynthesis from the tryptophan biosynthetic pathway in Arabidopsis thaliana. Plant J., 2000, 24, 327-333. 14. KLEE, H.J., HORSCH, R.B., HINCHEE, M.A., HEIN, M.B., HOFFMANN, N.L., The effects of overproduction of two Agrobacterium tumefaciens T-DNA auxin biosynthetic gene products in transgenic petunia plants. Genes Dev., 1987,1, 86-96. 15. ROMANO, C.P., COOPER, M.L., KLEE, H.J., Uncoupling auxin and ethylene effects in transgenic tobacco and Arabidopsis plants. Plant Cell, 1993, 5, 181-189. 16. ROMANO, C.P., ROBSON, P.R., SMITH, H., ESTELLE, M., KLEE, H., Transgenemediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-l hypocotyl elongation and axrl auxin-resistant mutants. Plant Mol. Biol, 1995,27, 1071-1083. 17. ZHAO, Y., CHRISTENSEN, S.K., FANKHAUSER, C, CASHMAN, J.R., COHEN, J.D., WEIGEL, D., CHORY, J., A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science, 2001, 291, 306-309. 18. TOBENA-SANTAMARIA, R., BLIEK, M., LJUNG, K., SANDBERG, G., MOL, J.N., SOUER, E., KOES, R., FLOOZY of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture. Genes Dev., 2002, 16, 753763. 19. HULL, A.K., VIJ, R., CELENZA, J.L., Arabidopsis cytochrome P450s that catalyze the first step of tryptophan-dependent indole-3-acetic acid biosynthesis. Proc. Natl. Acad. Sci. USA, 2000, 97, 2379-2384.

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20. MIKKELSEN, M.D., HANSEN, C.H., WITTSTOCK, U., HALKIER, B.A., Cytochrome P450 CYP79B2 from Arabidopsis catalyzes the conversion of tryptophan to indole-3acetaldoxime, a precursor of indole glucosinolates and indole-3-acetic acid. J. Biol. Chem., 2000,275,33712-33717. 21. ZHAO, Y., HULL, A.K., GUPTA, N.R., GOSS, K.A., ALONSO, J., ECKER, J.R., NORMANLY, J., CHORY, J., CELENZA, J.L., Trp-dependent auxin biosynthesis in Arabidopsis: Involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes Dev., 2002,16,3100-3112. 22. DELARUE, M., PRINSEN, E., ONCKELEN, H.V., CABOCHE, M., BELLINI, C, Sur2 mutations of Arabidopsis thaliana define a new locus involved in the control of auxin homeostasis. Plant J., 1998,14, 603-611. 23. BAK, S., FEYEREISEN, R., The involvement of two p450 enzymes, CYP83B1 and CYP83A1, in auxin homeostasis and glucosinolate biosynthesis. Plant Physiol, 2001,127, 108-118. 24. BARLIER, I., KOWALCZYK, M., MARCHANT, A., LJUNG, K., BHALERAO, R., BENNETT, M., SANDBERG, G., BELLINI, C, The SUR2 gene of Arabidopsis thaliana encodes the cytochrome P450 CYP83B1, a modulator of auxin homeostasis. Proc. Natl. Acad. Sci. USA, 2000, 97, 14819-14824. 25. BOERJAN, W, CERVERA, M.T., DELARUE, M., BEECKMAN, T., DEWITTE, W., BELLINI, C, CABOCHE, M., VAN ONCKELEN, H., VAN MONTAGU, M., INZE, D., Superroot, a recessive mutation in Arabidopsis, confers auxin overproduction. Plant Cell, 1995,7, 1405-1419. 26. KING, J.J., STIMART, D.P., FISHER, R.H., BLEECKER, A.B., A mutation altering auxin homeostasis and plant morphology in Arabidopsis. Plant Cell, 1995, 7, 2023-2037. 27. MIKKELSEN, M.D., NAUR, P., HALKIER, B.A., Arabidopsis mutants in the C-S lyase of glucosinolate biosynthesis establish a critical role for indole-3-acetaldoxime in auxin homeostasis. Plant J., 2004, 37, 770-777. 28. GRUBB, CD., ZIPP, B.J., LUDWIG-MULLER, J., MASUNO, M.N., MOLINSKI, T.F., ABEL, S., Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis. Plant J., 2004, 40, 893-908. 29. BARTLING, D., SEEDORF, M., SCHMIDT, R.C., WEILER, E.W., Molecular characterization of two cloned nitrilases from Arabidopsis thaliana: Key enzymes in biosynthesis of the plant hormone indole-3-acetic acid. Proc. Natl. Acad. Sci. USA, 1994, 91, 6021-6025. 30. HILLEBRAND, H., BARTLING, D., WEILER, E.W., Structural analysis of the nit2/nitl/nit3 gene cluster encoding nitrilases, enzymes catalyzing the terminal activation step in indole-acetic acid biosynthesis in Arabidopsis thaliana. Plant Mol. Biol., 1998, 36, 89-99. 31. NORMANLY, J., GRISAFI, P., FINK, G.R., BARTEL, B., Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded bytheNITl gens. Plant Cell, 1997,9, 1781-1790. 32. SEO, M., AKABA, S., ORITANI, T., DELARUE, M., BELLINI, C, CABOCHE, M., KOSHIBA, T., Higher activity of an aldehyde oxidase in the auxin-overproducing superrootl mutant of Arabidopsis thaliana. Plant Physiol, 1998,116, 687-693.

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33. POLLMANN, S., NEU, D., WEILER, E.W., Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone, indole-3-acetic acid. Phytochemistry, 2003, 62, 293-300. 34. LECLERE, S., RAMPEY, R.A., BARTEL, B., IAR4, a gene required for auxin conjugate sensitivity in Arabidopsis, encodes a pyruvate dehydrogenase El alpha homolog. Plant Physiol, 2004,135, 989-999. 35. HUTZINGER, O., KOSUGE, T., Microbial synthesis and degradation of indole-3-acetic acid. 3. The isolation and characterization of indole-3-acetyl-epsilon-L-lysine. Biochemistry, 1968, 7, 601-605. 36. GLASS, N.L., KOSUGE, T., Cloning of the gene for indoleacetic acid-lysine synthetase from Pseudomonas syringae subsp. savastanoi. J. Bacterial., 1986,166, 598-603. 37. ROMANO, C.P., HEIN, M.B., KLEE, H.J., Inactivation of auxin in tobacco transformed with the indoleacetic acid-lysine synthetase gene of Pseudomonas savastanoi. Genes Dev., 1991,5,438-446. 38. WEIJERS, D., SAUER, M., MEURETTE, O., FRIML, J., LJUNG, K., SANDBERG, G., HOOYKAAS, P., OFFRINGA, R., Maintenance of embryonic auxin distribution for apical-basal patterning by PIN-FORMED-dependent auxin transport in Arabidopsis. Plant Cell, 2005,17,2517-2526. 39. LJUNG, K., HULL, A.K, KOWALCZYK, M., MARCHANT, A., CELENZA, J., COHEN, J.D., SANDBERG, G., Biosynthesis, conjugation, catabolism and homeostasis of indole-3-acetic acid in Arabidopsis thaliana. Plant Mol. Biol, 2002, 49, 249-272. 40. STASWICK, P.E., SERBAN, B., ROWE, M., TIRYAKI, I., MALDONADO, M.T., MALDONADO, M.C., SUZA, W., Characterization of an Arabidopsis enzyme family that conjugates amino acids to indole-3-acetic acid. Plant Cell, 2005,17, 616-627. 41. STASWICK, P.E., TIRYAKI, I., ROWE, M.L., Jasmonate response locus JAR1 and several related Arabidopsis genes encode enzymes of the firefly luciferase superfamily that show activity on jasmonic, salicylic, and indole-3-acetic acids in an assay for adenylation. Plant Cell, 2002,14, 1405-1415. 42. SZERSZEN, J.B., SZCZYGLOWSKI, K., BANDURSKI, R.S., iaglu, a gene from Zea mays involved in conjugation of growth hormone indole-3-acetic acid. Science, 1994, 265, 1699-1701. 43. JACKSON, R.G., KOWALCZYK, M., LI, Y., HIGGINS, G., ROSS, J., SANDBERG, G., BOWLES, D.J., Over-expression of an Arabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterisation of transgenic lines. Plant J., 2002, 32, 573-583. 44. CHEN, F., D'AURIA, J.C., THOLL, D., ROSS, J.R., GERSHENZON, J., NOEL, J.P., PICHERSKY, E., An Arabidopsis thaliana gene for methylsalicylate biosynthesis, identified by a biochemical genomics approach, has a role in defense. Plant J., 2003, 36, 577-588. 45. ZUBIETA, C, ROSS, J.R., KOSCHESKI, P., YANG, Y., PICHERSKY, E., NOEL, J.P., Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family. Plant Cell, 2003,15, 1704-1716. 46. QIN, G., GU, H., ZHAO, Y., MA, Z., SHI, G., YANG, Y., PICHERSKY, E., CHEN, H., LIU, M., CHEN, Z., QU, L.J., An indole-3-acetic acid carboxyl methyltransferase regulates Arabidopsis leaf development. Plant Cell, 2005, 17, 2693-2704.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Twelve

AUXIN BIOLOGY AND BIOSYNTHESIS Jessica Calio, Yuen Yee Tarn,1 and Jennifer Normanly* Plant Biology Graduate Program, and Dept. of Biochemistry and Molecular Biology, University of Massachusetts, Amherst MA 01003 1

Current address: Children's Hospital Oakland Research Institute, Oakland CA 94609 * Author for correspondence, email: normanly(g),biochem.umass.edu

Introduction Auxin Biosynthesis from a Biological Perspective: Auxin Homeostasis Auxin Signaling Auxin Forms Gradients Auxin Inactivation Auxin Biosynthesis Auxin Biosynthesis from an Analytical Perspective IAA is a Low Abundance Compound Mutants and Stable Isotope Labeling to Study IAA Biosynthesis IAA Inter-Conversion to IBA and Role of IBA Cross-Talk with Other Pathways Summary and Future Directions

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INTRODUCTION Work in a variety of plant systems demonstrates a role for auxin biosynthesis in many aspects of plant growth and development.1"5 This chapter will discuss recent advances in understanding auxin biosynthesis from both a biological and analytical perspective. Indole-3-acetic acid (IAA) is the most abundant of the naturally occurring auxins, and the terms auxin and IAA are used interchangeably throughout.

AUXIN BIOSYNTHESIS FROM A BIOLOGICAL PERSPECTIVE: AUXIN HOMEOSTASIS From a biological perspective, auxin biosynthesis is just one component of auxin homeostasis. From embryogenesis through senescence, auxin is central to all aspects of plant growth and development. Auxin levels vary dramatically throughout the body and life of the plant, forming gradients that are a primary component of its action.6"11 The concentration of auxin in a plant varies both temporally and spatially in response to changing environmental and developmental cues and through intricate and apparently redundant regulatory networks. These networks include the biosynthesis, inactivation, transport, and inter-conversion pathways that regulate IAA levels, referred to as auxin homeostasis. Auxin response pathways are distinct from auxin homeostasis pathways; however, they clearly interact with each other and with other metabolic and signaling networks (Fig. 12.1).12"14

K5 SYNTHESIS

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OTHER METABOLIC NETWORKS

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OTHER SIGNALING NETWORKS

Figure 12.1: Model for auxin homeostasis showing inputs and outputs to the cellular IAA pool.

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Auxin Signaling One general model for auxin signaling involves the binding of IAA to a high affinity receptor, which causes a complex network of signaling events, resulting in changes in growth and development. From initial observations that application of auxin to excised hypocotyls results in rapid cell expansion2 and that auxin causes rapid and specific alterations in the levels of numerous transcripts,15 it is now clear that there are complex signaling cascades involving several gene classes that are important in auxin-mediated growth and developmental processes.16"18 Specifically, the AUX/IAA gene family of auxin regulatory proteins, initially characterized in soybean and pea, contains four conserved domains that function in dimerization and transcriptional activation and repression.19 The AUX/IAA genes encode short-lived nuclear proteins that can interact with themselves and with members of the ARF (auxin response factor) gene family.20"22 ARFs comprise a plant-specific family of 22 DNA-binding proteins that recognize Auxin Response Elements (AuxREs) and control auxin-regulated transcription. SAURs or small auxin up-regulated RNAs, of which there are 70 in Arabidopsis, are rapidly induced (2-5 minutes) after auxin application.19 The GH3 gene family with 19 members in Arabidopsis is also rapidly induced by auxin,19'23> 24 one of which is an IAA inactivating enzyme.25 The recent discovery of the F-box auxin receptor, TIR1 6> 27 is a significant advance in the auxin signaling field. TIR1 is believed to control the ubiquitindependent degradation of AUX/IAA proteins in auxin-responsive gene expression.26' 27 Several classes of well-known signal transduction proteins, including kinases,28"31 phosphatases,32'33 heterotrimeric G-proteins34 and RAC-like GTPases,1 ' 3 5 have also been implicated in auxin response. Additionally, a microRNA has been identified that specifically targets several ARFs.36 Many studies have established that auxin response is intricately tied to other response pathways,37 such as light, 12 ' 38 jasmonic •JQ

acid,

4 0 4 4

other plant hormones,

i i

41

and second messengers.

Auxin Forms Gradients Uniquely among plant hormones, auxin exhibits long distance polar transport (PAT), mediated by carrier proteins for both influx (PIN) and efflux (AUX1). ' 4 7 Localization of PIN genes correlates with auxin distribution,48 and vesicle targeting seems to play a role in positioning these efflux carriers.49 Putative regulators of PAT include the P1NOID gene, which encodes a protein-serine/threonine kinase,50 and which may also act as a negative regulator of auxin signaling.29 Auxin may feedback regulate transport by inhibiting endocytosis and, thus, the cycling of PIN proteins, resulting in their accumulation at the plasma membrane. Additionally, ATPbinding cassette (ABC) multi drug resistance proteins have been implicated in auxin transport.49'52 Gradients are clearly important for auxin action, and several have been

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demonstrated quantitatively. For example, in young tobacco leaves these gradients are also spatially correlated with higher levels of IAA near the petiole and lower levels near the tip.53 Muller et al.54 found that young expanding leaves have higher IAA levels than fully formed leaves, and specifically the base has more IAA than tip, confirming Ljung's work.53 Higher levels of IAA associated with the vasculature supports the hypothesis that auxin is involved in vein differentiation. The polar transport of auxin (PAT) is crucial in maintaining an auxin gradient in leaves. Weijers et al, showed that neither increasing the rate of IAA synthesis nor changing the rates of IAA conjugation had an effect on IAA gradients or embryo patterning except when PAT was altered.55 An auxin gradient has been demonstrated in Arabidopsis roots, with higher levels of IAA at the root shoot junction and lower levels at the distal end.56 High levels of IAA synthesis have been observed in roots,53 specifically the most apical section,57 and in fact the apical section also has a gradient, which is reversed. Excised roots are capable of making IAA although the concentration gradient is decreased compared to intact roots.5 Therefore, there must be two pools of IAA, one that is transported to the shoot and one that is synthesized in the root apex. It appears that auxin acts as a morphogen in developing tissues. For example, Reinhardt et al. have shown that proteins that control auxin transport also regulate phyllotaxus, and the IAA gradient that has been demonstrated in the cambium could be important in regulating wood formation.58 These cells are sensing IAA from the PAT system rather than synthesizing IAA. Auxin Inactivation IAA occurs in a variety of forms in planta; either free, which is generally accepted to be the biologically active form of the molecule, or conjugated to a variety of molecules, from single amino acids, sugars, or myo-inositol to more complex macromolecules including peptides, proteins, and glycans. 59 ' 60 Conjugates are an integral part of IAA homeostasis because they act as a storage form of IAA that can be rapidly hydrolyzed to yield free IAA. In Arabidopsis most of the IAA is amide-linked to amino acids and peptides while a small amount of IAA is esterlinked to sugars or myo-inositol.61' 2 The enzymes that metabolize IAA have been challenging to isolate from any plant species by classical biochemical approaches, with IAA glucose forming enzyme being one of the few identified in this manner.63 IAA conjugate hydrolases have been identified through genetic screens , and are apparently sufficiently functionally redundant to compensate for each other. Mutants in which only one member of this gene family is affected do not have noticeable "auxin" phenotypes (i.e., phenotypes that are classically associated with excess or insufficient auxin), however, a triple mutant, altered in three IAA hydrolases exhibits phenotypes consistent with a perturbation to auxin homeostasis.65 The enzymes responsible for

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amino acid conjugation of IAA have been particularly recalcitrant to purification; however, a gene in the GH3 family, corresponding to an activity that couples amino acids to IAA was identified recently through a more circuitous route than classic enzymological approaches.25 Auxin Biosynthesis IAA synthesis occurs throughout the plant, with newly fertilized embryos, young leaves, and roots exhibiting high IAA synthesis activity.4 The biosynthetic pathways for IAA (Fig. 12.2) can be classified as tryptophan (Trp)-dependent (TrpD) if IAA is derived via metabolism of Trp, or as Trp-independent (Trp-I) if IAA is derived from an early indolic precursor of Trp, most likely indole-3-glycerol phosphate (IGP).66 These two general routes of IAA synthesis can be distinguished in vivo by stable isotope labeling studies that examine incorporation of label from an early non-specific precursor into IAA and comparing that to incorporation of label directly from Trp. For example, dual labeling of Lemna gibba with [ 5N]anthranilate (ANA) and [2Hs]Trp revealed that the two IAA biosynthetic pathways were differentially utilized depending upon temperature. Double labeling studies in a variety of plant species have consistently demonstrated the differential utilization of Trp-D- and Trp-I-IAA synthesis pathways at critical times in plant development. In addition to the effect of temperature on IAA pathway utilization, a switch from TrpD- to Trp-I-IAA synthesis occurs during carrot somatic embryogenesis,68' 69 and during tomato fruit growth.70 Conversely, a change from Trp-I- to Trp-D-IAA biosynthesis has been measured in Scots pine seedlings and following wounding in bean seedlings. The myb transcription factor ATR1 is important in regulating IAA biosynthesis. The dominant overexpressor up-regulates Trp gene expression, and results in elevated IAA levels, most likely derived from the IAOx pathway that is dependent upon CYP79B2 and CYP79B3 activity (Fig. 12.2).73 This same pathway is also necessary for the elevated IAA levels observed in the surl (John Celenza, personal communication) and surf1' mutants. While labeling studies reveal the two general routes to IAA, neither Trp-D nor Trp-I pathways are completely defined in plants. Numerous pathways exist "on paper," in that putative intermediates have been identified as native compounds in plants, enzyme activities have been identified in various plant species, and/or genes encoding these enzymes have been identified, but most of these pathways are incompletely characterized with respect to intermediates, enzymes and genes.2"4 More importantly, none of these pathways has either temporal or spatial "assignments" within a given plant species. Our goal is to fill in the gaps in terms of genes, enzymes, and intermediates in the IAA synthesis pathways that we do know about, identify any unknown IAA synthesis pathways, and characterize the use of these pathways in the plant throughout development. We have been focusing upon one Trp-D pathway, the indole-3-acetaldoxime (IAOx) pathway, named after the

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branch point compound, which can be converted to IAA or indole glucosinolates (IGs). We use mutants and stable isotope labeling, both in a reverse genetics approach to confirm or negate the role of a particular gene in IAA synthesis, or in a forward genetics approach, whereby new IAA synthesis genes are identified in screens of mutant populations for alterations in IAA levels and pathway utilization.

chorismate

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IAA

Figure 12.2: IAA biosynthetic pathways in Arabidopsis thaliana. Dashed lines indicate no known gene or enzyme activity identified in Arabidopsis. ANA = anthranilate, PANA = 5-phosphoribosylanthranilate, CADP = l-(ocarboxyphenylamino)-l-deoxyribulose-5-phosphate, IGP = indole-3-glycerol phosphate, TRP = tryptophan, IAM = indole-3-acetamide, IPA = indole-3-pyruvic acid, IAAld = indole-3-acetaldehyde, IAOx = indole-3-acetaldoxime, S-IAH-L-cys = S-(indolylacetohydroximoyl)-L-cysteine, indole-3-T-OH = indole-3-thiohydroximate, IG = indole-3-methylglucosinolate, TRM = tryptamine. See also Woodward and Bartel.2

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AUXIN BIOSYNTHESIS FROM AN ANALYTICAL PERSPECTIVE IAA Is a Low Abundance Compound IAA has been the subject of intense study using a wide variety of disciplinary approaches. From an analytical perspective, IAA has several characteristics that have resulted in its general classification as "difficult". It is very labile, present in low amounts, and its recovery is unpredictable. From a metabolic perspective, IAA analysis is complicated by the fact that it can be derived from multiple biosynthetic pathways within an organism (Fig. 12.2). We have relied on mass spectrometry and isotope dilution analysis in the development of protocols that allow us to obtain absolutely quantitative information on IAA pool sizes, synthesis and turnover rates, and metabolic fate. Mutants and Stable Isotope Labeling to Study IAA Biosynthesis We have focused on the IAOx pathway in Arabidopsis because of its apparent localization in the root,57 rendering it amenable to study in vivo by stable isotope labeling. IAOx is an important branch point in Trp metabolism. It can be derived from Trp through two routes, one postulated74 and one confirmed (Fig. 12.2).75'76 In the postulated IAOx pathway, Trp is first converted to tryptamine (Trm), and then the flavin monooxygenase (FMO) YUCCA, of which there are at least 9 homologues in Arabidopsis, converts Trm to N-hydroxyl-Trm. This step has been shown in vitro only,74 and Trm has not yet been identified as a native compound in Arabidopsis. Arabidopsis mutants that overexpress 3 of 9 YUCCA genes exhibit not only classical auxin overproducing phenotypes, but also have 50% more free IAA compared to wild type, and microarray data show upregulation of auxin-responsive genes.7 Similarly, overexpression of the petunia ortholog of YUCCA, FLOOZY, results in high auxin phenotypes including epinastic leaves and long root hairs.77 In the confirmed IAOx pathway, Trp is converted to IAOx by either of two cytochrome P450s, CYP79B2 and CYP79B3.78'79 Expression patterns reveal that CYP79B2 and CYP79B3 are present in the root meristem and where lateral roots are formed.57 Transgenic Arabidopsis that overexpress CYP79B2 under control of the CaMV 35S promoter (CYP79B2-OEX) have free IAA levels that are slightly but significantly elevated. While this overexpressor does not exhibit auxin phenotypes unless Trp is supplied in the medium, microarray analysis reveals that auxin inducible genes are up-regulated in this line.76 Conversely, a double knockout (TDNA insertion mutants in each of these two CYP79B genes) has slightly but significantly reduced IAA levels,57'76 and the rate of IAA synthesis in the roots is decreased.57 Similarly to the CYP79B2 OEX line, the double knockout line does not have an obvious auxin phenotype. These subtle phenotypes are not unexpected

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given the presumed redundancy of the IAA synthesis pathways. Visual screens for auxin phenotypes have yielded only a smattering of auxin overproducers, and these have tended to be involved in a branch of the IAOx pathway, leading to IGs.80'81 Identifying auxin auxotrophs has been even less successful, presumably because a true auxin auxotroph would be inviable. Therefore, while we are able to quantify precisely as little as a two-fold difference in IAA levels between plant lines, we have resorted to dual labeling studies to tease out the utilization of IAA biosynthesis pathways, rather than looking only at IAA levels. Our hypothesis is that dual labeling studies employed in a mutant screen would be more likely to reveal new genes involved in IAA homeostasis. We have tested the efficacy of dual labeling experiments for reverse and forward genetic screens using the CYP79B2 OEX line. Figure 12.3 shows the ratio of the percent [15N]IAA enrichment to the percent [2Hs]IAA enrichment over the course of the labeling study. The experiment revealed a different utilization of the two IAA synthesis pathways in wild type (wt) and the CYP79B2 OEX (OEX) line. Trp-dependent pathway utilization appears to account for most IAA production in the OEX line (the ratio of percent enrichment of IAA from [15N]ANA to enrichment of IAA from [2Hs]Trp is low) compared to wt where labeling of IAA from [15N]ANA clearly exceeds labeling from [2Hs]Trp. Although free IAA levels in CYP79B2 OEX were only slightly higher than wild type,76 we were able to show that Trp-D-IAA synthesis was up-regulated and responsible for the observed increases in IAA in CYP79B2 OEX. The steps between IAOx formation and IAA are less clear. The only IAOxmetabolizing genes identified so far encode the cytochrome P450s CYP83B1 and CYP83A1, which catalyze the first committed step in IG synthesis.82"84 The current model is that IAOx can be converted to indole-3-acetaldehyde, (IAAld), indole-3acetonitrile (IAN), or indole-3-acetaldoxime-N-oxide (Fig. 12.2) although specific enzymes and genes have yet to be identified. Nitrilases with in vitro activity towards indole-3-acetonitrile (IAN) have been found in plants of the Cruciferae, Graminae and Musaceae families ' as well as tobacco.87 Arabidopsis has four genes encoding nitrilases, NITl-4^ All four nitrilases have been shown to convert IAN to IAA in vitro, although NIT4 most likely acts in cyanide detoxification and probably does not play a role in IAA biosynthesis.89

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hours Figure 12.3: Dual labeling of Arabidopsis wild type and CYP79B2 OEX (OEX) seedlings. Seven day old seedlings were transferred to liquid medium containing 20uM each [15N]ANA and [2H5]TRP and incubated for the times indicated. Samples were harvested (three replicates for each line and time point), IAA was isolated and the amounts of [15N]IAA, [2H5]IAA, and unlabeled IAA were determined by GC-MS. Shown here are the ratios of [15N]IAA to [2Hs]IAA in the wild type and OEX lines at each time point.

Overexpressing the nitrilases in either tobacco or Arabidopsis does not cause any obvious growth phenotypes unless IAN is supplied in the medium, which is interesting given the high endogenous levels of IAN in Arabidopsis.90' 91 The only reported IAN resistant mutations are in the NITl gene, but nitl plants do not have altered levels of endogenous IAN or IAA.91 No changes in IAA synthesis rates were observed in whole seedlings or excised roots of nitl mutants.57 The observation that the nitl mutant maintains normal IAA levels can be explained in two ways. Either exogenous IAN is metabolized only by NITl, but NIT2 and/or NIT3 can substitute for NITl in planta, IAN is not the normal substrate of nitrilases in plants, or another factor is required for nitrilase to convert IAN to IAA in v/vo.88'92 An alternate route for IAOx to IAA could be via IAAld (Fig. 12.2). Activities converting IAOx to IAAld have been described for both Avena and Chinese cabbage 93 ' 94 but genes corresponding to these activities have yet to be identified.

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IAA Inter-Conversion to IBA and Role oflBA Although IAA is the most commonly studied natural auxin, indole-3-butyric acid (IBA) is a native compound in Arabidopsis that exhibits a number of auxin activities particularly with respect to roots. 2 ' 64 IBA and IAA are inter-converted by a variety of plants, so IBA and IAA conjugates may play overlapping roles in the plant,64 with regard to inactivation of IAA or IBA could act as an auxin directly. A number of mutants insensitive to the auxin effects of IBA have been characterized,64 and some exhibit developmental defects. It would be interesting to know if IBA is derived from IAA that was synthesized by a specific pathway, either Trp-D or Trp-I. Dual labeling studies that examine incorporation of label from IAA precursors into IBA should help to address this question. Cross-Talk with Other Pathways Cross talk between the plant hormone signaling pathways provides a mechanism by which a small number of compounds can affect a large number of processes, and at the same time multiple hormones can coordinate a single response.37 Auxin rapidly regulates cytokinin biosynthesis through signal transduction pathways, while cytokinin regulates auxin biosynthesis more slowly through developmental changes.95 The ways in which auxin and cytokinin regulate each other involves a complex network of synergistic functions, as in the regulation of cell cycle, antagonistic functions as seen in lateral root formation, and additive functions Auxin has also been shown to interact with the hormone ethylene. Stepanova et al, showed that wei2 and wei 7 mutants are root-specific ethyleneinsensitive mutants that may be involved in ethylene-dependent IAA synthesis. This is consistent with previous work demonstrating that auxin is a key regulator in ethylene response.9 ' 9 7 Lateral root (LR) initiation is dependent on the presence of auxin in the root apex, and modulation of the auxin signal that promotes lateral root development appears to be controlled through proteolysis of two key factors, the KRP2 (a CDK inhibitor) protein and the transcriptional activator NAC1. Auxin also modulates the effect of gibberellins on root growth. Studies of lateral root formation have provided evidence of cross-talk between auxin and the cell cycle. There is a strong correlation between cell division and high levels of IAA, as auxin appears to influence cell cycle dependent processes, such as the function of telomerase in telomeres and the anaphase promoting complex (APC).100 In addition to lateral root formation, auxin signaling also has an important role in the vasculature. Differentiation of vascular tissue such as vessels and sieve tubes is a result of controlled changes in not only the site of IAA synthesis, but also in the concentration, and this is in coordination with cytokinin, gibberellin, and

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ethylene.101 For example, incomplete vascular systems are a consequence of diminished auxin signal transduction.17 Considerable evidence suggests that plant responses to stress perturb indolic pathways. Accumulation of the indolic phytoalexin camalexin, induced by abiotic elicitors, plant pathogens, and spontaneous lesions in the cell death mutant acd2, is accompanied by the coordinate induction of mRNAs for the Trp biosynthesis pathway enzymes,102 and a range of Tip-derived secondary products accumulate following infection of Arabidopsis by Pseudomonas.103 In maize, biotic stress also impacts indolic metabolism.104'105 Two gene products, BX1 and IGL, produce free indole for defense related secondary metabolites.104' 106 This indole is formed independently of Trp biosynthesis via a TSa-like indole synthase reaction, which differs from microbial Trp synthesis in which indole is an intermediate that is not released from the Trp synthase a2p2 complex.107 There is an uncharacterized IGLlike gene in the Arabidopsis genome, and it is tempting to speculate a role for this gene in the Trp-I pathway. The availability of T-DNA knockouts of this gene combined with dual labeling studies should confirm or negate a role in IAA synthesis.

SUMMARY AND FUTURE DIRECTIONS In order to more clearly differentiate among the multiple predicted IAA synthesis pathways, future analysis needs to involve the quantification of levels and synthesis rates of more metabolites than just IAA. Furthermore, the clear evidence for cross-talk between auxin homeostasis pathways and other hormonal or metabolic networks necessitates broader metabolite profiling to get at these interactions. Over the years we and others have established methods to quantify a number of IAA precursors and metabolites,61'108"113 and the availability of LC-MS in the repertoire of analytical tools means that more metabolites can be quantitatively profiled. The rate-limiting step is availability of internal standards.114 Several groups have developed methods to increase the number of small molecule regulatory compounds that can be quantified, but this number is still fairly small.54'115"1 7 In addition to the need for more internal standards, cellular resolution down to the subcellular level is the next big technological challenge associated with the characterization of auxin homeostasis.

ACKNOWLEDGEMENTS The authors wish to thank John Celenza for suggestions on the manuscript. Y.Y Tarn was supported by funding from NSF MCB 9870798 and NSF DBI 0077769 to J.N., and J. Calio was supported by funding from USDA 02-03555 to J.N.

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BAK, S., TAX, F., FELDMANN, K., GALBRAITH, D., FEYEREISEN, R., CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis., Plant Cell, 2001,13, 101-111. BAK, S., FEYEREISEN, R., The involvement of two P450 Enzymes, CYP83B1 and CYP83A1 in auxin homeostasis and glucosinolate biosynthesis., Plant Physiol., 2001, 127, 108-118. HEMM, M., RUEGGER, M., CHAPPLE, C , The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes., Plant Cell, 2003,15, 179-194. THIMANN, K., MAFIADEVAN, S., Nitrilase 1. occurrence, preparation, and general properties of the enzyme., Arch. Biochem. Biophys., 1964,105, 133-141. PARK, W. J., KRIECHBAUMER, V., MOLLER, A., PIOTROWSKI, M., MEELEY, R. B., GIERL, A., GLAWISCHNIG, E., The nitrilase ZmNIT2 converts indole-3acetonitrile to indole-3-acetic acid., Plant Physiol, 2003,133, 794-802. DOHMOTO, M , TSUNODA, H., ISAJI, G., CHIBA, R., YAMAGUCHI, K., Genes encoding nitrilase-like proteins from tobacco., DNA Research, 2000, 7, 283-289. NORMANLY, J., BARTEL, B., Redundancy as a way of life-IAA metabolism., Curr. Opin. Plant Biol, 1999, 2, 207-213. PIOTROWSKI, M , SABINE, S., WEILER, E., The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode b-cyano-L-alanine hydratase/nitrilase., J. Biol. Chem., 2001, 276, 2616-2621. SCHMIDT, R.-C, MULLER, A., HAIN, R., BARTLING, D.,WEILER, E. W., Transgenic tobacco plants expressing the Arabidopsis thaliana nitrilase II enzyme., Plant J., 1996,9,683-691. NORMANLY, J., GRISAFI, P., FINK, G., BARTEL, B., Arabidopsis thaliana mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT 1 gene., Plant Cell, 1997,9, 1781-1790. XU, P., NARASIMHAN, M., SAMPSON, T., COCA, M., HUH, G.-H., ZHOU, J., MARTIN, G., HASEGAWA, P., BRESSAN, R., A nitrilase-like protein interacts with GCC box DNA-binding proteins involved in ethylene and defense responses., Plant Physiol, 1998, 118, 867-874. RAJAGOPAL, R., LARSEN, P., Metabolism of indole-3-acetaldoxime in plants., Planta, 1972,103,45-54. HELMLINGER, J., RAUSCH, T., HILGENBERG, W., A soluble protein factor from Chinese cabbage converts indole-3-acetaldoxime to IAA., Phytochemistry, 1987, 26, 615-618. NORDSTROM, A., Cytokinins in Arabidopsis, tools, pathways and interaction with auxin., PhD Thesis, Department of Forest Genetics and Plant Physiology. 2004, Swedish University of Agricultural Sciences: Umea. ALONSO, J., STEPANOVA, A., SOLANO, R., WISMAN, E., FERRARI, S., AUSUBEL, F. M., ECKER, J. R., Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis., Proc. Natl. Acad. Sci. U. S. A., 2003,100, 2992-2997. COLLETT, C , HARBERD, N., LEYSER, O., Hormonal interactions in the control of Arabidopsis hypocotyl elongation., Plant Physiol., 2000,124, 553-562.

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CASIMIRO, I., BEECKMAN, T., GRAHAM, N., BHALERAO, R., ZHANG, H., CASERO, P., SANDBERG, G., BENNETT, M., Dissecting Arabidopsis lateral root development., Trends in Plant Sci., 2003, 8, 165-171. 99. BIRNBAUM, K., BENFEY, P., Network building: transcriptional circuits in the root., Curr. Opin. Plant Biol., 2004, 7, 582-588. 100. VANNESTE, S., MAES, L., DE SMET, I., HIMANEN, K., NAUDTS, M., INZE, D., BEECKMAN, T., Auxin regulation of cell cycle and its role during lateral root initiation. Physiol. Plant., 2005,123, 139-146. 101. ALONI, R., The induction of vascular tissues by auxin. In: Plant Hormones Biosynthesis, Signal Transduction, Action! (P. J. Davies, ed.), Kluwer Academic Publishers, Dordrecht. 2004, pp. 471-492 102. ZHAO, J., LAST, R. L., Coordinate regulation of the tryptophan biosynthetic pathway and indolic phytoalexin accumulation in Arabidopsis., Plant Cell, 1996, 8, 2235-2244. 103. HAGEMEIER, J., SCHNEIDER, B., OLDHAM, N. J., HAHLBROCK, K., Accumulation of soluble and wall-bound indolic metabolites in Arabidopsis thaliana leaves infected with virulent or avirulent Pseudomonas syringae pathovar tomato strains., Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 753-758. 104. FREY, M., CHOMET, P., E, G., STETTNER, C , GRUN, S., WINKLMAIR, A., EISENREICH, W., BACHER, A., MEELEY, R., BRIGGS, S., SIMCOX, K., GIERL, A., Analysis of a chemical plant defense mechanism in grasses., Science, 1997, 277, 696-699. 105. MELANSON, D., CHILTON, M. D., MASTERS-MOORE, D., CHILTON, W. S., A deletion in an indole synthase gene is responsible for the DIMBOA-deficient phenotype of bxbx maize., Proc. Natl. Acad. Sci. U. S. A., 1997, 94, 13345-13350. 106. FREY, M., STETTNER, C , PARE, P., SCHMELZ, E., TUMLINSON, J., GIERL, A., An herbivore elicitor activates the gene for indole emission in maize. Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 14801-14806. 107. RUVINOV, S., YANG, X.-J., PARRIS, K., BANIK, U., AHMED, S., MILES, E., SACKETT, D., Ligand-mediated changes in the tryptophan synthase indole tunnel probed by Nile Red fluorescence with wild type, mutant and chemically modified enzymes., J. Biol. Chem., 1995, 270, 6357-6369. 108. LJUNG, K., SANDBERG, G., MORITZ, T., Methods for plant hormone analysis. In: Plant Hormones: Biosynthesis, Signal Transduction, Action! (P. Davies, ed.), Kluwer Academic Publishers, Dordrecht. 2004, pp. 671-694 109. SUTTER, E. G., COHEN, J. D., Measurement of indolebutyric acid in plant tissues by isotope dilution gas chromatography-mass spectrometry analysis., Plant Physiol., 1992, 99, 1719-1722. 110. TAM, Y. Y., NORMANLY, J., Determination of indole-3-pyruvic acid levels in Arabidopsis thaliana by gas chromatography-selected ion monitoring-mass spectrometry.,./ Chromatogr. A, 1998,800, 101-108. 111. ILIC, N., NORMANLY, J., COHEN, J., Quantification of free plus conjugated indole3-acetic acid in Arabidopsis requires correction for the non-enzymatic conversion of indolic nitriles., Plant Physiol, 1996, 111, 781-788.

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112. COHEN, J., BALDI, B., SLOVIN, J., 13C6 [benzene ring] indole 3 acetic acid: A new internal standard for quantitative mass spectral analysis of indole 3 acetic acid in plants., Plant Physiol., 1986,80, 14-19. 113. CHEN, K.-H., MILLER, A. N., PATTERSON, G. W., COHEN, J. D., A rapid and simple procedure for purification of indole-3-acetic acid prior to GC-SIM-MS analysis., Plant Physiol., 1988, 86, 822-825. 114. TRETHEWEY, R., Metabolite profiling as an aid to metabolic engineering in plants. Curr. Opin. Plant Biol, 2004, 7, 196-201. 115. JANDER, G., Application of a high-throughput HPLC-MS/MS assay to Arabidopsis mutant screening, evidence that threonine aldolase plays a role in seed nutritional quality., Plant J., 2004, 39, 465-475. 116. CHIWOCHA, S., ABRAMS, S., AMBROSE, A., CUTLER, A., LOEWEN, M., ROSS, A., KERMODE, A., A method for profiling classes of plant hormones and their metabolites using liquid chromatography-electrospray ionization tandem mass spectrometry: an analysis of hormone regulation of thermodormancy of lettuce (Lactuca sativa L.) seeds., Plant J., 2003, 35, 405-417. 117. SCHEMELZ, E., ENGELBERTH, J., ALBORN, H., O'DONNELL, P., SAMMONS, M., TOSHIMA, H., TUMLINSON, J., Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants., Proc. Natl. Acad. Sci. U. S. A., 2003, 100, 10552-10557.

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Recent Advances in Phytochemistry, vol. 40 John T. Romeo (Editor) © 2006 Elsevier Ltd. All rights reserved.

Chapter Thirteen

TRANSLATIONAL OPPORTUNITIES IN PLANT BIOCHEMISTRY Cecilia A. Mclntosh Department of Biological Sciences East Tennessee State University Box 70703 Johnson City, Tennessee 37614

Email: mcintosc(a),etsu.edu

Introduction International Natural Products Repository Network Inflammatory Pathway Platforms and Plant Natural Products Transcription Factor Over-Expression for Metabolite Manipulation Heterologous Sesquiterpene Production Platforms New Technologies for Metabolomics Profiling Summary

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INTRODUCTION Great strides have been made in plant science research in recent years, and some of these relate directly to efforts using genomic, proteomic, and metabolomic approaches. While these approaches have provided the ability to address increasingly complex questions, many challenges remain. These challenges include (but are not limited to) concerns related to: high throughput techniques and the requisite issues of processing large numbers of samples and minimizing sample preparation; developing analytical methods that mesh accuracy, reliability, and high sensitivity; natural product profiling and development of complex analytical software; development of in vivo test systems; and availability of identified source plant material and reference compounds. Some of these challenges are long-standing and others are emerging as inroads in technology used to ask questions of increasing complexity. Added to the above hurdles are those that result from fostering interdisciplinary approaches. Several agencies, including the U.S. National Science Foundation, actively promote and support integrative research addressing these questions. The make-up of the NSF divisions that support the integrative programs, as well as a degree of interagency cooperation, reflects the interdisciplinary requirements of the research itself/'5 The following is a review of invited talks given at the La Jolla symposium that focused on resources and techniques. The symposium provided information on developing opportunities and/or methodologies to meet challenges in proteomic/metabolomic research. Speakers included: Troy Smillie (University of Mississippi), Bryan Greenhagen (Allylix, Inc.), Sekhar Boddupalli (Galileo Pharmaceuticals), Fabricio Medina-Bolivar (Arkansas Biosciences Institute at Arkansas State University), and David Weil (Agilent Technologies). Their purpose was to present new technologies, applications, and/or opportunities with relevance to phytochemistry and plant biochemistry research. Two of the topics refer to platforms or production platforms. In this sense, a platform refers to research and development and/or production and manufacturing requirements and may include the: research and development strategy, requisite hardware and software, biotechnology, production plan, etc. Due to the diversity of topics, it is not possible to do a thorough literature review of each area to accompany presentation of new approaches, but citations of representative articles, reviews, or books have been included.

INTERNATIONAL NATURAL PRODUCTS REPOSITORY NETWORK There are thousands of natural products made throughout the plant kingdom. Research addressing questions or testing hypotheses relating to chemistry,

TRANSLATIONAL OPPORTUNITIES OPPORTUNITIES IN TRANSLATIONAL IN PLANT PLANT BIOCHEMISTRY BIOCHEMISTRY 309 biochemistry, metabolism, pharmacology, pharmacognosy, physiology, nutrition, etc. depends upon the continued isolation and structural identification of novel compounds. Obtaining reliable information on the identification of source materials and the availability of reference compounds is not always straightforward. Only a relative handful of compounds are available from commercial sources. Information on methods for extraction, purification, and identification of natural products are scattered throughout the literature. One research problem for phytochemists that has been under discussion for several years is the issue of how to sift through all of the sources to obtain information on any particular compound and related compounds. Dr. Troy Smillie presented on-going efforts by the University of Mississippi to address these challenges. The University of Mississippi established their Research Institute of Pharmaceutical Sciences in 1964 which led to the launch of the Thad Cochran Research Center housing the National Center for Natural Products Research in the 1990's. This is a large center comprised of several research faculty and staff from various academic departments in the university as well as U.S. Department of Agriculture scientists. The center has several research and development programs for natural products discovery and development (including both terrestrial plant and marine products) as well as medicinal plant research. The Natural Product Repository currently has more than 14,000 samples of extracts, derived fractions, and pure compounds in its inventory. Ongoing collection efforts yield over 1000 additional samples per year with geographic representation that includes the Americas, Africa, Papau New Guinea, and India. Collaborations with other institutions provide access to further samples for bioassay screening. Natural products chemists at the Center continue to isolate compounds based on their own current projects. Plant physiologists and researchers in genomics, proteomics, and metabolomics have an increasing need for natural products for use as substrates or molecular probes, and these are often difficult to obtain. To assist in these efforts, the university is in the process of establishing an International Natural Products Repository Network (INPRN).7 The goal of the INPRN is to expand research interactions within the scientific community, and it was conceived to serve as a research tool for investigators in various fields by improving access to natural products that are critical reagents but may be unavailable from a commercial source or, if available, may be prohibitively expensive. An international steering committee is guiding network development. There are two components of the INPRN. There will be both a physical and a virtual repository of natural products that will be available to investigators via a webpage interface. The organizational chart is shown in Figure 13.1. One major requirement of such a large-scale effort is the development of a database to serve the program. Requirements of the database are that it be internet ready, secure, easy to use, robust and scalable, and able to handle multiple relations while remaining

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INPRN In-house Repository

INPRN Virtual Repository Member/Institute

INPRN Web Enabled Database and Discussion Forum

Program Member Requests

Figure 13.1: Organization of INPRN (provided by T. Smillie).

affordable. The center is nearing completion of this phase (T. Smillie, personal communication). Information that will be available within the database includes: source material, common name and synonyms, IUPAC nomenclature, class of compound, molecular form/weight, structure, available quantity, spectroscopic information, stability information, toxicity/hazard warnings, storage conditions, and isolation techniques. Samples with no known intellectual property issues are accepted; donations are actively encouraged, and some have already occurred. This is an excellent altruistic opportunity for individual scientists to make significant contributions to research by outreach on a larger scale. All samples donated will have the donor's identity associated with the sample in the database. Contributions to the physical repository have specific requirements as to the minimum amount needed and the

TRANSLATIONAL IN PLANT PLANT BIOCHEMISTRY BIOCHEMISTRY 311 TRANSLATIONAL OPPORTUNITIES OPPORTUNITIES IN nature of the requisite storage conditions; contributions to the virtual repository, of course, are not required to adhere to those same guidelines. Multiple submissions of the same material from different investigators are encouraged by the INPRN. It is important to note that the INPRN is not a business, and it will be run as a non-profit entity. This fosters altruistic collaborations for the advancement of science in general. Participation is open to educational, governmental, and other non-profit research groups; direct interactions with commercial partners will be discouraged. Contact information and updates are available through the INPRN homepage.7

INFLAMMATORY PATHWAY PLATFORMS AND PLANT NATURAL PRODUCTS Many challenges are shared by the fields of rational medicinal chemistry and natural products chemistry. Limited chemical diversity and limited emphasis on functional biology versus target-based optimization for drug discovery are coupled to on-going efforts to discover new natural products. Expectations of chemical diversity as a corollary to biodiversity are not always borne out, and reproducing natural chemistry in a laboratory setting can be difficult. Obtaining a renewable supply of source material as well as controlling the expenses incurred in tooling up to identify potential drugs adds to the challenge. Dr. Sekhar Boddupalli of Galileo Pharmaceuticals (www, galileopharm.com) shared information on new innovations to address such issues. Some approaches are geared toward meshing functional chemistry with functional biology to identify novel starting points. Advantages of using nature-derived chemistry from food sources include a lower risk of toxicity, a higher probability of efficacy, and the proprietary advantages to "first-in-class" drugs. First-in-class drugs are those that possess novel characteristics leading to new approaches/targets for treatment of a condition. Disadvantages of using naturederived chemistry from food sources include: the chemical complexity of foods, which leads to the problem that not all of the chemical content of food is known or "mapped;" the potential of off-target effects; and the possibility of poor pharmacokinetics. The specific example presented is related to discovery and development of first-in-class inhibitors against inflammation and metabolic disease targets, specifically those that are inhibitors of lipoxygenases (LO). These could be applied to the treatment of diseases mediated by inflammation such as asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease (IBD), cardiovascular disease, and osteoporosis. Galileo Pharmaceuticals has a proprietary Conserved Inflammatory Pathway (CIP) modulator technology platform that enables rapid identification of novel small molecules acting against a broad range of inflammatory targets. The approach may be applicable to other biological problems.

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Information from genomics as well as other data indicate that plants and humans have conserved inflammatory pathways, e.g., pathogen and lipoxygenaseinduced signaling pathways. In mammalian systems, signal transduction leads to production of isoprostanes and prostaglandins, while jasmonates and phytoprostanes are produced in plants (Fig. 13.2). 8 It is well established that treating plant cell cultures with various stressors can lead to production of protective phytochemicals.15 Many of these compounds have activity in mammalian systems, likely due to pathway conservation. Galileo took advantage of the ability to elicit production of potential anti-inflammatory compounds by the systematic exposure of a broad range of plant species to a variety of stress conditions. They then screened the small molecule libraries in functional mammalian cell- and target-based assays, and they have identified novel, drug-like small molecules that modulate therapeutically relevant targets such as lipoxygenases, kinases, and nuclear receptors. By using this approach, they have established a proprietary set of active compounds, new chemical entities, acting as CIP modulators (Fig. 13.3).16 Plants

Mammals

esterifiedlinoleate

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Figure 13.2: Bioactive Lipids: Conserved Inflammatory Pathways (used by permission of Galileo Pharmaceuticals).

'

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TRANSLATIONAL OPPORTUNITIES OPPORTUNITIES IN PLANT PLANT BIOCHEMISTRY BIOCHEMISTRY 313 313 TRANSLATIONAL mm

f

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Conserved Stress-induced pathways between plants & mammals Plant cell culture library: 85% of taxonomic orders

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Figure 13.3: Galileo Discovery Platform: Strategy for Development (used by permission of Galileo Pharmaceuticals).

The development of platforms for rational investigation of chemicals may also prove beneficial for compounds with biological activities other than mammalian drugs. Adaptation of this methodology may find application in studies of plantplant, plant-microbe, or plant-insect interactions as well as in studies of plant metabolism and physiology.17"20

TRANSCRIPTION FACTOR OVER-EXPRESSION FOR METABOLITE MANIPULATION Dr. Fabricio Medina-Bolivar of the Arkansas Biosciences Institute and the Dept. of Biological Sciences at Arkansas State University presented recent results from investigations into use of transcription factors to manipulate metabolite biosynthesis in hairy root cultures as part of his work on the application of metabolic engineering for the discovery of Pharmaceuticals from plants. One of his research interests is the identification of specialized metabolites produced in tobacco that

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could lead to the treatment of Parkinson's disease. This work started as collaborative research with Dr. Neal Castagnoli Jr. of the Harvey W. Peters Center for the Study of Parkinson's Diseases and Disorders of the Nervous System at Virginia Tech. Epidemiology data have shown that tobacco smokers have lower incidents of Parkinson's disease, and it has been postulated that this may be related to the lower levels of monoamine oxidase (MAO) activity in the brain of smokers.21 Dr. MedinaBolivar is using elicitation in tobacco hairy roots as means to produce chemicals found in tobacco smoke and discover novel MAO inhibitors. Hairy roots are useful as bioproduction systems for specialized metabolites as well as recombinant proteins, providing genetic stability and containment.22'23 Dr. Medina-Bolivar's research group is over-expressing transcription factors that may regulate specialized metabolic pathways. These are being expressed with promoters that have been studied in his laboratory and shown to be highly active and inducible in tobacco hairy roots (Fig. 13.4). Ultimately, genetically engineered hairy roots will be analyzed for production of novel metabolites and tested in bioassays. This novel approach may lead to the discovery of pharmaceutically important therapeutic drugs.

Figure 13.4: Hairy roots elicited with cooper sulfate. The dark area produced in the root tips upon elicitation is used to determine the efficacy of the elicitor. (used by permission of F. Medina-Bolivar).

TRANSLATIONAL OPPORTUNITIES OPPORTUNITIES IN TRANSLATIONAL IN PLANT PLANT BIOCHEMISTRY BIOCHEMISTRY 315 HETEROLOGOUS SESQUITERPENE PRODUCTION PLATFORMS Dr. Bryan Greenhagen of Allylix, Inc. shared general strategies that are being used to develop platforms for production of heterologous sesquiterpenes. Intellectual property issues preclude presentation of specific details. However, the overall scheme for developing model systems for pathways and for producing compounds is dependent upon understanding structure(s) of the biocatalyst(s) involved, obtaining the genes for the enzymes/biocatalysts, inserting the gene into a production microbe, producing the compound through fermentation, and optimizing activity of the compound through final chemical modification. The production host must be well suited to production of the compound of interest. For example, production of sesquiterpenes would require a host with metabolism that results in a high flux of carbon into farnesyl pyrophosphate to enable reconstruction of the pathway. A key to success is to evolve the biosynthetic potential by mapping enzyme active sites by mutational analysis and identification of change-in-function mutations. This has the potential to lead to enantio-specific product engineering.28 Once structural elements controlling specificity are known, combinatorial engineering of the proteins can be initiated. Advances in the development of biosynthetic production platforms are dependent upon thorough kinetic analyses of enzymes and consideration of the complexities of cellular biosynthesis, as well as development of the actual production process.29

NEW TECHNOLOGIES FOR METABOLOMIC PROFILING One of the most important current hurdles facing metabolomics research is relieving the bottlenecks of sample preparation and bioinformatics. Samples for metabolomic analysis are usually complex and may contain thousands of compounds. Many compounds share chemical properties, which presents problems for manual extraction of compounds of interest.3 Dr. David Weil of Agilent Technologies presented information on new technology developed to address these issues. The approach used is to leverage or adapt advances in proteomics technology (e.g., high throughput capacity, sensitivity, reduced experimental variation, robust bioinformatics) for use in metabolomics.30"31 Important factors to consider in developing technology are sample preparation, separation, sensitivity of the method, reduction of experimental variation, and integration of informatics. Capillary HPLC can provide high resolution separation of similar compounds. Current solutions developed by Agilent Technologies involve coupling an Agilent Capillary HPLC system with an Agilent LC/MSD TOF mass spectrometer for nano-scale analyses.30' 32'33 The system includes software ("Mass Hunter" and "Mass Profiler") to expedite the informatics

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aspects.32"33 This system is capable of 32 attamole resolution although routine analysis is at the femtomole range. Details of the system along with sample data are available at the company's web site.32"33

SUMMARY Genomic approaches and related technologies have widened the spectra of experimental questions for which the potential of obtaining answers exists.34"35 These questions include those important for basic sciences as well as applied sciences such as pharmaceutical or food science. Proteomic and metabolomic investigations have brought additional requirements for new and/or improved technology. Continued progress in development of biological systems that can be manipulated, production of custom enzymes, development of screening platforms and strategies, and innovations in analytical technology will be critical to on-going success in the field. Additional contributions to scientific advancement in the "omics" related fields will be fostered by sharing of samples of and/or information on phytochemicals through the International Natural Products Repository Network.

ACKNOWLEDGEMENTS Thanks to all speakers for contributing to an exciting symposium session. Special thanks to Dr. Medina-Bolivar, Dr. Boddupalli, and Dr. Smillie for providing materials to aid in writing this symposium summary. REFERENCES 1. National Science Foundation Emerging Frontiers home page http://www.nsf.gov/ div/index.j sp?org=EF 2. National Science Foundation NSF-NIST (Interaction in Chemistry, Materials Research, Molecular Biosciences, Bioengineering, and Chemical Engineering) home page http://www.nsf.gov/funding/pgm_summ.jsp?pims_id=5665&org=MCB 3. National Science Foundation Biochemical Engineering and Biotechnology home page http://www.nsf.gov/funding/pgm summ.jsp?pims id=13368&org=BES&from=home 4. National Science Foundation: Interagency Opportunities in Metabolic Engineering http://www.nsf.gov/pubs/2005/nsfD5502/nsfD5502.htm 5. National Science Foundation Quantitative Systems Biotechnology and Post Genomic Engineering http://www.nsf.gov/eng/bes/biochemdetail.isp 6. BARTON, D., NAKANISHI, K. (eds-in-chief), METH-COHN, O. (exec, ed.), Comprehensive Natural Products Chemistry. Elsevier, 1999, vol. 1-9. 7. International Natural Products Repository Network home page http://inprn.org/ 8. MUSIEK, E.S., YIN, H., MILNE, G.L., MORROW, J.D., Recent advances in the biochemistry and clinical relevance of the isoprostane pathway. Lipids, 2005, 40, 987994.

TRANSLATIONAL IN PLANT PLANT BIOCHEMISTRY BIOCHEMISTRY 317 TRANSLATIONAL OPPORTUNITIES OPPORTUNITIES IN 9. MONTUSCHI, P., BARNES, P.J., ROBERTS, L.J. II., Isoprostanes: Markers and mediators of oxidative stress. FASEBJ., 2004,18, 1971-1800. 10. GERSHENSON, J., 2002. Secondary metabolites and plant defense. In: Plant Physiology (L. Taiz and E. Zeiger, eds,), Sinauer Associates, Inc., Sunderland, Massachusetts. 2002. pp 283-308. 11. CREELMAN, R.A., MULLET, J.E., Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Phys. Plant Mol. Biol, 1997, 48, 355-381. 12. SALZMAN, R.A., BRADY, J.A., FINLAYSON, S.A., BUCHANAN, C D . , SUMMER, E.J., SUN, F., KLEIN, P.E., KLEIN, R.R., PRATT, L.H., CORDONNIER-PRATT, M.M., MULLET, J.E., Transcriptional profiling of sorghum induced by methyl jasmonate, salicylic acid and aminocyclopropane carboxylic acid reveals cooperative regulation and novel gene responses. Plant Physiol, 2005,138,352-368. 13. LOEFFLER, C , BERGER, S., GUY, A., DURAND, T., BRINGMANN, G., DREYER, M., VON RAD, U., DURNER, J. MUELLER, M.J., Bl-phytoprostanes trigger plant defense and detoxification responses. Plant Physiol, 2004,137, 328-340. 14. LI, X., SCHULER, M.A., BERENBAUM, M.R., Jasmonate and salicylate induce expression of herbivore cytochrome P450 genes. Nature, 2002, 419, 712-715. 15. GUNDLACH, H., MULLER, M.J., KUTHCEN, T.M., ZENK, M.H., Jasmonic acid is a signal transducer in elicitor-induced plant cell cultures. Proc. Natl. Acad. Sci. USA, 1992,89,2389-2393. 16. BODDUPALLI, S., Conserved inflammatory pathway (CIP) modulator platform (nonconfidential summary). PSNA symposium summary provided by Galileo Pharmaceuticals. 2005, pp 1-3. 17. ROMEO, J.T. (ed.), Chemical Ecology and Phytochemistry of Forest Ecosystems. Rec. Adv. Phytochem., vol. 39, Elsevier, 2005, 307 p. 18. ROMEO, J.T., DOWNUM, K.R., VERPOORTE, R. (eds.), Phytochemical Signals and Plant-Microbe Interactions. Rec. Adv. Phytochem., vol. 32, Plenum Press, 1998, 254 p. 19 ROMEO, J.T. (ed), Integrative Phytochemistry: From Ethnobotany to Molecular Ecology. Rec. Adv. Phytochem., vol. 37, Pergamon (Elsevier), 2003, 329 p. 20. ROMEO, J.T. (ed), Phytochemicals in Human Health, Protction, Nutrition, and Plant Defense. Rec. Adv. Phytochem., vol. 33, Kluwer (Plenum), 1999, 432 p. 21. CASTAGNOLI K., MURUGESAN, T., Tobacco leaf, smoke and smoking, MAO inhibitors, Parkinson's disease and neuroprotection; are there links? Neurotoxicology, 2004,25,279-291. 22. MEDINA-BOLIVAR, F., CRAMER, C , Production of recombinant proteins in hairy roots cultured in plastic sleeve bioreactors. In: Recombinant Gene Expression: Reviews and Protocols. P. Balbas and A. Lorence, (eds.). Humana Press, Totowa. 2004, pp. 351363. 23. MEDINA-BOLIVAR, F., FLORES, H., Root culture and natural products: "Unearthing" the hidden half of plant metabolism. Plant Tissue Culture and Biotechnology, 1995, 1, 59-74. 24. LORENCE, A., WOFFENDEN, B.J., SMITH, M., NESSLER, C.L., MEDINABOLIVAR, F., Over-expression of transcription factors to manipulate specialized metabolite biosynthesis. (http://www.psna-online.org/PSNAabst05.pdf; page 96) 25. Commerce Lexington press release, (http://www.lexicc.com/companies/3350-

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18307%20eBus%20ad-Allvlix.pdf) 26. Life Sciences and High-Tech Financial Forum 2005. http://www.connect.org/programs/ lifesciencesff/presenterprofiles.htm 27. Allylix home page, http://www.allylix.com 28. GREENHAGEN, B., CHAPPELL, J., Molecular scaffolds for chemical wizardry: learning nature's rules for terpene cyclases. Proc. Natl. Acad. Sci. USA, 2001, 98, 13479-13481. 29. TAKAHASHI, S., ZHAO, Y., O'MAILLE, P.E., GREENHAGEN, B.T., NOEL, J.T., COATES, R.M., CHAPPELL, J., Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes. J. Biol. Chem., 2005, 280, 3686-3696. 30. MILLER, B., LI, X., FJESDSTED, J., KINCAID, R., CHAKEL, J., WEIL, D., Differential detection of metabolites using Mass Hunter and Mass Profiler. (http://www.psna-online.org/PSNAabstQ5.pdf: page 87) 31. WELLS, D.A., WEIL, D.A., Directions in automated sample preparation of proteins. PharmaGenomics, 2003, Nov-Dec, 42-54. (http://www.forumsci.co.il/HPLC/ Protein sample_prep.pdf) 32. http://www.agilent.com 33. Agilent Technologies product note. Turning samples into answers. http://www.chem.agilent.com/temp/radD793C/00054960.pdf 34. ROMEO, J.T. (ed), Secondary Metabolism in Model Systems. Rec. Adv. Phytochem., vol. 38, Elsevier. 2004, 270 p. 35. ROMEO, J.T., DIXON, R.A. (eds), Phytochemistry in the Genomics and Post-Genomics Eras. Rec. Adv. Phytochem., vol. 36, Pergamon (Elsevier). 2002, 258 p.

INDEX a/p-hydrolase family, 122, 124 a-Acids, 184, 187, 193 P-Acids, 192, 193 p-D-glucosidase (SGD), 61, 62 P-glucuronidase (GUS), 57, 59, 196, 258 activity, 59, 196 P-ketoacyl-ACP, 120, 122, 123, 127 A9-tetrahydrocannabinol (THC), 181, 186, 187, 190-192 ABC transporters, 69 Acne, 145 Acyl carrier protein (ACP), 120, 122124, 127, 136, 165 Acylphloroglucinol glucosides, 184 AdoMet-dependent methyltransferase, 228, 232, 236, 240, 246 Affinity chromatography, 72 Aflatoxin, 167 Agrobacterium tumefaciens, 272 Airborne mycoses, 27 Aldehyde dehydrogenase, 277 Aldehyde oxidase 1 (AO1), 277 Alfalfa (Medicago sativa), 56, 195 Alkaloids, 2, 3, 8, 12, 13, 24, 26, 27, 31, 33, 36, 37, 39, 41, 43-47, 58, 59, 61, 63-65, 68-70, 122 accumulation, 58, 68, 69 benzylisoquinoline, 64, 67, 69, 71 biosynthesis, 2, 3, 8, 12, 33, 45, 58, 59,65 clavine, 27, 39 corynanthe, 12 ergot, 23-27, 31,33, 36-47 pyrrolizidine (PAs), 58, 62, 63

319 319

quinolizidine (QAs), 63 terpenoid indole (TIA), 2, 3, 8, 10, 13,59,61,62 translocation, 69 Alkyl resorcinols, 134, 142 Alkyl sterols, 221 Allelopathic, 158, 171, 174 Alternaria brassicae, 146 Alternaria carthami, 146 Amides of lysergic acid, 24, 26, 27, 38,41 Aminotransferases, 86, 87, 91, 101, 103, 105, 107 prephenate (PNT), 85-87, 91, 101, 103,105, 107, 110 Anacardiaceae, 132, 134, 142, 151 Anacardic acids, 132, 133, 135-137, 139-142, 144-146, 148, 149, 151, 168 bioactivity, 135, 144, 145 biosynthesis, 136, 137, 140, 144 saturated, 133 Analgesic, 64, 186 Ancestral trait, 44 Anchusa officinalis, 87 Anti-anxiety, 187 Anti-arrhythmic, 3 Antibacterial activity, 145 Anti-cancer, 3, 144, 148, 149 activity, 144, 148, 149 Anticholinergic drugs, 58 Anticonvulsive, 187 Antiemetic, 186 Antiestrogenic activity, 151 Antifungal, 146, 168 Anti-inflammatory, 184, 187, 312 Anti-malarial, 3

320

INDEX INDEX

Antimicrobial, 62, 64, 68, 167 alkaloids, 68 phytoalexins, 167 Antinausea, 187 Antioxidants, 187 Anti-tumor activity, 148; see also Anti-cancer Aphid, 118, 132,133 Apocynaceae, 2 Appetite stimulant, 186 Arabidopsis, 57, 72, 85-87, 89, 91, 95, 96, 99-101, 103, 105, 107, 110, 199, 242, 253,255,257-266, 272, 274, 275, 277, 279, 281, 282, 289, 290, 293-297 A. thaliana, 86, 95, 199, 231, 255, 265,282 genes, 87, 99, 101, 105, 107, 264 genome, 72, 91, 107, 262, 297 mutants, 258, 272, 275, 293 Arabidopsis esterase family, 261, 262, 264 ARF (auxin response factor), 289 Arginine decarboxylase (ADC), 59 Arogenate dehydratase (ADT), 85, 86, 87,91,107, 110 Arogenate dehydrogenase (AD), 87 Ascomycete, 24 Asexual fungi, 25 Aspergillosis, 27 Aspergillus fumigatus, 23, 24, 26, 27,

31,33,36,37,38,40,43-45,47 Aspirin, 132, 149, 151 Asteraceae, 62, 63 Atropa belladonna, 59 Atropine, 58 AtSABATH enzyme, 257, 261, 265 Auxin, 105, 258, 261, 264, 272, 274279, 281, 282, 288-290, 293, 296, 297; see also Indoleacetic Acid (IAA) ARF (auxin response factor), 289

Auxin Response Elements (AuxREs), 289 biosynthesis, 272, 274-278, 282, 288,291,296 biosynthesis genes, 274, 289 conjugation, 278 gradient, 258, 264, 290 homeostasis, 105, 277, 282, 288, 290,297 homeostasis pathways, 288, 297 inducible genes, 274, 293 overproduces, 294 overproduction mutant, 272, 274, 276, 277 regulatory proteins, 289 signaling, 275, 289, 290, 296 tryptophan-dependent biosynthesis, 272, 274 tryptophan-independent biosynthesis pathway, 272 Auxin overproduction mutant, 272, 274, 276, 277 Avena, 295 Bacteria, 68, 86, 87, 93, 100, 144, 145, 167, 199, 265, 272, 274, 278, 279 gram negative, 144 gram positive, 144 pathogenic, 145, 240, 242 Bacterial IAA-lysine synthase (iaaL), 271,278,279 Basil, 55, 56, 119, 195, 198 Benzoic acid (BA), 63, 257 Benzoquinone sorgoleone, 158; see also Sorgoleone Benzoyl-CoA, 168 Benzylisoquinoline alkaloids (BIA), 64, 67, 69, 71 Berberine, 64, 65, 68, 69 Berberis stolonifera, 65 Biodiversity, 116, 311

INDEX INDEX

Bioengineering, 144, 151; see also Genetic engineering Bioinformatics, 72, 116, 315 Biosynthesis, 2-4, 8, 12, 33, 45,, 55, 56, 58, 59, 65, 67, 68, 70-72, 85-87,91, 101,103,105,107, 110,136,137,140,144,159, 163, 167, 168, 174, 187, 190-193, 198,201,202,212,213,216, 220, 247, 272, 274-278, 282, 288, 291,294,296 alkaloids, 2, 3, 8, 12, 33, 45, 58, 59, 65 anacardic acids, 136, 137, 140, 144 auxin, 272, 274-278, 282, 288, 291, 296 cannabinoid, 187, 190-192 flavonoids, 72, 193 indole-3-acetic acid (IAA), 276, 277,291,294 monoterpenes, 55, 198, 201 phenylalanine, 56, 71, 85-87, 91, 101,103, 105, 107, 110 sanguinarine, 67, 68, 70 secologanin, 4 sorgoleone, 159, 163, 167, 168, 174 sterols, 212, 213, 216, 220, 247 terpene indole alkaloids (TIA), 3, 12 tropane alkaloids (TPA), 59 xanthohumol, 192, 193, 202 Bitter acids, 184, 187, 192, 193, 196, 198,200,201 a-acids, 184, 187, 193 p-acids, 192, 193 BLAST searches, 91, 101, 162 Boraginaceae, 62 Breast cancer, 148, 149, 151 Brewing, 180, 182, 186, 193 C4 plants, 54 Candida utilis, 145

321

Cannabaceae, 179, 180, 187, 195, 201 Cannabinoid, 181, 187, 190-192, 202 biosynthesis, 187, 190-192 Cannabis, 179-181, 184, 186, 187, 190,192,195, 196,198,201,202 C. sativa, 180 C. ruderalis, 180 trichomes, 196, 202 Carboxyl group, 145, 190, 255, 278, 279,281 Cardiovascular disease, 220, 311 Cardols, 134, 142, 145, 148 Carvone, 55 Caryophyllene, 181, 182 Cashew (Anacardium occidentale), 132,133,144,145,148 Cashew apple, 145, 148 Cathenamine, 10, 13 CBi receptor, 186 CB2 receptor, 186 cDNA libraries, 2, 56, 136, 144, 159, 160,190, 196, 198,199,275 Cell-to-cell transport, 262, 265 Cell cultures, 2, 61, 68, 69, 70, 99, 262,312 Cell proliferation, 63, 144, 149, 151 Cell sap analysis, 57 Cellular compartmentalization, 54; see also Compartmentalization Cereal, 158 Cervix carcinoma, 148, 149 Chalconaringenin, 192, 193, 195 Chalcone isomerase, 193, 199, 200 Chanoclavine, 36, 43 Chemical diversity, 116,311; see also Diversification phenotypic, 116, 122, 127 Chemopreventative, 144, 151 Chemotherapeutic, 144, 151 Chinese cabbage, 295 Cholesterol, 212, 213, 216, 217, 220, 221,224,225,242,246

322

INDEX INDEX

Chorismate mutase (CM), 85, 87, 100 Chorismate synthase, 87, 95 chsjil, 193,200 Cinnamate-4-hydroxylase (C4H), 71, 86 Clarkia breweri, 257, 258, 259 Claviceps, 23-25, 27, 36 C. africana, 25, 27, 36, 38, 40, 43 C.fusiformis, 25, 27, 33, 38, 40, 47 C/wupa//, 25, 41, 42, 47 C. purpurea, 24, 27, 31, 33, 36-45 C. sorghi, 36 Clavicipitaceae, 24 Clavicipitaceous fungus, 26 Clavine alkaloids, 27, 39 C-methylation, 213, 217, 224-226, 228,229,231,240,247 Cocaine, 58 Codeine, 65 Codeinone reductase (COR), 65, 67, 71 Codon usage bias, 45 Colletotrichum capsici, 146 Co-localization, 68, 71, 72; see also Compartmentalization Colorado potato beetle {Liptinotarsa decemlineata), 118, 146, 148, 151 Colupulone, 184, 192 Companion cells, 67, 69 Comparative genomics, 86 Compartmentalization, 43, 54, 57, 61, 62,63,72 subcellular, 55, 59, 63, 69, 72 Complexes, 2, 12, 24, 26, 27, 38-43, 46, 54, 56, 71, 72, 91, 93, 124, 134, 135, 142,171,217,225,231,272, 289,290,296,297,308,315 Condensed tannins, 182 Conifers, 56 Conserved inflammatory pathways, 312 Coptis japonica, 65, 69

Corn earworm (Heliothis zed), 118 Corynanthe alkaloids, 12 Cover crop, 158, 171 COX-1, 149 COX-2, 149, 151 Crop plants, 144, 151, 174 Cruciferae, 294 Curvularia lunata, 146 Cyanogenic glucosides, 8, 62 Cyanogenic glycoside, 71 CYP79B2, 271, 275, 276, 291, 293, 294 CYP79B3,276,291,293 CYP80B1, 64, 65, 67, 69, 70 CYP83B1,276,294 Cysteine rich proteins, 199 Cytochrome P450s, 40, 55, 62, 71, 72, 159, 162, 163, 193, 195, 276, 293, 294 Cytokinin, 296 Cytosol-localized mevalonate pathway, 55, 61 Cytotoxicity, 149 Defense responses, 57, 260, 262; see also Pest resistance, Plant defense against insects, 55, 260 against predators, 62 plant, 54, 57, 58, 68, 260, 265 Dehydroquinate synthase (DHQS), 85, 91,93,95,96 Deoxy-D-arabino-heptulosonate 7 phosphate synthase (DHS), 63, 85, 95,96 Deoxyhumulone, 192, 193 Desmethyl sterol, 213, 221, 225 Desmethylxanthohumol, 186, 193, 195,202 Deterrents, 62 feeding, 62 Detoxification, 199, 294 Developmental control, 68

INDEX INDEX

323

Developmental defects, 220, 275, 279, 296 Dextrans, 70 DHQD/SDH genes, 85, 96 DHQS genes, 85 DHS genes, 85, 95 Digit aria sanguinalis, 158 Dihydroergot alkaloids, 27, 36 Dihydrostilbenes, 181 Dimethylallylpyrophosphate (DMAPP), 37, 55, 59, 192, 193, 195,201 Diterpenes, 56 Diverging pathways, 43 Diversification, 43, 93, 100, 191 profiles, 43 DMATrp synthase, 31, 33, 37 DMATrp synthase gene, 31 DNA-chip database, 265 Dopamine, 25, 64 Drugs, 181,187, 191, 195,289,311, 312,313,314 anticholinergic, 58 discovery, 311

dihydroergot alkaloids, 27, 36 pathway, 24, 27, 31, 33, 37, 38, 41, 43,47 Ergotism, 24, 26 Eschscholzia californica (California poppy), 64 Estrogenic activities, 187 Estrogens, 150 Ethylene, 254, 296, 297 Ethylene-dependent IAA synthesis, 296 Eugenol, 56 Euglena gracillis, 145 Eupatorium, 62, 63 E. cannabinum, 63 Evolution, 44, 47, 86, 89, 134, 144, 212, 216, 220, 226, 228, 231, 240, 244, 246, 247 Evolutionary analysis, 212; see also Phylogenetic Expressed sequence tag (EST), 56, 86, 93, 96, 99, 100, 101, 103, 105, 107, 115,118-120,144,157, 159, 160, 162, 163, 166, 168, 174, 179, 190, 196, 198-202

E. coli, 6, 110, 120, 163, 168, 264 Elymoclavine, 38, 40 Endophytes, 25, 26, 39, 47 ER receptors, 151 Erg6p mutant, 242 Ergine, 41-43, 47 Ergonovine, 41-43 Ergopeptines, 24, 26, 27, 38-43 Ergosterol, 213, 216, 220, 221, 224, 225, 226, 242, 247 pathway, 220, 224 Ergot, 23-27, 31,33, 36-47 poisoning, 25 Ergot alkaloids, 23-27, 31, 33, 36-47 gene cluster, 36, 37, 38, 42, 43, 45, 47

Fabaceae, 62 Farnesoic acid (FA), 259, 260, 265 Farnesoic acid carboxyl methyltransferase (FAMT), 259, 260 Fatty acid, 72, 119, 120, 124, 127, 133-136, 140, 142, 151, 159, 162, 163,165, 166, 174,181,198,279 Fatty acid desaturase gene, 136 Fatty acid desaturases, 72, 159 F-box auxin receptor, 289 Fecosterol, 228, 229, 242 Feeding deterrents, 62 Fermentation cultures, 26 Festuclavine, 38 Fir {Abies spp.), 56

324

INDEX INDEX

First-in-class inhibitors, 311 Flavonoids, 56, 71, 72, 86, 180, 181, 182, 191, 193,200 biosynthesis, 72, 193 FLOOZY, 275, 293 Floral volatiles, 124, 254, 262 Food preference, 148 Forage, 25 Fruit fly (Drosophila melanogaster), 42 Fully sequenced genomes, 110 Fumigaclavines, 36 Functional annotation, 93 Fungal elicitation, 56 Fungal pathogens, 144, 146 Fungi, 24, 25, 27, 33, 38, 39, 41-43, 45,46,47,86,87,93,100,145, 167, 212, 216, 220, 224, 226, 236, 246,247,265 asexual, 25 clavicipitaceous, 26 endophytes, 25, 26, 39, 47 endophytic, 25 imperfect, 24 Fungus-specific modification, 44 Fusarium oxysporum, 146 Fusarium udum, 146 GAMT1,258,265 GAMT2, 258, 265 Gene clusters, 27, 31, 33, 43, 47; see also Gene families ergot alkaloid, 36-38, 42, 43, 45, 47 shikimate pathway genes, 85, 86, 91,92,93,95 Gene families, 86, 87, 89, 91, 93, 107, 110,231 SMT gene subfamilies, 246 Gene knockout analyses, 31,47 Genetic engineering, 174, 220; see also Bioengineering Genetic manipulation, 116

Genome Arabidopsis, 72, 91, 107, 262, 297 fully sequenced, 110 poplar, 89, 91, 99, 100, 101, 105, 110 rice, 86, 91, 101 Genomics, 2, 36, 86, 87, 110, 149, 180,282,308 comparative, 86 structural, 86 Geraniaceae, 132 Geraniol, 56, 59, 61 Geraniol synthase, 56 Geranium, 132, 133, 135-137, 140, 142, 144, 146, 147, 149, 151 Geranium trichome system, 137 Geranyl diphosphate, 190, 191 Geranyl diphosphate synthase (GPPS), 55,191 Geranyltransferase, 191 Gerbera hybrida, 190 GH3 proteins, 279 Gibberella fujikuroi, 225 Gibberellin, 296 Ginkgo, 133, 135, 144, 145 Ginkgo biloba, 135 Ginkgoaceae, 132 GL7 mutant, 221 Glandular trichomes, 55, 56, 116, 118, 127, 132, 135-137, 142, 146, 147, 192, 193, 195, 196, 198, 201, 202; see also Trichomes Glucosinolates, 8, 57, 277, 292 Glucosyltransferase, 277 Glutathione transferases, 199 G-proteins, 289 Gradients, 55, 68, 69, 93, 258, 264, 288, 290 Graminae, 294 Grasses, 24, 168 Gravitropic responses, 281 Green flourescent protein (GFP), 57

325

INDEX INDEX Green manure, 158 Green peach aphids {Myzus persicae), 118 Guttiferae, 180 Gyrodinium cohnii, 145 Hairy roots, 59, 313, 314 Hard resin, 184, 186 Heart disease, 220, 311 Hedgehog protein, 221 Hemp, 180, 181, 187 Herbicides, 158 Heterologous expression, 33, 110, 163 Hevea brasiliensis, 57, 122 Hexadecadienoic acid, 166 Histone acetyltransferase (HAT), 149 Homospermidine, 63 Homospermidine synthase (HSS), 63 Honeybee {Apis mellifera), 42 Hop, 179, 180, 182, 184, 186, 187, 192,193,195,196,198,199, 200202 proteins, 201 resins, 184 Hopanoids, 216, 217, 225, 246 Horizontal gene transfer, 45 Hormones, 42, 254, 260, 265, 272, 274, 289, 296 Host fitness, 26 Human health, 134 Human pathogen, 24, 26 Humulone, 180, 182, 184, 192, 195 Humulus, 179, 180 H. lupulus, 179, 180, 182,200 Hydrangea macrophylla, 190 Hydrolytic enzymes, 58 Hyoscyamine, 59 Hydroxy-6-alkybenzoic acids, 132 Hydroxylase, 55, 65 Hydroxyphenylacetaldehyde (4HPAA), 64 Hydroxytryptamine (5-HT), 25

Hyoscyamus niger, 59 IAA glucose ester (IAGlc), 281 IAA methyl ester (MelAA), 257, 262, 264,281 IAA-Lys, 271,278, 279 iaaM gene, 271, 274 iaaM overexpression, 274, 276 IAA-specific methyltransferase (IAMT1),281 IAA-sugar conjugation, 281 IAGlu, 281 Idioblasts, 58, 61,62 Immunocytochemical analyses, 55 Immunolocalization, 63, 69, 72 Imperfect fungus, 24 Increased appetite, 186 Indole, 2-4, 6-8, 12, 13, 37, 59, 257, 265, 272, 274-279, 291, 294, 296, 297; see also Terpene indole alkaloids Indole-3-acetaldehyde, 277, 294 Indole-3-acetaldoxime (IAOx), 275,

276,277,291,294 pathway, 291,293 Indole-3-acetamide, 272, 274, 278 Indole-3-acetic acid (IAA), 257, 264, 265, 271, 272, 274, 276-279, 281, 287-291, 293-297; see also Auxin biosynthesis, 276, 277, 291, 294 ethylene-dependent IAA synthesis, 296 Indole-3-butyric acid (IBA), 258, 287, 296 Indolic glucosinolate biosynthesis pathway, 277 Inflammation, 311 Inflammatory bowel disease (IBD), 311 Inhibit larvae, 148 Inhibit prostaglandin synthase, 149 Inhibiting spore germination, 146

326

INDEX INDEX

Inter-conversion pathways, 288 Interdisciplinary approaches, 308 Internal secretory systems, 57 Intracellular transport, 55, 69 Isoflavone O-methyltransferase (IOMT), 72 Isopentenyl diphosphate (IPP), 55, 59, 201 Isopiperitenol dehydrogenase (IPD), 55 Isoprenoid biochemistry, 216 Isoprostanes, 312 Isothiocyanates, 57 Jasmonates, 56, 122, 254, 312 methyljasmonate (MeJA), 254, 262, 263,264,265 Jasmonic acid (JA), 254, 257, 262265,281,289 Kinases,91,99,289, 312 Knockout mutants, 27, 31, 33, 39, 41, 47, 264,293,297 LA1777 database, 120 Lamiaceae, 55 Lanosterol, 213, 216, 217, 220, 221, 224,225,229,231 Lateral root formation, 296 Latex, 57, 58 Latex proteins (MLPs), 58 Laticifers, 57, 58, 61, 62, 67, 69-71 Laurate, 135 Leafhopper (Empoasca fabae), 56 Ligand-dependent transcription factors, 150 Linalool synthase, 56 Lipoxygenase, 148, 312 Lipoxygenase-induced signaling pathways, 312 Liver cancer, 148 Liver toxins, 62

Loganin, 61 Lolium arundinaceum, 26 Long distance transport, 289, 290 Long distance polar transport (PAT), 289, 290 Lonicera tatarica, 7 Loss-of-function mutants, 275 LPS1 -encoding gene, 40, 41 Lupinus albus, 63 Lupinus polyphyllus, 63 Lupulone, 184, 192 Lycopersicon esculentum, 87, 199; see also Tomato Lysergic acid, 24, 27, 38-42 amides, 24, 26, 27, 38, 41 Lysergyl peptide synthetase (LPS1, LPS2), 31,39-42,44 Macromolecular complexes, 71 Maize, 281, 297 Marijuana, 180, 181, 186 Medicinal plants, 2, 144, 148 Metabolic channels, 71 Metabolic disease, 311 Metabolic engineering, 12, 195, 202, 313; see also Genetic engineering Metabolomic, 308, 315, 316 profiling, 118, 127 Metallothioneins, 199 Methicillin-resistant Staphylococcus aureus (MRSA), 145 Methyl IAA (MelAA), 258, 262, 264, 281 Methylation, 33, 39, 59, 61, 193, 195, 201, 202, 213, 220, 224-226, 228, 229, 231, 240, 244, 247, 254, 261, 264,265,281 Methylerythritol 4-phosphate (MEP) pathway, 55, 61, 195, 198,201 Methylesters, 135, 254, 261, 265 Methyljasmonate (MeJA), 254, 262, 263,264, 265

INDEX INDEX Methyljasmonate esterase (MJE), 262 Methylketone synthase 1 (MKS1), 115, 118, 120, 122-127 Methylketones, 115, 118, 120, 123, 124, 125, 127, 198 biosynthetic pathway, 118, 124 Methylsalicylate (MeSA), 254, 257, 261-264 Methyltransferases, 59, 64, 65, 72, 159,162, 163, 174,192,195,212, 257-261,266 AdoMet-dependent, 232, 236, 240, 246 AtSABATH, 265 famesoic acid carboxyl (FAMT), 259,260 IAA-specific (IAMT1), 281 isoflavone 0-methyltransferase (IOMT), 72 iV-methyltransferase, 33 O-methyltransferase, 64, 65, 72, 159, 162,163, 174,195,200,201 SABATH, 257 salicylic acid carboxyl (SAMT), 257-259 sterol (SMT), 211-213, 216, 220, 225,226,228,229,231,232, 236, 240, 242, 244, 246 Mevalonate pathway, 55, 61 Microarray, 86, 93, 264, 293 analysis, 264, 293 expression profiling, 86, 93 Mint, 55, 119, 195 Model plants, 86 Model system, 73,282, 315 Molecular recognition, 212, 246 Monoamine oxidase (MAO), 314 inhibitors, 314 Monolignol, 86, 93, 96, 99 Monooxygenase activity, 38, 42 Monoterpenes, 3, 55, 56, 72 biosynthesis, 55, 198, 201

327 oxygenated, 56 Monoterpene iridoid secologanin, 3 Monoterpenoids, 53, 59, 61 Monoterpenoid indole alkaloids, 61 Morinda citrifolia, 95 Morphine, 64, 65, 67, 68, 71 Morphine-specific branch pathway, 71 Morphogen, 290 Multi drug resistance proteins, 289 Multienzyme complex, 54, 71, 124 Multifunctional activities, 166 Multifunctional enzymes, 39, 72 Musaceae, 294 Muscle relaxant, 64 Mutants, 27, 47, 110, 123, 200, 220, 221, 232, 236, 242, 244, 257, 258, 264, 272, 274-278, 290, 291, 292, 293, 295-297 Arabidopsis, 258, 272, 275, 293 auxin overproduction, 272, 274, 276, 277 knockout, 27, 31, 33, 39, 41, 47, 264,293, 297 loss-of-function, 275 superroot, 271, 276, 277, 291 Mutational analysis, 315 Mycotoxins, 24, 167 Myristic acid, 136, 139, 140 Myristicaceae, 132 Myrosinases, 57 Natural insecticide, 127 Natural Product Repository, 309 Natural Products Repository Network (INPRN), 307, 309, 311,316 Natural rubber (cw-l,4-polyisoprene), 57 Neotyphodium, 24-27, 31, 36, 38, 39, 41-43,47 N. coenophialum, 26, 39, 41 Neurospora crassa, 145

328

INDEX INDEX

Nicotiana, 59, 87, 195, 199; see also Tobacco N. tabacum, 59, 199 Nictonic acid (NA), 174, 261, 265 Nitrilases, 277, 294, 295 JV-methyltransferase, 33 Nonribosomal peptide pathways, 12 Norcoclaurine, 64 Novel alkaloid derivatives, 13 iV-oxide, 62 Obstructive pulmonary disease (COPD), 311 Old yellow enzyme, 36 Oleoresin, 56 Oleoresin terpenes, 56 Olivetolic acid, 190-192 O-methyltransferases, 64, 65, 72, 159, 162,163,174,195,200,201 Onobrychis viciifolia, 71 Opium poppy (Papaver somniferum), 58,64,65,67,69-71,73 Orchidaceae, 62 Ornithine decarboxylase (ODC), 59 Oryza, 85, 86; see also Rice O. sativa, 102,231 Osteoporosis, 311 Overexpress, 293 Overexpression lines, 174, 258, 274, 276,281 Oxygenated monoterpenes, 56 Ozoroa insignis, 132, 148 Ozoroa mucronata, 132, 149 P450 enzyme, 2 P450 monooxygenase, 38, 42, 159; see also Cytochrome P450s Palmitic acid, 140 Palmitoleic acid, 166 Papaveraceae, 65 Parasitic wasps, 261 Paspalum spp., 25

Pathogenic bacteria, 145, 272, 274 Pathogens, 24, 26, 55, 68, 95, 96, 100, 144, 145, 195,297,312 bacteria, 145, 272, 274 fungal, 144, 146 human, 24, 26 soil-borne, 68 Pathways, 2, 8, 10-13, 24, 27, 31, 33, 36-39, 41, 43, 44, 46, 47, 54-56, 59, 61, 63-65, 67, 68, 71-73, 85-87, 89, 91,93,95,96,100,101, 103, 107, 110,118-120,124,127, 151, 159, 163,174,192, 193, 195,198,200, 201, 212, 213, 216, 217, 220, 221, 224, 225, 236, 244, 246, 247, 261, 262, 272, 274, 276-278, 282, 291, 293,294,296,297,312,315 bacteria, 145, 272, 274 conserved inflammatory, 312 cytosol-localized mevalonate, 55, 61 diverging, 43 ergot alkaloid, 24, 27, 31, 33, 37, 38,41,43,47 ergosterol, 220, 224 indole-3-acetaldoxime (IAOx), 291, 293 indolic glucosinolate biosynthesis, 277 inter-conversion, 288 lipoxygenase-induced signaling, 312 methylerythritol 4-phosphate (MEP), 55, 61, 195, 198,201 methylketone, 118, 124 mevalonate, 55, 61 morphine-specific branch, 71 nonribosomal peptide, 12 phenylpropanoid, 56, 71, 86, 91, 93, 96,103,110 plastid-localized MEP, 61

INDEX INDEX

shikimate, 85-87, 91, 93, 95, 101, 102, 107,110,124 shunt, 39, 43 sterol,213,216,220 trp-I, 291,297 Pea, 199, 289 Pearl millet (Pennisetum glaucum), 25,38 Peltate glands, 55, 119 Penicillium griseofulvum, 145 Penicillium paxilli, 37 Penicillium spp., 37 Peppermint (Mentha xpiperita), 55, 198 Peptide synthetases, 31, 39-42, 44 Peptides, 2, 12, 24, 27, 31, 39-42, 44, 290 Peroxidases, 38 Pest resistance, 56, 132, 133, 136, 148; see also Defense responses Pest-susceptible, 132, 133 Petunia, 124, 274, 275, 293 Pharmaceutical agents, 2, 61 Pharmacological activity, 64, 187 Phenolic lipids, 134, 135, 141, 142, 144, 145, 151, 167, 168 Phenolics, 58, 86, 134, 135, 141, 142, 144,145, 151,167,168,180,202 terpenophenolic, 179, 180, 182, 184, 186, 195,196,201,202 Phenotypic diversity, 116, 122, 127; see also Chemical diversity Phenylalanine, 39, 71, 85-87, 96, 99, 100,101,103, 105, 107,110,242 biosynthesis, 56, 71, 85-87, 91, 101, 103, 105,107,110 Phenylalanine ammonia-lyase (PAL), 71,86,95,96 Phenylpropanoid genes, 93, 103 Phenylpropanoid metabolism, 86, 93, 99

329

Phenylpropanoid pathway, 56, 71, 86, 91,93,96,103,110 Phenylpropene volatiles, 55 Phloem, 56-59, 61-63, 68, 105 Phloroglucinol, 180, 181, 191 Phospholipids, 220, 225 Phylogenetic, 87, 91, 99, 101, 103, 107, 168,212 primitive enzyme, 217, 244 reconstructions, 87 trees, 91, 101 Phytoprostanes, 312 Phytosterols, 212, 213, 217, 220, 221, 224-226, 228, 231, 246, 247; see also Sterols Phytothora cactorum, 220 Phytotoxins, 158 PI126449 database, 119, 120 Pictet-Spengler reaction, 7, 13 PIN genes, 289 Pine (Pinus spp.), 56, 291 Pistachio {Pistacia vera), 132 Plant defense, 54, 57, 58, 68, 260, 265; see also Defense responses Plastid-localized MEP pathway, 61 Platforms, 202, 308, 313, 315, 316 Polyclonal antibody, 125 Polyketide synthases, 72, 124, 134, 135, 141, 142, 144, 151, 159, 162, 163, 167, 168, 174, 190, 192, 193, 201,202 type III, 134,135, 141, 144, 151,

159, 167, 168,190, 192,200 Polyketides, 2, 12, 72, 124, 134, 135, 141, 142, 144, 151, 159, 162, 163, 167, 168, 174, 180-182, 184, 187, 190,192,193,201,202 Polyphenolics, 182 Poor milk production, 26 Poplar genome, 89, 91, 99-101, 105, 110

330

INDEX INDEX

Prenylation, 33, 37, 187, 191, 192, 193,201,202 chalcones, 186 flavones, 182 flavanones, 186 prenylflavonoids, 201 transferase, 37, 55 Prephenate aminotransferase (PNT), 85-87, 91, 101, 103, 105, 107, 110 Primary metabolism, 54, 93, 127 Primitive enzyme, 217, 244 Proanthocyanidins, 182 Promoter-GUS fusion, 59, 258 Propionibacterium acnes, 145 Prostaglandins, 148, 149, 151, 312 Protein receptors, 246, 254 Protein S (CrPS), 61 Protein sequences, 64, 91, 122, 162, 166,168,201,262 Protein structure studies, 142 Protein-serine/threonine kinase, 289 Proteome, 127 Proteomics, 253 Prototheca wickerhamii, 240 Protozoa, 145, 212, 216, 226, 240, 244,246,247 Pseudomonas, 272, 279, 297 P. syringae, 272, 279 Psychoactive drug, 180, 181, 187 Putrescine, 59 Putrescine iV-methyltransferase (PMT), 59 Pyrrolizidine alkaloids (PAs), 58, 62, 63 Quantitative trait, 125, 127 Quantitative trait loci (QTL), 118, 125,127 Quinolizidine alkaloids (QAs), 63 RACE (Rapid Amplification of cDNA Ends), 168

Rauwolfia serpentina, 4, 8 Real time RT-PCR, 163, 168, 174, 193,258,264 Receptor protein, 216 Redundancy, 272, 293 Redundant regulatory networks, 288 Regiospecificities, 72 Resin, 53,56, 184, 186, 196 ducts, 56 Resistance, 56, 69, 131-133, 136, 144146, 148, 151, 159, 254, 257, 261, 263, 264, 274, 275, 278, 289, 295 pest, 56, 132, 133, 136, 148 system acquired (SAR), 254, 261, 262 Resorcinol, 159, 163, 167, 168, 174 Retrobiosynthetic NMR analysis, 159 Reverse genetics, 2, 292 Reverse transcriptase-PCR, 63 Rhizosphere, 158 Rhus semialata, 145 Rice, 85, 86, 89, 91, 95, 96, 99, 101, 103, 105,107, 110, 168, 171; see also Oryza genome, 86, 91, 101 Root hairs, 158-160, 163, 166, 168, 171, 174,275,293 Root phenotypes, 276 Rosa hybrida, 163 Rubiaceae, 2 Rutaceae, 65 Rye (Secale cereale), 24 Ryegrass (Lolium perenne), 26, 39, 43 SABATH methyltransferases, 257 Saccharomyces cerevisiae, 33, 145, 163, 229, 231; see also Yeast S-adenosyl methionine (SAM), 59, 65, 159,195,200,254,281 Salicylic acid (SA), 132, 145, 149, 254,257,262,265,281

INDEX INDEX Salicylic acid carboxyl mehthyltransferase (SAMT), 257259 Salt tolerance, 261 Sanguinarine, 64, 65, 67, 68, 69, 70 biosynthesis, 67, 68, 70 Saturated anacardic acids, 133 SAURs (small auxin up-regulated RNAs), 289 S-cells, 57 Scopolamine, 58, 59 Screening platforms, 316 Secologanin biosynthesis, 4 Secolysergine, 39, 43 Secondary metabolism, 2, 8, 54, 191 Semecarpus anacardium, 148, 149 Senecio, 62, 63 Sequestration, 70, 71 Serotonin, 25 Sesquiterpene, 55, 181 Shared genes, 44, 315 Shikimate, 85-87, 90-93, 95, 96, 99101,103,107,110,124 pathway, 85-87, 91, 93, 95, 101, 102, 107,110,124 pathway genes, 85, 86, 91-93, 95 Shikimate kinase (SK), 85, 91, 93, 99, 179 Shunt pathways, 39, 43 Sieve elements, 57, 67, 69-71 Signaling, 216, 221, 254, 258, 260262, 265, 275, 288, 289, 296, 312 auxin, 275, 289, 290, 296 mitochondrial translocation, 64 molecules, 221, 254, 258, 260-262, 265 pathways, 296, 312 perception, 254 transduction, 61, 255, 262, 289, 296,297,312 Single-cell EST database approach, 118

331 Sitosterol, 213, 216, 217, 224, 226, 247 SMT enzymes, 216, 220, 225, 226, 231,232,240 SMT gene subfamilies, 246 Soil-borne pathogens, 68 Solanaceae, 58 Solarium habrochaites, 118 Sorghum {Sorghum vulgare), 24, 25, 27,71, 158, 159, 163, 165, 166, 168,171, 174 Sorghum bicolor, 87, 158-160, 174 Sorgoleone, 157-159, 163, 165-168, 171,174 accumulation, 174 biosynthesis, 159, 163, 167, 168, 174 Soybean, 226, 236, 242, 244, 289 Spearmint (Mentha spicata), 55 Specialized metabolism, 116, 127 Spider mite {Tetranuchus urticae), 118,132,133 Spruce (Picea spp.), 56 Squalene, 213, 216, 217, 220, 225 Stable isotope labeling, 291-293 Staphylococcus aureus, 145 Stem-boring insects, 56 Sterols, 212, 213, 216, 217, 220, 221, 224, 225, 226, 228, 229, 231, 232, 236, 240, 242, 244, 246, 247; see also Phtyosterols biosynthesis, 212, 213, 216, 220, 247 desmethyl,213,221,225 diversity, 225 ergosterol, 213, 216, 220, 221, 224, 225,226, 242, 247 evolution, 212, 246 fecosterol, 228, 229, 242 lanosterol, 213, 216, 217, 220, 221, 224,225,229,231 pathway, 213, 216, 220

332

INDEX INDEX

zymosterol, 228, 229, 231, 232, 242,244, 247 Sterol methyltransferase (SMT), 211213, 216, 220, 225, 226, 228, 229, 231, 232, 236, 240, 242, 244, 246 Stilbenecarboxylates, 142, 144 Stilbenes, 71, 134, 142, 144, 168, 181, 182, 190, 191 Storage, 62, 63, 118, 127, 140, 186, 290,310,311 Streptococcus mutans, 145 Stress, 26, 58, 59, 171,297, 312 biotic, 297 tolerance, 26, 261 Strictosidine, 4, 6-8, 10, 12, 13, 61, 62 Strictosidine glucosidase, 8, 13 Strictosidine synthase (STR), 4, 6, 7, 13,61,62 Structural diversity, 180, 187; see also Chemical diversity Structural genomics, 86 Structure-activity, 212, 221, 225 Strychnos, 12 Strychnos nux vomica, 12 Subcellular compartments, 55, 59, 63, 69, 72; see also Compartmentalization Substrate channeling, 124 Subtraction techniques, 2 Sulfur, 57 Superroot mutants, 271, 276, 277, 291 surl,211,291 sur2, 276, 277 Suppression of cyclooxygenase-2 gene transcription, 187 Sweet basil (Ocimum basilicum), 55, 195, 198 Symplastic transport, 69, 70 Synergism, 145 System acquired resistance (SAR), 254,261,262

Tachycardia, 186 Tall fescue, 26 Tall fescue endophyte toxicosis, 26 Targeting, 55, 72, 103,289 target-based assays, 312 target-based optimization, 311 Technologies, 2, 308, 316 analytical, 316 Terpene indole alkaloids (TIA), 2, 3, 8, 10, 13, 59, 62 biosynthesis, 3, 12 Terpene synthases, 56, 198, 200, 201 Terpenophenolic, 179, 180, 182, 184, 186, 195, 196,201,202 Thalictrum flavum (meadow rue), 64, 65, 68, 69 Thalictrum glaucum, 69 Thalictrum minus, 65 Three dimensional structure, 127 Tobacco, 72, 87, 96, 118, 122, 262, 264, 274, 290, 294, 295, 313, 314; see also Nicotiana Tobacco hornworm {Manduca sextd), 118 Tobacco mosaic virus (TMV), 262 Tomato, 86, 87, 95, 93, 95, 96, 99, 100,116,118, 120,122,125,127, 198,200,262,265,291 Tooth decay, 145 Toxicosis, 24, 26 Trans-anti stereochemistry, 213, 217 Trans-cinnamic acid, 71 Transcriptional controls, 71, 100, 289 ligand-dependent factors, 150 Transcriptome, 86, 174, 198 Transgenic plants, 247, 263, 264, 274, 279,281 Translocation of alkaloids, 69 Transport, 54, 55, 63, 68-70, 105, 158, 262,265,281,288-290 cell-to-cell, 262, 265 intracellular, 55, 69

333

INDEX INDEX long distance polar (PAT), 289, 290 symplastic, 69, 70 Traumatic resin ducts (TRD), 56 Treating diabetes, 187 Tree, 89, 93, 99, 103, 107, 134, 168 Trichomes, 53-56, 115, 116, 118, 119, 127, 131-133, 135-137, 139, 140, 142, 144, 146, 147, 151, 179, 190193, 195,196, 198, 199,201,202, 257; see also Glandular trichomes Tridecanone (2TD), 118, 119, 125 Trigonelline, 261, 265 Tropane alkaloids (TPA), 58, 59 biosynthesis, 59 Tropinone, 59 Trp-I pathways, 291, 297 Trp-independent (Trp-I), 291, 296, 297 Trypanosma brucei, 240 Tryptamine, 3, 4, 6, 7, 61, 62, 274, 293 Tryptophan, 3, 4, 7, 24, 33, 37, 59, 61, 86, 87, 93, 258, 272, 274, 275, 278, 291 Type III polyketide synthase, 134, 135, 141, 144, 151, 159, 167, 168, 190, 192,200 Tyrosine, 7, 64, 86, 87, 93, 100, 101, 242

UGT74B1,271,277 Ulcers, 145 Undecanone (2UD), 118, 119 Velvetleaf (Abutilon theophrasti), 111 Vinblastine, 3, 59, 61 Vincristine, 3, 59, 61 Wheat (Triticum aestivum), 24 Whitefly, 132, 133 Wood, 93, 95, 99-101, 103, 105, 107, 290 Wounding, 56, 58, 100, 257, 291 Xanthohumol, 186, 187, 192, 193, 195,198,200-202 biosynthesis, 192, 193, 202 X-ray crystallographic, 191 Yeast, 72, 91, 110, 145, 166, 220, 221, 224-226, 228, 229, 232, 236, 240, 242, 244, 246, 247, 275; see also Saccharomyces cerevisiae YUCCA genes, 275, 293 Zymosterol, 228, 229, 231, 232, 242, 244,247

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