Pectins and Their Manipulation (Sheffield Biological Siences)

Pectins and their Manipulation

GRAHAM B. SEYMOUR J. PAUL KNOX, Editors

Blackwell Publishing

Pectins and their Manipulation

Sheffield Biological Sciences A series which provides an accessible source of information at research and professional level in chosen sectors of the biological sciences. Series Editors: Professor Jeremy A. Roberts, Plant Science Division, School of Biosciences. University of Nottingham. Professor Peter N.R. Usherwood, Molecular Toxicology Division. School of Biosciences, University of Nottingham. Titles in the series: Stress Physiology in Animals Edited by P.H.M. Balm Seed Technology and its Biological Basis Edited by M. Black and J.D. Bewley Leaf Development and Canopy Growth Edited by B. Marshall and J.A. Roberts Environmental Impacts of Aquaculture Edited by K.D. Black Herbicides and their Mechanisms of Action Edited by A.H. Cobb and R.C. Kirkwood The Plant Cell Cycle and its Interfaces Edited by D. Francis Meristematic Tissues in Plant Growth and Development Edited by M.T. McManus and B.E. Veit Fruit Quality and its Biological Basis Edited by M. Knee Pectins and their Manipulation Edited by Graham B. Seymour and J. Paul Knox

Pectins and their Manipulation Edited by GRAHAM B. SEYMOUR Department of Plant Genetics and Biotechnology Horticulture Research International Warwick, UK and

J. PAUL KNOX Centre for Plant Sciences University of Leeds UK

Blackwell Publishing

CRC Press

© 2002 by Blackwell Publishing Ltd Editorial Offices: Osney Mead. Oxford OX2 OEL. UK Tel: +44 (0)1865 206206 108 Cowley Road. Oxford OX4 1JF. UK Tel: +44 (0)1865 791100 Blackwell Munksgaard. Norre Sogade 35. PO Box 2148. Copenhagen. DK-1016. Denmark Tel: +45 77 33 33 33 Blackwell Publishing Asia, 54 University Street. Carlton, Victoria 3053. Australia Tel: +61 (0)3 9347 0300 Blackwell Verlag, Kurfurstendamm 57. 10707 Berlin. Germany Tel: +49 (0)30 32 79 060 Blackwell Publishing, 10 rue Casimir Delavigne. 75006 Paris, France Tel: +33 1 53 10 33 10 ISBN 1-84127-228-0 Published in the USA and Canada (only) by CRC Press LLC 2000 Corporate Blvd.. N.W. Boca Raton, FL 33431, USA Orders from the USA and Canada (only) to CRC Press LLC USA and Canada only: ISBN 0-8493-9789-8 The right of the Author to be identified as the Author of this Work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988. without the prior permission of the publisher. This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher

cannot assume responsibility for the validity of all materials or for the consequences of their use. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation, without intent to infringe. First published 2002 A catalogue record for this title is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress Set in 101/2/12pt Times by Thomson Press (India) Ltd Printed and bound in Great Britain by Bookcraft Ltd. Midsomer Norton. Bath

Preface The physical, biochemical and functional properties of pectins are of interest to a diverse range of scientists working in areas from plant biology to food science. This book sets out to provide state-of-the-art reviews of key areas relating to the structure and function of pectins in both foods and developing plant systems. The book covers not only the chemical structure, biosynthesis and degradation of these important biopolymers in plants, but also their biophysical properties, their links to other wall components and their cell and developmental biology. Pectins are the most structurally complex polysaccharides in plant cell walls and determining their chemical structure and precise biological roles still provides a significant challenge. However, in the last decade the information available on pectin structure has widened considerably, and our understanding of the structure-function relationships of pectins in the context of plant cell walls is beginning to derive a major impetus from the development of new methodologies and the molecular and genetic dissection of the biological basis of plant growth. Pectins remain at the heart of these cell wall related processes and discoveries of both biological and commercial significance will surely follow a more precise understanding of these intriguing, complex polymers in their biological context. Overall, our aim has been to bring together in one volume information reflecting the current status of pectin research, to provide a basis for those interested in the manipulation of pectin properties in plant and food systems. J. Paul Knox Graham B. Seymour

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Contributors

Dr Gert-Jan W. M. van Alebeek Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands Dr Jacques A. E. Benen

Department of Agrotechnology and Food Sciences, Laboratory of Microbiology, Wageningen University, Dreijenlaan 2, 6703 HA Wageningen, The Netherlands

Dr Michael C. Jarvis

Department of Chemistry, Glasgow University, Glasgow G12 8QQ, UK

Dr J. Paul Knox

Centre for Plant Sciences, University of Leeds, Leeds LS2 9JT, UK

Dr Debra Mohnen

Complex Carbohydrate Research Center, and Department of Biochemistry and Molecular Biology, The University of Georgia, 220 Riverbend Road, Athens GA 30602-4712, USA

Professor Andrew J. Mort

Department of Biochemistry and Molecular Biology, 246 Noble Research Center, Oklahoma State University, Stillwater OK 74078-3035, USA

Dr Claus Rolin

CPKelco Aps, DK-4623 Lille Skensved, Denmark

Dr Henk A. Schols

Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands

Dr Graham B. Seymour

Department of Plant Genetics and Biotechnology, Horticulture Research International, Wellesbourne, Warwick CV35 9EF, UK

Viii

CONTRIBUTORS

Dr Gregory A. Tucker

School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LEI 2 5RD, UK

Dr Jean-Paul Vincken

Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands

Dr Alphons G. J. Voragen

Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, Bomenweg 2, 6703 HD Wageningen, The Netherlands

Contents

1

2

3

The chemical structure of pectins HENK A. SCHOLS and ALPHONS G. J. VORAGEN

1

1.1 Introduction 1.2 Chemical structure of pectins 1.2.1 Structural elements 1.3 Chemical stability of pectins 1.4 Pectins as food ingredients 1.5 Pectins as'bioactive'compounds 1.6 Methodology in pectin research References

1 2 6 18 19 21 22 25

Interactions between pectins and other polymers ANDREW J. MORT

30

2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Introduction An overview of cell wall models Experimental approaches to the study of covalent crosslinks between polymers What types of crosslinks to expect Historical perspective on crosslinks to pectins Evidence in 1973 for a xyloglucan-pectin linkage and a pectin-protein linkage Testing the 1973 cell wall model Recent evidence for the existence of some crosslinking between pectin and hemicellulose 2.9 Ester linkages between pectin and other polymers 2.10 Recent evidence for the existence of some crosslinking between pectin and extensin 2.11 Considering crosslinks and cell wall dynamics 2.12 Miscellaneous interactions of pectins with other polymers 2.12.1 Covalent interactions 2.12.2 Noncovalent interactions 2.13 General conclusions Acknowledgements References

30 30 31 33 33 34 35

Biosynthesis of pectins DEBRA MOHNEN

52

3.1 3.2 3.3 3.4

52 53 53 59

Introduction What is the structure of newly synthesized pectin? Subcellular location of pectin synthesis Synthesis of the nucleotide-sugar substrates required for pectin synthesis

38 41 42 44 45 45 46 47 47 47

X

4

CONTENTS

3.4.1 Undine diphosphate-a-D-galacturonic acid (UDP-GalA) 3.4.2 Undine diphosphate-b-L-arabinose (UDP-L-Ara) 3.4.3 Undine diphosphate-b-L-rhamnose (UDP-L-Rha) 3.4.4 Undine diphosphate-a-D-galactose (UDP-Gal) 3.4.5 Undine diphosphate-ct-D-glucuronic acid (UDP-GlcA) 3.4.6 Undine diphosphate-a-D-xylose (UDP-Xyl) 3.4.7 Guanosine diphosphate-b-L-fucose (GDP-Fuc) 3.4.8 Undine diphosphate-a-D-apiose (UDP-apiose) 3.4.9 Guanosine diphosphate-b-L-galactose (GDP-Gal) 3.4.10 XXX-Kdo. XXX-Dha and XXX-aceric acid 3.5 Glycosyltransferases involved in pectin biosynthesis 3.5.1 Synthesis of homogalacturonan 3.5.2 Synthesis of substituted homogalacturonans 3.5.3 Synthesis of rhamnogalacturonan I (RG-I) 3.6 Future directions and resources for studying pectin biosynthesis Acknowledgements References

62 63 64 65 66 68 68 70 71 71 72 73 78 82 87 88 88

Biophysical properties of pectins MICHAEL C. JARVIS

99

4.1 Introduction 4.2 The mechanical properties of biopolymer gels 4.2.1 Gel structure 4.2.2 Mechanisms for the deformation of gels under stress 4.2.3 Single-chain mechanics 4.2.4 Junction zones under mechanical stress 4.3 Mechanochemistry of the component chains of pectins 4.3.1 Chain conformation 4.3.2 Single pectic chains under tension 4.3.3 Chain aggregation and the potential formation of junction zones 4.3.4 Covalent crosslinks 4.4 Conclusions: pectic gels under stress References

5

Cell and developmental biology of pectins J. PAUL KNOX 5.1 Introduction to pectin biology 5.2 Tools and approaches for the analysis of pectins in planta 5.3 Pectins and the cell wall 5.3.1 Pectic polysaccharides and matrix properties 5.3.2 Pectin and cell wall architecture 5.4 Pectins and cell processes 5.4.1 Pectins, metabolism and signalling 5.4.2 Cell proliferation 5.4.3 Cell expansion 5.4.4 Cell differentiation 5.4.5 Pectins and the intercellular matrix: cell adhesion 5.5 Prospects References

99 103 103 104 105 107 111 111 113 115 118 121 123

131 131 132 134 134 135 138 138 139 139 142 143 145 146

CONTENTS

6

Modification and degradation of pectins

Xi

150

GREGORY A. TUCKER and GRAHAM B. SEYMOUR

7

6.1 Introduction 6.2 Pectin-degrading enzymes 6.2.1 Pectinesterase 6.2.2 Polygalacturonase 6.2.3 Pectate lyase 6.2.4 Pectin acetylesterase 6.2.5 b-Galactosidase and a-arabinosidase 6.2.6 Rhamnogalacturonase and minor pectinases 6.2.7 Peroxidase 6.3 Pectin modification during plant development 6.3.1 Fruit ripening 6.3.2 Abscission 6.3.3 Growth 6.4 Functional analysis of pectinases 6.5 Application of gene silencing techniques 6.6 Use of mutants 6.7 Conclusion and prospects References

150 150 153 154 155 156 157 157 158 158 15 8 162 162 163 164 166 167 168

Microbial pectinases JACQUES A. E. BENEN, JEAN-PAUL VINCKEN and GERT-JAN W. M. VAN ALEBEEK

174

7.1 7.2

Introduction Polygalacturonases 175 7.2.1 Exopolygalacturonases 7.2.2 Endo-xylogalacturonan hydrolase 7.2.3 Endopolygalacturonases 7.3 Pectate and pectin lyases 7.3.1 Exo-pectate lyases and oligogalacturonan lyase 7.3.2 Endo-pectate lyases 7.3.3 Pectin lyases 7.4 Heterogalacturonases 7.4.1 Endo-acting rhamnogalacturonan-degrading enzymes 7.4.2 Rhamnogalacturonan rhamnohydrolase 7.4.3 Rhamnogalacturonan galacturonohydrolase 7.4.4 Synergy in rhamnogalacturonan degradation 7.5 Pectic esterases 7.5.1 Pectin methylesterases 7.5.2 Pectin homogalacturonan acetylesterases 7.5.3 Rhamnogalacturonan acetylesterases References

174 179 181 181 190 193 193 197 202 203 207 208 208 208 209 211 213 215

xii

8

CONTENTS

Commercial pectin preparations GLAUS ROLIN

222

8.1 Production 8.1.1 Raw materials 8.1.2 Production process for high-ester pectin 8.1.3 Production process for low-ester and amidated pectin 8.1.4 Standardization 8.2 Commercial definitions and standards 8.2.1 'Pectin'as defined by national and international authorities 8.2.2 Vocabulary of terminology and concepts 8.3 General properties 8.3.1 Composition 8.3.2 Acidic properties 8.3.3 Interactions 8.3.4 Chemical stability 8.3.5 Parameters influencing thickening and gelling 8.4 Handling advice 8.4.1 Making an aqueous solution 8.4.2 Mixing pectin with other ingredients 8.4.3 Making gels 8.4.4 Avoiding degradation—storing pectin 8.5 Uses 8.5.1 Sweet, fruit-flavored gels 8.5.2 Use with fermented or acidified milk 8.5.3 Stabilization of oil-in-water emulsions 8.5.4 Fat replacement 8.5.5 Pharmaceutical uses Acknowledgements References

222 222 223 224 224 225 225 226 227 227 228 229 229 229 232 232 233 234 235 235 235 236 238 238 238 239 239

Index

242

1

The chemical structure of pectins Henk A. Schols and Alphons G. J. Voragen

1.1

Introduction

Pectins are common to the cell walls of higher plants and contribute to many cell wall functions. Cell walls determine the size and shape of cells and, consequently, the integrity and rigidity of plant tissues. In addition, pectins play a role in ion transport and water retention, they determine the pore size of cell walls and they are involved in defence mechanisms against infections by plant pathogens, wounding, and stress (Bacic et al., 1988). The specific functions of pectins in distinct parts of cell walls or plant tissues are strongly influenced by the amount and nature of the pectic molecules present. The structure of pectins is further influenced by enzymatic and chemical modification reactions during the growth of plants, during the ripening and storage of fruits and as a result of the processing of fruits and vegetables. The structure of pectic molecules therefore depends on many parameters and is subject to considerable change. In addition to all these functions in living tissues, pectins are also of commercial interest, as they are used as gelling agents in the manufacture of jams, jellies, marmalades and confectionery and for the stabilisation of acidified dairy drinks (Voragen et al, 1995; May, 2000). The raw materials used to extract pectins on an industrial scale are usually by-products from the food industry, such as citrus peel and apple pomace. Commercial pectin preparations are generally more than 70% galacturonic acid by weight, as the extraction conditions specifically remove the greatest part of the neutral side chains present in native pectin (Voragen et al, 1995; May, 2000; Thibault and Ralet, 2001). Pectins also play a nutritional role as soluble or insoluble components of a 'dietary fibre-rich diet'. They are reported to have the potential to lower blood cholesterol levels, to affect glucose metabolism, to act against diarrhoea, and to function as detoxicants, as regulators and protectants of the gastrointestinal tract, as immune system stimulants and as anti-ulcer and anti-nephrotic agents (Endress, 1991; Yamada, 1996, 2000; and references in Voragen et al, 1995). Previous books on pectins include those of Walter (1991) and Visser and Voragen (1996).

2

PECTINS AND THEIR MANIPULATION

1.2 Chemical structure of pectins The term pectin covers a diverse group of associated polysaccharides from the primary cell walls and intercellular regions of plants. The major sugar residue of which most pectins are composed is D-galacturonic acid, which is present in an a-(l—>4)-linked linear chain in which varying proportions of the acid groups are esterified with methanol (Figure 1.1). In commercially available pectin, the galacturonic acid content is usually over 75%, while the extent of methyl-esterification may vary between 30 and 80%. To control the rheological and physical characteristics of pectin when it is used as a food ingredient, it is often derivatised to amidated pectin in which the OCH3 group at C-6 is replaced by an NH2 group (May, 2000). When dealing with pectins in plant materials, the term pectic substances is commonly used to include associated neutral sugar side chains (arabinans, galactans and arabinogalactans), which are linked to rhamnogalacturonan segments within the pectin molecule. Other structural elements of pectin include rhamnogalacturonan, xylogalacturonan and apiogalacturonan (Voragen et al, 1995, 2001). The term 'protopectin' is often used to designate the native pectin fractions in cell walls that cannot be extracted by nondegradative methods but this term will not be used further in this chapter.

Figure 1.1 a-D-Galacturonic acid, the predominant building block in pectins. The location of methylesterification as found in native pectins is indicated, and the location of the amide group, as present in industrially modified amidated pectins, is also shown. Arrows indicate the potential for degradation by b-elimination in the ester form. Acetyl groups may be present at O-2 and/or O-3. (May 2000.)

THE CHEMICAL STRUCTURE OF PECTINS

3

In the 1980s Albersheim's group described the presence of highly substituted rhamnogalacturonan regions (consisting of alternating rhamnose and galacturonic acid moieties) carrying side chains of mainly arabinose and galactose residues (McNeill et al., 1980, 1982). This rhamnogalacturonan I (RG-I) was released after digestion of suspension-cultured sycamore cells with a crude mixture of pectin-degrading enzymes. A general representation of native pectins is shown in Figure 1.2; this has developed from the work of De Vries and colleagues (De Vries et al., 1982; De Vries, 1988), who used pure and well-characterised enzymes to study the structure of apple pectin extracted under mild conditions. After enzymatic degradation of the galacturonan backbone of these pectins, the polymeric fragments obtained were analysed and were found to vary in the ratio of uronic acid to neutral sugar. These nondegraded parts of homogalacturonan segments linked to one or more rhamnogalacturonan elements were further fractionated by sizeexclusion chromatography, and it was concluded that the pectin was organised more regularly than expected. The neutral sugars in pectins are concentrated in blocks of highly substituted rhamnogalacturonan ('hairy') regions in which only a relatively small part of the uronic acids is present. These hairy regions are separated by ('smooth') regions consisting of only D-galactosyluronic residues. The proportion of 'smooth' to 'hairy' regions can vary greatly depending on the type of tissue or its developmental state. Using rhamnogalacturonan specific hydrolases, it has been shown that highly ramified RG-blocks exist with long neutral sugar side chains in addition to segments of ~ 30 residues of alternating rhamnose and galacturonic acid sequences in which some rhamnosyl residues carry single unit galactose substitution at O-4 (Schols et al, 1990b; Schols and Voragen, 1994, 1996). Some rhamnogalacturonan preparations (e.g. from sycamore cells) were even fully resistant to degradation by RG hydrolases, demonstrating the absence of less highly substituted RG segments. The availability of rhamnogalacturonan hydrolase and lyase also resulted in the recognition of other structural elements of pectin such as xylogalacturonan (Schols et al., 1995). Table 1.1 summarises the various structural elements described so far and these elements are discussed below. The relative amounts and the precise chemical fine structures of the various elements within a pectin molecule are likely to depend on the origin and developmental stage of the plant material and the function of the cell wall involved (Schols and Voragen, 1996). No plant materials or even tissues appear to be the same in terms of pectin fine structure, Some data are provided in Table 1.2. Indeed, it is also shown that, for certain tissues, some structural elements are not present at all. For example, whereas homogalacturonan elements are absent from the cell walls of soybean, xylogalacturonan is abundant (Huisman et al., 200 1b). Aspects of the cell and developmental biology of pectins are discussed in Chapters 5 and 6.

4

PECTINS AND THEIR MANIPULATION

Figure 1.2 Schematic structure of pectin, including the smooth region (homogalacturonan) and the hairy region; various structural elements of pectin are also shown. Occurrence, amount and chemical fine structure of the individual segments may vary significantly depending on the source of the pectin and its developmental stage (Schols and Voragen, 1996; Voragen et al, 2001). Gal, galactose; GalA, galacturonic acid; Rha, rhamnose; Ara, arbainose Fuc, fucose; Xyl, xylose; DHA, 3-deoxy-D-lyxo2-heptulosaric acid; KDO, 2-keto-3-deoxy-D-mannooctulosonic acid; Api, apiose; AceA. aceric acid; GlcA, glucuronic acid, Ac. acetyl group; Me, methyl ester; 4-O-Me, 4-O-methyl ether.

THE CHEMICAL STRUCTURE OF PECTINS

5

Table 1.1 Structural elements present in pectic substances. Possible variations within an individual element are given Structural element

Diversity based on

Homogalacturonan

• Length of the homogalacturonan segment between individual rhamnose residues • Degree and distribution of methyl-esterification • Degree and distribution of acetyl esterification

Rhamnogalacturonan/RG-I

• Nature of neutral and acidic sugars present in side chains • Length, sugar, and linkage composition and degree of branching of side chains • Distribution of side chains over the alternating rhamnose(1 —>4)-galacturonic acid backbone • Rhamnose:galacturonic acid ratio? • Degree and distribution of acetyl esterification, methyl esters present

RG-II

• • • • •

Very conserved structure Proportion of rare sugars such as 0-Me-xylose, KDO, DHA. Distribution of neutral sugars in the side chains Number, type and distribution of uronic acids in the (side) chains Attachment and distribution of RG-II chains over the pectic molecule

Xylogalacturonan

• • • •

Degree of xylose substitution, length of short xylose side chains Other sugars (e.g. fucose) present in side chains Degree of methyl-esterification (and acetylation?) Distribution of substituents over the backbone

Arabinan

• Size, degree and type of branching, distribution of branches over backbone • Polymer attached to (pectin, arabinogalactan)

Arabinogalactan I

• • • • •

Size, ratio of arabinose to galactose, other sugars in side chains? Linkages present, internal arabinofuranose residues in backbone? Terminal arabinopyranose in side chains? Distribution of substituents over the backbone Attachment to and distribution over pectin molecule

Arabinogalactan II

• • • • •

Size, ratio of arabinose to galactose, other sugars in side chains? Linkages present Distribution of substituents over the backone Protein content? Attachment to other pection (or protein) and distribution over molecule

Apiogalacturonan

• • • •

Degree of apiose substitution Length of apiose chains Methyl-esterification? Distribution of substituents over the backbone

Adapted from Schols and Voragen (1996). Relevant references are given in the description of the various structural elements.

6

PECTINS AND THEIR MANIPULATION

Table 1.2 Occurrence and proportion of the various structural elements of pectin in apple, sugar beet and soy

Total polysaccharide (w/w, % dry matter) Pectic substances (% of total) Structural element (% of pectic substances) Homogalacturonan Xylogalacturonan Rhamnogalacturonan II Rhamnogalacturonan backbone Arabinan I I Arabinogalactan I 1 Arabinogalactan II

Soybean meal

Sugar beet pulp

Apple

16 59

67 40

20 42

0 21 4 15

29 < 1 4 8 46 12

36 4 10 4 27 20 0

60

Based on Voragen et al. (2001).

7.2.1

Structural elements

1.2.1.1 Homogalacturonan The major structural differences between various commercial pectins (in essence homogalacturonans) and the differences in functionality of the homogalacturonan segments in plant tissues are explained by the level of methyl esters present at C-6 of the galacturonic acid residues and the distribution of these esters over the galacturonan backbone (Figure 1.3). Usually, the degree of methylesterification (DM) is expressed as moles of methanol present per 100 moles of galacturonic acid. When the DM is 50 or higher, pectins are called high-methoxy 1 (HM) pectins, whereas the term low-methoxyl (LM) pectins indicates a DM < 50. This distribution is very complex because the methyl ester distribution may vary at an intramolecular level (within one molecule) and at an intermolecular level (over various pectin molecules within a mixture) (De Vries et al.. 1983a). As a consequence, the distribution of the methyl esters over the galacturonan backbone may differ significantly, resulting in the different physical behaviours of different pectin molecules. Within a single molecule, the distribution of ester groups may vary from a rather random distribution in which all esters are 'spread out' over the polymer to a more blockwise distribution in which long stretches of nonesterified galacturonic acid residues are interspersed with segments that are almost completely methyl-esterified (Voragen et al.. 1995; Daas et al., 2000a,b). Since it is believed that homogalacturonans are synthesised completely methyl-esterified, the distributions found in specific pectins will depend on the action of endogenous enzymes such as pectin methylesterases (which may cause a blockwise distribution) or by the conditions under which the raw material is processed or the pectin is extracted (Voragen et al.. 1995). It has been found that the distribution of methyl esters over the backbone varies much more than can be covered by terms such as regular, random or blockwise (Figure 1 A).

Figure 1.4 Schematic representation of three DM 50 pectins with different methyl ester distributions and having different physical properties. Methyl-esterified and nonesterified galacturonic acid are represented by black and open circles respectively, (Daas et al., 2000b.)

8

PECTINS AND THEIR MANIPULATION

The distribution of esterified galacturonic acid residues in fractions obtained from carefully extracted lemon albedo, and from commercially extracted lemon and apple pectin preparations appears to be far from random (De Vries, 1988; Kravtchenko et al, 1993; Mort et al, 1993; Daas et al, 2000a,b). In addition to methyl-esterification, depending on the origin of the pectin, galacturonic acid moieties may be substituted with acetyl groups at O-2 or O-3 (e.g. sugar beet pectin, potato pectin) (Voragen et al, 1995), although it has recently been found that pectins from many more sources may be acetylated to some level (Schols, unpublished results). 1.2.1.2 Rhamnogalacturonan Rhamnogalacturonan is a general name for the rhamnose-rich and galacturonic acid-rich regions of pectin molecules, which consist mainly of repeating units of alternating a-(l 2)-linked rhamnosyl and a-(l—>4)-linked galacturonosyluronic acid residues (Figure 1.5). Such regions of alternating rhamnose and galacturonic acid residues isolated from suspension-cultured sycamore cells were found to be as long as 100-300 repeats and were named rhamnogalacuturonan I (RG-I) (McNeill et al, 1980, 1984; Albersheim et al, 1996). It is thought that the size of the RG-I backbone depends on the plant tissue and on environmental conditions. Using selective hydrofluoric acid (HF) hydrolysis conditions at low temperatures, Komalavitas and Mort (1989) demonstrated that RG-I from carrot, cotton, tobacco and tomato is acetylated at mainly O-3 of the galacturonic acid moieties. This acetylation of rhamnogalacturonans from various fruit and vegetable tissue is confirmed by others (Voragen et al, 1995; Schols and Voragen, 1996). NMR studies revealed that acetyl substitution in apple pectin rhamnogalacturonans occurs mainly as double substitution at O-2 and O-3 of the galacturonic acid residues (Schipper and Schols, unpublished results). However, Kouwijzer et al (1996) calculated that both the O-2 and O-3 positions are energetically favourable, with the most important contribution coming from an acetyl group at O-2. So far, no evidence has been published indicating that methyl esters are present on galacturonic acid residues within the rhamnose-galacturonic acid repeats of RG-I. Their presence would have been revealed by the HF hydrolysis studies as mentioned above (Komalavilas and Mort, 1989). Rhamnogalacturonans obtained after extensive degradation by homogalacturonan-degrading enzymes often consist of unequal amounts of rhamnose and galacturonic acid (ratio of rhamnose to galacturonic acid ranging from 0.05 to 1) (Schols and Voragen, 1996). However, in spite of considerable effort, it has not been demonstrated that more than one galacturonic acid residue could intersperse two rhamnose residues in the RG-I backbone and it is suggested that the 'excess' of galacturonic acid residues are remnants of the nondegradable homogalacturonan segment (Zhan et al, 1998). Depending on the origin of the cell walls examined (type of cell wall, tissue, etc.), 20-80% of the rhamnose residues are branched with side chains attached

Figure 1.5 The alternating structure of rhamnogalacturonan segments of pectin. The number, length and structure of the side chains attached to O-2 and/or O-3 of the rhamnose residue may vary significantly on the origin of the pectin. The acetyl groups may be substituted at O-2, O-3 or both O-2 and O-3 of the galacturonic acid residues.

10

PECTINS AND THEIR MANIPULATION

to O-3 and/or O-4. The length of these side chains can vary from one single galactose residue up to chains of 50 residues or more composed of arabinose (e.g. sugar beet), galactose (e.g. flax, garlic, onion) or both (e.g. soy, potato) (references in Voragen et al., 1995). For pectins from certain sources, it has been shown that ester-linked ferulic acid is present in the neutral sugar side chains (e.g. in sugar beet and suspension-cultured spinach cell walls) (Fry, 1982; Rombouts and Thibault, 1986). Taking into account all possible variations, it can be stated that rhamnogalacturonans are a family of complex structures with 'only' the rhamnogalacturonan backbone in common. This complex nature of rhamnogalacturonans is further underlined by the fact that the various side chains and single sugar residue substitutions are thought not to be randomly distributed over the rhamnogalacturonan backbone (Schols and Voragen, 1994). 1.2.1.3 Xylogalacturonans Using rhamnogalacturonan-degrading enzymes on apple pectic material, Schols et al. (1995) isolated a polymeric xylogalacturonan region consisting of a linear homogalacturonan with single-unit substituents of xylose linked to the O-3 of part of the galacturonic acid moieties (Figure 1.6). The amount of b-xylose substitution of xylogalacturonans may vary significantly, depending on their origin (e.g. apple, pea hulls, carrot, watermelon, soybeans, cocoa) (Weightman et al., 1994; Schols et al., 1995; Kikuchi et al., 1996; Yu and Mort, 1996; Renard et al., 1997; Redgewell and Hansen 2000; Huisman et al., 2001b). Also, the amount and distribution of methyl esters present within the xylogalacturonan segment may vary significantly, since for apple pectin various populations of xylogalacturonans with a rather distinctive methyl ester level have been reported (Schols et al.. 1995). Furthermore, the presence of (small amounts of) fucose in xylogalacturonans has been described, while it is also suggested in literature that side chains of more than one xylose (additional xylose and/or fucose residues) may exist in pea xylogalacturonan (Renard et al., 1997). 1.2.1.4 Apiogalacturonan Apiogalacturonans are reported to be present in duckweed (Hart and Kindel, 1970). They may have both mono- and di-apiosyl side chains attached to the galacturonan backbone, although little is known about their distribution over the backbone. 1.2.1.5 Rhamnogalacturonan II or highly branched galacturonan (HBG) Rhamnogalacturonan II (RG-II or HBG) was first identified by Albersheim's group (Spellman et al., 1983) after isolation from suspension cultured sycamore cells and consists of a backbone of about nine a-(l->4)-linked galacturonosyl residues carrying four side chains containing a number of rare sugars (e.g. apiose, aceric acid, KDO and DHA) (Figure 1.2). Subsequently. RG-II has

Figure 1.6 Schematic structure of a xylogalacturonan. The position of methyl esters is chosen arbitrarily.

12

PECTINS AND THEIR MANIPULATION

been found in many other plant tissues and seems to have a rather conserved structure. This structural element might be involved in the crosslinking of two pectin molecules within the cell wall through a borate diester (Pellerin el al., 1996; Vidal et al., 2000). 1.2.1.6 Arabinans Arabinans are branched homoglycans composed mainly of a backbone of a-L(1—>5)-linked arabinofuranosyl residues and in native form are substituted with a-arabinofuranosyl residues at the O-2 and/or O-3 position (Figure 1.7). Such a substitution can be a single arabinosyl residue, but also longer (branched) chains can be present, mostly one to three arabinosyl residues (Figure 1.7), resulting in complex structures (see the reviews of Whitaker (1984), Beldman et al. (1997) and Voragen el al. (1995). Arabinan side chains may also occur as substituents of arabinogalactans and will be discussed later. Arabinans have been described to be present in cell walls from apples, sugar beet, rapeseed, carrot, cowpea, azukibean and soybean, rape, mustard, lemon, grape, cabbage, onion, potato and others. Arabinans can have molecular weights up to l0 kDa and are commonly covalently linked to galacturonic acid-rich polymers. Although arabinans have been isolated in a pure form, the extraction conditions used may have resulted in chemical (b-elimination, chain peeling) or enzymatic degradation of any attached pectin structure (Voragen et al., 1995; Beldman et al., 1997 and references therein). 1.2.1.7 Galactans Galactans and arabinogalactans in which the galactan backbone is substituted with various amounts of arabinose or galactose have been found in a broad range of higher plants (Clarke et al., 1979; Fincher et al. ,1983). Aspinall (1973) classified the arabinogalactans into type I and type II arabinogalactans, where both types may be present as side chains of pectin or as a single polymer. Type II arabinogalactans may also be present as side chains of a protein segment and as such are referred to as arabinogalactan proteins (AGPs). 1.2.1.8 A rabinogalactan type I Arabinogalactans of type I are quite common in various types of plant tissues and consist of a (1—>4)-linked linear chain of b-D-galactopyranosyl residues. Pure galactans have been isolated from lupin, potato and tabacco, for example, although usually type I galactans are substituted with short chains of a-(l->5)arabinofuranosyl residues attached through the O-3 positions). O-6 substitution of the galactan backbone with b-galactose is also found. Figure 1.8 shows the schematic structure of type I arabinogalactans. Depending on the means of extraction, remnants of the pectic backbone may still be present. However, care should be taken when drawing conclusions in cases where uronides are

Figure 1.7 Schematic structure of a highly branched a-L-(l 5)-arabinan.

Figure 1.8 Schematic structure of a type I fi-( I—»4)-linked arahinogalactan.

THE CHEMICAL STRUCTURE OF PECTINS

15

found within a given arabinogalactan preparation: galactan type I having singleunit galacturonic acid substitution through O-6 has been reported in tamarack wood (Jiang and Timell, 1972). Recently, Huisman et al. (200la) used specific enzymes to degrade soybean galactan type I and used chromatography and mass spectrometry to demonstrate the presence of rather unusual structures containing sequences of (l-»4)-linked galactose residues bearing an arabmopyranose residue as the nonreducing terminal residue, and a mixture of linear oligosaccharides constructed of (l->4)-linked galactose residues interspersed with an internal (l-»5)-linked arabinofuranose residue. 1.2.1.9 Arabinogalactan type 11 Arabinogalactans of type II are highly branched polysaccharides with ramified chains of |3-D-galactopyranose residues joined by 1 ->3 and 1 —>6 linkages. They are more common in plants than type I and have been reported in leaves, stems, roots, floral parts, seeds and media of suspension cultured cells. The hypothetical structure of a type II arabinogalactan is shown in Figure 1.9. In general, the interior backbone of a type II galactan consists mainly of (3-( 1 ->3)-linked galactose moieties; (3-(l ->6)-linked galactopyranosyl residues occur mainly in the exterior chains, which may be terminated with an L-arabinopyranosyl residue. The (1 ->3)-galactan may be branched through O-6 with arabinofuranose residues or (to a lesser extent) with arabinopyranose units, while also longer a-( 1 -> 3)-linked arabinose chains may be present (Figure 1.9). In general, galactose is more abundantly present than arabinose, although the ratio arabinose to galactose may differ significantly. Arabinogalactans of type II may be present within the ramified regions of pectins but are also often reported to be linked to AGPs, of which gum arabic from Acacia Senegal is a well known example. It should be emphasised that many variations with respect to the model shown in Figure 1.9 have been reported, including substitution with glucuronic acid (Ponder and Richards, 1997; Majewska-Sawka and Northnagel, 2000). Although recently it has been suggested that a covalent linkage may exist between pectin and AGP (Oosterveld et al., 2002), no unambiguous evidence has been presented. Perez and co-workers have used molecular modelling methods to derive conformational structures of the various pectic subunits (Perez et al, 2000). Figure 1.10 shows the three-dimensional representation of three different neutral side chains of pectins. The arabinan side chains are attached at an angle of about 50° to the rhamnogalacturonan backbone and form a 'herring bone' type of structure. Arabinogalactans type I are orthogonal to the rhamnogalacturonan backbone, resulting in a 'persil-mill' structure, while the galactan chains possesses a helical structure with an almost perfect fivefold symmetry. The lateral (1 -^3)-linked |3-galactan type II chain has a fairly open helical structure with low symmetry. Although little is known about the precise consequences of having different pectins structures present in different (parts of) plant tissues or

Image Not Available

18

PECTINS AND THEIR MANIPULATION

cell walls, these findings provide insight into the possible 'packing' or density of the side chains of the rhamnogalacturonan backbone. The arabinan substitution leaves plenty of unoccupied space for additional short O-3- and O-2substituted branches to the arabinan backbone. The arabinan side chains of the arabinogalactan I tend to form a semicircle around the backbone and the attachment of the galactan to the rhamnogalacturonan leaves ample unoccupied space to allow for further substitution with arabinofuranoses. The modelling of arabinogalactan II predicts that there will be no steric hindrance even when infinitely long (1^3)-linked ^-linked galactan side chains are substituted with infinitely long (1—>6)-linked galactan side chains, which in turn are substituted by (l-^3)-linked a-arabinofuranoses (Perez et al, 2000).

1.3 Chemical stability of pectins The behaviour of pectins as food thickeners and gelling agents, and obviously also their behaviour within the plant cell wall, is determined to an important extent by the molecular weight of the pectin. Therefore, it is necessary to recognise that pectin molecules are not stable under all conditions. Pectin solutions are most stable at pH 3—4, even at higher temperatures. At lower pH, even at low temperature, esters are removed and the neutral sugar side chains are hydrolysed (references in Voragen et al., 1995). Linkages between galacturonic acid residues are more stable than linkages involving neutral sugars. Arabinosylfuranose linkages are particular sensitive to low pH. Ester linkages present in the pectin can be cleaved rather easily at alkaline conditions and low temperatures (e.g. 0.05 M NaOH; 0-4°C; 2-6 h) without influencing the molecular weight significantly. Release of methyl esters at low temperatures already starts at pH 8 and will accelerate rapidly with increasing pH. No distinct difference has been reported for the stability of methyl esters and acetyl esters. It should be mentioned here that feruloyl ester linkages (as might be present in the side chains of pectins) require much more alkaline conditions for saponification (0.5 M NaOH; 16 h at room temperature). In competition with the saponification reaction, a p-eliminative depolymerisation reaction may occur, even at a pH slightly higher than 7 at room temperature. At elevated temperatures, this degradation takes even place under slightly acidic conditions (pH 5), resulting in a dramatic shift in the molecular weight. This ^-elimination takes place only at a glycosidic linkage on the nonreducing side of a methyl-esterified galacturonic acid residue. This depolymerization reaction can easily be monitored spectrophotometrically at 235 nm, owing to the formation of an unsaturated bond at the nonreducing end of the degradation products. Kravtchenko et al. (1992) reported the splitting of 50% of the glycosidic bonds of a high-methoxyl pectin backbone at pH 5 and 115:C in the ^-elimination promoting sodium citrate buffer. As will be mentioned

THE CHEMICAL STRUCTURE OF PECTINS

19

below, ^-elimination is an important factor in the application of pectins in food manufacture and causes loss in firmness of plant tissue during the processing of fruit and vegetables (Van Buren, 1991).

1.4 Pectins as food ingredients As mentioned above, pectins are extracted from citrus peel and apple pomace on an industrial scale to be used as a gelling and thickening agent for food applications. The extraction conditions are in the range of pH 1.5-3,60-100°C, 0.5-6h (Thibault and Ralet, 2001). Pectin is recovered from the extract by alcoholic precipitation followed by a washing procedure to remove contaminants such as acid, sugars, polyphenols and other alcohol-soluble material. The degree of methyl-esterification (DM) depends on the starting material as well as on the extraction conditions used and can be decreased to a desired DM by saponification under conditions of low pH and rather mild temperatures (Voragen et al., 1995; Thibault and Ralet, 2001). This treatment may yield highly methoxylated (HM) pectin (DM 55-75%) or low-methoxylated (LM) pectin (DM 20-45%). HM pectins will gel only in the presence of sugars or other co-solutes at a pH < 3.2, at which the free carboxyl groups are not ionised. LM pectins will gel mainly through their interaction with calcium (or other bivalent) ions. Although less important for HM pectins, the calcium sensitivity of pectins is governed by the amount and distribution of unesterified galacturonic acids over the pectic backbone. Commercial pectins can also be amidated through treatment of HM pectin with ammonia, resulting in the replacement of a OCHi group by an amide group. This replacement is not complete and also results in the formation of carboxyl groups. Typical values for DM and degree of amidation (DAm) for LM amide pectin are 30 and 20, respectively. The calcium sensitivity of such pectins is much lower than that of normal LM pectins and the gelling behaviour can be controlled more easily (May, 2000). Within all the main groups of pectins mentioned (HM, LM and LMA pectins), major variations may exist in the functionality of pectins having the same DM and galacturonic acid content, and even similarity of other characteristics as neutral sugar content and molecular weight. This phenomenon, which complicates the industrial application of pectins, has been studied recently by several groups making use of new spectrometric and spectroscopic techniques (Daas et al., 1998, 1999, 2000b; Korner et al, 1998, 1999; and references in those papers). The number of galacturonic acids present in nonesterified blocks, the size of these blocks and even the distribution of these blocks over the pectin backbone may be quite different from one pectin to another. These differences have been quantified for a number of industrial pectins, and the differences within the pectin preparations were visualised by making a so-called similarity tree (Daas et al., 2001). It can be seen from Figure 1.11 that using such a similarity tree

THE CHEMICAL STRUCTURE OF PECTINS

21

pectins can be divided into three groups, but in addition differences between pectins within each group are visualized. These variations are assumed to be a consequence of both the 'characteristics' of the starting material used and the conditions of the extraction process. Processing of fruits and vegetables often includes a heating step and this may lead to a chemical degradation of pectins. Obviously, the effect of heating depends strongly on the type of tissue, the cell wall composition and its architecture. For the p-eliminative depolymerisation, the pH is of major importance since this process is rather pH dependent. In particular, the softening of vegetable tissue during processing (pH around 6-6.5) can be attributed to ^-elimination. This depolymerisation may result in the loss of appropriate textural properties and is therefore an undesired quality defect, although cooking of vegetables also results in a less raw appearance, making the product palatable and tasty. The traditional way of inactivating endogenous enzymes often includes a blanching step (5-15 min at 100°C). Recently other methods for dealing with pectin-degrading enzymes have been presented (references in Voragen et al, 1995; Siliha et al, 1996). For tissues containing endogenous pectin methylesterase but (almost) no polygalacturonase, an activation of the pectin methylesterase at 60°C for about 30 min (long-time, low-temperature blanching) before the final inactivation of the enzyme at higher temperatures may result in a significant increase of the tissue firmness (Siliha et al., 1996). This firming is caused by the de-esterification of pectin followed by calcium-pectate gel formation.

1.5

Pectins as 'bioactive' compounds

Pectins are considered to be beneficial to human health in general, partly owing to their function as dietary fibre (either soluble and insoluble). Pectins (especially the neutral sugar side chains) are reported to have a high water-holding capacity as compared to cereal fibres (Hwang and Kokini, 1992; Hwang et al., 1993; references in Voragen et al., 1995). No precise structural requirements have been reported. Negatively charged pectins (i.e. low-esterified pectins) may bind bivalent metal ions (e.g. Ca2+ and heavy metals), reducing the uptake by the human body (Endress, 1991, and references therein). Cholesterol-lowering effects have also been reported for isolated pectins (Truswell and Beynen 1992). Although it is suggested that one of the mechanisms involved is the binding of bile acids through Ca2+ bridges (Hoagland, 1989), it is concluded by Endress that the degree of methyl-esterification had no effect on the cholesterol-lowering effect of pectins (Endress, 1991, and references therein). Along with other carbohydrate components, pectins are fermented in the colon, with formation of short-chain fatty acids. It has been shown that nonmethyl-esterified pectins were more rapidly fermented than methyl-esterified

22

PECTINS AND THEIR MANIPULATION

pectins (Dongowski and Lorenz 1998). Van Laere et al. (2000) studied the in vitro fermentation of various pectic substances (arabinogalactan type I, arabinan, rhamnogalacturonan, homogalacturonan) by selected bacterial strains isolated from human and porcine faeces. The groups of Bacteroides and Clostridium were shown to ferment most of the substrates. Homogalacturonans and rhamnogalacturonans were resistant to fermentation by Bifidobacteria and Lactobacillus spp., while arabinan and arabinogalactan type I were well fermented. It was concluded that the chemical structure of the various structural elements determines the fermentability and utilisation by selected bacterial strains, but no general trends can be observed (Van Laere et«/., 2000) Pectins have also been shown to possess a variety of pharmacological activities such as immunostimulating activity, anti-metastatic activity, anti-ulcer activity and anti-nephrotic activity (Yamada, 1996, 2000; Yu et al., 2001) and much attention has been paid to elucidating the precise structures responsible. Many of the described bioactivities have been found to be correlated to the ramified rhamnogalacturonan regions of pectin, while the complex RG-H element has also been shown to be responsible for some bioactivities (Yamada. 1996).

1.6 Methodology in pectin research Various extraction and fractionating procedures have been developed for the purpose of following changes to pectins during growth, ripening, storage or processing of plant materials (Selvendran and Ryden, 1990; Voragen et al.. 1995, 2001). These procedures usually start with a clean-up step of the source material to inactivate endogenous enzymes and to remove interfering compounds such as sugars, amino acids, organic acids, starch, proteins, nucleic acids and polyphenols to yield cell wall material. This cell wall material is then sequentially extracted under conditions that correspond to the release of specific groups of pectins (Selvendran and Ryden, 1990; Voragen et al.. 1995. 2001; Figure 1.12). Such an extraction procedure can reveal changes within the cell wall architecture (solubility and extractibility of polysaccharides) and changes in specific populations of polysaccharides as a result of ripening or processing that can not be seen from a simple sugar composition analysis of the whole isolated cell wall material. This is illustrated in the analysis of olives for oil production and of table olives, where the effect of ripening stage was found to be completely different for the different classes of olives (Vierhuis et al.. 2000; Mafra et al.. 2001). In general, extracts obtained are fractionated to homogeneity using sizeexclusion and/or anion-exchange chromatography. Some useful remarks when working with pectins in solution, for example, can be found in Mort et al. (1991).

THE CHEMICAL STRUCTURE OF PECTINS

23

Purified cell wall material (CWM) Extraction with cold/hot water or with buffer solutions

—*

Water-soluble pectins

4 Residue Extraction with cold/hot solutions —> of chelating agents: sodium hexametaphosphate ammonium oxalate, EDTA, CDTA e.g. 0.05 M buffer, pH 4.8-5, 4 h, 20-25°C

Chelator-soluble pectins (calcium-bound pectins)

4 Residue Extractions with cold/ambient 0.05 M sodium carbonate (+ NaBH4)

—^

Carbonate-soluble pectins (pectins bound by ester linkages and hydrogen bonding)

—>

Pectins bound by oxidative coupling

—^

Acid-soluble pectins (released by splitting of acid-labile glycosidic linkages)

—>

Alkali-soluble pectins (pectins bound by ester linkages and hydrogen bonding)

4 Residue Extraction with sodium chlorite-acetic acid mixture

Alternative route CWM Extraction with hot dilute acids (pH 2.5, 70°C, 30 min)

4 Residue Extraction with cold dilute sodium hydroxide 0.05 M NaOH, 0°C, 16 h

Figure 1.12 Scheme for the extraction of different pectin populations from plant cell wall material (Voragenefa/., 1995).

For structure elucidation of such homogeneous fractions, sugar and glycosidic linkage composition and anomeric configuration of sugar residues are determined. Strategies for establishing the fine structure further include fragmentation with pure, well-defined and specific enzymes (or with less specific chemical reactions) and fractionation to homogeneity of the fragments in the digests (Lau etal., 1988; Franssen etal., 2000; Huisman etal., 200Ib). These fragments often fit within the analytical ranges of advanced NMR and mass spectroscopic techniques and these techniques allow the establishment of the absolute structure,

24

PECTINS AND THEIR MANIPULATION

including the presence and distribution of substituents. With the information obtained in such approaches, tentative structures of the structural elements of pectic substances as described in Figure 1.2 and Table 1.1 have been constructed. Some of the strategies and procedures used in elucidation of oligosaccharide structures will be illustrated through the separation and identification of oligogalacturonic acids. As discussed above, a more detailed characterisation of pectins having a completely different functionality has been given by Daas et al (1998, 1999, 2000a). Rather than employing the frequently used highperformance anion-exchange chromatography (HPAEC) methods at pH 12 that result in an excellent separation of oligogalacturonic acids, Daas et al. used a HPAEC method at pH 5 (as previously described by Hotchkiss and Hicks, 1990; Hotchkiss et al., 1996) to obtain additional information about the methyl ester groups present (Daas et al., 1998). They were able to separate and to identify the various degradation products present in a polygalacturonase digest (PG from Kluyveromycesfragiles} with respect to degree of polymerisation and number of methyl esters present. Matrix-Assisted Laser Desorption/Ionisation Time of Right Mass Spectrometry (MALDI-TOF MS) was a very valuable tool for determining the number of methyl esters present on each oligomer (Daas et al, 1998). In subsequent publications, Daas et al. (2000) used various new parameters (such as degree of blockiness, ratio between nonesterified versus esterified oligomers released) to characterise the various pectins. At the same time, Korner et al. (1998, 1999) used MALDI-TOF MS and nanoelectrospray ionization ion trap mass spectrometry to locate methyl-esterified galacturonic acid residues in oligomers up to a degree of polymerization of 10 present in a complex mixture obtained after pectin digestion by polygalacturonase and pectin lyase. The data sets obtained were used to draw conclusions about the structure of the parent pectins as well as on the mode of action of the enzymes (Korner et al., 1998, 1999; Limberg et al., 2000a,b). Also post-source decay fragmentation on a MALDI-TOF mass spectrometer was useful in elucidating the precise structure of esterified uronides (Van Alebeek et al., 2000b; Kester et al., 2000). A similar approach of using highly specific enzymes in combination with chromatographic and spectrometric techniques to elucidate the structure of complex arabinogalactans of type I has been described (Huisman et al., 2001 a). Although enzymes are often recognised to be much more selective for polysaccharide degradation, special applications using acid hydrolysis for the characterisation of complex polysaccharides have been published by the group of Mort. To study the distribution of methyl esters over a pectin backbone, first the esterified galacturonic acids are converted to galactose by reduction with sodium borohydride, then the glycosidic linkages of the resulting galactose residues are cleaved selectively by liquid HF solvolysis. Separation and quantification of the resulting galacturonic acid-containing oligomers reveals the proportion of each stretch of contiguous nonesterified galacturonic acid residues in the original pectin (Mort et al, 1993). The same group also described the

THE CHEMICAL STRUCTURE OF PECTINS

25

selective hydrolysis of various different neutral sugar linkages with anhydrous liquid HF at low temperatures (0 to -60°C) (Mort, 1983). Other useful methods for the characterisation of polysaccharides and enzyme digests derived from them are be found in Schols and Voragen (2002).

References Albersheim, P., Darvill, A.G., O'Neill, M.A., Schols, H.A. and Voragen, A.G.J. (1996) An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 47-55. Aspinall, A.O. (1973) Carbohydrate polymers of plant cell walls, in Biogenesis of Plant Cell Wall Polysaccharides (ed. F. Loewus), Academic Press, New York, pp. 95-115. Bacic, A., Harris, P. and Stone, B. (1988) Structure and function of plant cell walls, in The Biochemistry of Plants, vol. 14, Carbohydrates, Academic Press, London, pp. 297-369. Beldman, G., Schols, H.A., Pitson, S.M., Searle-van Leeuwen, M.J.F. and Voragen, A.G.J. (1997) Arabinans and arabinan degrading enzymes, in Advances in Macromolecular Research, vol. 1. (ed. R.J. Sturgeon), JAI Press, London, pp. 1-64. Carpita, N.C. and Gibeault, D.M. (1993) Structural models of primary cell walls in flowering plants: consistency of molecular structure with the physical properties of the wall during growth. Plant J., 3, 1-30. Clarke, A.E., Anderson, R.L. and Stone, B.A. (1979) Form and function of arabinogalactans and arabinogalactan proteins. Phytochemistry, 18, 521-540. Daas, P.J.H., Arisz, P.W., Schols, H.A., De Ruiter, G.A. and Voragen, A.G.J. (1998) Analysis of partially methyl-esterified galacturonic acid oligomers by high-performance anion-exchange chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Anal. Biochem., 257, 195-202. Daas, P.J.H., Meyer-Hansen, K., Schols, H.A., De Ruiter, G.A. and Voragen, A.G.J. (1999) Investigation of the non-esterified galacturonic acid distribution in pectin with endopolygalacturonase. Carbohydr. Res., 318, 135-145. Daas, P.J.H., Voragen, A.G.J. and Schols, H.A. (2000a) Determination of the distribution of nonesterified galacturonic acid in pectin with endo-polygalacturonase, in Gums and Stabilisers for the Food Industry 10 (eds PA. Williams and G.O. Phillips), Royal Society of Chemistry, Cambridge, pp. 3-18. Daas, P.J.H., Voragen, A.G.J. and Schols, H.A. (2000b) Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase. Carbohydr. Res., 326, 120-129. Daas, P.J.H., Boxma, B., Hopman, A.M.C.P, Voragen, A.G.J. and Schols, H.A. (2001) Nonesterified galacturonic acid sequence homology of pectins. Biopolymers, 58, 1—8. De Vries, J. (1988) Repeating units in the structure of pectins, in Gums and Stabilisers for the Food Industry 4 (eds G.O. Philips, D.J. Wedlock and PA. Williams), IRL Press, Oxford, pp. 25-29. De Vries, J.A., Rombouts, F.M., Voragen, A.G.J. and Pilnik, W. (1982) Enzymatic degradation of apple pectins. Carbohydr. Polym., 2, 25-33. De Vries, J.A., Rombouts, F.M., Voragen, A.G.J. and Pilnik, W. (1983a) Distribution of methoxyl groups in apple pectic substances. Carbohydr. Polym., 3, 245-258. De Vries, J.A., Rombouts, P.M., Voragen, A.G.J. and Pilnik, W. (1983b) Comparison of the structural features of apple and citrus pectic substances. Carbohydr. Polym., 4, 89-101. Dongovski, G. and Lorenz, A. (1998) Unsaturated oligogalacturonides are generated by in vitro treatment of pectin with human faecal flora. Carbohydr. Res., 314, 237-244.

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Endress, H.-U. (1991) Non-food uses of pectins, in The Chemistry and Technology of Pectin (ed. R.H. Walter), Academic Press, San Diego, pp. 251-268. Fincher, G.B., Stone, B.A. and Clarke, A.E. (1983) Arabinogalactan proteins: structure, biosynthesis and function. Annu. Rev. Plant Physioi, 34, 47-70. Franssen, C.T.M., Haseley, S.R., Huisman, M.M.H., el al. (2000) Studies on the structure of a lithiumtreated soybean pectin: characteristics of the fragments and determination of the carbohydrate substituents of galacturonic acid. Carbohydr. Res., 328, 539-547. Fry, S.C. (1982) Phenolic components of the primary cell wall: feruloylated disaccharides of D-galactose and L-arabinose from spinach polysaccharides. Biochem. J.. 203, 493-504. Hart, D.A. and Kindel, P.K. (1970) A novel reaction involved in the degradation of apiogalacturonans from lemna minor and the isolation of apibiose as a product. Biochemistry, 9, 2190-2196. Hoagland, P.O. (1989) Binding of dietary anions to vegetable fiber. J. Agric. Food Chem.. 37, 1343-1347. Hotchkiss, A.T. and Hicks, K.B. (1990) Analysis of oligogalacturonic acids with 50 or fewer residues by high-performance anion-exchange chromatography and pulsed amperometric detection. Anal, Biochem., 184, 200-206. Hotchkiss, A.T, Bahtimy, K.E1. and Fishman, M. (1996) Analysis of pectin structure by HPAEC-PAD. in Modern Methods of Plant Analysis, vol. 17. Plant Cell Wall Analysis (eds H.F. Linskens and J.F. Jackson), Springer Verlag, Berlin, pp. 129-146. Huisman, M.M.H., Briill, L.P., Thomas-Oates, J.E., Haverkamp, J., Schols, H.A. and Voragen, A.G.J. (200la) The occurrence of internal (l-S)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances. Carbohydr. Res.. 330, 103-114. Huisman, M.M.H., Fransen, C.T.M., Kamerling, J.P., Vliegenthart, J.F.G., Schols. H.A. and Voragen. A.G.J. (2001b) The CDTA-soluble pectic substances from soybean meal are composed of rhamnogalacturonan and xylogalacturonan but not homogalacturonan. Biopolymers. 58. 279-294. Hwang, J. and Kokini, J.L. (1992) Contribution of the side branches to rheological properties of pectins. Carbohydr. Polym., 19, 41-50. Hwang, J., Ryun, Y.R. and Kokin, J.L. (1993) Sideschains of pectins: some thougths on their role in plant cell walls and foods. Food Hydrocolloids. 7, 39-53. Jiang, K.J. and Timell, T.E. (1972) Polysaccharides in compression wood of tamarack (Larix laricina) IV. Constitution of an acidic galactan. Sven. Paperstidn.. 75, 592-594. Kester, H.C.M., Benen, J.A.E., Visser, J., et al. (2000) Tandem mass spectrometric analysis ofAspergillus niger pectin methylesterase: mode of action on fully methyl-esterified oligogalacturonates. Biochem. J., 346, 469^74. Kikuchi. A., Edashige, Y., Ishii, T. and Satoh, S. (1996) A xylogalacturonan whose level is dependent on the size of cell clusters is present in the pectin from cultured carrot cells. Planta-Heidelberg., 200. 369-372. Komalavilas. P. and Mort, A.J. (1989) The acetylation at O-3 of galacturonic acid in the rhamnose-rich portion of pectins. Carbohydr. Res., 189, 261-272. Komer, R., Limberg, G., Mikkelsen, J.D. and Roepstorff, P. (1998) Characterization of enzymatic pectin digests by matrix-assisted laser desorption/ionization mass spectrometry. J. Mass Spectmm.. 33. 836-842. Korner, R., Limberg, G., Christensen, T.M.I.E., Mikkelsen, J.D. and Roepstorff. P. (1999) Sequencing of partially methyl-esterified oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities. Anal. Chem.. 71. 1421-1427. Kouwijzer, M., Schols, H.A. and Perez, S. (1996) Acetylation of rhamnogalacturonan I and homogalacturonan: theoretical calculations, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14. Elsevier Science, Amsterdam, pp. 57-65. Kravtchenko. T.P., Arnould, I., Voragen, A.G.J. and Pilnik, W. (1992) Improvement of the selective depolymerization of pectic substances by chemical p-eliminaton in aqueous solution. Carbohydr. Polym.. 19. 237'-242.

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Kravtchenko, T.P., Penci, M., Voragen, A.G.J. and Pilnik, W. (1993) Enzymatic and chemical degradation of some industrial pectins. Carbohydr. Polym., 20, 195-205. Lau, J.M., McNeil, M., Darvill, A.G. and Albersheim, P. (1988) Treatment of rhamnogalacturonan I with lithium in ethylenediamine. Carbohydr. Res., 168, 245-274. Limberg, G., Korner, R., Buchholt, H.C., Christensen, T.M.I.E., Roepstorff, P. and Mikkelsen, J.D.G. (2000a) Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. Niger. Carbohydr. Res., 327, 293-307. Limberg, G., Korner, R., Buchholt, H.C., Christensen, T.M.I.E., Roepstorff, P. and Mikkelsen, J.D. (2000b) Quantification of the amount of galacturonic acid residues in block sequences in pectin homogalacturonan by enzymatic fingerprinting with exo- and endo-polygalacturonase II from Aspergillus niger. Carbohydr. Res., 327, 321-332. Mafra, I., Lanzab, B., Reisa, A., et al. (2001) Effect of ripening on texture, microstructure and cell wall polysaccharide composition of olive fruit (Olea europaea). Physiol. Plant, 111, 439-447. Majewska-Sawka, A. and Nothnagel, E.A. (2000) The multiple roles of arabinogalactan proteins in plant development. Plant Physiol., 122, 3-9. May, C.D. (2000) Pectins, in Handbook of Hydrocolloids (eds G.O. Phillips and P.A. Williams). Woodhead Publishing, Cambridge, pp. 169-188. McNeil, M., Darvill, A.G. and Albersheim, P. (1980) Structure of plant cell walls. X. Rhamnogalacturonan I, a structurally complex pectic polysaccharide in the walls of suspension-cultured sycamore cells. Plant Physiol., 66, 1128-1134. McNeil, M., Darvill, A.G. and Albersheim, P. (1982) Structure of plant cell walls. XII. Identification of seven differently linked glycosyl residues attached to O-4 of the 2.4-linked L-rhamnosyl residues of rhamnogalacturonan I. Plant Physiol., 70, 1586-1591. McNeil, M., Darvill, A.G., Fry, S.C. and Albersheim, P. (1984) Structure and function of the primary cell walls of plants. Annu. Rev. Biochem., 53, 625—663. Mort, AJ. (1983) An apparatus for safe and convenient handling of anhydrous, liquid hydrogen fluoride at controlled temperatures and reaction times. Application to the generation of oligosaccharides from polysaccharides. Carbohydr. Res., 122, 315-321. Mort, A.J., Moerschbacher, B.M., Pierce, M.L. and Maness, N.O. (1991) Problems encountered during the extraction, purification, and chromatography of pectic fragments, and some solutions to them. Carbohydr. Res., 215, 219-227. Mort, A.J., Qiu, F. and Maness, N.O. (1993) Determination of the pattern of methyl-esterification in pectin: distribution of contiguous nonesterified residues. Carbohydr. Res., 247, 21-35. Oosterveld, A.O., Voragen, A.G.J. and Schols, H.A. (2002) Characterization of hop pectins shows the presence of an arabinogalactanprotein. Carbohydr. Polym., in press. Pellerin, P., Doco, T, Vidal, S., Williams, P., Brillouet, J.M. and O'Neill, M.A. (1996) Structural characterization of red wine rhamnogalacturonan II. Carbohydr. Res., 290, 183-197. Perez, S., Mazeau, K. and Herve du Penhoat, C. (2000) The three-dimensional structures of the pectic polysaccharides. Plant Physiol., 38, 37-55. Ponder, G.R. and Richards, G.N. (1997) Arabinogalactan from Western larch Part III: alkaline degradation revisited, with novel conclusions on molecular structure. Carbohydr. Polym., 34. 251-261. Redgwell, R.J. and Hansen, C.E. (2000) Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans. Planta-Berlin, 210, 823-830. Renard, C.M.G.C., Weightman, R.M. and Thibault, J.F. (1997) The xylose-rich pectins from pea hulls. Int. J. Biol. Macromol., 21, 155-162. Rombouts, F.M. and Thibault, J.F. (1986) Feruloylated pectic substances from sugar-beet pulp. Carbohydr. Res., 154, 177-187. Schols, H.A. and Voragen, A.G.J. (1994) Occurence of pectic hairy regions in various plant cell wall materials and their degradability by rhamnogalacturonase. Carbohydr. Res., 256, 83-95.

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Schols, H.A. and Voragen, A.G.J. (1996) Complex pectins: structure elucidation using enzymes, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 3-19. Schols, H.A. and Voragen, A.G.J. (2002) Pectic polysaccharides, in Food Enzymology (eds J.R. Whitaker and A.G.J. Voragen), in press. Schols, H.A., Posthumus, M. A. and Voragen, A.G.J. (1990a) Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process. Carbohydr. Res., 206, 117-129. Schols, H.A., Geraeds, C.C.J.M., Searle-van Leeuwen, M.F., Kormelink, F.J.M. and Voragen, A.G.J. (1990b) Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr. Res., 206, 105-115. Schols, H.A., Bakx, E.J., Schipper, D. and Voragen, A.G.J. (1995) A xylogalacturonan subunit present in the modified hairy regions of apple pectin. Carbohydr. Res., 279, 265-279. Selvendran, R.R. and Ryden, P. (1990) Isolation and analysis of plant cell walls, in Methods in Plant Biochemistry, vol. 2, Carbohydrates (eds P.M. Dey and J.B. Harborne), Academic Press, London. pp. 549-579. Siliha, H., Jahn, W. and Gierschner, K. (1996) Effect of a new canning process on cell wall pectic substances, calcium retention and texture of canned carrots, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 495-508. Spellman, M.W., McNeil, M., Darvill, A.G. and Albersheim, P. (1983) Characterization of a structurally complex heptasaccharide isolated from the pectic polysaccharide rhamnogalacturonan II. Carbohydr. Res., 122, 131-153. Thibault, J.F. and Ralet, M.C. (2001) Pectins, their origin, structure and function, in Advanced Dietary Fibre Technology (eds B.V. McCleary and L. Prosky), Blackwell Science, London, pp. 369-378. Truswell, A.S. and Beynen, A.C. (1992) Dietary fibre and plasma lipids: potential for prevention and treatment of hyperlipidaemias, in Dietary Fibre—A Component of Food-Nutritional Function in Health and Disease (eds T.F. Scheizer and C.A. Edwards), Springer Verlag, Berlin, p. 21. Van Alebeek, G.J.W.M., Zabotina, O., Beldman, G., Schols, H.A. and Voragen, A.G.J. (2000a) Esterification and glycosydation of oligogalacturonides: examination of the reaction products using MALDI-TOF MS and HPAEC. Carbohydr. Polym., 43, 39-46. Van Alebeek, G.J.W.M., Zabotina, O., Beldman, G., Schols, H.A. and Voragen, A.G.J. (2000b) Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. / Mass Spectrom., 35, 831-840. Van Buren, J.P. (1991) Function of pectin in plant tissue structure and firmness, in The Chemistry and Technology of Pectins (ed. R.H. Walter), Academic Press, San Diego, pp. 1-22. Van Laere, K.M.J., Hartemink, R., Bosveld, M., Schols, H.A. and Voragen, A.G.J. (2000) Fermentation of plant cell wall derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria. J. Agric. Food Chem., 48, 1644-1652. Vidal, S., Doco, T, Williams, P., et al. (2000) Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain. Carbohydr. Res., 326, 277-294. Vierhuis, E., Schols, H.A., Beldman, G. and Voragen, A.G.J. (2000) Isolation and characterisation of cell wall material from olive fruit (Olea europaea cv koroneiki) at different ripening stages. Carbohydr. Polym., 43, 11-21. Visser, J. and Voragen, A.G.J. (eds) (1996) Progress in Biotechnology 14: Pectins and Pectinases, Elsevier. Amsterdam. Voragen, A.G.J., Pilnik, W., Thibault, J.-F, Axelos, M.A.V. and Renard, C.M.C.G. (1995) Pectins, in Food Polysaccharides and Their Applications (ed. A.M. Stephen), Marcel Dekker, New York, pp. 287-339. Voragen, A.G.J., Beldman, G. and Schols, H.A. (2001) Chemistry and enzymology of pectins, in Advanced Dietary Fibre Technology (eds B.V. McCleary and L. Prosky), Blackwell Science. London, pp. 379-398.

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Walter, R.H. (1991) The Chemistry and Technology of Pectins, Academic Press, San Diego. Weightman, R.M., Renard, C.M.G.C. and Thibault, J.F. (1994) Structure and properties of the polysaccharides from pea hulls. Part I: Chemical extraction and fractionation of the polysaccharides. Carbohydr. Polym., 24, 139-148. Whitaker, J.R. (1984) Pectic substances, pectic enzymes and haze formation in fruit juices. Enzyme Microb. TechnoL, 6, 341-348. Yamada, H. (1996) Contribution of pectins on health care, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 173-190. Yamada, H. (2000) Bioactive plant polysaccharides from Japanese and Chinese traditional herbal medicines, in Bioactive Carbohydrate Polymers (ed. B.S. Paulsen), Kluwer Academic, Dordrecht, pp. 15-24. Yu, L. and Mort, A.J. (1996) Partial characterisation of xylogalacturonans from cell walls of ripe watermelon fruits: inhibition of endopolygalacturonase activity by xylosylation, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 79-98. Yu, K.W., Kiyohara, H., Matsumoto, T., Yang, H.C. and Yamada, H. (2001) Characterisation of pectic polysaccharides having intestinal immune system modulating activity from rhizomes of Atractylodes lancea DC. Carbohydr. Polym., 46, 147—156. Zhan, D., Janssen, P. and Mort, A.J. (1998) Scarcity of complete lack of single rhamnose residues interspersed within the homogalacturonan regions of citrus pectin. Carbohydr. Res., 308, 373-380,

2

Interactions between pectins and other polymers Andrew J. Mort

2.1 Introduction The structures of the various regions of pectins were reviewed in Chapter 1. This chapter will address the question of whether pectins are covalently attached to other cell wall polymers and, if they are, the nature of the linkage between the pectin and the other polymer and the regions of the pectin that are involved. Because pectins are often fairly loosely defined, the structural components included under the term 'pectin' will be stated explicitly. A molecule of pectin may be considered to contain almost any combination of the following regions or sections: (1) homogalacturonan (HG), (2) xylogalacturonan (XGA). (3) rhamnogalacturonan I (RG-I), (4) rhamnogalacturonan II (RG-II), (5) (3(1—^4)-galactan, (6) «-(!-> 5 )-arabinan, (7) 3,4- and 3,6-linked arabinogalactans. These different regions or sections never seem to occur by themselves in a distinct molecule unless the pectin from which they were derived has been degraded enzymically or chemically. The unifying feature of pectin is the presence of a-(l—>4)-linked galacturonic acid. Pectins are a major component (50% or more) of primary cell walls of most dicots and many monocots, and a significant component (~10%) of the walls of the graminaceous monocots. Thus, their interactions with other cell wall polymers are of extreme importance in understanding the function of cell walls. 2.2 An overview of cell wall models In the early 1970s, it was proposed that all cell wall polymers were interconnected into an extensive network (Keegstra et al., 1973) reminiscent of, but more complex than, the peptidoglycan of bacterial cell walls. However, recent models (McCann and Roberts, 1991; Carpita and Gibeaut, 1993) of primary cell walls of plants suggest that there is no covalent linkage between the pectins and the other cell wall polymers or do not consider the possibility. Possible covalent linkages between pectins and other cell wall polymers are omitted because there is no consensus about their identity or even their existence. Whether right or wrong, reasonable models can be made without them. A unifying feature of all of the models, both old and new, is a noncovalent, but very strong, interaction between sections of the hemicellulosic xyloglucan (XG) chains and cellulose

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microfibrils. The exact molecular details of the interaction between the XG and cellulose are not known; it is certainly a strong interaction, but it can be disrupted by alkaline conditions (Bauer etal, 1973; Hayashi, 1989; Vincken and Voragen, 1995). McCann et al. (1990,1992) found that the XG chains are long enough that they can span the region between two cellulose chains and interact with both, thus forming intercellulose crosslinks. Carpita and Gibeaut (1993) refer to the XG-cellulose network as the load-bearing network. In the models, pectins are shown as unlinked, wavy lines (McCann and Roberts, 1991) or as components interacting with each other (as a second network) through Ca2+ crossbridges (invoking the well known egg-box model (Powell etal., 1982)) between regions of sparsely esterified homogalacturonans (Carpita and Gibeaut, 1993). Recently it has become clear that pectins also interact with each other by borate ester crosslinks between apiose residues in the RG-II regions (Ridley et al, 2001). Although the hydroxyproline (Hyp)-rich cell wall structural protein extensin (Lamport, 1977) was incorporated in a key position crosslinking to pectin in the 1973 model, it is downplayed or absent in modern models. However, Carpita and Gibeaut do include it in their model as a third network, an independent crosslinking of protein to protein.

2.3

Experimental approaches to the study of covalent crosslinks between polymers

Evidence for or against covalent interactions between pectins and other polymers largely falls into two classes: (1) co-extraction of pectin and the other polymer under conditions that would be expected to solubilize only one of them unless that one were holding the other in the wall by being linked to it; (2) co-chromatography of solubilized pectins along with the other polymer under conditions that ought to separate the two polymers if they were not covalently attached to each other. Both of these lines of evidence are open to other interpretations, such as polymer entanglement in the first case and anomalous chromatography in the second. Ultimate proof of a covalent crosslink between pectin and another polymer will come from experiments in which the entire structure of a molecule containing a fragment of pectin crosslinked to a fragment of another polymer is determined. A second-best experiment would be to show that treatment of a pectin-polymer complex with an enzyme exhibiting monospecific activity or with a chemical method exhibiting monospecific activity cleaves the link between them without affecting the backbone of the pectin or that of the other polymer. Of course this approach necessitates the availability of pure enzymes or the crosslink having a unique chemical susceptibility. A big problem in identifying crosslinks by structural characterization is that they do not need to be very abundant in the walls. To form a three-dimensional

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crosslinked network there need only be a minimum of two crosslinks per polymer molecule. As an illustration, let us assume the polymer and the pectin both have a molecular weight of 100000 (~600 sugar residues) and that the crosslink consists of, say, 10 sugars; then a crosslinking section would make up only ~ 1 % of the mass of the complex. After specific degradation of the pectin and other polymer with enzymatic or chemical methods, the desired fragment should be only about 2-3% of the initial complex. To isolate adequate amounts of the desired crosslinked fragment for characterization, a large amount of starting complex will be needed, or the methods used to identify the fragment will need to be very sensitive. The situation may be worse than this because the crosslinking fragments may be heterogeneous. Very few crosslinks between cell wall polymers have been conclusively identified. To the author's knowledge, there is only one clear-cut example of isolation and characterization of a crosslink between polysaccharides in cell walls. Ishii (1991) succeeded in isolating and characterizing a fragment containing a diferulic acid bridging two trisaccharides digested out of arabinoxylan with the crude enzyme mixture Driselase. To achieve this, Ishii started with 100kg of bamboo shoots from which he isolated 400 g of bamboo cell walls. He reported a final yield, after Driselase digestion and several chromatographic separations, of only 23 |jig crosslinked arabinoxylan fragment per gram of cell wall. That is 9.2 mg total material for characterization from 100kg of plant material. Several factors contributed to Ishii's success: (1) It had been suggested for many years that arabinoxylans could be crosslinked via diferulic acid (Markwalder and Neukom, 1976), and diferulic acid can be isolated from cell walls after saponification. (2) In previous work, Fry (1982b) had found that the crude enzyme preparation Driselase does not hydrolyze phenolic acid ester-sugar linkages to any great extent but does degrade most cell wall polysaccharides, thus solubilizing oligosaccharides esterified to phenolic acids. Ishii and Hiroi (1990) had previously isolated various oligosaccharides from arabinoxylan of bamboo esterified to monomeric phenolic acids using the same approach. They found that there was only 1.5 mg of total phenolic acids per gram of cell wall and that they could recover only 16 fig of coumaroyl arabinoxylan fragment, and 39 fig of feruloylated XG fragment per gram of wall. The molar ratio of /7-coumaric acid:ferulic acid:diferulic acid in the bamboo walls was 1:2.8:0.06. The best example of success with the second approach—cleaving the crosslink without affecting the crosslinked polymers—is the demonstration that two RG-II molecules obtained from a wide range of species are often crosslinked together by borate esters (Ishii et ai, 1999). It has not been possible to characterize the complete structure of the crosslinked RG-II structure because of its large size (MO 000 Daltons); however, the crosslink between the two RG-II segments can be cleaved by a 30-minute treatment with 0.1 M HC1 at room temperature (Kobayashi et al., 1996). These conditions can reasonably be believed to nor cleave any glycosidic bonds. The crosslink can be regenerated by incubation

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of the monomeric segments in pH 3.5 phthalate buffer along with 15 mM boric acid for three days at room temperature (Ishii et al, 1999). 2.4 What types of crosslinks to expect Fry (1986) reviewed all of the types of crosslinks that had been suggested to occur between cell wall polymers. Those applicable to pectin include a glycosidic linkage from the reducing end of the pectin molecule to extensin, a glycosidic linkage from the reducing end of XG to the pectin, ester links from the carboxylic acid groups of some of the galacturonic acid (GalA) residues to other poly saccharides, crosslinks between phenolic acids esterified to side chain residues of the pectin and other polymers, and ionic interactions between acidic groups of pectins and basic groups of proteins. These are types of crosslinks suggested for primary, growing cell walls. Walls that are lignifying during secondary wall formation, during maturation, or in response to infection or wounding probably incorporate phenolic acid esters on their pectins into the lignin (Whitmore, 1978). Recently Fry's group has suggested the possibility of direct amide linkages between pectins and proteins and has synthesized galacturonosyl-lysyl amides as model compounds to help test for them (Perrone et al., 1998). There is evidence consistent with the presence of each of the types of interactions listed. However, none of them has been conclusively identified. 2.5

Historical perspective on crosslinks to pectins

The first model of primary plant cell walls that attempted to provide a relatively complete picture, at the molecular level, of what all of the wall polysaccharides and proteins are like and how they interact with each other was published in 1973 (Keegstra et al., 1973). This was a groundbreaking achievement. However, the model contained more detail than the data could support, and has subsequently been shown to be incorrect in several features. It stimulated a great interest in cell wall structure and spawned many theories of how cell walls grow. Collection of the data to build the model was made possible by the combination of several key advances in carbohydrate analysis. Albersheim's group had adapted hydrolysis with trifluoroacetic acid and formation of alditol acetates for the quantitative analysis of monosaccharide compositions of polysaccharides by gas-liquid chromatography (GLC) (Albersheim et al., 1967). Hakamori (1964) had established the use of sodium hydride and dimethyl sulfoxide, along with methyl iodide, for essentially complete methylation of polysaccharides in a single treatment for sugar linkage analysis. Bjorndal et al (1970) had perfected the use of gas chromatography-mass spectrometry (GC-MS) to identify the pattern of methyl and acetyl groups on the partially methylated alditol acetates used to characterize the methylated polysaccharides.

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In addition, English et al. (1972) had purified an endopolygalacturonase (EPG) from Colletotrichum lindemuthianum, which allowed very specific and extensive digestion of cell walls for solubilization of fragments for characterization. Large enough quantities of relatively uniform primary cell walls were available because of the establishment of a prolific, stable suspension culture of sycamore maple (Lamport, 1964). Of course, most of the structures proposed for the polysaccharides in the walls had been partially characterized before by such groups as those of Aspinall, Tipson, Hirst, and Jones (Stephen, 1983) from bulk plant material and gums, but had never before been put into a cohesive model of a cell wall. In the 1973 model, pectin was proposed to be linked covalently to both the hemicellulose XG and to the cell wall protein extensin.

2.6 Evidence in 1973 for a xyloglucan-pectin linkage and a pectin-protein linkage Purified EPG digestion of sycamore walls solubilized a little less than 50% of the pectin (if we count HG, RG-I, RG-II, arabinan, galactan and arabinogalactans as pectin) along with small amounts of XG. (The authors of the model did not know at that time of the existence of distinct regions in pectins, so they proposed a more uniform structure of pectin in which there were alternating GalA-Rha disaccharides and GalA^io-Rha oligosaccharides in the backbone with neutral sugar side chains branching from the Rha residues.) From their results one can estimate that only about 10% of the XG was solubilized by the EPG, and about 50-70% of the XG released then bound to DEAE-Sephadex and eluted with pectin, indicating a linkage between XG and pectin, since XG by itself is neutral. Arabinan, galactan and arabinogalactan were possible candidates for the crosslink between the XG and pectin in this fraction. However, since mild acid hydrolysis (0.01 N TFA for 1 h at 102°C), which would cleave arabinofuranosyl linkages, did not appear to break the linkage between the XG and pectin, it was proposed that a crosslink with a galactan backbone was involved (Talmadge etal, 1973). After (but not before) the EPG digestion of the walls, 8 M urea solubilized a rather small proportion of the wall containing both XG and pectin. Urea is a chaotropic agent and was proposed to disrupt some of the binding of XG to cellulose, thus releasing some XG linked to pectin. Extraction of EPG-treated walls with 0.5 M NaOH (with 100 mM NaBH4 to prevent the peeling reaction (Green et al., 1977)) solubilized 16% of the original wall weight (much more material than did urea), which contained almost equal proportions of pectin and XG. Digestion of EPG-pretreated walls with a purified endoglucanase also solubilized a mixture of XG fragments and pectin, accounting for ~ 15% of the walls. About two-thirds of this extract bound to DEAE-Sephadex and there was co-elution of sugars characteristic of pectins and XG (Bauer et al.. 1973).

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35

Ninety percent of the material in acidic fractions of the urea extract bound to purified cellulose, indicating that the acidic part of the extract was somehow interacting with cellulose, presumably by being linked to the XG. A smaller proportion (35%) of the acidic part of the base extract also bound to cellulose (Bauer etaL, 1973). Probably for the sake of simplicity, in the model of cell walls resulting from these experiments, all of the XG was proposed to be covalently linked to the pectin, and all of the pectin linked to XG. The linkage was proposed to be a glycosidic linkage between the reducing end of XG and pectin side chains. However, there was not really any way to estimate what fraction of the XG or pectin must be linked to each other. Another part of the cell wall model proposed by Keegstra et al. (1973) was a linkage between the pectin and the cell wall protein extensin. The evidence for this was less direct than that for the XG-pectin link. There was a heavy reliance on the structure of a Hyp-rich glycoprotein in the medium around the cultured cells because it was extremely difficult to obtain adequate amounts of the actual extensin (with polysaccharides attached) from cell walls. It is probably not worth reviewing the evidence involving the extracellular protein here because Pope (1977) showed that it was distinct from extensin, in fact being an arabinogalactan protein. There was some direct evidence of a covalent linkage between pectin and extensin. Pronase solubilized 2% and 4% of the wall, respectively, from EPG-treated walls and EPG/cellulase-treated walls. Almost half of what was released by pronase from the EPG/cellulase-treated walls bound to a DEAE Sephadex column and contained sugars corresponding to those expected in the Hyp arabinosides of extensin, and arabinogalactan, galactan and rhamnogalacturonans of pectin. The linkage was proposed to be from the reducing end of the rhamnogalacturonan to a (3->6)-linked arabinogalactan side chain on the protein.

2.7 Testing the 1973 cell wall model Probably because it was so detailed and based on tissue-culture walls, the model of Keegstra et al. (1973) was soon contradicted. Unfortunately, several subsequent authors considered the initial wall model with an almost all-ornothing approach. Albersheim (1975) took out the linkage between pectin and extensin because evidence of the linkage had been derived mostly from an extracellular protein that was actually not extensin. Cell walls of many parts of plants are not rich in the amino acid Hyp, which is considered indicative of extensin. In fact, tissueculture cell walls, along with those of roots, are unusually rich in extensin, containing on the order of 1-3% Hyp by weight. Most studies of cell walls from plants have been done on aerial parts, and pea hypocotyl cell walls, for example, are only -0.3% Hyp (Klis, 1976).

36

PECTINS AND THEIR MANIPULATION

Monro etal. (1976) listed various results of their experiments on solubilizing cell wall polymers from lupin and mung bean hypocotyls that were inconsistent with a strict adherence to the model proposed by Keegstra and colleagues: 1.

Extraction of pectins did not solubilize very much extensin. It should have done so if the only thing crosslinking extensin to the insoluble cellulose is pectin. 2. 10% KOH at room temperature solubilized hemicellulose without solubilizing pectin. This should not happen if all of the hemicellulose is linked to pectin and the pectin is insolubilized by its crosslink to cellulose via the hemicellulose. 3. 6 M guanidine thiocyanate did not solubilize all of the hemicellulose of the walls as one would expect if only the noncovalent interactions to cellulose hold it into the walls. 4. After extraction with 6 M guanidine thiocyanate, cold alkaline conditions, which would cleave ester bonds, did solubilize more hemicellulose, indicating the participation of such alkali-labile covalent linkages in holding some of the hemicellulose in the wall. 5. In the 1973 model linking the reducing end of XG to pectin and linking the reducing end of pectin to extensin, the most alkali-labile link would be the glycosidic link to the serine hydroxyl. Cold alkaline conditions extracted hemicellulose without causing the extensive ^-elimination of serine that would have to occur if the link between extensin and an arabinogalactan crosslink to the hemicellulose via pectin were being broken. 6. Room-temperature alkaline extraction after the cold extraction did solubilize extensin and caused extensive ^-elimination of serine. However, there appeared to be two stages in the extraction. Hemicellulose was solubilized rapidly followed by a slower solubilization of Hyp-containing protein. 7. Some polysaccharides and proteins were not solubilized by the warm alkali, indicating a strong, covalent interaction between them and cellulose. The model Monro and colleagues proposed to substitute for the earlier one was less detailed in its descriptions of interactions between polymers (Monro et al., 1976). They proposed mostly noncovalent interactions between pectins, hemicellulose, and extensin. Some unidentified alkali-labile linkages between hemicellulose and pectins were suggested. (To date, alkali-labile linkages between hemicelluloses and pectins have never been isolated or convincingly demonstrated.) They also suggested a possible warm-alkali-labile covalent linkage between extensin and hemicellulose. A partial explanation for some of the contradictory results to do with extensin solubilization was provided by Mort and Lamport (1977), who found that even

INTERACTIONS BETWEEN PECTINS AND OTHER POLYMERS

37

solubilization of all of the polysaccharides, including cellulose, of tomatoculture cell walls did not solubilize the extensin. Fry (1982a) identified an unusual amino acid in acid hydrolysates of walls as isodityrosine and proposed that this formed crosslinks between extensin molecules. However, Epstein and Lamport (1984) could only find isodityrosine linking what used to be two tyrosine residues within the same peptide. Thus, it formed an mframolecular crosslink. Several groups have shown that extensin precursors do seem to form crosslinks between molecules, but the exact nature of the crosslink has proved elusive (Schnabelrauch et al., 1996). The presence of a covalent linkage between XG and pectin even in sycamore suspension-culture cell walls was called into question by Darvill et al. (1980) in a review on wall structure. The authors stated, without giving experimental detail, that they had been unable to obtain large quantities of XG linked to pectin; although they left open the possibility that small amounts may be linked. Subsequent models of cell walls stress noncovalent interactions between cell wall polymers. McCann and Roberts (1991) presented a model for onion epidermal cell walls based on electron microscopy of replicas of the walls after fast-freeze, deep-etch, rotary shadowing after various extractions following the scheme of Redgewell and Selvendran (1986). Extraction with CDTA (cyclohexanediaminetetraacetic acid) and Na2CC>3 to remove calcium ions and cleave ester linkages removed most of the pectin but almost none of the hemicellulose, after which the researchers could see what they presumed to be XG crosslinks between the cellulose fibrils (McCann et al., 1990). Thus, it appeared that XGs by themselves form crosslinks between cellulose microfibrils. Estimation of the length of individual KOH-extracted XG molecules by rotary shadowing and electron microscopy showed that they were long enough to link between cellulose fibrils (McCann et al., 1992). Crosslinking by XG between cellulose microfibrils need not involve other polymers in order to make a network. Talbott and Ray (1992) found that they could extract essentially all of the polyuronide from cell walls of pea hypocotyl sections with 20 mM ammonium oxalate at 70°C, pH 4.0, while extracting essentially no hemicellulose. Subsequent 4 M KOH extraction solubilized 32% of the original weight of the walls, and the 27% remaining was designated a-cellulose. In contrast to most reports, the hemicellulose contained a high proportion of arabinose and galactose, indicating the presence of arabinan and galactan, or arabinogalactan. There was no further analysis of the a-cellulose fraction, although 27% of the weight of the walls being cellulose is at the high end of what is reported for primary walls (Carpita and Gibeaut, 1993). Because of their ability to solubilize pectin and hemicellulose completely and separately, they asserted that there were no covalent linkages between hemicellulose and pectin. Linkages between extensin and pectin in the pea walls were not even investigated because of the low amount of the protein in the walls (Klis, 1976).

38

PECTINS AND THEIR MANIPULATION

Over a period of about 25 years, Selvendran's group has investigated the polysaccharide composition of a wide variety of crop plant parts using a sequential extraction scheme designed to cause the least possible degradation of polymers as they are solubilized along with the most complete extraction. Sugars characteristic of pectins are invariably found in most fractions, as opposed to the single fraction found by Talbott and Ray. This makes analysis of the pectin very complex because each fraction has somewhat different proportions of various polymers in it, and after an additional fractionation of a particular extract by ionexchange or gel-permeation chromatography, one ends up with large numbers of samples to analyze and interpret. In many instances there is co-chromatography of some hemicellulose along with the pectin, providing evidence for crosslinking between pectin and XG (Stevens and Selvendran, 1984a,b; Selvendran, 1985: Redgwell and Selvendran, 1986; Gooneratne et al, 1994), or pectin and xylan (Waldron and Selvendran, 1992).

2.8

Recent evidence for the existence of some crosslinking between pectin and hemicellulose

In recent years, what appears very convincing evidence for the existence of a covalent link between a portion of XG and pectin has been provided. Femenia and colleagues in a study of changes in cell walls of cauliflower tissues during maturation (Femenia et al, 1998a,b; 1999) found that, after removal of most of the pectin by extraction with water, CDTA and Na2CC>3,0.5 M KOH solubilized a mixture of XG, xylan, phenolics and pectin. Ion-exchange chromatography of the extract gave a neutral fraction and lesser amounts of acidic fractions. It appeared that the acidic fractions were complexes of xylan, XG, pectin and polyphenolics. One fraction containing the potentially crosslinked complex was found to be reduced in molecular weight by purified EPG or xylanase. The authors suggested that the complex was formed during lignification as the stems matured. Ferulic acid was found to be esterified to about 1 in 60 of the sugar residues of primary cell wall pectin from spinach cell suspension cultures (Fry. 1983). The sites of attachment appeared to be the nonreducing ends of arabinan and galactan side chains of the pectin. Pectins extracted from sugar beet pulp are well-known to be feruloylated on arabinosyl side chains (Oosterveld et al., 2001). Ferulic acid on pectin may serve as sites for diphenolic crosslinks (as suggested by Fry to occur between pectin monomers) and/or sites where lignification during wall maturation may take place. Thompson and Fry (2000) found that 30% of the XG of suspension-cultured rose cells appeared to be very strongly associated with pectin. Intact walls were extracted with 6M NaOH (containing 1% w/v NaBH 4 to prevent peeling) and

INTERACTIONS BETWEEN PECTINS AND OTHER POLYMERS

39

the resulting extract, after dialysis, was fractionated on the anion exchanger QSepharose under various conditions. With a step gradient of aqueous pyridine acetate, only ~40% of the XG eluted at low ionic strength, and 55% needed between 0.7 M and 1.4 M acetate for elution. If 8 M urea was incorporated into the buffer to disrupt hydrogen bonding, 35% of the XG still needed this high salt concentration for elution. The acidic nature of the XG-pectin complex was retained after an additional base treatment, but could be partially abolished by treating with a cloned EPG, or an endoarabinanase or a galactanase from the Megazyme Company. The acidic character of some of the XG-pectin complex was greatly diminished by EPG. Although results from treatment with the endoarabinanase or galactanase were taken to implicate involvement of arabinan and galactan in the crosslinking, Mort's group has found that the endoarabinanase and galactanase from Megazyme contain small amounts of endoglucanase, which releases XG fragments from XG-pectin complexes (see below). The XG-pectin complex obtained by Thompson and Fry also co-electrophoresed on glass fiber paper. In addition, the complex bound to filter paper, presumably by way of XG hydrogen bonding to the cellulose. Thompson and Fry have provided good evidence for some of the XG crosslinking with some of the pectin in rose cell walls, but the link is not yet identified. This author's group has been performing similar experiments using cotton suspension-cultured cells as the source of cell walls. If cotton walls are digested with EPG (from Megazyme), about one-quarter of the weight of the walls is solubilized, but only about one-eighth of the rhamnose. Thus most of what is often called RG-I or the 'hairy region' of the pectin is not released from the walls by cleavage of the majority of the HG. Following digestion by EPG, cellulase solubilized the XG and the RG (An, 1991). It is interesting that, although cellulase does not degrade RG, digestion of cellulose and XG by cellulase released RG from the walls. Following digestion by EPG with a 24% KOH extraction, instead of cellulase, also solubilized the pectin and the majority of the XG (El Rassi et al., 1991). Despite such a harsh treatment, chromatography indicated a linkage between some of the XG and the RG. Fractionation on Poros D or a Dionex PA 1 column of the XG and RG co-extracted by KOH showed there to be two classes of XG: neutral and acidic (Fu, 1999). Both classes gave rise to the same expected XG fragments when treated with the purified endoglucanase available from Megazyme. XG oligomers are identified by labeling them with aminonaphthalene trisulfonic acid (ANTS) and subjecting them to capillary electrophoresis. By quantitative analysis of the total sugar content and relative molar compositions of the two classes of XG-containing fractions, approximately 50% of the XG in cotton walls is found to be linked to an acidic polymer. From the sugar composition of the acidic fraction, one can deduce that, in addition to XG, it consists of RG with galactose- and arabinose-containing side

40

PECTINS AND THEIR MANIPULATION

chains along with xylogalacturonan (XGA). If the acidic fraction is adsorbed to microcrystalline cellulose, 85-90% of the XG binds, along with 50% of the RG-XGA. This is taken to indicate that half of the RG in the 24% KOH extract is not linked to XG. Elution of the cellulose with 1 M NaOH releases a little less than half of the bound sugar, including XG, RG and XGA. This group has not yet been successful in efforts to identify the linkage between the XG and pectin. Two approaches have been taken. One is to find a specific method to cleave the crosslink, thus identifying it by the specificity of the cleavage method. The other is to try to degrade the crosslinked fragment into a small enough piece that it can be fully characterized, containing both a portion of the pectin and one of the XG. Both methods involve use of very specific cleavage methods. Unfortunately, these are not easy to come by. The commercially available enzymes directed against cell wall polysaccharides are all purified from culture filtrates of organisms growing on some kind of plant cell wallderived material. The organisms are producing multiple enzymes, so the enzyme one wants must be separated from all of the other enzymes completely if it is to be monospecific. The endoarabinanase and galactanase from Megazyme have been tested and found to contain small amounts of endoglucanase activity. The assay involves preparing a mixture of XG fragments that are dimers of the repeat units, labeling their reducing ends with ANTS, exposing the labeled substrates to the enzyme, and analyzing the products by capillary zone electrophoresis. Both enzymes convert the dimers to monomers (Fu, 1999). When the acidic fraction of the XG-RG complex extracted from cotton was treated with the endoarabinanase purchased from Megazyme, 30-40% of the XG then behaved as neutral on a PA 1 anion-exchange column, but this enzyme also caused an apparent molecular weight decrease of tamarind XG. There was a similar effect with the galactanase. The use of liquid hydrogen fluoride (HF) at —73°C was tried to cleave furanoses specifically, but it was found that the small proportion of nonspecific cleavage of the XG backbone was enough to invalidate any conclusions about furanosyl linkages being key in connecting the XG to pectin. To overcome the problem of specificity, the attempt has been made to express cloned cell wall polysaccharide-degrading enzymes in the yeast Pichiapastoris. This expression system was chosen for the following reasons: 1. 2. 3. 4.

Many of the cloned enzymes are from fungi, so they may be best expressed in a fungus. Also, many of the enzymes are glycosylated, so fungal glycosylation is perhaps necessary for the action of some of them. The system purchased is designed to cause secretion of the expressed protein fused to a His6 tag for ease of purification using a nickel column. Testing of the medium from nontransformed Pichia showed no endoglucanase activity against the XG oligomer tested as described above. Pichia is not known to be able to use plant cell walls as a carbon source. Culturing of Pichia is not demanding and the medium is inexpensive.

INTERACTIONS BETWEEN PECTINS AND OTHER POLYMERS

41

When the endoarabinanase of Bacillus subtilis cloned into Pichia was used to try to break the link between XG and pectin, it failed, although the cloned enzyme was perfectly capable of digesting a-(l-*5)-linked arabinofuranan (Fu et al., 2001). The crosslink, therefore, is not a linear arabinan, but it could be a highly branched arabinan resistant to the endoarabinanase. Work is in progress to clone an arabinosidase for debranching arabinans. Some progress has been made on trying to digest the crosslinked fragment into a small enough piece to be characterized. If the XG-RG complex is taken after desorption from cellulose by 1 M NaOH and digested with endoglucanase and rhamnogalacturonase, a fraction is obtained that has a molecular weight of approximately 15 000 and, from its sugar composition, appears still to contain fragments of RG, XGA and XG. Two-dimensional NMR spectroscopy of this fraction confirms that it contains all three components (Fu, 1999). It appears that this link between XG and pectin occurs near the junction between RG and XGA. No signals for phenolics were observed in the NMR spectra. It is necessary to acquire additional specific enzymes to finally degrade this complex to a small enough piece (~20 sugar residues) to be able to identify what it is that links the XG to the pectin. The yield of the 15 000 Da complex has been approximately 4 mg/g of cell wall. The yield of a 20-residue fragment would be less than one milligram from an experiment of the same scale (starting with one gram of cell walls). Two or three milligrams would be a good amount for complete characterization by NMR.

2.9 Ester linkages between pectin and other polymers There have been repeated suggestions that pectins may link to each other and to other polymers by esterification through the carboxyl groups of the GalA residues (Fry, 1986). Perhaps the strongest evidence for this comes from the extractability by Na2CC>3 of some pectins that are not solubilized by chelators or chaotropic agents (Selvendran, 1985). It is proposed that the carbonate is cleaving ester linkages. Such ester linkages have never been directly demonstrated, perhaps because they would be quite labile and not easy to isolate; although oligomers containing methyl esters of GalA are certainly easy enough to isolate. Maness et al. (1990) optimized conditions for reduction of esters of GalA to galactose using strongly buffered, cold, concentrated sodium borohydride. They compared the GalA and galactose content of samples before and after the reduction to estimate the degree of esterification of the GalA carboxyl groups in a sample. They presumed that there would only be methyl-esterification in natural pectins, so the result would indicate the pectin's degree of methyl-esterification. Methanolysis, trimethylsilylation, and gas chromatography were used for the quantitation.

42

PECTINS AND THEIR MANIPULATION

Kim and Carpita (1992) modified the procedure, incorporating activation with a carbodiimide and sodium borodeuteride reduction. First, the esterified GalA was reduced with sodium borodeuteride to dideutero-galactose and the sample was split into two. In one portion the GalA carboxyl groups were activated with the carbodiimide and reduced to dideutero-galactose with borodeuteride. Both portions were hydrolyzed and converted to alditol acetates for analysis by GC-MS. By comparing the amount of dideutero-galactose in the samples from the single and double reductions, the authors estimate the percentage of GalA involved in ester linkage to its carboxyl group. Colorimetric analysis of methanol in the sample allowed calculation of the degree of methyl-esterification. For maize pectin (Kim and Carpita, 1992) up to one-third of the esters were inferred to be non-methyl esters and in tobacco-culture cell walls (McCann et al., 1994). up to 60% of the esters were determined to involve an alcohol other than methanol. Brown and Fry (1993) also looked for non-methylesters of galacturonic acid. They digested radiolabeled pectin from spinach suspension-cultured cells with Driselase and used ID and 2D paper chromatography to look for spots that did not co-chromatograph with the expected monomer, dimer and trimer of GalA. They had shown the Driselase could completely convert methyl-esterified oligomers of GalA to GalA monomers plus methanol. They found several spots that had different migration behavior, but they did not identify their structures. They estimated that about 5% of the GalA was esterified to an alcohol other than methanol. Needs et al. (1998), using the same borodeuteride reduction method developed by Kim and Carpita, found only 8% of the GalA as non-methyl esters in a carrot root wall preparation, but were unsuccessful in isolating oligosaccharides containing them from a Driselase digest of the walls.

2.10

Recent evidence for the existence of some crosslinking between pectin and extensin

After the work of Monro, Penny and Bailey (1976), there have been only a few reports on connections of pectins to cell wall proteins. Much more attention has been given to isodityrosine-like crosslinks formed directly between protein molecules. (Although these also have not been chemically characterized.) Mort (1978) found that after removal of around 80% of the pectin from tomato suspension-culture walls with a 2 h boiling ammonium oxalate treatment, oxidation with acidic sodium chlorite (which depolymerizes lignin and other polyphenolics and destroys diphenolics) solubilized a high proportion of the extensin protein along with a considerable amount of pectin. The pectin and about one-third of the extensin fragments could not be separated by any separation method tested, but because the chlorite oxidation made the extensin fragments

INTERACTIONS BETWEEN PECTINS AND OTHER POLYMERS

43

acidic by converting the abundant lysine residues to a-amino adipic acid, ionexchange chromatography could not be relied on to separate acidic pectin from the protein. All other methods of separation such as gel-permeation chromatography and density-gradient ultracentrifugation gave ambiguous results because there was a continuum of sizes or densities of the pectin and protein that overlapped. O'Neill and Selvendran (1980) and Ryden and Selvendran (1990) obtained similar results using a milder chlorite treatment, except that a smaller proportion of the extensin appeared to be linked to pectin in runner-bean pod parenchymous tissue. From the poor solubility and low sugar percentage of the extensin-pectin fraction, they suggested that the linkage between the protein and pectin was via polyphenolics that had not been destroyed by the chlorite treatment. In more recent work, Qi et al. (1995) investigated the effectiveness of various treatments of cotton suspension-culture cell walls for solubilizing extensin fragments and the nature of the fragments released by these treatments. Only when they used treatments that included a proteolysis step (in this case trypsinization) was much extensin (indicated by Hyp) solubilized. This was as expected from the insolubility of tomato extensin even in totally deglycosylated cell walls (Mort and Lamport, 1977), which was taken to indicate that the protein was crosslinked via direct protein-protein crosslinks or protein-phenolic-protein crosslinks (Wilson and Fry, 1986). For the most effective solubilization of Hyp, the following sequence of treatments was used: 1. 2. 3. 4. 5.

Digest the walls with EPG to remove most of the HG. Digest this residue with cellulase to remove hemicellulose and cellulose. Treat the residue with HF at —73°C to remove most of the arabinosecontaining side chains from the Hyp. Wash the residue with ammonium bicarbonate buffer, pH 7.6, to remove ionically bound sugars. Digest the residue with trypsin in ammonium bicarbonate buffer.

The trypsinization step in this sequence solubilized about 50% of the cotton extensin along with sugars expected to be in RG and XGA. One-third of the peptide material behaved on size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as though it had a very high molecular weight and co-chromatographed with the pectin. The other two-thirds of the peptide material appeared to have molecular weights less than 30 kDa by SDS-PAGE and was associated with very little pectin. It is difficult to explain these results without suggesting that there is some covalent crosslinking of extensins to pectin. From its stability to HF at —73°C, we can conclude that the crosslink is not a linear or branched arabinan. No subsequent progress has been made on identifying the nature of this crosslink.

44

2.11

PECTINS AND THEIR MANIPULATION

Considering crosslinks and cell wall dynamics

Weighing the arguments for and against there being covalent linkages between pectins and XG and between pectins and cell wall proteins, it seems that no single experimental system can reflect the diverse requirements that cell walls must fulfill. Models are easier to draw and explain if they are simple, but there is a wide range of species, tissues, developmental states, and environmental conditions to describe, so there is no need for cell walls to be the same in all cases. A cell's wall is in a continuous state of flux, particularly as a cell divides, expands, or matures. During cell wall formation and turnover, crosslinkages are presumably made and broken. As a cell wall is being formed, most new polymers are secreted from the inside of the cell probably by way of vesicles coming from Golgi. We do not know which polysaccharides are linked together when the vesicles are still on their way to the wall. For instance, is the RG region of pectin linked to the HG region? Are arabinan and galactan side chains already on the RG regions? Once a vesicle's contents enter the walls, how are they incorporated? Fry and his co-workers have investigated the role of XG endotransglycosylase in inserting new XG into old XG polymers by density shifts using 13C density-labeled walls followed by feeding of 12Cand tritium-labeled glucose to identify newly incorporated XG (Thompson and Fry, 2001). Tritium-labeled XG is rapidly incorporated into the previously formed, denser XG. These experiments allowed the authors to conclude that XG endotransglycosylase is involved in the incorporation of new XG into existing walls. It is also involved in restructuring of the wall. Fry's group tried to detect a polygalacturonan endotransglycosylase activity but failed to find it (GarciaRomera and Fry, 1994). No other similar experiments investigating incorporation of other polysaccharides or protein into the wall appear to have been published. It is known that there is an abundance of carbohydrate-hydrolyzing enzymes in cell walls (Zhang el al., 1996; Cosgrove, 1999). These include a variety of glycosidases, methylesterases, cellulases and EPGs. Enzymes needed for linking polysaccharides together would not have been detected because there is not yet a convenient assay for them. In addition to carbohydrate-modifying enzymes, there are peroxidases, phosphatases (Kaneko, 1999) and probably proteases (Jones and Mullet, 1995). Changes in activities of enzymes in the cell wall are undoubtedly reflected in restructuring of the wall. A large drop in the amount of EPG activity in the intercellular spaces of expanding cotton cotyledons was found as the expansion rate decreased (Zhang, 1998). During ripening of some fruits there is a massive induction of pectin-degrading enzymes and, in some cases, of cellulases (Fischer and Bennett, 1991; Chapter 6). Thus, cell walls are probably in a continuous state of flux.

INTERACTIONS BETWEEN PECTINS AND OTHER POLYMERS

2.12

45

Miscellaneous interactions of pectins with other polymers

2.12.1 Covalent interactions A group of proteins that may be covalently linked to pectin are the wallassociated kinases (WAKs) (Wagner and Kohorn, 2001). These are proteins that have an intracellular kinase domain and an extracellular N-terminal domain interacting with the cell wall. WAKs are solubilized from cell walls by boiling in SDS/dithiothreitol, but can also be released by digestion of the walls with a purified EPG. The proteins solubilized by either method react in Western blots with monoclonal antibodies directed against HG. Nothing is known about the nature of the linkage between the pectin and the protein. Perrone et al. (1998) have suggested that it is possible that there are amide linkages between the carboxylic acid groups of GalA residues in pectins and amino groups of proteins. They synthesized both a- and s-amides between GalA and lysine and linked multiple e-groups of lysines to polygalacturonic acid. The linkages were stable to various proteases and Driselase. They labeled suspension-culture cells with [14C]glucuronic acid to produce pectins containing [14C]GalA; after digestion of the walls with Driselase, mild acid hydrolysis to remove arabinose side chains from hydroxyproline residues in extensin, and then proteases, they found some radiolabeled GalA that behaved cationically, indicating that it was linked to a positively charged moiety such as a peptide. These compounds have not yet been identified. Walls can become heavily lignified during secondary wall formation and as a wound or disease resistance response. Whitmore (1978) has found that the lignin formed artificially in pine tissue-culture cell walls by incubating them with coniferyl alcohol and H2O2 incorporates both carbohydrate and hydroxyprolinecontaining protein. Grabber et al. (1995) did similar experiments in maize cell suspension-culture walls. They incubated purified walls with an H262-generating system along with coniferyl alcohol and synapyl alcohol. They analyzed the content of phenolic acid ester monomers and dimers that could be solubilized from the resulting lignified walls by room-temperature NaOH, representing ester-linked phenolics, and by 4 N NaOH at 170°C, representing ether-linked phenolics. Only 20% of the ferulic acid originally present in the cell walls remained as such after the reaction. Products from only 40% of the original ferulic acid esters could be identified in what was recovered by the combination of both hydrolyses after the lignification. We can conclude from these experiments that, during natural lignification, ferulic acids esterified to pectins and tyrosine residues in cell wall protein will become incorporated into lignin along with their associated polymer. There was a recent report of incorporation of coniferyl alcohol into a ligninpectin complex by the dehydrogenation polymerization (DHP) reaction in the

46

PECTINS AND THEIR MANIPULATION

presence of pectin (Cathala et al., 2001). In the DHP reaction, conifery 1 alcohol, or other lignin precursor, is converted to a free radical by loss of a proton and an electron induced by peroxidase. These free radicals then couple to form lignin. It was proposed that in the presence of pectin some of the carboxylic acid groups of the GalA residues react with the free radicals to form esters. Polymerization of phenolic material by the DHP reaction in the presence of pectin decreases the proportion of polymer soluble in dioxane-water (9:1, v/v). A large increase in the percentage of the phenolic material synthesized in the presence of pectin that could be solubilized by dioxane-water (9:1, v/v) after alkali treatment gave support to the ester linkage hypothesis. It seems likely that pectin-lignin crosslinks might form this way in maturing cell walls. 2.12.2 Noncovalent interactions It has repeatedly been suggested that pectins with a low degree of esterification will interact ionically with the multiple lysine and histidine residues spaced along extensin (Cassab and Varnner, 1988; Kieliszewski and Lamport, 1994). This hypothesis has been tested by MacDougall et al. (2001) by looking at the ability of carrot extensin, synthetic extensin peptide fragments and polyarginine or poly lysine to form gels with pectin or to modify Ca2+-induced pectin gels. A short heptamer VHHYKYK found in the carrot extensin and the polycationic peptides formed strong gels with pectin, whereas a longer peptide APEHHYKYKSPPPPKHFPAPEHHYKYKYKS did not reliably form gels, and extensin did not. Extensin did, however, cause precipitation of the pectin if it was mixed directly with it at neutral pH. The same was true for the peptides. Thus, to form a gel the pectin solution was acidified, mixed with the peptide. and then brought to pH 6 by adding a strong buffer solution. Pectins have also been implicated in noncovalent interactions with other proteins. Baldwin et al. (1993) found that the arabinogalactan protein they were investigating bound to spots of pectin on nitrocellulose membrane. Binding of the arabinogalactan protein was visualized using antibody to the protein. No function for the binding was shown. In studies of pollen tube adhesion to styles, Lord's group (Mollet et al., 2000) has implicated two macromolecular components as being involved. One is a small 9kDa protein that they called stigma/stylar cysteine-rich adhesin: the other appears from the conditions used to extract it from stylar tissue, its behavior on gel-permeation and ion-exchange chromatography and its sugar composition to be an HG-rich pectin. Both the small protein and the pectin are needed for maximal adhesion. The small protein and the pectin bind to each other in vitro. It has recently been proposed that, at the point of biosynthesis, pectin and glucuronoarabinoxylan are noncovalently bound to a protein or proteins that

INTERACTIONS BETWEEN PECTINS AND OTHER POLYMERS

47

cause them to bind to XGs (Rizk et al., 2000) in a pH-dependent manner. The authors hypothesize that the interaction may play a role in cell wall assembly. 2.13

General conclusions

It certainly appears that a portion of pectin is covalently attached to other polymers in the cell wall. The proportion of the pectin that is linked to other polymers probably varies from plant to plant, from tissue to tissue, and during development, and the natures of the linkages are unknown. Until the crosslinks have been structurally characterized they will remain mysterious and controversial. Progress in isolating and identifying crosslinks has been very slow and arduous, so very few groups have been working on them. With the availability of a wide range of cloned, monospecific enzymes in the near future, and the increased sensitivity of NMR and MS, now is the time for rapid progress to occur. Acknowledgements I am very happy to acknowledge the financial support of the DOE, NSF, and USDA for the work done in my laboratory. I thank Dr Margaret Pierce for her help in preparing and clarifying the manuscript.

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Cassab, G.I. and Varnner, J.E. (1988) Cell wall proteins. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39. 321-353. Cathala, B., Chabbert, B., Joly, C., Dole, P. and Monties, B. (2001) Synthesis, characterisation and water sorption properties of pectin-dehydrogenation polymer (lignin model compound) complex. Phytochemistry, 56, 195-202. Cosgrove, D.J. (1999) Enzymes and other agents that enhance cell wall extensibility. Annu. Rev. Plant Physiol. Plant Mol. Biol., 50, 391^17. Darvill, A., McNeil, M., Albersheim, P. and Delmer, D. (1980) The primary cell walls of flowering plants, in The Biochemistry of Plants, vol. 1 (ed. N.E. Tolbert), Academic Press, New York, pp. 92-162. El Rassi, Z., An, J., Tedford, D. and Mort, A. (1991) High performance reversed-phase chromatography mapping of pyridylamino derivatives of xyloglucan oligosaccharides. Carbohydr. Res.. 215. 25-38. English, P.D., Maglothin, A., Keegstra, K. and Albersheim, P. (1972) Cell wall-degrading endopolygalacturonase secreted by Colletotrichum lindemuthianum. Plant Physiol., 49. 293-298. Epstein, L. and Lamport, D.T.A. (1984) An intramolecular linkage involving isodityrosine in extensin. Phytochemistry, 23, 1241-1246. Femenia, A., Garosi, P., Roberts, K.. Waldron, K.W., Selvendran. R.R. and Robertson. J.A. (1998a) Tissue-related changes in methyl-esterification of pectic polysaccharides in cauliflower (Brassica oleracea L. van botrytis) stems. Planta, 205, 438-444. Femenia. A., Waldron, K.W., Robertson, J.A. and Selvendran, R.R. (1998b) Compositional and structural modification of the cell wall of cauliflower (Brassica oleracea L. var. botryis) during tissue development and plant maturation. Carbohydr. Polym., 39, 101-108. Femenia, A., Rigby, N.M., Selvendran, R.R. and Waldron. K.W. (1999) Investigation of the occurrence of pectic-xylan-xyloglucan complexes in the cell walls of cauliflower stem tissues. Carbohydr. Polym., 39, 151-164. Fischer, R.L. and Bennett, A.B. (1991) Role of cell wall hydrolases in fruit ripening. Annu. Rev. Plant Physiol. Plant Mol. Biol., 42, 675-703. Fry, S.C. (1982a) Isodityrosine, a new crosslinking amino acid from plant cell-wall glycoprotein. Biochem.J., 204, 449^55. Fry, S .C. (1982b) Phenolic components of the primary cell wall: feruloy lated disaccharides of D-galactose and L-arabinose from spinach polysaccharide. Biochem. J., 203, 493-504. Fry, S.C. (1983) Feruloylated pectins from the primary cell wall: their structure and possible functions. Planta, 1ST, 111-123. Fry, S.C. (1986) Crosslinking of matrix polymers in the growing cell walls of angiosperms. Annu. Rev. Plant Physiol., 37, 165-186. Fu, J. (1999) Extraction and characterization of xyloglucan-rhamnogalacturonan crosslinked complex in cotton suspension cell walls. PhD thesis, Oklahoma State University, Stillwater. OK. Fu, J., Prade, R. and Mort, A. (2001) Expression and action pattern of Botryotinia fuckeliana (Botrytis cinerea) rhamnogalacturonan hydrolase in Pichia pastoris. Carbohydr. Res., 330. 73-81. Garcia-Romera, I. and Fry, S.C. (1994) Absence of transglycosylation with oligogalacturonides in plant cells. Phytochemistry, 35, 67-72. Gooneratne, J., Needs, P.W., Ryden, P. and Selvendran, R.R. (1994) Structural features of the cell walls polysaccharides of mung bean Vigna radiata. Carbohydr. Res.. 265. 61-77. Grabber, J.H., Hatfield, R.D., Ralph, J., Zon, J. and Amrhein. N. (1995) Ferulate crosslinking in cell walls isolated from maize cell suspensions. Phytochemistry, 40. 1077-1082. Green, J.W., Pearl, I.A., Hardacker, K.W., Andrews. B.D. and Haigh. F.C. (1977) The peeling reaction in alkaline pulping. Tappi J., 60, 120-125. Hakamori, S.-I. (1964) A rapid permethylation of glycolipids. and polysaccharides catalyzed by methylsulfinyl carbanion in dimethylsulfoxide. /. Biochem. (Tokyo), 55. 205-208. Hayashi, T. (1989) Xyloglucans in the primary cell wall. Annu. Re\: Plant Physiol. Plant Mol. Biol.. 40. 139-168.

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Ishii, T. (1991) Isolation and characterization of a diferuloyl arabinoxylan hexasaccharide from bamboo shoot. Carbohydr. Res., 219, 15-22. Ishii, T. and Hiroi, T. (1990) Linkage of phenolic acids to cell wall polysaccharides of bamboo shoot. Carbohydr. Res., 206, 297-310. Ishii, T, Matsunaga, T., Pellerin, P., O'Neill, M.A., Darvill, A.G. and Albersheim, P. (1999) The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently crosslinked dimer. J. Biol. Chem., 247, 13098-13104. Jones, J.T. and Mullet, J.E. (1995) A salt- and dehydration-inducible pea gene, CyplSa, encodes a cell-wall protein with sequence similarity to cysteine proteases. Plant Mol. Biol., 28, 1055-1065. Kaneko, T.S. (1999) Plant cell wall acid phosphatase. Curr. Top. Plant Biology, 1, 105-111. Keegstra, K., Talmadge, K., Bauer, W.D. and Albersheim, P. (1973) The structure of plant cell walls. III. A model of the walls of suspension-cultured sycamore cells based on the interconnections of the macromolecular components. Plant Physiol., 51, 188-196. Kieliszewski, M.J. and Lamport, D.T.A. (1994) Extensin: repetitive motifs, functional sites, posttranslational codes, and phylogeny. Plant J., 5, 157-172. Kim, J.-B. and Carpita, N.C. (1992) Changes in esterification of the uronic acid groups of cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiol., 98, 646-653. Klis, P.M. (1976) Glycosylated seryl residues in wall protein of elongating pea stems. Plant Physiol., 57,224-226. Kobayashi, M., Match, T. and Azuma, J. (1996) Two chains of rhamnogalacturonan II are crosslinked by borate-diol ester bonds in higher plant cell walls. Plant Physiol., 110, 1017-1020. Lamport, D.T.A. (1964) Cell suspension cultures of higher plants, isolation and growth energetics. Exp. Cell Rex., 33, 195-206. Lamport, D.T.A. (1977) Structure, biosynthesis and significance of cell wall glycoproteins, in Recent Advances in Phytochemistry (eds F.A. Loewus et al.), Plenum, New York, pp. 79-115. MacDougall, A.J., Brett, G.M., Morris, V.J., Rigby, N.M., Ridout, M.J. and Ring, S.G. (2001) The effect of peptide-pectin interactions on the gellation behaviour of plant cell wall pectin. Carbohydr. Res., 335, 115-126. Maness, N.O., Ryan, J.D. and Mort, A.J. (1990) Determination of the degree of methyl-esterification of pectins in small samples by selective reduction of esterified galacturonic acid to galactose. Anal. Biochem., 185, 346-352. Markwalder, H.U. and Neukom, H. (1976) Diferulic acid as a possible crosslink in hemicelluloses from wheat germ. Phytochemistry, 15, 836-837. McCann, M.C. and Roberts, K. (1991) Architecture of the primary cell wall, in The Cytoskeletal Basis of Plant Growth and Form (ed. C.W. Lloyd), Academic Press, New York, pp. 102-129. McCann, M.C., Wells, B. and Roberts, K. (1990) Direct visualization of crosslinks in the primary plant cell wall. /. Cell ScL, 96, 323-334. McCann, M.C., Wells, B. and Roberts, K. (1992) Complexity in the spatial localization and length distribution of plant cell-wall matrix polysaccharides. J. Microsc., 166, 123-136. McCann, M.C., Shi, J., Roberts, K. and Carpita, N.C. (1994) Changes in pectin structure and localization during the growth of unadapted and NaCl-adapted tobacco cells. Plant J., 5, 773-785. Mollet, J.-C., Park, S.-Y, Nothnagel, E.A. and Lord, E.M. (2000) A lily stylar pectin is neccessary for pollen tube adhesion to an in vitro stylar matrix. Plant Cell, 12, 1737-1749. Monro, J.A., Penny, D. and Bailey, R.W. (1976) The organization and growth of primary cell walls of lupin hypocotyl. Phytochemistry, 15, 1193-1198. Mort, A.J. (1978) Partial characterization of extensin by selective degradation of cell walls. PhD thesis, Michigan State University, East Lansing, MI. Mort, A.J. and Lamport, D.T.A. (1977) Anhydrous hydrogen fluoride deglycosylates glycoproteins. Anal. Biochem., 82, 289-309. Needs, P.W., Rigby, N.M., Colquhoun, I.J. and Ring, S.G. (1998) Conflicting evidence for non-methyl galacturonoyl esters in Daucus carota. Phytochemistry, 48,11-11.

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O'Neill, M.A. and Selvendran, R.R. (1980) Glycoproteins from the cell wall of Phaseolus coccineus. Biochem. J., 187, 53-63. Oosterveld, A., Pol. I.E., Beldman, G. and Voragen. A.G.J. (2001) Isolation of feruloylated arabinans and rhamnogalacturonans from sugar beet pulp and their gel forming ability by oxidative crosslinking. Carbohydr. Polym., 44. 9-17. Pen-one, P.. Hewage. C.M., Sadler. I.H. and Fry, S.C. (1998) N-a- and /V-e-D-galacturonoyl-Llysine amides: properties and possible occurrence in plant cell walls. Phytochemistry. 49. 1879-1890. Pope, D.G. (1977) Relationships between hydroxyproline-containing proteins secreted into the cell wall and medium by suspension-cultured Acer pseudoplatinus cells. Plant Physioi.. 59. 894—900. Powell, D.A., Morris, E.R., Gidley. M.J. and Rees. D.A. (1982) Conformations and interactions of pectins. II. Influence of residue sequence on chain association in calcium pectate gels. J. Moi Bioi. 155, 517-531. Qi, X., Behrens, B.X.. West, P. and Mort. A.J. (1995) Solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures. Evidence for a covalent crosslink between extensin and pectin. Plant Physioi.. 108, 1691-1701. Redgwell, R.J. and Selvendran, R.R. (1986) Structural features of cell-wall polysaccharides of onion Allium cepa. Carbohydr. Res., 157. 183-199. Ridley, B., O'Neill, M.A. and Mohnen, D. (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry. 57, 929-967. Rizk, S.E., Abdel-Massih, R.M., Baydoun. E.A. and Brett. C.T. (2000) Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea. Planta. 211. 423-429. Ryden, P. and Selvendran, R.R. (1990) Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus). Biochem. J.. 269. 393^402. Schnabelrauch, L.S.. Kieliszewski, M.. Upham. B.L., Alizedeh, H. and Lamport. D.T.A. (1996) Isolation of pI-4.6 extensin peroxidase from tomato cell suspension cultures and identification of Val-Tyr-Lys as putative intermolecular crosslink site. Plant J.. 9, 477^489. Selvendran, R.R. (1985) Developments in the chemistry and biochemistry of pectic and hemicellulosic polymers. J. Cell Sci. Suppl., 2. 51-58. Stephen. A.M. (1983) Other plant polysaccharides. in The Polysaccharides. vol. 2 (ed. G.O. Aspinall). Academic Press, New York, pp. 97-193. Stevens. B.J.H. and Selvendran, R.R. (1984a) Structural features of cell-wall polymers of the apple. Carbohydr. Res., 135. 155-166. Stevens, B.J.H. and Selvendran, R.R. (1984b) Structural features of cell-wall polysaccharides of the carrot. Carbohydr. Res., 128. 321-333. Talbott, L.D. and Ray, P.M. (1992) Molecular size and separability features of pea cell wall polysaccharides. Plant Physioi., 98, 357-368. Talmadge, K., Keegstra, K., Bauer, W.D. and Albersheim, P. (1973) The structure of plant cell walls. I. The macromolecular components of the walls of suspension-cultured sycamore cells with a detailed analysis of the pectic polysaccharides. Plant Physioi., 51, 158-173. Thompson, J.E. and Fry, S.C. (2000) Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells. Planta, 211, 275-286. Thompson, J.E. and Fry, S.C. (2001) Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells. Plant J., 26, 23-34. Vincken, J.P.. Keizer, A. de. Beldman, G. and Voragen, A.G.J. (1995) Fractionation of xyloglucan fragments and their interaction with cellulose. Plant Physioi., 108, 1579-1585. Wagner, T.A. and Kohom, B.R. (2001) Wall-associated kinases are expressed throughout plant development and are required for cell expansion. Plant Cell, 13, 303-318. Waldron, K.W. and Selvendran, R.R. (1992) Cell wall changes in immature Asparagus stem tissue after excision. Ph\tochemistr\, 31. 1931-1940.

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5I

Whitmore, F.W. (1978) Lignin-carbohydrate complex formed in isolated cell walls of callus. Phytochemistry, 17, 421-425. Wilson, L.G. and Fry, J,C. (1986) Extensin—a major cell wall glycoprotein. Plant Cell Environ., 9. 239-260. Zhang, Z. (1998) Changes in homogalacturonans, polygalacturonase activities, and cell wall linked proteins during cotton cotyledon expansion. PhD thesis, Oklahoma State University, Stillwater. OK. Zhang, Z., Pierce, M.L. and Mort, A.J. (1996) Detection and differentiation of pectic enzyme activity in vitro and in vivo by capillary electrophoresis of products from fluorescent labeled substrate. Electwphoresis, 17, 372-378.

3

Biosynthesis of pectins Debra Mohnen

3.1

Introduction

Pectin is the most structurally complicated polysaccharide in the plant cell wall. Accordingly, the study of its synthesis is complex owing to the large number of enzymes required. At least 12 activated sugar substrates are required for pectin synthesis. The known activated sugars are nucleotide-sugars, although the possibility that lipid-linked sugars may be involved cannot be ruled out. The regulation of the synthesis of the activated sugar substrates is likely to be important in the overall regulation of pectin synthesis. Based on the known structure of pectin, at least 14 distinct enzyme activities are required to synthesize the activated sugar substrates and 58 distinct glycosyl-, methyl- and acetyltransferases are required to synthesize the complicated family of polymers known as pectin. While progress has been made in characterizing some of the pectin biosynthetic enzymes in crude or partially purified plant homogenates, in no case has a single enzyme been completely characterized in regard to enzyme structure, subcellular location, protein sequence, gene identity and enzyme regulation. Several recent comprehensive reviews on pectin structure (Mohnen, 1999; Ridley et al., 2001), synthesis (Mohnen, 1999; Ridley et a/., 2001) and function (Ridley et al., 2001; Willats et al., 2001 a) and on nucleotidesugar interconversion pathways (Reiter and Vanzin, 2001) have been published and should be consulted for detailed background information. In addition, several general reviews on cell wall synthesis (Gibeaut, 2000; Reid, 2000; Dhugga, 2001; Perrin et al, 2001) and on progress and strategies to identify wall biosynthetic genes (Keegstra and Raikhel, 2001; Perrin et a/., 2001) and glycosyItransferases (Henrissat et a/., 2001; Keegstra and Raikhel, 2001) have recently been published. The goal of this review is to summarize our present level of understanding of pectin synthesis. The first part of the review outlines our understanding of the subcellular location of pectin synthesis. The nucleotide-sugars required for pectin synthesis are then introduced and progress towards understanding the mechanism, site(s) and regulation of their synthesis is reviewed. Finally, a list of the glycosyl-, methyl- and acetyltransferases required for pectin synthesis is provided and progress towards identifying and characterizing these enzymes is summarized. It is hoped that this review will provide a foundation to facilitate the identification of the pectin biosynthetic genes and the development of better molecular tools to study pectin biosynthesis.

BIOSYNTHESIS OF PECTINS

53

3.2 What is the structure of newly synthesized pectin? One of the challenges of studying pectin synthesis is that we do not know the structure of de novo synthesized pectin. For example, the detailed structural characterization of pectin isolated from the wall (O'Neill et al, 1990; Mohnen, 1999; Ridley et al., 2001) has led to the identification of the family of complex polysaccharides known as homogalacturonan (HGA), rhamnogalacturonan I (RG-I) and the substituted galacturonans such as the ubiquitous rhamnogalacturonan II (RG-II) (Ridley etal, 2001), and the less prevalent xylogalacturonan (Schols et al, 1990, 1995; Yu and Mort, 1996), and apiogalacturonan (Hart and Kindel, 1970; Watson and Orenstein, 1975). However, we do not yet know whether these polysaccharides are synthesized as one polymer or whether they are synthesized as individual polymers that become interconnected during or following their insertion into the wall (see Figure 3.1). Furthermore, we do not know whether the structural differences found in each of the polysaccharides isolated from the wall, such as variations in the degree and pattern of methyland acetyl-esterification of homogalacturonan (Willats et al., 200Ib), arise in the wall (i.e. in mum by wall-localized enzymes) or whether the differences occur, at least in part, during synthesis. This uncertainty makes it challenging to propose models for how pectin is synthesized and to predict which enzymes are required intracellularly for synthesis, rather than being required extracellularly for in mum modeling of pectin. For the purpose of this review, the working hypothesis is that homogalacturonan can be synthesized as in independent polymer. It is also proposed that substituted galacturonans such as ubiquitous RG-II (Ridley et al., 2001) and the other less prevalent xylogalacturonan (Schols et al., 1990, 1995; Yu and Mort, 1996) and apiogalacturonan (Ridley et al., 2001) can be synthesized as modified versions of homogalacturonan. Finally, it is hypothesized that RG-I is synthesized as an independent polymer that may, or may not, be covalently linked to homogalacturonan or to substituted galacturonans (see Figure 3.1). It must be stressed, however, that no unequivocal evidence is available to support the hypothesized independence of RG-I and HGA synthesis or of the dependence of RG-II synthesis on HGA. One of the goals, or outcomes, of current research in pectin synthesis should be the elucidation of how the three main pectic polymers, HGA, RG-I and RG-II, are linked together during synthesis.

3.3

Subcellular location of pectin synthesis

Several lines of evidence indicate that pectin is synthesized in the Golgi and transported to the wall via membrane-bound vesicles. The plant Golgi apparatus is a dynamic series of membrane-bound vesicles and stacks that function in the biosynthesis pectins and hemicellulose, in the glycosylation of proteins

BIOSYNTHESIS OF PECTINS

33

and in the synthesis of lipids (Staehelin and Moore, 1995; Nebenfiihr and Staehelin, 2001). The Golgi vesicles move along actin filaments via myosin motors (Nebenfiihr et al., 1999) and it is believed that this movement targets the transport of pectin and other macromolecules to the cell wall. The autoradiographic identification of radiolabeled polysaccharides in Golgi cisternae and their chase from the Golgi to the cell wall using non-radiolabeled precursors (Northcote and Pickett-Heaps, 1966; Northcote, 1970) was among the first evidence that pectin is synthesized in the Golgi. For example, Golgienriched fractions isolated from cells grown in the presence of radiolabeled glucose contained radioactive galactose (Gal), arabinose (Ara) and galactosyluronic acid (GalA, galacturonic acid), the major glycosyl residues found in the pectic poly saccharides (Stoddart and Northcote, 1967; Harris and Northcote, 1971). The identification of pectin-specific carbohydrate epitopes in the Golgi, via immunocytochemistry of thin cell sections using anti-pectin antibodies (Moore et al., 1991; Staehelin and Moore, 1995; Willats et al., 2000), provides further evidence that pectins are synthesized in the Golgi apparatus (Staehelin and Moore, 1995) and suggests that specific pectic carbohydrate epitopes are sublocalized in the Golgi. Studies using antibodies reactive to HGA-like and the RG-I-like epitopes suggest that the syntheses of homogalacturonan (HGA) and rhamnogalacturonan I (RG-I) begin in the cis-Golgi (Lynch and Staehelin, 1992; Zhang and Staehelin, 1992; Staehelin and Moore, 1995) and continue into the medial Golgi (Moore etal, 1991; Zhang and Staehelin, 1992; Staehelin and Moore, 1995) with more extensive branching taking place in the trans-Golgi cisternae (Zhang and Staehelin, 1992; Staehelin and Moore, 1995). Furthermore, studies using antibodies reactive against relatively unesterified HGA (JIMS (VandenBosch etal, 1989; Knox etal., 1990); PGA/RG-I (Moore etai, 1986; Moore and Staehelin, 1988; Lynch and Staehelin, 1992); 2F4 (Liners et al, 1989)) and relatively esterified HGA (JIM7 (Knox et al, 1990)) suggest that HGA becomes methyl-esterified in the medial and trans-Golgi (Vian and Roland, 1991; Liners and Van Cutsem, 1992; Zhang and Staehelin, 1992; Sherrier and VandenBosch, 1994; Staehelin and Moore, 1995), and is transported to the plasma membrane in vesicles as a highly methyl-esterified polymer that is inserted into the wall (Carpita and Gibeaut, 1993; Liners et al, 1994; Dolan et al, 1997). The de-esterification of HGA by pectin methylesterases (Micheli, 2001) in the wall or at cell plate (Dolan et al, 1997) produces more acidic HGA (Stoddart and Northcote, 1967; Shea et al, 1989; Liners and Van Cutsem, 1992; Li et al, 1994; Marty et al, 1995). Such a spatial partitioning of HGA esterification and de-esterification is supported by the localization of esterified HGA throughout the cell wall (Fujiki et al, 1982; Knox et al, 1990; Vian and Roland, 1991; Liners and Van Cutsem, 1992; Liners et al, 1994; Sherrier and VandenBosch, 1994; Marty et al, 1995; Dolan et al, 1997; Willats et al, 200la), the localization of relatively unesterified HGA in the middle lamella,

58

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and the frequently observed absence of unesterified HGA epitopes in the transGolgi vesicles. In spite of such results, however, the fact that some cell types show a different localization of unesterified HGA, such as melon callus cells (Vian and Roland, 1991) that contain unesterified HGA in the trans-Golgi, suggests that HGA can be inserted into the wall in a relatively unesterified state in some cells. This calls into question the general belief that HGA is necessarily synthesized in a highly esterified form (Knox et al., 1990; Casero and Knox, 1995). It is important to realize that specific pectic epitopes localize to different Golgi compartments in different cell types (Knox et al., 1990: Lynch and Staehelin, 1992; Casero and Knox, 1995; Staehelin and Moore. 1995), suggesting that pectin synthesis may differ in different cell types, in different species, at different points during development, or even at different locations in the same wall (Stacey et al., 1995; Willats et al.. 1999; McCartney et al., 2000; Orfila and Knox, 2000; Willats et al, 2000). However, since the absence of a specific carbohydrate epitope may be due to masking of the epitope rather than to a lack of the synthesis of the epitope, it is necessary to confirm carbohydrate-epitope immunocytochemistry results with localization studies on the biosynthetic enzymes themselves in order to make conclusions regarding the mechanism of biosynthesis. The first enzymatic evidence for the location of a pectin biosynthetic glycosyltransferase was the localization of HGA-galacturonosyltransferase (GalAT) to the Golgi and the demonstration that its catalytic site faces the Golgi lumen (Sterling et al., 2001). Most of the GalAT activity in pea (Pisum sativum) colocalizes in linear and discontinuous sucrose gradients with the Golgi marker latent UDPase and is separated from the endoplasmic reticulum, mitochondria and plasma membrane (Sterling et al., 2001). The catalytic site of GalAT was shown to reside within the lumen of the Golgi, since GalAT activity was reduced by treatment with proteinase K only if Golgi membranes were first permeabilized with detergent (Sterling et al., 2001). The enzymes that methyl-esterify HGA have also been localized to the Golgi, further confirming the location of pectin synthesis. Tobacco HGA methyltransferase (HGA-MT) activity localizes to the Golgi and the enzyme's catalytic site was shown to face the Golgi lumen (Goubet and Mohnen, 1999a). The localization of roughly half of the pectin methyltransferase activity in flax to the Golgi (Vannier et al., 1992; Bourlard et al., 1997b) provides further evidence that HGA methyl-esterification occurs in the Golgi. The location of pectin synthesis in the Golgi leads to the question of where the nucleotide-sugar substrates are synthesized and of how the substrates gain access to the enzyme. It has been proposed that the nucleotide-sugars required for pectin synthesis are synthesized on the cytosolic side of the Golgi and transported into the Golgi lumen by specific nucleotide-sugannucleoside monophosphate antiporters (Sterling etal, 2001) (see Figure 3.2a). While this model is consistent with the topology of some nucleotide-sugar biosynthetic enzymes in animals

BIOSYNTHESIS OF PECTINS

59

(Berninsone and Hirschberg, 2000) and plants (Schroeder and Hagiwara, 1989; Munoz et al, 1996; Neckelmann and Orellana, 1998; Baldwin et al, 2001), there are also indications that some nucleotide biosynthesis enzymes, such as UDP-glucuronic acid decarboxylase (Hayashi et al., 1988; Kearns etal., 1993), may actually reside in the Golgi (see Figure 3.2b). Thus, until the subcellular location of the enzymes is confirmed experimentally, two models for the location of the nucleotide-transforming enzymes must be considered (Figure 3.2). For those nucleotide-sugars that are synthesized in the cytosol, it has been shown in both animals (Capasso and Hirschberg, 1984) and plants (Munoz et al., 1996; Wang et al, 1997; Neckelmann and Orellana, 1998; Baldwin et al, 2001) that transport occurs via nucleotide-sugannucleoside monophosphate antiporters that reside in the endoplasmic reticulum or Golgi membranes. As shown in Figure 3.2a, nucleotide-sugars that are synthesized on the cytosolic side of the Golgi are predicted to be transported into the Golgi lumen by specific nucleotidesugarnucleoside monophosphate antiporters. The channeling of nucleotidesugars from the cytosol into the Golgi may be facilitated by nucleotide-sugar binding proteins (Faik et al, 2000). Once the nucleotide-sugar is transported into the Golgi it is used as a substrate by a pectin biosynthetic glycosyltransferase that transfers the glycosyl residue onto a growing polymer. The released nucleoside diphosphate (NDP) is hydrolyzed by a Golgi-localized nucleotide -S'-diphosphatase (NDPase) (Orellana et al, 1997) into NMP and inorganic phosphate. The nucleoside monophosphate is then transported out of the Golgi by a nucleotide-sugannucleoside monophosphate antiporter.

3.4

Synthesis of the nucleotide-sugar substrates required for pectin synthesis

It is commonly accepted that nucleotide-sugars are the immediate substrates for the glycosyItransferases that drive pectin biosynthesis. Radiolabeled nucleotidesugars have been used in vitro as substrates to assay a number of pectin biosynthetic glycosy Itransferases including al,4-galacturonosyltransferase (Sterling et al, 2001; Takeuchi and Tsumuraya, 2001) and galactosyltransferase (Geshi et al, 2000; Peugnet etal, 2001). Figure 3.3 shows a summary of the nucleotidesugar interconversion pathways that are believed or proposed to be the primary pathways for the synthesis of the activated sugars required for pectin synthesis (Feingold and Avigad, 1980; Feingold and Barber, 1990; Mohnen, 1999; Gibeaut, 2000; Reiter and Vanzin, 2001; Ridley et al., 2001). Although the interconversion pathways are likely the main pathways for the synthesis of the nucleotide-sugars, there is a set of alternative pathways for the synthesis of the NDP-sugars known as the salvage pathway. In the salvage pathway L-Ara, D-Gal, D-Man (mannose), D-GalA, D-GlcA, L-Rha, L-Fuc (fucose), D-Glc (glucose) and D-Xyl (xylose) are recycled from the wall by conversion into sugar-1-phosphates

BIOSYNTHESIS OF PECTINS

6!

by the action of C-1-kinases (Hassid et al, 1959; Hassid, 1967; Feingold and Avigad, 1980; Feingold and Barber, 1990). The resulting sugar-1-phosphates are transformed into nucleotide-sugars by pyrophosphorylases that transfer the nucleoside monophosphate (NMP) from a nucleoside triphosphate (NTP) onto the phosphate of the sugar-1 -phosphate with the release of pyrophosphate: sugari-P + NTP -» NDP-sugar + PPj (Hassid et al, 1959). The so-called salvage pathway is thought to function in the re-utilization of glycosyl residues following turnover of the wall. The synthesis of UDP-GlcA from GlcA-l-P derived from myoinositol (the myoinositol pathway) (Hassid, 1967; Feingold and Barber, 1990) also uses, in part, some of the enzymes from the salvage pathway. The genes for some of the enzymes in the salvage pathway have been identified, including galactokinase (AGK1 and GAL1) (Kaplan et al., 1997; Sherson et al., 1999), arabinose kinase (ARA1) (Sherson et al., 1999), and GDP-D-mannose pyrophosphorylase (also referred to mannose-1 -phosphate guanylyltransferase) (VTC1 and CF77) (Keller et al, 1999; Lukowitz et al., 2001). Although the amount of each type of glycosyl residue required for wall synthesis will depend on the exact structure of pectin being synthesized in that cell type at that specific point of development, a comparison of the glycosyl residue composition of pectins isolated from walls of sycamore cell suspensions (Eberhard et al., 1989), tobacco cell suspensions (Mohnen et al, 1996) and Arabidopsis leaves (Zablackis et al, 1996) (see Table 3.1) gives an indication of the relative proportion of the different glycosyl residues in pectin. The most abundant glycosyl residue in pectin is D-GalA followed by, depending on the species and cell type, L-Ara, L-Rha, D-Gal, D-GlcA and D-Xyl. The other glycosyl residues required for pectin synthesis (i.e. L-Fuc, L-Gal, D-Apiose, L-aceric acid, D-Kdo (ketodeoxymanno-octulopyranosylonic acid), and D-Dha (deoxylyxo-heptulopyranosylaric acid)) represent ~ 1 % or less of normalized mol% of pectin (Ridley et al, 2001). The nucleotide-sugars involved in pectin synthesis (Mohnen, 1999; Ridley et al, 2001) and in general cell wall synthesis (Feingold and Avigad, 1980; Feingold and Barber, 1990; Gibeaut, 2000) and the molecular genetics of plant nucleotide-sugar interconversion pathways (Reiter and Vanzin, 2001) have been reviewed. The reader is directed to these reviews for additional background information. Here, only the salient features of the biosynthetic pathways will be presented. Hexose phosphates made directly from carbohydrate products of photosynthesis or from transported sucrose or stored starch are the precursors for the nucleotide-sugars (see Figure 3.3). As mentioned above, while it has traditionally been held that the nucleotide-sugars are synthesized on the cytosolic side of the Golgi, there is evidence that some nucleotide-sugars, such as UDPXyl, may at least in part be synthesized in the Golgi lumen (Hayashi et al, 1988). The nucleotide-sugars required for pectin synthesis will be described in the order of the abundance of their respective glycosyl residues in pectin, as based on the composition of the three different pectins shown in Table 3.1.

62

PECTINS AND THEIR MANIPULATION

Table 3.1 Comparison of the glycosyl residue composition2 of pectic polysaccharidesb released from tobacco, sycamore and Arabidopsis walls Normalized mol% Glycosyl residue

3

Galacturonic acid Arabinose Rhamnose Galactose Glucuronic acid Xylose Fucose Unknownf Mannose

Sycamore suspension

56.0 15.2 7.4 11.6 5.0 1.5 1.2 1.8

0

Tobacco suspension11

Arabidopsis leavese

74.2 2.6 6.4 1.4 7.5 0.5 1.0 6.5 0

-72.1 6.6 9.8 6.5 0.3 2.0 0.7 0

a

Glycosyl residues < \% of total are not shown. Pectic polysaccharides released from total walls by digestion with endopolygalacturonase. c Data from Eberhard et al. (1989). d Data from Mohnen et al. (1996). e Calculated from data in Zablackis et al. (1996). f An unidentified uronic acid. b

3.4.1 Uridine diphosphate-Qi-D-galacturonic acid (UDP-GalA) UDP-GalA is a substrate for the synthesis of all the pectic polymers (i.e. HGA, RG-I, RG-II). The main route for synthesis of UDP-GalA in the plant is the 4-epimerization of UDP-GlcA catalyzed by UDP-glucuronate 4-epimerase (UDP-GlcA 4-epimerase) (EC 5.1.3.6) (Neufeld et al., 1958; Feingold et al.. 1960; Ankel and Tischer, 1969; Feingold and Avigad, 1980; Mitcham et al.. 1991; Liljebjelke et al, 1995; Eidson et al., 1996). The reaction is believed to proceed through a 4-keto intermediate and may require a tightly bound NAD^ coenzyme (Feingold and Avigad, 1980). UDP-glucuronate 4-epimerase activity has been recovered from plant homogenates as both soluble and membranebound enzyme (Feingold et al.. 1960; Liljebjelke et al., 1995).

The biochemically best-characterized plant UDP-GlcA 4-epimerase is that from the blue green alga Anabaena flos-aquae (Gaunt et al., 1974). The Anabaena UDP-GlcA 4-epimerase has a Km for UDP-GlcA of 37 p. M, a pH optimum of 8.5, and an equilibrium constant of 2.6 in the direction of UDPGalA formation (Gaunt et al., 1974). Crude extracts from many different plant species have been shown to have UDP-GlcA 4-epimerase activity and have been used to make radiolabeled UDP-GalA via the 4-epimerization of UDPGlcA radiolabeled in the sugar (Neufeld et al., 1958; Feingold et al., 1960; Mitcham et al., 1991; Liljebjelke et al.. 1995) or in the nucleotide (Orellana

BIOSYNTHESIS OF PECTINS

63

and Mohnen, 1999) moiety. UDP-[14C]GalA has also been synthesized for in vitro use by the enzymatic oxidation of UDP-[14C]Gal to UDP-[14C]GalA (Rao and Mendicino, 1976; Kelleher and Bhavanandan, 1986; Basu etal, 2000) with a yield of >90% UDP-[14C]GalA using a simple polyethyleneiniine (PEIcellulose) column chromatography purification step (Basu et al., 2000). We find it necessary to purify the UDP-[I4C]GalA synthesized from UDP-[14C]Gal by high-pressure liquid chromatography (HPLC) to remove contaminants that inhibit al,4-galacturonosyltransferase activity. With the HPLC purification step we obtain ~27% yield of UDP-[l4C]GalA (J. Sterling and D. Mohnen, unpublished results). The UDP-GlcA 4-epimerase from plants has not been purified to homogeneity, nor has its gene been cloned. However, a gene (Capl3) for a bacterial UDP-GlcA 4-epimerase from Streptococcus pneumoniae type 1 has been identified (Munos et al, 1999). The bacterial enzyme has a Km for UDPGlcA of 240 [JtM, a pH optimum of 7.5, an equilibrium constant of 1.3 in the direction of UDP-GalA, and Mr of 80,000. The bacterial enzyme appears to require a tightly bound NAD+ for activity (Munos et al., 1999). Reiter and Vanzin (2001) report that BLAST searches of the Ambidopsis genome yields six predicted coding regions with high degrees of sequence similarity to the CapU gene from Streptococcus pneumoniae. Definitive evidence that these putative UDP-GlcA epimerase genes encode functional UDP-GlcA 4-epimerase has not yet been reported. 3.4.2

Uridine diphosphate-fi-L-arabinose (UDP-L-Ara)

UDP-L-Ara is a substrate for the synthesis of RG-I and RG-II. UDP-L-Ara is formed by the 4-epimerization of UDP-Xyl catalyzed by UDP-arabinose 4epimerase (EC 5.1.3.5) (Feingold et al., 1960; Fan andFeingold, 1970; Feingold and Avigad, 1980; Robertson et al, 1995). UDP-arabinose 4-epimerase activity has been identified in paniculate preparations from multiple plant species (Feingold and Avigad, 1980).

A partially purified UDP-xylose 4-epimerase from wheat germ (Fan and Feingold, 1970) was shown to have a pH optimum of 8.0, an apparent Km of 1.5 mM for UDP-Xyl and 0.5 mM for UDP-L-Ara, and an equilibrium constant of 0.8 in the direction of UDP-Ara (Fan and Feingold, 1970). A comparable equilibrium constant of 1.0 has been reported for the UDP-xylose 4-epimerase from mung bean (Feingold et al., 1960). If NAD+ is required for the reaction, it must be tightly bound to the enzyme (Feingold and Avigad, 1980). The wheat germ UDP-xylose 4-epimerase (Fan and Feingold, 1970; Feingold and Avigad, 1980) has been used to synthesize non-radiolabeled and radiolabeled UDP-^-L-arabinopyranose (Pauly et al, 2000) for use for in vitro wall polymer

64

PECTINS AND THEIR MANIPULATION

biosynthesis studies. The Ara in the synthesized UDP-Ara is in the pyranose form, while the predominant form of arabinose is arabinofuranose in the wall polysaccharides, proteoglycans, arabinans and arabinogalactan proteins (Hassid et al., 1959; Feingold and Avigad, 1980; Carpita, 1996). It is not known when the mutorotation occurs; however, it is believed to occur during polysaccharide biosynthesis, presumably by arabinosyltransferase(s) that catalyze ring rearrangement before formation of the glycosidic bond (Carpita, 1996). An Arabidopsis mutant (mur4) has been identified that has reduced membranebound UDP-xylose 4-epimerase activity in the leaves, cotyledons and flowers (Burget and Reiter, 1999). 3.4.3

Undine diphosphate-$-L-rhamnose (UDP-L-Rha)

L-Rhamnose is a component of RG-I and RG-II. Plants such as tobacco (Barber, 1963), mung bean (Barber, 1962), Silene dioica (Kamsteeg et al., 1978), pummelo (Citrus maxima) (Bar-Peled et al., 1991) and Chlorella pyrenoidasa (Barber and Chang, 1967) can convert UDP-D-Glc to UDP-L-rhamnose in an NADH-dependent reaction (reviewed in Feingold and Avigad, 1980; Feingold and Barber, 1990). UDP-4-keto-6-deoxy-D-Glc is an intermediate in the conversion (Barber, 1963; Barber and Chang, 1967; Kamsteeg et al., 1978). UDPRha has been shown to be a substrate in plants for the rhamnosylation of secondary metabolites such as flavonoid-glycosides (Bar-Peled et al., 1993) and it is assumed that UDP-L-Rha is the nucleotide-sugar substrate for the synthesis of RG-I and RG-II. However, it should be noted that it has not yet been experimentally confirmed that UDP-Rha is the substrate for RG-I and RGII synthesis. A biosynthetic scheme for the synthesis of UDP-Rha in plants has been proposed (Kamsteeg et al., 1978; Feingold and Avigad, 1980; Mohnen. 1999) based on the rfb genes-encoded pathway for the synthesis of dTDPrhamnose from dTDP-glucose in bacteria (Stevenson et al.. 1994). The proposed pathway is shown in equation 3.

Based on the bacterial pathway, the proposed pathway for UDP-Rha synthesis in plants begins with the conversion of UDP-D-Glc to UDP-4-keto-6-deoxy-Glc catalyzed by UDP-glucose 4,6-dehydratase (EC 4.2.1.76). The UDP-4-keto6-deoxy-Glc is subsequently epimerized to UDP-4-keto-6-deoxy-L-mannose

BIOSYNTHESIS OF PECTINS

65

by UDP-4-keto-L-rhamnose 3,5-epimerase. Finally, the UDP-4-keto-6-deoxyL-mannose is reduced to UDP-L-rhamnose by UDP-4-ketorhamnose reductase. None of these enzymes has been purified to homogeneity or cloned in plants. It is also not clear how many enzymes would encode the required enzyme activities. Blast searches of the Arabidopsis genome database using E. coli dTDP-Lrhamnose biosynthetic genes (E. coli has unique genes for each of the three required enzymatic activities for dTDP-rhamnose synthesis) have identified three Arabidopsis genes with significant sequence similarity to dTDP-D-glucose 4,6-dehydratase (Reiter and Vanzin, 2001). Based on the primary sequence of these genes, it has been proposed that they may each encode all three enzymatic activities required for UDP-Rha synthesis (Reiter and Vanzin, 2001). Blast searchers have also identified other Arabidopsis genes with sequence similarity to individual E. coli genes that encode only one of the enzymatic activities required for dTDP-Rha synthesis (see Reiter and Vanzin, 2001). However, direct proof that any of these Arabidopsis putative UDP-Rha biosynthetic genes actually encodes a UDP-Rha biosynthetic protein has not yet been reported. 3.4.4 Uridine diphosphate-a-D-galactose (UDP-Gal) UDP-Gal is a substrate for the synthesis of RG-I and RG-II. UDP-Gal is formed from UDP-Glc by a 4-epimerization catalyzed by UDP-Glc 4-epimerase (EC 5.1.3.2) (Fan and Feingold, 1969; Feingold and Avigad, 1980). The reaction mechanism includes an enzyme-bound UDP-4-keto-hexose intermediate (Maxwell, 1957; Maitra and Ankel, 1971; Wee and Frey, 2001) which binds the enzyme approximately 100 times more tightly than UDP-Glc (Feingold and Avigad, 1980; Wee and Frey, 2001).

The structure of the enzyme from E. coli has been determined by X-ray crystallography (Bauer et a/., 1992). The bacterial enzyme comprises two identical 39.5 kDa subunits (Wilson and Hogness, 1969; Bauer etal., 1992), each of which binds a NAD+ cofactor (Bauer et al., 1992). Each subunit folds into a distinct N-terminal domain, primarily responsible for NAD+/NADH positioning, that has a seven-stranded parallel fi-pleated sheet flanked on either side by ot-helices. The small C-terminal motif is responsible for binding the UDP-sugar (Thoden et al., 1996a, 1996b; Thoden and Holden, 1998). The active site is located between the two domains. The size of the enzyme varies in different species, as does the tightness by which the enzyme binds NAD+. For example, the bovine enzyme is a monomer of 40 kDa that requires exogenous NAD+ for activity while the UDP-D-Glc 4-epimerase from Candida pseudotropicalis is made up of two identical 60 kDa subunits each of which contains one tightly bound NAD+ (Maxwell, 1957; Geren and Ebner, 1977; Feingold and Avigad, 1980).

66

PECTINS AND THEIR MANIPULATION

The UDP-Glc 4-epimerase from leaves of Vicia faba is a soluble cytoplasmic protein with a pH optimum of 8.8 and an apparent Km for UDP-Gal of 95 jiM (Konigs and Heinz, 1974). A UDP-Glc 4-epimerase purified from wheat germ extract has Mr of 100000 and requires NAD+ for activity (Fan and Feingold, 1969; Feingold and Avigad, 1980). The tightness of binding of NAD+ to plant UDP-Glc 4-epimerase appears to be species-specific (Feingold and Avigad. 1980) and both soluble and membrane-bound activities have been recovered in plants (Feingold and Avigad, 1980). An Arabidopsis gene for UDP-Glc 4epimerase (UGE1) has been cloned and expressed in E. coli (Dormann and Benning, 1996). The Arabidopsis expressed protein encodes a 39 kDa protein with a broad pH optimum from 7.0 to 9.55 and an apparent Km for UDP-Glc of 110 M^M (Dormann and Benning, 1996). A cDNA encoding a predicted 39kDa putative UDP-Glc 4-epimerase from pea (Pisum sativum) that has 92% sequence homology to the Arabidopsis gene has also been cloned (Lake et a!., 1998) although no characteristics of the enzyme have been reported. Two cDNAs that encode UDP-Glc epimerases of 39.3 and 38.4 kDa from developing seeds of guar (Cyamopsis tetragonoloba) endosperm have also been reported (Joersbo et al, 1999). Analysis of the Arabidopsis genome has led to the identification of four coding regions with significant sequence identity to UGE1 (Reiter and Vanzin, 2001), two of which (UGE2 and UGE3) encode functional UDP-Glc epimerases (Reiter and Vanzin, 2001). 3.4.5

Undine diphosphate-oi-D-glucuronic acid (UDP-Glc A)

UDP-D-Glucuronic acid is the likely substrate for the incorporation of GlcA into RG-II and into some side branches of RG-I. UDP-GlcA is produced either by the oxidation of UDP-Glc catalyzed by UDP-Glc 6-dehydrogenase (Feingold et al., 1960; Feingold and Avigad, 1980) or by the uridylation of Glc-l-P via the myoinositol pathway (Feingold and Avigad, 1980).

UDP-Glc 6-dehydrogenase (EC 1.1.1.22) catalyzes the 4-electron oxidation of UDP-Glc at C-6 and the reduction of two moles of NAD+ (Feingold and Avigad, 1980; Feingold and Franzen, 1981). The reaction is ordered: beginning with binding of UDP-Glc, followed by binding of NAD"1" (Feingold and Avigad, 1980; Hempel et al, 1994; Campbell et al, 2000), reduction of the first bound NAD+ and release of the first NADH. This is followed by binding of the second NAD+, reduction and release of the second NADH and finally release of the UDP-GlcA (Feingold and Avigad, 1980; Campbell et al, 1997). Bovine UDPGlc 6-dehydrogenase consists of six 52 kDa subunits (Zalitis and Feingold, 1969; Gainey et al., 1972; Franzen et al, 1978; Feingold and Avigad, 1980: Jaenicke et al., 1986; Hempel et al., 1994) with one mole of substrate bound per

BIOSYNTHESIS OF PECTINS

67

two moles of enzyme (i.e. 'half-of-the-site' behavior) (Franzen et al, 1978; Hempel et al., 1994). In contrast, UDP-Glc 6-dehydrogenase from E. coll consists of two identical subunits of 50 kDa each (Schiller et al., 1976; Feingold and Franzen, 1981). The X-ray crystal structure of UDP-glucose dehydrogenase from the bacteria Streptococcus pyogenes has been solved (Campbell et al., 2000). The S. pyogenes UDP-Glc dehydrogenase appears to exist as either a monomer or dimer in solution. Each monomer consists of two discrete a/|3 domains connected by a long a-helix. Each domain consists of a core (3-sheet sandwiched between a-helices. The N-terminal domain contains a sixstranded parallel p-sheet that binds NAD+ (Campbell et al., 2000). UDPGlc 6-dehydrogenases have been purified 1000-fold from pea ((Strominger and Mapson, 1957), 12-fold from germinating lily (Lilium longiflorum) pollen (Davies and Dickinson, 1972), 62-fold from soybean nodules (Stewart and Copeland, 1998), and 341-fold from elicitor-treated French bean (Phaseolus vulgaris L) cell suspensions (Robertson et al., 1996). The apparent Km values for UDP-Glc for the different enzymes were 70|xM, 300 jxM, 50|iM, and 5.5mM, respectively, and the apparent Km values for NAD+ were 115|iM, 400 |iM, 120 |iM and 20 |xM. The soybean enzyme has a pH optimum of 8.4, a native molecular mass of 272 kDa, and a subunit molecular mass of 47 kDa, suggesting that the enzyme functions as a hexamer (Stewart and Copeland, 1998). All known eukaryotic UDP-Glc 6-dehydrogenases are cooperatively inhibited by UDP-Xyl, suggesting a feedback inhibition of the enzyme by UDP-Xyl (Feingold and Avigad, 1980; Campbell et al, 1997). A cDNA clone for UDP-Glc 6-dehydrogenase from soybean that is highly homologous to the cloned bovine UDP-GlcDH gene (Hempel et al., 1994) encodes a protein with a predicted molecular mass of 52.9 kDa (Tenhaken and Thulke, 1996). The soybean gene has a conserved NAD+-binding site motif and contains the catalytic Cys residue (Hempel et al., 1994; Tenhaken and Thulke, 1996). An Arabidopsis gene (UGD) encoding UDP-Glc dehydrogenase has been identified and its gene expression has been studied using (3-glucuronidase and green fluorescent protein reporter constructs (Seitz et al., 2000). Three additional putative UDP-Glc dehydrogenases have been identified in Arabidopsis via sequence analysis (Reiter and Vanzin, 2001). The four genes share 83-93% sequence identity (Seitz et al., 2000). The protein with UDP-Glc 6-dehydrogenase activity that was purified from French bean (Robertson et al., 1996) does not share the characteristics of the cloned UDP-GlcDH from soybean (Strominger and Mapson, 1957) or Arabidopsis (Seitz et al, 2000). The putative UDP-GlcDH from French bean (Robertson et al, 1996) has a molecular mass of 40 kDa, a high apparent Km of 5.5 mM for UDP-Glc, co-purifies with alcohol dehydrogenase activity and is preferentially located in cells that make secondary walls. It is unclear whether the 40 kDa protein from French bean represents a bona fide. multifunctional UDP-GlcDH preferentially expressed during secondary wall synthesis (Robertson et al., 1996), or whether it is an alcohol dehydrogenase

68

PECTINS AND THEIR MANIPULATION

that can oxidize UDP-Glc in vitro but plays little or no role in the formation of UDP-GlcA in planta. 3.4.6

Uridine diphosphate-ct-D-xylose (UDP-Xyl)

UDP-Xyl is the expected substrate for the synthesis of xylogalacturonan and RG-II. UDP-Xyl is produced by the decarboxylation of UDP-GlcA catalyzed by UDP-GlcA carboxylase (EC 4.1.1.35) (Feingold et al., 1960; Feingold and Avigad, 1980; Hayashi etal, 1988; Hannapel, 1991).

UDP-GlcA decarboxylase contains a tightly-bound NAD+ and catalyzes a reaction that proceeds via a UDP-4-keto-hexose intermediate (Feingold and Avigad, 1980). Partially purified UDP-GlcA decarboxylase from wheat germ (John et al., 1977) has a pH optimum of 7.0 and consists of two 210kDa isoenzymes that do not require exogenous NAD+ for activity. Both isozymes are activated by low (< 100 |iM) concentrations of UDP-GlcA, indicating cooperative allosteric regulation by UDP-GlcA (John et al., 1977). The apparent Km values of the fully activated wheat germ isozymes for UDP-GlcA were 0.18 mM. and 0.53 mM, respectively. Both UDP-GlcA decarboxylase isozymes are allosterically inhibited by UDP-Xyl (John etal., 1977). UDP-GlcA decarboxylase is recovered as both a soluble and membrane-bound enzyme (Hayashi et al, 1988). In soybean, the membrane-bound UDP-GlcA carboxylase has a pH optimum of 6.0-7.5 and an apparent Km of 240 \jM for UDP-GlcA (Hayashi et al.. 1988) while the soluble UDP-GlcA carboxylase has an apparent Km of 700 |iM (Hayashi et al., 1988). At least some of the UDP-GlcA carboxylase activity has been reported to reside within the lumen of the Golgi (Hayashi et al., 1988). The first gene for UDP-GlcA decarboxylase was identified in Cryptococcus neoformans (Bar-Peled et al., 2001). The C. neoformans gene encodes a 46.5 kDa protein that catalyzes the production of UDP-Xyl from UDP-GlcA. The enzyme has an apparent Km for UDP-GlcA of ~700jiM. a pH optimum of 7.5 and is inhibited by NADH, UDP and UDP-Xyl. Three Arabidopsis genes shown to encode functional UDP-D-glucuronate decarboxylases (accessions AF387787, AF387788, AF387789) have been identified (M. Bar-Peled. unpublished). The purification and partial sequencing of UDP-D-glucuronate decarboxylases from pea has led to the identification of a pea gene (accession BAB40967) that encodes a functional UDP-GlcA decarboxylase (see Reiter and Vanzin, 2001). 3.4.7 Guanosine diphosphate-fi-L-fucose

(GDP-Fuc)

GDP-L-Fuc is the likely substrate for the synthesis of RG-II and for some of the side branches of RG-I. Soluble enzyme preparations from various plant

BIOSYNTHESIS OF PECTINS

69

species can convert GDP-D-Man to GDP-L-Fuc (Liao and Barber, 1971). The synthesis of GDP-L-Fuc using enzyme preparations from Phaseolus vulgaris requires NADPH or NADH, occurs at a pH optimum of 6.9-7.8, has an apparent Km for GDP-D-Man of 160|JiM (Liao and Barber, 1971) and an apparent molecular mass of 120 kDa (Liao and Barber, 1972). The reaction proceeds via the C-4 oxidation and C-6 reduction of GDP-Man catalyzed by GDPD-Man 4,6-dehydratase (EC 4.2.1.47) (Liao and Barber, 1971; Feingold and Avigad, 1980). The product formed, GDP-4-keto-6-deoxy-D-mannose, appears to tightly bind a GDP-4-keto-6-deoxy-D-Man 3,5-epimerase, which converts it to a GDP-4-keto-6-deoxy-L-galactose intermediate (Feingold and Avigad, 1980; Bonin et at, 1997) that is then reduced by a GDP-4-keto-L-fucose reductase activity to yield GDP-L-fucose (Feingold and Avigad, 1980). Although it was previously thought that the final two enzyme activities might reside on separate proteins, it has been shown in humans (Sullivan et al, 1998) and in transgenic Saccharomyces cerevisiae harboring the E. coli genes (Mattila et al.., 2000) that a single enzyme, GDP-keto-6-deoxymannose 3,5-epimerase4-reductase, catalyzes both the epimerization and reduction steps. The first enzyme in the pathway, GDP-D-Man-4,6-dehydratase, is inhibited by GDPfucose (Sullivan et al, 1998; Kornfeld and Ginsburg, 1966), indicating feedback inhibition.

The Ambidopsis mutant murl is defective in the synthesis of L-fucose in the aerial parts of the plant (Reiter et al, 1993). The MUR1 gene encodes a 41.9kDa GDP-D-mannose 4,6-dehydratase (Bonin et al, 1997). A second Ambidopsis gene, GMD1, encodes a second GDP-D-mannose 4,6-dehydratase that is highly expressed in roots (Bonin et al., 1997). TheArabidopsis gene GER1 encodes a GDP-keto-6-deoxymannose 3,5-epimerase-4-reductase (Bonin and Reiter, 2000). A second putative GDP-keto-6-deoxymannose 3,5-epimerase-4reductase gene, GER2, with 88% amino acid identity to GER1 has been identified by DNA sequence analysis (Reiter and Vanzin, 2001). The dwarf phenotype associated with murl-I and murl-2 was shown to be due to a substitution of Lgalactose for the L-fucose and 2-0-methyl-L-galactose for 2-0-methyl-L-fucose in RG-II. This change in RG-II structure results in a reduction in the amount of borate crosslinked RG-II dimer (O'Neill et al., 2001) that is present in the walls and leads to the dwarfism. This result provides unequivocal proof that the pectic polysaccharide RG-II is essential for normal plant growth (O'Neill et al, 2001).

70

3.4.8

PECTINS AND THEIR MANIPULATION

Uridine diphosphate-a-D-apiose (UDP-apiose)

UDP-apiose is a substrate for the synthesis of the species-specific substituted galacturonan apiogalacturonan that is found in some aquatic monocotyledonous plants such as Spirodela polyrrhiza (Watson and Orenstein, 1975) and Lemna minor (Hart and Kindel, 1970). Apiogalacturonan is a homogalacturonan in which D-apiose or apiobiose (D-Api/-pl,3-D-apiose) are attached to O-2 or O3 of HGA. UDP-apiose is also the likely substrate for the synthesis of RG-II (O'Neill etal., 2001; Ridley etal., 2001). UDP-apiose is formed by a decarboxylation and rearrangement of UDP-GlcA catalyzed by a NAD+-dependent UDPapiose/UDP-Xyl synthase (Wellmann and Grisebach, 1971; Baron et al.. 1973; Kindel and Watson, 1973; Watson and Orenstein, 1975; Matern and Grisebach. 1977; Feingold and Barber, 1990). UDP-Xyl is also a product of the in vitro enzymatic reaction (Matern and Grisebach, 1977) with UDP-apiose:UDP-Xyl ratios of 1.4 reported (Matern and Grisebach, 1977). The UDP-apiose synthase and UDP-Xyl synthase activities could not be separated in a 1400-fold purified protein preparation, leading to the suggestion that a single multifunctional protein is responsible for both activities (Wellmann and Grisebach, 1971; Matern and Grisebach, 1977). However, it has more recently been suggested that xylose may be an artificial product recovered in in vitro reactions (Gardiner et al., 1980) and the name UDP-apiose synthase has been used for the enzyme (Gardiner et al., 1980). It is believed that UDP-apiose synthesis occurs via the formation of an L-tf/reo-4-pentosulose intermediate, common to both UDP-apiose and UDP-Xyl formation, followed by ring contraction and epimerization (Matern and Grisebach. 1977).

Partially purified UDP-apiose/UDP-Xyl synthase from Lemna minor has optimum activity at M mM NAD+ and a pH of 8.0-8.3 (Kindel et al., 1911). Partially purified UDP-apiose/UDP-Xyl synthase from parsley (Matern and Grisebach, 1977) is composed of an 86 kDa protein consisting of two identical 44 kDa subunits and a 65 kDa protein consisting of two identical 34 kDa subunits (Matern and Grisebach, 1977). The 86 kDa protein contains all the enzyme activity, binds 0.5 mol of UDP-GlcA per mol of protein and, in the presence of UDP-GlcA, binds 0.5 mol NAD+ per mol of catalytic protein (Matern and Grisebach, 1977). The 65 kDa protein is enzymatically inactive but was reported to be required for stability of the 86 kDa protein (Matern and Grisebach. 1977). UDP-D-glucose and UDP-methyl-D-GlcA are competitive inhibitors of UDPapiose/UDP-Xyl synthase (Gebb etal.. 1975).

BIOSYNTHESIS OF PECTINS

71

3.4.9 Guanosine diphosphate-$-L-galactose (GDP-Gal) GDP-L-Gal is a possible substrate for the L-Gal in RG-II and for the L-Gal that is substituted for the L-Fuc (Zablackis etal., 1996) in xyloglucan synthesized in Arabidopsis murl mutants. Murl mutants have a mutation in GDP-D-mannose 4,6-dehydratase and, thus, have reduced amounts of L-Fuc in the aerial portions of the plant that is replaced with L-Gal (Zablackis et al, 1996). It has been proposed that GDP-D-Man is converted to GDP-L-Gal by GDP-D-mannose 3,5epimerase (Feingold and Avigad, 1980). GDP-D-mannose 3,5-epimerase

The reversible 3,5-epimerization of GDP-Man has been reported using extracts from Chlorella pyrenoidosa (Hebda et al, 1979; Hebda and Barber, 1978). The partially purified Chlorella GDP-D-mannose 3,5-epimerase had a molecular mass of lOOkDa, a broad pH optimum centering at 8.1, an apparent Km of 96|xM for GDP-D-Man and 97 \)M for GDP-L-Gal, and an equilibrium constant of 2.9 in the direction of GDP-Man (Hebda et al, 1979). 3.4.10 XXX-Kdo, XXX-Dha and XXX-aceric acid The identity and biosynthetic pathways for the activated glycosyl donors of the Kdo, Dha and aceric acid in RG-II have not been experimentally established in plants. In contrast, a great deal of information is available regarding the synthesis in bacteria of cytidine 5/-monophosphate-3-deoxy-D-ma/zwo-octulosonate (CMP-Kdo), the activated donor of the Kdo found in lipopolysaccharides and other extracellular bacterial poly saccharides (Unger, 1981; Raetz, 1990; Pazzani et al, 1993; Baasov and Kohen, 1995; Rosenow et al, 1995; Jelakovic et al, 1996; Jelakovic and Schulz, 2001). Assuming that plants use CMP-Kdo to synthesize RG-II, and that they synthesize CMP-Kdo using a similar pathway as bacteria, the following pathway is proposed. D-Ribulose 5-phosphate is isomerized to D-arabinose 5-phosphate by D-arabinose-5-phosphate isomerase (Unger, 1981). The D-arabinose 5-phosphate is condensed with phosphoenolpyruvate by Kdo-8-phosphate synthetase (2-dehydro-3-deoxyphosphooctonate aldolase) to form Kdo 8-phosphate (2-dehydro-3-deoxy-D-octonate 8-phosphate) (Unger, 1981; Doong et al, 1991). The 8-phosphate is removed from Kdo 8-phosphate by Kdo-8-phosphate phosphatase to produce Kdo and inorganic phosphate (Unger, 1981). Finally, CMP-Kdo and pyrophosphate are formed from cytidine 5'-triphosphate (CTP) and Kdo by CMP-Kdo synthetase (Unger, 1981). Kdo8-phosphate synthetase has been identified in multiple plant species and has been partially purified from spinach (Doong et al, 1991). Kdo-8-phosphate

72

PECTINS AND THEIR MANIPULATION

synthetase has a pH optimum of 6.2, an apparent Km of 270 [iM for arabinose 5-phosphate and an apparent Km of 35 jiM for phosphoenolpyruvate (Doong et al., 1991). A cDNA from pea has been identified that encodes a 31.7kDa functional Kdo-8-phosphate synthetase when expressed in E. coli (Brabetz et al, 2000). The expressed pea enzyme has a pH optimum of 6.1. The identity and the biosynthetic pathway in plants for the activated glycosyl donor for Dha (3-deoxy-D-lyxo-2-heptulosaric acid) is not known. However, it has been proposed that 3-deoxy-D-arab/no-heptulosonate 7-phosphate synthase could catalyze the condensation of phosphoenolpyruvate with threose to generate a precursor of Dha (Doong et al., 1992). A cytosolic form of 3deoxy-D-arabmo-heptulosonate 7-phosphate synthase with a wide substrate specificity has been identified in plants (Doong et al., 1992). cDNAs encoding plastidic 3-deoxy-D-ara£wo-heptulosonate 7-phosphate synthases involved in the shikimate pathway leading to aromatic secondary metabolism have been identified from potato (Solanwn tuberosum L.) (Dyer et al., 1990) and other plant species (Herrmann, 1995). An alternative route for the synthesis of the activated donor of Dha might be the interconversion of CMP-Kdo to CMPDha through oxidation and decarboxylation reactions. There is no information available regarding the nature or mode of synthesis of the activated donor for aceric acid.

3.5 Glycosyltransferases involved in pectin biosynthesis Elucidating the mechanism by which polysaccharides are synthesized is a challenging endeavor. Polymer synthesis involves at least three stages: initiation, elongation and termination. While nothing is known about the initiation and termination of pectin synthesis, considerable effort has been directed towards identifying and characterizing glycosyltransferases that presumably catalyze the elongation of pectin. Such glycosyltransferases transfer a glycosyl residue from an activated sugar donor (e.g. a nucleotide-sugar) either onto endogenous pectic acceptors in the Golgi or onto exogenous oligosaccharide or polysaccharide pectic acceptors in in vitro reactions using permeabilized Golgi or microsomes or detergent-solubilized enzymes. Although it has recently been proposed to be useful to make a distinction between the terms glycosyltransferase and glycan synthase (Perrin et al., 2001) when discussing polysaccharide synthesis, it appears premature at this time to make this distinction for the pectin biosynthetic enzymes. Glycan synthases have been defined as enzymes that synthesize the backbone of a polysaccharide (i.e. the a 1,4-linked D-galactosyluronic acid back bone of HGA or the alternating [a-D-GalA-l,2-a-L-Rha-l,4->] backbone of RG-I). It is often presumed that such enzymes should act processively, that is, catalyze multiple rounds of catalysis before releasing their oligosaccharide/polysaccharide acceptor. It is likely that such processivity. if it occurs

BIOSYNTHESIS OF PECTINS

73

for pectin synthesis, could require protein complexes. Such complexes may, or may not, remain intact in the homogenates and fractions used for studying pectin synthesis. Since both RG-I and the substituted regions of HGA (e.g. RG-II) are highly branched, it is not clear that it would be more favorable for overall biosynthetic rates and structure fidelity for pectin synthesis to occur processively or distributively. No evidence for in vitro processivity of pectin synthesis has yet been reported. For these reasons all the pectin biosynthetic enzyme activities identified to date are defined in this review as glycosyltransferases. If these glycosyltransferases are eventually shown to act processively when associated with other polypeptides in complexes, then it is recommended that the appropriate glycan synthase name be used for the holoenzyme complex. Alternatively, if the glycosyltransferases identified to date are eventually shown to act processively under specific reaction conditions (e.g. in the presence of specific cofactors or substrates), then it is recommended that these enzymes be named their corresponding glycan synthase, as put forward by Perrin et al. (2001). As mentioned above, it is not known whether all of the pectic polysaccharides are synthesized as a single polysaccharide or whether separate populations of polymers are synthesized. It is likely that the substituted galacturonan named RG-II is synthesized in the same polymer that contains contiguous regions of HGA, since RG-II isolated from cell walls is extended on both ends by regions of HGA. It is also possible that some HGA is synthesized without substituted HGA regions. It is less clear whether the syntheses of HGA and RG-I occur on the same polysaccharide chain or whether they occur as independent biosynthetic events. Work from the groups of Voragen (Schols et ai, 1995) and Mort (Yu and Mort, 1996), suggesting that RG-I is covalently attached to xylogalacturonan-like regions, indicates that RG-I and HGA may exist as a single polymer. In the following discussion, the enzymes activities required for the synthesis of pectin will be divided into those required for HGA synthesis, substituted galacturoronan synthesis (including RG-II and xylogalacturonan), and RG-I synthesis. 3.5.1 Synthesis of homo galacturonan Homogalacturonan (HGA) is a partially methyl-esterified and acetylated homopolymer of od,4-linked D-galactosyluronic acid (Ridley et al., 2001). It is unclear how much the degree of polymerization (DP) of HGA varies within pectin; however, a DP range of 72-100 has been reported (Thibault et al., 1993). As shown in Table 3.2, the synthesis of HGA requires at least one homogalacturonan al,4-galacturonosyltransferase (HGA-GalAT) (also referred as polygalacturonate: al,4-galacturonosyltranferase), a homogalacturonanmethyltransferase (HGA-MT) (also referred to as pectin methyltransferase), and a homogalacturonan 3-O-acetyltransferase (HGA-AT).

74

PECTINS AND THEIR MANIPULATION

Table 3.2 Glycosyltransferase activities required for HGA biosynthesis" Enzyme b Type of transferase

Acceptor substrate

Enzyme activity

Reference for structure

GlycosylD-GalAT

*GalAal.4-GalA

al.4-GalAT

O'Neill etal. (1990)

GalAal.4-GalA<,,,

HGA-MT

Goubeletal. (1998): Vznnieretal. (1992): Kaussf/a/. (1967)

GalAal.4-GalA,, M

HGA-AT

Ishii(1997):DeVries*>/a/. (1986): Ishii( 1995): Rombouts and Thibault (1986)

MethylHGA methyltransferase (HGA-MT) AcetylHGA: GalA 3-0acetyltransferase (HGA-AT) a

Adapted from Ridley et al. (2001). All sugars are D sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to the glycosyl residue on the left* of the indicated acceptor. b

3.5.1.1 Homogalacturonan galacturonosyltransferase (HGA-GalAT) Membrane-bound a 1,4-galacturonosyltransferase (GalAT) activity has been identified and partially characterized in mung bean (Villemez et al., 1966; Kauss and Swanson, 1969), tomato (Lin et al., 1966), turnip (Lin et al., 1966). and sycamore (Bolwell et al., 1985), tobacco suspension (Doong et al., 1995). radish roots (Mohnen et al., 1999), enriched Golgi from pea (Sterling et al., 2001), Azuki bean (Vigna angularis) (Takeuchi and Tsumuraya, 2001) and Arabidopsis (Sterling and Mohnen, unpublished results) (see Table 3.3). The Gal AT from pea has been localized to the Golgi (Sterling et al., 2001) with its catalytic site facing the lumenal side of the Golgi, (Sterling et al., 2001). These results provide the first direct enzymatic evidence that the synthesis of HGA occurs in the Golgi. In in vitro reactions, GalAT adds [14C]GalA from UDP-[14C]GalA (Liljebjelke et al., 1995) onto endogenous acceptors in microsomal membrane preparations to produce radiolabeled products of large molecular mass (i.e. ~105kDa in tobacco microsomal membranes (Doong et al., 1995) and >500kDa in pea Golgi (Sterling et al., 2001)). The cleavage of up to 89% of the radiolabeled product into GalA, digalacturonic acid (diGalA) and trigalacturonic acid (triGalA) following exhaustive hydrolysis with a purified endopolygalacturonase confirmed that the product synthesized by tobacco GalAT was largely HGA. The product produced in vitro in tobacco microsomes is ~50% esterified (Doong et al., 1995), while the product produced in pea Golgi did not appear to be esterified (Sterling et al., 2001). These results suggest that the degree of methyl-esterification of newly synthesized HGA may be species-specific and that methyl-esterification occurs after the synthesis of at least a short stretch of HGA. GalAT activity in detergentpermeabilized microsomes from etiolated azuki bean seedlings adds [ 14 C]GalA

7S

BIOSYNTHESIS OF PECTINS

Table 3.3 Comparison of catalytic constants and pH optimum of HGA otl,4-galacturonosyltransferases a ' b Apparent Km for UDP-GalA Enzyme11 a

Ga!AT GalAT GalAT GalAT GalAT GalAT GalAT (sol)c GalAT (per)'1

Plant source Mung bean Mung bean Pea Pea Sycamore Tobacco Tobacco Azuki bean

Vmm

(H.M)

pH optimum

(pmol mg~' min "" ' )

1.7 n.d. n.d.e n.d. 770 8.9 37 140

6.0 n.d. 6.0 n.d. n.d. 7.8 6.3-7.8 6.8-7.8

-4700 n.d. n.d. n.d. 9

150 290 2700

Reference Villemez et al. (1966) Crombie and Reid (2001 ) Gumming and Brett (1986) Sterling et al. (2001) Bol well etal. (1985) Doong era/. (1995) Doong and Mohnen ( 1 998) Takeuchi and Tsumuraya (2001)

"Adapted from Mohnen (1999). Un!ess indicated, all enzymes are measured in paniculate preparations. c (sol): detergent-solubilized enzyme. d (per): detergent-permeabilized enzyme. e n.d.: not determined. b

from UDP-[14C]GalA onto acid-soluble polygalacturonate (PGA) exogenous acceptors (Takeuchi and Tsumuraya, 2001). Treatment of the radiolabeled product with a purified fungal endopolygalacturonase yielded GalA and diGalA, confirming that the activity identified was GalAT. The azuki bean enzyme has a broad pH range of 6.8-7.8 and a surprisingly high specific activity of 1300-2000pmol mg"1 min" 1 , especially considering the large amount (3.14.1 nmol mg"1 min"1) of polygalacturonase activity that was also present in the microsomal preparations. As with the product made by tobacco, no evidence for the processive transfer of galactosyluronic acid residues onto the acceptor was obtained (see below). GalAT can be solubilized from membranes with detergent (Doong and Mohnen, 1998). Solubilized GalAT adds GalA onto the nonreducing end (Scheller et al., 1999) of exogenous HGA acceptors of degrees of polymerization >10 (Doong et al., 1995). The bulk of the HGA elongated in vitro by solubilized GalAT from tobacco membranes (Doong and Mohnen, 1998) and detergent-permeabilized Golgi from pea (Sterling et al., 2001) is elongated by a single GalA residue. These results suggest that solubilized GalAT in vitro acts nonprocessively, (i.e. distributively). The lack of in vitro processivity of the solubilized GalAT may indicate that the enzyme does not synthesize HGA in a processive manner in vivo, or it may be an artifact due to the dissociation of a required biosynthetic complex or cofactor(s)/substrate(s) during solubilization of the enzyme. Attempts to recover processive in vitro GalAT activity by using alternative pectic acceptors or high concentrations (up to lOmM) of

76

PECTINS AND THEIR MANIPULATION

UDP-GalA were not successful (H.F. Quigley and D. Mohnen, unpublished results; Ridley et al, 2001). There is also no evidence that the inclusion of the methyl donor S-adenosylmethionine (Takeuchi and Tsumuraya, 2001; Kauss and Swanson, 1969; Doong et al, 1995) and/or the acetyl donor acetyl-CoA promotes the processivity of Gal AT (H.F. Quigley and D. Mohnen, unpublished results; Ridley et al., 2001). Thus, the question of whether or not Gal AT in vivo is processive remains to be resolved. The gene for HGA-GalAT has not yet been identified, although efforts to purify the enzyme are on-going in several laboratories. 3.5.7.2 HGA methyltransferase (HGA-MT) The methyl-esterification of HGA at the C-6 carboxyl group is catalyzed by HGA methyltransferase (HGA-MT). Although the enzyme has been referred to as pectin methyltransferase, the term HGA-MT is preferred to distinguish HGA-MT from the enzymes that methylate RG-I or RG-II. HGA-MT has been identified in microsomal membranes from mung bean (Kauss et al, 1967. 1969; Crombie and Reid, 1998), flax (Vannier et al, 1992; Schaumann et al. 1993), tobacco (Goubet et al., 1998), and soybean (Ishikawa et al.. 2000) (see Table 3.4). Membrane-bound HGA-MTs from flax (Bruyant-Vannier et al, 1996; Bourlard et al., 1997) and tobacco (Goubet and Mohnen, 1999b) have been solubilized using detergent. Two apparent HGA-MT isozymes, PMT5 and PMT7. from flax have been reported with pH optima of 5.0 and 6.5, respectively (Bourlard et al, 2001). Efforts to purify these apparent isozymes resulted in the identification of a small additional polypeptide with HGA-MT activity designated PMT18. The isoelectric points and apparent molecular masses of PMT5, PMT 7 and PMT18 were 5.8-6.5, 8.7-9.2 and 4.0-4.5, and 40, 110 and 18kDa, respectively (Bourlard et al, 2001). It is proposed that the 18kDa protein is a subunit of the 40 and HOkDa proteins, based on the appearance of the larger proteins when PMT 18 is rechromatographed by sizeexclusion chromatography and by the appearance of an 18 kDa band when PMT5 and PMT7 are separated by SDS-polyacrylamide gel electrophoresis (Bourlard etal, 2001). Furthermore, photoaffinity labeling of PMT5 and PMT7 with [3H]5"-adenosylmethionine yielded a single labeled band at 18 kDa. No information on the gene encoding HGA-MT is available. HGA-MT is localized to the Golgi (Vannier et al, 1992; Bourlard et al, 1997b; Baydoun etal, 1999; Goubet and Mohnen, 1999a) with its catalytic site facing the Golgi lumen (Goubet and Mohnen, 1999a). Available biochemical evidence suggests that at least a small stretch of HGA is synthesized prior to its methylation by HGA-MT in the Golgi. This conclusion is based on studies with intact membranes which show that UDP-GalA stimulates HGA-MT activity (Kauss and Swanson, 1969; Goubet et al, 1998) and from studies with detergent-permeabilized membranes and solubilized HGA-MT which show that

BIOSYNTHESIS OF PECTINS

77

Table 3.4 Comparison of catalytic constants and pH optimum of HGA methyltransferasesa

Enzyme1''0 HGA-MT

b

Plant source Mung bean

Apparent Km for SAMd (|iM)

pH optimum

e Vmax

59

6.6-7.0

2.7f

Apparent molecular mass (kDa) _

PMT

Flax

10-30

6.8

n.d. h

-

PMT(soI)*

Flax

0.5

7.1'or 5.5J

n.d.

_

HGA-MT HGA-MT (sol)

Tobacco Tobacco

38 18

7.8 7.8

49 7.3

-

PMT-MT PMT-MT5k PMT-MT7k PMT-MT 18k

Soybean Flax Flax Flax

230 n.d. n.d. n.d.

6.8 5.0 6.5 n.d.

1360 n.d. n.d. n.d.

40 110 18

Reference Kauss and Hassid ( 1967); Kauss <>?«/. (1969); Kauss era/. (1967) Schaumann et al. (1993); Vanniere/a/. (1992) Bourlard et al. (1997a,b); Bruyant-Vannier et al. (1996) Goubetetal. (1998) Goubet and Mohnen (1999b) Ishikawa et al. (2000) Bourlard et al, (2001) Bourlard et al. (2001) Bourlard et al. (2001)

a

Adapted from Ridley et al. (2001). Unless indicated, all enzymes are measured in paniculate preparations. C A11 enzymes are thought to be HGA-MT. However, since some authors use the previous name (pectin methyltransferase) it is included for clarity. d Apparent Km forS-adenosylmethionine. e Knax inpmol min" 1 mg~' protein. ' Vmax is calculated from data in Kauss et al. (1969). g (sol): detergent-solubilized enzyme. h n.d.: not determined. 'From Bruyant-Vannier et al. (1996). 'From Bourlard et al. (1997a,b). k Purified enzymes. h

polygalacturonic or pectin are acceptors for HGA-MT in vitro. Some of the HGA-MTs in detergent-permeabilized membranes from flax and soybean show a preference for partially esterified pectin (Bourlard et al., 1997b; Ishikawa et al., 2000; Bourlard et al, 2001) over polygalacturonic acid. These results suggest that multiple HGA-MTs may exist that differ in their specificity for HGA of differing degrees of methylation. Such HGA-MTs may be preferentially involved in the initial methylation of HGA or in the methylation of more highly esterified HGA. 3.5.1.3 HGA acetyltransferase (HGA-AT) The Gal A residues in HGA may, depending upon the species, be partially Oacetylated at C-2 or C-3 (Ishii, 1995; Ishii, 1997). Pectin 0-acetyltransferase activity has been identified in microsomes from suspension-cultured potato cells (Pauly and Scheller, 2000). The incubation of potato microsomes with

78

PECTINS AND THEIR MANIPULATION

[14C]acetyl-CoA yielded a salt/ethanol precipitable product from which approximately 8% of the radioactivity could be solubilized by treatment with endopolygalacturonase and pectin methylesterase. These results suggest that 8% of the radiolabeled acetate was transferred either onto HGA or onto solubilized RGII or RG-I fragments. It remains to be shown whether the described activity represents HGA-AT or an enzyme that acetylates one of the other pectic polysaccharides that may be covalently linked to HGA. 3.5.2 Synthesis of substituted homogalacturonans There have been no reports of a systematic effort to study the glycosyltransferases required for the synthesis of RG-II, the most structurally complicated polysaccharide in the cell wall. As shown in Table 3.5, at least 24 transferase activities are likely required to synthesize RG-II. It is possible that the apiosyltransferase identified in the studies of apiogalacturonan synthesis in Lemna is related to, or is the same apiosyltransferase(s) as that required for RG-II synthesis (see below), however, this remains to be shown. 3.5.2.1 Rhamnogalacturonan-U methyltransferase (RG-II-MT) The GalA in the HGA backbone of RG-II may be partially methyl-esterified. A detergent-solubilized pectin methyltransferase activity from suspensioncultured flax cells was identified that could transfer methyl groups from 5-adenosylmethionine onto RG-II isolated from wine (Bourlard et a/., 1997a). The addition of RG-II to enzyme reactions gave a 7-fold stimulation of methyltransferase activity above the level obtained in the absence of exogenous acceptor. The radiolabeled product had a size similar to RG-II monomers and RG-II dimers (Bourlard et al., 1997; Ridley et al., 2001). It has not yet been shown where in RG-II the methyl group was added. The identified methylation could represent methyl-esterification of the HGA backbone of RG-II. Alternatively, it could represent methyl-etherification of RG-II since RG-II contains methyl groups on non-galacturonic glycosyl residues (e.g. 2-0-methylxylose and 2-0methylfucose (Darvill et al., 1978; O'Neill et al., 1996)) of side chain residues. More research is required to determine the exact location of the methylation and of the identity of the potentially novel enzyme activity reported. 3.5.2.2 Apiogalacturonan apiosyltransferase The substituted galacturonan known as apiogalacturonan is produced in some aquatic monocotyledonous plants (Hart and Kindel, 1970; Watson and Orenstein, 1975). Apiogalacturonan has apiose or apiobiose (D-Api/-pl,3-D-apiose) attached to O-2 or O-3 of HGA (Hart and Kindel, 1970; Watson and Orenstein, 1975). Table 3.6 shows some of the transferase activities likely to be required to synthesize apiogalacturonan. The anomeric configuration of the glycosidic linkage of apiose to HGA may be in the (3 configuration (Watson and Orenstein. 1975). It is not known how apiogalacturonan is related to RG-II. which has

BIOSYNTHESIS OF PECTINS

79

Table 3.5 Glycosyltransferase activities likely to be required for RG-1I biosynthesis1'b Enzyme d Type of glycosyl transferase

RG-II side chainc

Parent polymer

Acceptor substrate

Enzyme activity

Reference for structure

D-GalAT D-GalAT

A

HGA/RG-II4 RG-II

*GalAal,4-GalA L-RhapU'-Apif

od,4-GalAT al,2-GalAT

D-GalAT

A

RG-II

L-RhapU'-Api/

{31,3-GalAT

L-RhaT

A,B

RG-II

Api/f51,2-GalA

pU'-L-RhaT

L-RhaT

C

RG-II

Kdo2,3-GalA

al,5-L-RhaT

L-RhaT

B

RG-II

L-Araa 1,4-Gal

al,2-L-RhaT

O'Neill et at. (1990) O'Neill etal (1997); Carpita and Gibeaui (1993) O'Neill et al. (1997); Carpita and Gibeaut (1993) O'Neill et al, (1997); Carpita and Gibeaut (1993) O'Neill et al. (2001, 1997); Carpita and Gibeaut (1993) O'Neill et al. (1997); Carpita and Gibeaut (1993)

L-RhaT L-GalT

B2 B

RG-II RG-II

L-Araa 1,4-Gal GlcAfJ 1,4-Fuc

pl,3-l.-RhaT al,2-L-GalT

D-GalT

B

RG-II

L-Ace/Acd,3-Rha

pl,2-Ga!T

l.-AraT

D

RG-II

pDha2,3-GalA

pl,5-L-Ara/T

L-AraT

B

RG-II

Galp 1,2-L-Ace/A

a 1,4-L-ArapT

L-AraT

B2

RG-II

L-Rhaal,2-L-Ara

pl,2-L-Ara/T

L-FucT

A

RG-II

L-RhapM,3'-Api/

al,4-L-FucT

L-FucT

B

RG-II

GalpU-L-AceA/

al,2-L-FucT

D-Api/T

A,B

RG-II

GalAal,4-GalA

pM,2-Api/T

O'Neill et al. (1997); Carpita and Gibeaut (1993) Vidal et al. (2000); O'Neill et al (1997); Carpita and Gibeaut (1993) O'Neill el al. (2001, 1997); Carpita and Gibeaut (1993) Vidal et al. (2000, 1997); Carpita and Gibeaut (1993) O'Neill et al. (1997); Carpita and Gibeaut (1993) O'Neill et al. (1997); Carpita and Gibeaut (1993) Vidal et al. (2000); O'Neill ef al. (1997); Carpita and Gibeaut (1993) O'Neill et al. (1997); Carpita and Gibeaut (1993)

80

PECTINS AND THEIR MANIPULATION

Table 3.5 (continued) Enzyme d I

Type of glycosyl transferase

RG-II side chain'-

Parent polymer

Acceptor substrate

Enzyme activity

Reference for structure

D-XylT

A

RG-II

L-Fucctl.4-L-Rha

al.3-XylT

D-GlcAT

A

RG-II

L-Fucal.4-L-Rha

pM.4-GlcAT

D-KdoT

C

RG-II

GalAal.4-GalA

2.3-KdoT

D-DhaT

D

RG-II

GalAal.4-GalA

p2.3-DhaT

L-Ace/A

B

RG-II

L-RhapM.3'-Api/

al.3-AceA/T

RG-II

D-Xylal,3-L-Fuc

RG-II

L-Fucal.2-D-Gal

RG-II

L-Fuca 1.2-D-Gal

RG-II

L-Ace/Aal.3-L-Rha

O'Neill etal. (1997): Carpita and Gibeaut (1993) O'Neill et al. (1997): Carpita and Gibeaut (1993) O'Neill et al. (2001. 1997): Carpita and Gibeaut(1993) O'Neill et al. (2001. 1997): Carpita and Gibeaut(1993) Vidal et al. (2000. 1997): Carpita and Gibeaut(1993) O'Neill et al. (1997): Carpita and Gibeaut (1993) O'Neill et al. (1997): Carpita and Gibeaut (1993) O'Neill et al. (1997): Carpita and Gibeaut (1993) Vidal cial. (2000. 1997): Carpita and Gibeaut (19931

Methyl-RG-II: xylose 2-Omethyltransferase RG-II:fucose 2-Omethyltransferase AcetylRG-II:fucose acetyltransferase RG-II:aceric acid 3-0acetyltransferase a

Adapted from Ridley et al. (2001). This list of glycosyltransferase activites is based on the most extended structure of RG-II (Ridley et al., 2001). Note that terminal pRha/>—>3oArap- in side chain B and the terminal pAra/—»2aRha/>- in side chain B are not present in all RG-II preparations (O'Neill et al.. 2001). c The side chain of RG-II that contains the specified glycosyl residue. See Ridley et al. (2001). O'Neill et al. (2001) for side chain structure. d All sugars are D sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to the glycosyl residue on the left* of the indicated acceptor. b

two of its four side branches attached to an HGA backbone by a pApi/ linked to the O-2 of HGA (Ridley et al., 2001). It is also not known whether the apiosyltransferases that synthesize RG-II are the same as those involved in apiogalacturonan synthesis since no studies specifically directed at the 31.2apiosyltransferase involved in RG-II synthesis have been reported. The in vivo

BIOSYNTHESIS OF PECTINS

81

Table 3.6 Some of the glycosyltransferase activities likely to be required for apiogalacturonan biosynthesis Enzyme a ,b Type of transferase

Acceptor substrate

Enzyme activity

Reference for structure

D-GalAT D-Api/T

*GaIAal,4-GalA GalAal,4-GalA

al,4-GalAT p],2-Api/T

D-Api/T

GalAal.4-GalA

pl,3-Api/T

D-Api/T

Api/pM,2-GalA

pl,3-Api/T

D-Api/T

Api/pl.3-GalA

pM,3-Api/T

O'Neills al. (1990) Hart and Kindel ( 1970); Watson and Orenstein (1975) Hart and Kindel (1970); Watson and Orenstein (1975) Hart and Kindel ( 1970); Watson and Orenstein (1975) Hart and Kindel (1970); Watson and Orenstein (1975)

a

All sugars are D sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to the glycosyl residue on the left* of the indicated acceptor.

b

synthesis of apiogalacturonan, however, has been studied in vegetative fronds of Spirodelapolyrrhiza (Longland et al., 1989) and a D-apiosyltransferase has been identified and characterized in cell-free particulate preparations from duckweed (Lemna minor) (Pan and Kindel, 1977). The apiosyltransferase transferred [!4C]apiose from UDP-[l4C]apiose onto endogenous acceptors in particulate membrane preparations from Lemna. The enzyme has a pH optimum of 5.7 and an apparent Km for UDP-apiose of 4.9 |xM (Pan and Kindel, 1977). Interestingly, the rate of apiosyltransferase activity was increased twofold by the inclusion of UDP-GalA in the reaction (Pan and Kindel, 1977) and the product synthesized in the presence of UDP-GalA bound more tightly to anion exchange resin than the product synthesized without UDP-GalA (Mascaro and Kindel, 1977). These results suggest either that the apiosyltransferase transfers apiose onto a growing HGA chain or that the amount of endogenous HGA substrate was limiting. The product was solubilized in 1 % ammonium oxalate, as expected for apiogalacturonans isolated from Lemna wall (Mascaro and Kindel, 1977) and the product was fragmented by treatment with a fungal pectinase as expected for apiogalacturonan. The fact that acid hydrolysis of the 14C-labeled product yielded [14Clapiose and [14C]apiobiose (Mascaro and Kindel, 1977) confirmed that an apiogalacturonan apiosyltransferase was identified. There are no reports of the purification of the enzyme. 3.5.2.3 Xylogalacturonan xylosyltransferase Xylogalacturonan is a region of HGA in which some of the GalA residues are substituted at O-3 with (3-D-xylose (see (Schols et al., 1995). Table 3.7 shows some of the transferases likely to be required for Xylogalacturonan synthesis. Xylosyltransferase activity was identified during studies of apiogalacturonan synthesis (Mascaro and Kindel, 1977; Pan and Kindel., 1977).

82

PECTINS AND THEIR MANIPULATION

Table 3.7 Glycosyltransferase activities likely required to synthesize the substituted galacturonan known as xylogalacturonan Enzyme3 Type of transferase

Acceptor substrate

Enzyme activity

Reference for structure

D-GalAT D-XylT

*GalAal,4-GalA Gal Aa 1 .4-Gal A

a 1. 4-Gal AT pM.3-XylT

O'Neill^/ al. (1990) Aspinall(1980):Yuand Mort (1996); Kikuchi et al. (1996):SchoIse/a/. (1995)

a

All sugars are D sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to the glycosyl residue on the left* of the indicated acceptor.

The product produced was not characterized in detail; however, at least some of the radioactive xylose appeared to be incorporated into apiogalacturonan and/or HGA. Thus, the enzyme may have been a xylogalacturonan xylosyltransferase. There have been no reports of the identification of the a 1,3-xylosyltransferase that transfers xylose from UDP-Xyl onto the L-Fuca 1,4-L-rhamnosyl portion of the side branch of RG-II (Ridley et al., 2001). 3.5.2.4 Other glycosyltransferases There are no reports of targeted studies for the glycosyltransferases that insert fucose, Kdo, Dha or aceric acid into pectins. However, success in identifying fucosyltransferase genes involved in the synthesis of hemicellulose could be useful in the study of pectin synthesis. Fucose is a component of RG-II and RG-I. No fucosyltransferase involved in the synthesis of either of these pectic polymers has knowingly been studied. However, an Arabidopsis gene for an a 1,2fucosyltranferase that fucosylates a side branch in the hemicellulose xyloglucan has been described (Perrin etal, 1999) that has 35-73.8% amino acid sequence identify to 10 other putative fucosyltransferase genes in Arabidopsis (Perrin et al., 2001). The possibility that one or more of these genes may encode fucosyltransferase^) involved in RG-I or RG-II synthesis remains to be investigated. 3.5.3 Synthesis of rhamnogalacturonan I (RG-I) RG-I is a family of polysaccharides with an alternating [—*4)-a-D-Gal/?A(1—>-2)-a-L-Rha/7-(l—»•] backbone in which roughly 20-80% of the rhamnoses are substituted by arabinans, galactans or arabinogalactans (Carpita and Gibeaut. 1993; Mohnen, 1999; Ridley etal., 2001). Table 3.8 shows a list of all the likely enzyme activities required to synthesize all the known structures of RG-I, based on the available structural information. There is considerable evidence from immunocytochemistry studies of plant tissues using antibodies against specific carbohydrate epitopes found in RG-I (Willats et al., 200la) that supports the view that the precise structure of the side chains of RG-I varies in a cell type- and

BIOSYNTHESIS OF PECTINS

83

Table 3.8 Glycosyltransferases 'required' for RG-I biosynthesisa Enzymeb

Type of glycosyltransferase

Parent polymer

Acceptor substrate

Enzyme activity

D-GalAT

RG-I

*L-Rhacd,4-GalA

ed,2-GalAT

D-GalAT L-RhaT

RG-I/HGAC RG-I

GalAal,2-L-Rha GaIAal,2-L-Rha

aI,4-GalAT al,4-L-RhaT

L-RhaT D-GalT

HGA/RG-I0 RG-I

GalAotl,4-GalA L-Rhaal,4-GalA

al,4-L-RhaT PM-GalT

D-GalT

RG-I

Galpl.4-Rha

pl,4-GalT

D-GalT

RG-I

Galpl,4-Gal

pl,4-GalT

D-GalT

RG-I

Galpl,4-Gal

Pl,6-Gall

D-GalT

RG-I/AGPd

Galpl,3-GaI

pl,3-GalT

D-GalT

RG-I/AGPd

Galpl,3-Gal

pl,6-GalT

D-GalT

RG-I/AGPd

Galpl,6-Galpl,3-Gal

pl,6-GalT

D-GalT L-AraT

RG-I RG-I

L-Ara/-l,4-Gal Galpl,4-Rha

1,5-GalT a 1 ,3-L-Ara/T

L-AraT

RG-I

L-Ara/al,3-Gal

a 1 ,2-L-Ara/T

L-AraT

RG-I

L-Ara/al,2-Ara

1,5-L-Ara/T

L-AraT L-AraT

RG-I RG-I

L-Rhaal,4-GalA L- Ara/a 1 ,5- Ara

1,4-Ara/T a 1,5-L-Ara/T

L-AraT

RG-I

L- Ara/a 1,5- Ara

al,2-L-Ara/T

L-AraT

RG-I

L- Ara/a 1,5- Ara

al,3-L-Ara/T

L-AraT

RG-I

L- Ara/a 1,3-Ara

al.3-L-Ara/T

L-AraT

RG-I

Galpl,4-Gal

al,3-L-Ara/T

Reference for structure O'Neill etal. (1990); Edaetal. (1986); Lau etal. (1985) O'Neill et al (1990); Edaetal. (1986); Lau etal. (1985) O'Neill etal. (1990); Lau et al. (1987) O'Neill etal. (1990); Lau etal. (1987) Morita(1965a,b); Aspinall et al. (1967); Stephen (1983); Aspinall(1980); O'Neill etal. (1990); Lau etal. (1987) O'Neill etal. (1990); Lau etal. (1987) Carpita and Gibeaut (1993) Carpita and Gibeaut (1993) Carpita and Gibeaut (1993) Huisman et al. (200 la) O'Neill etal. (1990); Lau etal. (1987) O'Neill etal. (1990): Lau etal. (1987) O'Neills al. (1990); L&uetal. (1987) Lau etal. (1987) Carpita and Gibeaut (1993) Carpita and Gibeaut (1993) Carpita and Gibeaut (1993) Carpita and Gibeaut (1993) Morita(1965a,b); Aspinall et al. (1967); Stephen (1983); Aspinall (1980);

84

PECTINS AND THEIR MANIPULATION

Table 3.8 (continued) Type of glycosyltransferase

Parent polymer

Enzyme11 Acceptor substrate

Enzyme activity

L-AraT

RG-I

L-Ara/-1.3-Gal

1.5-L-Ara/T

L-AraT

RG-I/AGP11

Galpl .6-Gal

otl.3-L-Ara/T

L-AraT

RG-I/AGP*1

Galpl .6-Gal

a 1 .6-L-Ara/T

L-AraT L-FucT

RG-I RG-I

Galpl .4-Gal Galpl .4-Gal

1.4-L-Ara/?T otl.2-L-Fuc/T

D-GlcAT D-GlcAT MethylRG-I:GlcA 4-0methyltransferase AcetylRG-I:GalA 3-0/2-O-acetyltransferase

RG-I RG-I

Gal.. Gal..

31.6-GlcAT 31.4-GlcAT

Reference for structure O'Neill etal. (1990): Carpita and Gibeaut (1993) Morita(1965a.b): Aspinall et at (1967): Stephen (1983): Aspinall (1980) Carpita and Gibeaut (1993) Carpita and Gibeaut (1993) Huisman er «/. (2001 a) O'Neill et at (1990): Lmetat (1987)

An etal. (1994) .\netal. (1994)

RG-I

GlcApl. 6-Gal

\netal. (1994)

RG-I

GalAal.2-L-Rhaal.4, ril

Lerouge et at (1993): Ishii(1997): O'Neill etal. (1990): Bacicetal. (1988): Komalavilas and Mort(1989)

a

Adapted from Ridley et at (2001). All sugars are D sugars and have pyranose rings unless otherwise indicated. Glycosyltranferases add to the glycosyl residue on the left* of the indicated acceptor. c Enzyme that may be required to make an HGA/RG-I junction. d Enzyme activity would also be required to synthesize arabinogalactan proteins (AGPs) (see Caspar et at. 2001). b

development-specific manner. Thus, it not expected that all of the biosynthetic enzyme activities shown in Table 3.8 will necessarily be expressed in any given cell type synthesizing a specific RG-I structure. 3.5.3.1 RG-I galactosyltransferase (GalT) The synthesis of the family of polysaccharides known as RG-I requires at least eight different galactosyltransferase (GalT) activities to synthesize the diverse side chain linkages found in the RG-I structures identified to date (see Table 3.8). Probable pl,4-GalT and |Jl,3-GalT activities were identified in early studies of microsomal preparations from mung bean (McNab et al., 1968; Panayotatos and

BIOSYNTHESIS OF PECTINS

85

Villemez, 1973). A more recent study confirmed that a |31,4-galactosyltranferase activity with a pH of optimum of 6.5 was present in mung bean microsomes based on sensitivity of the product to digestion with endo-|31,4-galactanase (Brickell and Reid, 1996). Galactosyltransferases have also been identified in paniculate homogenates (Goubet and Morvan, 1993; Goubet and Morvan, 1994) and solubilized enzyme (Goubet, 1994) from flax (Linum usitatissimum L.). Flax GalTs solubilized from microsomes with detergent transferred [3H]Gal from UDP-[3H]Gal onto exogenous RG-I-enriched and pectic (31,4-galactan acceptors (Peugnet et al., 2001) to yield a radiolabeled product of high molecular mass. Interestingly, the pH optimum for transfer onto lupin pectic (31,4-galactan (i.e. pH 6.5) was different from the pH optimum for transfer of Gal onto an endopolygalacturonase-treated RG-I-enriched fractions from flax (i.e. two optima: pH 6.5 and 8.0) (Peugnet et al., 2001). Cleavage of the large radiolabeled product with either rhamnogalacturonan I hydrolase (RGase A) or with rhamnogalacturonan I lyase (RGase B) resulted in a fragmentation of the radiolabeled product into lower molecular mass material that, at least in part, eluted as expected for small oligomers (i.e. DP 3-6) (Peugnet et al., 2001). This result confirmed that the GalTs added Gal onto RG-I (Peugnet et al., 2001). The fragmentation of at least a portion of the radiolabeled product with (31,4-endogalactanase further demonstrated that at least some of the GalT activity represented ^1,4-galactosyltransferase (Peugnet et al, 2001). The GalT at pH 8.0 has an apparent Km of 460 |iM for UDP-Gal. The characteristics of the GalT at pH 8.0 are consistent with an enzyme that adds galactose onto short galactan side branches of RG-I. An RG-I :(31,4-galactosyltransferase was identified and partially characterized from potato suspension cultured cells (Geshi et al., 2000). The potato GalT transfers [14C]Gal from UDP-[14C]Gal onto endogenous acceptor(s) in the membranes to produce a >500 kDa product. The product can be fragmented by endo-p 1,4-galactanase into [14ClGal and [14C]galactobiose. Treatment of the intact radiolabeled product with rhamnogalacturonase A, an endohydrolase that cleaves the glycosidic linkage between the GalA and the Rha in the RG-I backbone (Azadi et al., 1995), yielded radiolabeled fragments between 50kDa and 180 kDa in size (Geshi et al., 2000). The subsequent treatment of the [14C]Gallabeled fragments with endo-|M ,4-galactanase yielded [14C]Gal and slightly larger fragments that themselves could be cleaved to [14C]Gal by a purified Pgalactosidase. These results suggest that potato microsomal membranes contain enzymes that both initiate and elongate {31,4-galactan side chains of RG-I. The potato ^1,4-GalT has a pH optimum of 6.0-6.5. A fenugreek gene for an a-D-1,6-galactosyltransferase involved in the synthesis of the hemicellulose galactomannan (Edwards et al., 1999) has been identified. Eight Arabidopsis genes with sequence similarity exist inArabidopsis (Perrin et al., 2001) and the possibility that one or more of these putative galactosyltransferases is involved in pectin synthesis remains to be explored.

86

PECTINS AND THEIR MANIPULATION

D-Galactose is a component of both RG-II and RG-I, while L-galactose is found only in RG-II. No information is available on the enzyme that adds L-Gal onto RG-II. 3.5.3.2 RG-I arabinosyltramferase RG-I and RG-II contain L-arabinose in multiple linkages (see Tables 3.5 and 3.8). Most of the arabinose is in the furanose ring form, although it has recently been reported that a terminal arabinose exists in the pyranose form in some RG-I side chains (Huisman et a/., 200la). Arabinosyltransferase activity has been identified in microsomes from mung bean (Phaseolus aureus) shoots (Odzuck and Kauss, 1972) and from bean (Phaseolus vulgaris) hypocotyl and callus (Bolwell and Northcote, 1981). Definitive evidence that these AraT activities are involved in pectin synthesis was not demonstrated (see Mohnen, 1999, for review). The arabinosyltransferase activity in bean hypocotyl and callus was primarily associated with enriched Golgi, and to a lesser extent with enriched endoplasmic reticulum (Bolwell and Northcote, 1983). The difficulty of studying arabinosyltransferases that specifically synthesize pectin, as opposed to other hemicellulosic polysaccharides or arabinogalactan proteins has been discussed (Nunan and Scheller, 2001). An approach that entails the use of detergent-solubilized microsomes and specific pectic oligo/polysaccharide acceptors has been reported to yield arabinosylation of pectic acceptors (Nunan and Scheller. 2001). 3.5.3.3 RG-I methyltransferase (RG-I-MT) A detergent-solubilized pectin methyltransferase (PMT) from flax has been reported to use an RG-I-enriched fraction as an exogenous acceptor (Bourlard et al., 1997a). Specifically, pectin methyltransferase activity was stimulated in the presence of an enriched RG-I fraction 1.5-fold to 1.7-fold above levels recovered using endogenous acceptor. The resulting radiolabeled product had a size similar to RG-I. It was not shown, however, where in RG-I the methylation occurred. Thus, it is not clear whether the methylation occurred on GalA in the RG-I backbone, or whether it occurred on possible HGA tails that may be covalently linked to RG-I. Also, it was not shown whether some of the methylation may have occurred on a non-galacturonic substituent in RG-I such as methylation at the 4-position of glucuronic acid in the side branches of RG-I (An et a/., 1994). The location on the polymer of the methylation in the RG-Ienriched fraction and, thus, the identity of the potentially novel enzyme activity, remains to be determined. 3.5.3.4 RG-I acetyltransferase (RG-I-AT) The GalA residues in the alternating [^4)-a-D-Gal/?A-( 1 -»• 2)-a-L-Rhap-( 1 -»• ] backbone of RG-I may be acetylated on C-2 and/or C-3 (Komalavilas and

BIOSYNTHESIS OF PECTINS

87

Mort, 1989). Microsomes from suspension-cultured potato cells (Pauly and Scheller, 2000) contain an RG-I acetyltransferase that transfers [14C]acetate from [14C]acetyl-CoA onto RG-I yielding a >500kDa radiolabeled product (Pauly and Scheller, 2000). The release of [14C]acetate following incubation of the radiolabeled product with a purified rhamnogalacturonan O-acetyl esterase, and the fragmentation of the product by rhamnogalacturonan lyase (RGase B) confirmed that the enzyme was an RG-I acetyltransferase (Pauly and Scheller, 2000). The RG-I acetyltransferase has an apparent Km for acetyl-CoA of 35 |xM, an apparent Kmax of 54pmol min^ 1 mg~~' protein and a pH optimum of 7.0, with 80% of activity recovered at pH 6.5-8.0.

3.6

Future directions and resources for studying pectin biosynthesis

The partial characterization and, in some cases, purification of selected pectin biosynthetic genes has provided a core of biochemical information on the enzymes. However, to significantly increase our understanding of when, where and how the enzymes interact to produce pectin, it is essential that the genes for the biosynthetic enzymes be identified (see Henrissat et al., 2001; Keegstra and Raikhel, 2001; Perrin et al., 2001; Reiter and Vanzin, 2001). The identification of the genes would provide primary structure information and allow the generation of antibodies against the biosynthetic enzymes that could be used for analysis of enzyme localization and provide a means to identify members of the expected biosynthetic protein complexes. The genes could also be used to express the enzymes for use in detailed kinetic, structure and function studies. The manipulation of the genes in transgenic plants should allow hypotheses regarding pectin structure-function in the plant to be tested and would facilitate the elucidation of how the individual biosynthetic enzymes interact to synthesize pectin. The identification of the pectin biosynthetic genes will likely occur using multiple strategies including enzyme purification, mutant identification and characterization, and DNA sequence/motif similarity computer searches. In all cases, however, the definitive identification of any pectin biosynthetic gene will require proof of enzyme activity. Thus, continued progress in identifying and making available the required nucleotide-sugar and oligo/polysaccharide substrates for the biosynthetic enzymes is essential. The National Science Foundation-funded Plant Cell Wall Biosynthesis Research Coordination Network, also referred to as WallBioNet (http://xyloglucan.prl.msu.edu), serves as an information center and resource for researchers studying plant cell wall biosynthesis. It provides a central location for updated information on progress in, and available tools for, studying wall biosynthesis research. It also partially supports the synthesis of rare and needed substrates and acceptors for wall biosynthesis that are made available to the scientific community.

88

PECTINS AND THEIR MANIPULATION

Acknowledgements I thank my colleagues at the CCRC for their helpful discussions. This effort was supported in part by NSF grant No. MCB-0090281, NRI competitive USDA award 2001-35318-11111 and DOE-funded center grant DE-FG05-93ER20097.

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Bauer, A.J., Rayment, I., Frey, P.A. and Holden, H.M. (1992) The molecular structure of UDP-galactose 4-epimerase from Escherichia coli determined at 2.5 A resolution. Proteins, 12, 372-381. Baydoun, E.A.H., Rizk, S.E. and Brett, C.T. (1999) Localisation of methyltransferases involved in glucuronoxylan and pectin methylation in the Golgi apparatus in etiolated pea epicotyls. J. Plant Physiol., 155, 240-244. Berninsone, P.M. and Hirschberg, C.B. (2000) Nucleotide-sugar transporters of the Golgi apparatus. Curr. Opin. Struct. BioL, 10, 542-547. Bolwell, G.P. and Northcote, D.H. (1981) Control of hemicellulose and pectin synthesis during differentiation of vascular tissue in bean (Phaseolus vulgaris) callus and in bean hypocotyl. Planta, 152, 225-233. Bolwell, G.P. and Northcote, D.H. (1983) Arabinan synthase and xylan synthase activities of Phaseolus vulgaris. Subcellular localization and possible mechanism of action. Biochem. J., 210, 497-507. Bolwell, G.P., Dalessandro, G. and Northcote, D.H. (1985) Decrease of polygalacturonic acid synthase during xylem differentiation in sycamore. Phytochemistry, 24, 699-702. Benin, C.P. and Reiter, W.-D. (2000) A bifunctional epimerase-reductase acts downstream of the MURl gene product and completes the de novo synthesis of GDP-L-fucose in Arabidopsis. Plant J.,21, 445-454. Bonin, C.P., Potter, I., Vanzin, G.F. and Reiter, W.-D. (1997) The MURl gene of Arabidopsis thaliana encodes an isoform of GDP-D-mannose-4,6-dehydratase, catalyzing the first step in the de novo synthesis of GDP-L-fucose. Proc. Natl. Acad. Sci. USA, 94, 2085-2090. Bourlard, T., Pellerin, P. and Morvan, C. (1997a) Rhamnogalacturonans I and II are pectic substrates for flax-cell methyltransferases. Plant Physiol. Biochem., 35, 623-629. Bourlard. T., Schaumann-Gaudinet, A., Bruyant-Vannier, M.-P. and Morvan, C. (1997b) Various pectin methyltransferase activities with affinity for low and highly methylated pectins. Plant Cell Physiol,, 38,259-267. Bourlard, T, Vannier, M.-P., Schaumann, A., Bruyant, P. and Morvan, C. (2001) Purification of several pectin methyltransferases from cell suspension cultures of flax. C. R. Acad. Sci. Paris, Science de la vie, 324, 335-343. Brabetz, W., Wolter, P.P. and Brade, H. (2000) A cDNA encoding 3-deoxy-D-manno-oct-2-ulosonate8-phosphate synthase of Pisum sativum L (pea) functionally complements a kdsA mutant of the Gram-negative bacterium Salmonella enterica. Planta, 212, 136-143. Brickell, L.S. and Reid, J.S.G. (1996) Biosynthesis in vitro of pectic (l-4)-p-D-galactan, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 127-134. Bruyant-Vannier, M.-P., Gaudinet-Schaumann, A., Bourlard, T. and Morvan, C. (1996) Solubilization and partial characterization of pectin methyltransferase from flax cells. Plant Physiol. Biochem., 34, 489-499. Burget, E.G. and Reiter, W.-D. (1999) The mur4 mutant of Arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose. Plant Physiol., 121, 283-389. Campbell, R.E., Sala, R.F., Van De Rijn, I. and Tanner, M.E. (1997) Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. J. BioL Chem., 272, 3416-3422. Campbell, R.E., Mosimann, S.C., Van De Rijn, I., Tanner, M.E. and Strynadka, N.C.J. (2000) The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation. Biochemistry, 39, 7012-7023. Capasso, J.M. and Hirschberg, C.B. (1984) Mechanisms of glycosylation and sulfation in the Golgi apparatus: evidence for nucleotide-sugar/nucleoside monophosphate and nucleotide sulfate/nucleoside monophosphate antiports in the Golgi apparatus membrane. Proc. Natl. Acad. Sci, USA, 81, 7051-7055. Carpita, N.C. (1996) Structure and biogenesis of the cell walls of grasses. Annu. Rev. Plant Physiol. Plant Mot. BioL, 47, 445-476.

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Orellana, A., Neckelmann, G. and Norambuena, L. (1997) Topography and function of Golgi uridine-5'-diphosphatase from pea stems. Plant Physiol., 114, 99-107. Orfila, C. and Knox, J.P. (2000) Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp. Plant Physiol., 122, 775-781. Pan, Y.-T. and Kindel, P.K. (1977) Characterization of paniculate D-apiosyl- and D-xylosyltransferase from Lemna minor. Arch. Biochem. Biophys., 183, 131-138. Panayotatos, N. and Villemez, C.L. (1973) The formation of a p"-( 1 -»4)-D-galactan chain catalysed by a Phaseolus aureus enzyme. Biochem. 7., 133, 263-271. Pauly, M. and Scheller, H.V. (2000) O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of rhamnogalacturonan 0-acetyl-transferase from potato suspension cultured cells. Planta, 210, 659-667. Pauly, M., Porchia, A., Olsen, C.E., Nunan, K.J. and Scheller, H.V. (2000) Enzymatic synthesis and purification of UDP-p-L-arabinopyranose, a substrate for the biosynthesis of a plant polysaccharides. Anal. Biochem., 278, 69-73. Pazzani, C., Rosenow. C., Boulnois, G.J., Bronner, D., Jann, K. and Roberts, I.S. (1993) Molecular analysis of region-1 of the Escherichia-coli K5 antigen gene-cluster—a region encoding proteins involved in cell-surface expression of capsular polysaccharide. J. Bacterial., 175, 5978-5983. Perrin, R., Wilkerson, C. and Keegstra, K. (2001) Golgi enzymes that synthesize plant cell wall polysaccharides: finding and evaluating candidates in the genomic era. Plant Mol. Bioi. 47, 115-130. Perrin, R.M., DeRocher, A.E., Bar-Peled, M., et al. (1999) Xyloglucan fucosyltransferase. an enzyme involved in plant cell wall biosynthesis. Science, 284, 1976-1979. Peugnet, I., Goubet, F, Bruyant-Vannier, M.-P. et al. (2001) Solubilization of rhamnogalacturonan I galactosyltransferases from membranes of a flax cell suspension. Planta, 213, 435-445. Raetz, C.R.H. (1990) Biochemistry of endotoxins. Annu. Rev. Biochem., 59, 129-170. Rao, A.K. and Mendicino, J. (1976) Preparation of UDP-D-[U- l4 C]galacturonic acid and UDP-D-[63 H]galactose with high specific activities. Analy. Biochem., 72, 400-406. Reid. J.S.G. (2000) Cementing the wall: cell wall polysaccharide synthesising enzymes. Curr. Opin. PlantBiol.,3,5\2-5\6. Reiter, W.-D. and Vanzin, G.F (2001) Molecular genetics of nucleotide sugar interconversion pathways in plants. Plant Mol. Biol, 47, 95-113. Reiter, W.-D., Chappie, C.C.S. and Somerville, C.R. (1993) Altered growth and cell walls in a fucose-deficient mutant ofArabidopsis. Science, 261, 1032-1035. Ridley, B.L., O'Neill, M.A. and Mohnen. D. (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry, 57, 929-967. Robertson, D., McCormack, B.A. and Bolwell, G.P. (1995) Cell-wall polysaccharide biosynthesis and related metabolism in elicitor-stressed cells of French bean (Phaseolus vulgaris L.). Biochem. J.. 306, 745-750. Robertson, D., Smith, C. and Bolwell, G.P. (1996) Inducible UDP-glucose dehydrogenase from French bean (Phaseolus vulgaris L.) locates to vascular tissue and has alcohol dehydrogenase activity. Biochem. J.. 313, 311-317. Rombouts, P.M. and Thibault, J.F. (1986) Sugar beet pectins: chemical structure and gelation through oxidative coupling, in Chemistry and Function of Pectins (eds M.L. Fishman and J. J. Jen). American Chemical Society, Washington, DC, pp. 49-60. Rosenow, C., Roberts, I.S. and Jann, K. (1995) Isolation from recombinant Escherichia-coli and characterization of CMP-Kdo synthetase, involved in the expression of the capsular K5 polysaccharide (K-CKS). FEMS Microbiol. Lett.. 125, 159-164. Schaumann. A., Bruyant-Vannier, M.-P, Goubet, F. and Morvan, C. (1993) Pectic metabolism in suspension-cultured cells of flax, Linum usitatissimum. Plant Cell Physiol.. 34. 891-897. Scheller, H.V., Doong, R.L.. Ridley, B.L. and Mohnen, D. (1999) Pectin biosynthesis: a solubilized galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDPgalacturonic acid onto the non-reducing end of homogalacturonan. Planta. 207. 512-517.

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Schiller, J.G., Lamy, F., Frazier, R. and Feingold, D.S. (1976) UDP-glucose dehydrogenase from Esherichia coli purification and subnit structure. Biochim. Biophys. Acta, 453, 418-425. Schols, H.A., Posthumus, M.A. and Voragen, A.GJ. (1990) Structural features of hairy regions of pectins isolated from apple juice produced by the liquefaction process. Carbohydr. Res., 206, 117-129. Schols, H.A., Bakx, E.J., Schipper, D. and Voragen, A.G.J. (1995) A xylogalacturonan subunit present in the modified hairy regions of apple pectin. Carbohydr. Res., 279, 265-279. Schroeder, J.I. and Hagiwara, S. (1989) Cytosolic calcium regulates ion channels in the plasma membrane of Viciafaba guard cells. Nature, 338, 427-430. Seitz, B., Klos, C., Wurm, M. and Tenhaken, R. (2000) Matrix polysaccharide precursors in Arabidopsis cell walls are synthesized by alternate pathways with organ-specific expresssoin patterns. Plant J., 21, 537-546. Shea, E.M., Gibeaut, D.M. and Carpita, N.C. (1989) Structural analysis of the cell walls regenerated by carrot protoplasts. Planta, 179, 293-308. Sherrier, DJ. and VandenBosch, K.A. (1994) Secretion of cell wall polysaccharides in Vicia root hairs. PlantJ., 5, 185-195. Sherson, S., Gy, I.,Medd, J., Schmidt, R.,etal. (1999)Thearabinosekinase,A/M7, gene of Arabidopsis is a novel member of the galactose kinase gene family. Plant Mol. Biol, 39, 1003-1012. Stacey, N.J., Roberts, K., Carpita, N.C., Wells, B. and McCann, M.C. (1995) Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements. PlantJ., 8, 891-906. Staehelin, L.A. and Moore, I. (1995) The plant Golgi apparatus: structure, functional organization and trafficking mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol., 46, 261-288. Stephen, A.M. (1983) Other plant polysaccharides, in The Polysaccharides, vol. 2 (ed. G.O. Aspinall), Academic Press, New York, pp. 97-193. Sterling, J., Quigley, H.F., Orellana, A. and Mohnen, D. (2001) The catalytic site of the pectin biosynthetic enzyme a-l,4-galacturonosyltransferase (Gal AT) is located in the lumen of the Golgi. Plant Physiol., 127, 360-371. Stevenson, G., Neal, B., Liu, D., et al. (1994) Structure of the O antigen of Escherichia coli K-12 and the sequence of its rfb gene cluster. J. Bacterial., 176, 4144-4156. Stewart, D.C. and Copeland, L. (1998) Uridine 5'- diphosphate-glucose dehydrogenase from soybean nodules. Plant Physiol., 116, 349-355. Stoddart, R.W. and Northcote, D.H. (1967) Metabolic relationships of the isolated fractions of the pectic substances of actively growing sycamore cells. Biochem. J., 105, 45-59. Strominger, J.L. and Mapson, L.W. (1957) Uridine diphosphoglucose dehydrogenase of pea seedlings. Biochem. J., 66, 567-572. Sullivan, F.X., Kumar, R., Kriz, R., et al. (1998) Molecular cloning of human GDP-mannose 4,6dehydratase and reconstitution of GDP-fucose biosynthesis in vitro. J. Biol. Chem., 273,8193-8202 (Abstract). Takeuchi, Y. and Tsumuraya, Y. (2001) In vitro biosynthesis of homogalacturonan by a membranebound galacturonosyltransferase from epicotyls of azuki bean. Biosci. Biotechnol. Biochem., 65, 1519-1527. Tenhaken, R. and Thulke, O. (1996) Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase. Plant Physiol., 112, 1127-1124. Thibault, J.-F., Renard, C.M.G.C., Axelos, M.A.V., Roger, P. and Crepeau, M.-J. (1993) Studies of the length of homogalacturonic regions in pectins by acid hydrolysis. Carbohydr. Res., 238, 271-286. Thoden, J.B. and Holden, H.M. (1998) Dramatic differences in the binding of UDP-galactose and UDPglucose to UDP-galactose 4-epimerase from Escherichia coli. Biochemistry, 37, 11469-11477. Thoden, J.B., Frey, PA. and Holden, H.M. (1996a) Crystal structures of the oxidized and reduced forms of UDP-galactose 4-epimerase isolated from Escherichia coli. Biochemistry, 35, 2557-2566.

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Thoden, J.B., Frey, PA. and Holden, H.M. (1996b) High-resolution X-ray structure of UDP-galactose 4-epimerase complexed with UDP-phenol. Protein Sci., 5. 2149-2161. Unger, P.M. (1981) The chemistry and biological significance of 3-deoxy-D-/mmno-2-octulosonic acid (KDO).Adv. Carbohydr. Chem. Biochem., 38. 323-388. VandenBosch, K.A., Bradley, D.J.. Knox, J.P., Perotto. S., Butcher. G.W. and Brewin. N.J. (1989) Common components of the infection thread matrix and the intercellular space identified by immunocytochemical analysis of pea nodules and uninfected roots. EMBO J., 8. 335-342. Vannier. M.P.. Thoiron, B., Morvan, C. and Demarty, M. (1992) Localization of methyltransferase activities throughout the endomembrane complex system of flax (Linum usitatissimum L) hypocotyls. Biochem. J.. 286, 863-868. Vian, B. and Roland, J.-C. (1991) Affinodetection of the sites of formation and of the further distribution of polygalacturonans and native cellulose in growing plant cells. Biol. Cell, 71. 43-55. Vidal. S., Doco, T. Williams, P., er al (2000) Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain. Carboh\dr. Res.. 326, 277-294. Villemez. C.L., Swanson, A.L. and Hassid. W.Z. (1966) Properties of a polygalacturonic acidsynthesizing enzyme system from Phaseolus aureus seedlings. Arch. Biochem. Biophys.. 116. 446-452. Wang. J., Dudareva, N., Bhakta, S.. Raguso, R.A. and Pichersky. E. (1997) Floral scent production in Clarkia breweri (Onagraceae) II. Localization and developmental modulation of the enzyme S-adenosyl-L-methionine:(iso)eugenol 0-methyltransferase and phenylpropanoid emission. Plant Physio!., 114, 213-221. Watson, R.R. and Orenstein. N.S. (1975) Chemistry and biochemistry of apiose. Adv. Carbohydr. Chem. Biochem., 31. 135-184. Wee. T.G. and Frey, PA. (2001) Studies on the mechanism of action of uridine diphosphate galactose 4-epimerase. II. Substrate-dependent reduction by sodium borohydride. J. Biol. Chem.. 248.33—40. Wellmann, E. and Grisebach. H. (1971) Purification and properties of an enzyme preparation from Lemna minor L. catalyzing the synthesis of UDP-apiose and UDP-D-xylose from UDP-D-glucuronic acid. Biochim. Biophys. Acta. 235. 389-397. Willats, W.G.T., Steele-King, C.G.. Marcus, S.E. and Knox, J.P. (1999) Side chains of pectin polysaccharides are regulated in relation to cell proliferation and cell differentiation. Plant J.. 20.619-628. Willats, W.G.T., Steele-king, C.G.. McCartney, L.. Orfila. C.. Marcus. S.E. and Knox. J.P. (2000) Making and using antibody probes to study plant cell walls. Plant Physiol. Biochem.. 38, 27-36. Willats, W.G.T., McCartney. L.. Mackie. W. and Knox. J.P. (2001 a) Pectin: cell biology and prospects for functional analysis. Plant Mol. Biol., 47. 9-27. Willats. W.G.T.. Orfila, C., Limberg, G.. et al. (2001b) Modulation of the degree and pattern of methylesterificaton of pectic homogalacturonan in plant cell walls. J. Biol. Chem.. 276. 19404-19413. Wilson, D.B. and Hogness, D.S. (1969) The enzymes of the galactose operon in Escherichia coli. II. The subunits of uridine diphosphogalactose 4-epimerase. J. Biol. Chem.. 244. 2132-2136. Yu. L. and Mort. A.J. (1996) Partial characterization of xylogalacturonans from cell walls of ripe watermelon fruit: inhibition of endopolygalacturona.se activity by xylosylation. in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen). Progress in Biotechnology 14. Elsevier Science. Amsterdam, pp. 79-88. Zablackis, E.. York. W.S.. Pauly. M.. et al. (1996) Substitution of L-fucose by L-galactose in cell walls of arabidopsis murl. Science. 272, 1808-1810. Zalitis. J. and Feingold, D.S. (1969) Purification and properties of UDPGDH from beef liver. Arch. Biochem. Biophys.. 132. 457^465. Zhang. G.F. and Staehelin. L.A. (1992) Functional compartmentation of the golgi apparatus of plant cells: immunocytochemical analysis of high-pressure frozen- and freeze-substituted sycamore maple suspension culture cells. Plant Physiol.. 99. 1070-1083.

4

Biophysical properties of pectins Michael C. Jarvis

4.1

Introduction

Pectins comprise up to two-thirds of the dry mass of the primary walls of plant cells. Since plant cell walls are among the most sophisticated structural materials known, it seems logical to explore the contribution of pectins to their strength and flexibility. This chapter will attempt to do that. To make it possible, some new ideas on hydrated biopolymer structures under mechanical stress will need to be introduced. It must immediately be stated, however, that direct experimental evidence on the mechanical role of pectins in vivo is at present limited and contradictory. Small-deformation suspension rheology of cell walls, from which polymers had been sequentially removed, gave little evidence that anything other than cellulose contributed to mechanical behaviour (Whitney et al., 1999). Similar experiments on artificial composites of bacterial cellulose and pectin (Chanliaud and Gidley, 1999) led to similar conclusions. On the other hand, using twodimensional infrared/mechanical spectroscopy Wilson et al. (2000) were able to demonstrate that pectic polysaccharides within hydrated cell walls do reorient reversibly under load, which is consistent with a potential load-bearing function although it does not clarify whether pectins carry a significant fraction of the total load. These experiments were concerned with stress-strain relationships in the plane of the cell wall, but there is also evidence that pectins may carry stresses at right angles to the wall plane, and mediate adhesion between cells (Jarvis, 1992, 1998). Also, thermal or enzymatic degradation of pectins in fruit and vegetables is commonly accompanied by important changes in texture (Kunzek et al., 1999). These latter observations encourage the search for a mechanical function for the pectic polymers, and it can be expected that much new information will emerge from experiments on the intact cell walls of transgenic plants altered in pectic composition (Pilling et al., 2000; Sorensen et al., 2000). In the meantime, we must make the best possible use of indirect evidence derived from the physical and gelling properties of extracted pectins. That will occupy most of this chapter. Caution is needed in such an indirect approach. Pectins isolated by extraction may not be the same as pectins inside a living plant. The gelling properties of extracted pectins have led to a widespread assumption that they are in the gel state (Table 4.1) also within the hydrated cell wall. Although it has not always been critically based, this assumption is broadly consistent with, for example.

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Table 4.1 Distinguishing between phases within the plant cell wall Solid Deforms more or less reversibly (elastically). although the elastic nature of the deformation may be obscured by the behaviour of other components when the whole cell wall deforms. No major change in volume during deformation. Equilibrium with the solution phase is controlled by the solubility product KSP, which is independent of the amount of the solid phase present. So molecules below the surface of the solid phase are considered as being isolated from the solution.

Gel Deformation is more or less reversible as for a solid, although its time dependence may differ, that is viscosity may be significant (for a more rigorous rheological definition see Ross-Murphy (1998)). Water may be squeezed out of a gel or taken up by it under suitable conditions, with corresponding changes in the volume of the gel phase. Equilibrium with the solution phase is controlled by the mean number of crosslinks between polymer molecules. A threedimensional network of polymer chains becomes infinite in extent, quite suddenly, when the mean number of crosslinks per chain exceeds two. This marks the phase transition from a polymer solution to a gel. Because a polysaccharide chain can be attached through a glycosidic linkage only at one end (the reducing end), it is not topologically possible for a polysaccharide. however complex or branched, to form a gel unless additional interchain links form. We call these crosslinks and they may be either covalent or noncovalent.

the NMR relaxation properties of pectins in mum (Ha et a/., 1997). A gel is a three-dimensional network of chains held together by covalent or noncovalent crosslinks, enclosing substantial amounts of water and possibly entrapping other polymers if these are too large to pass through the pores in the network. The relationships between the structure of gels and their properties will be explored later in this chapter. For the moment it is sufficient to state that the crosslinks that establish a gel network can be either single covalent bonds, or regions in which two or more polymer chains associate laterally, rather as they might associate in a fibrous crystalline solid (Figure 4.1). There are two clear problems in using isolated pectins in the gel state as models for pectins in situ: one concerns the nature of the crosslinks, and the other arises from the possibility that the other polysaccharides within the cell wall influence network formation there by pectins. In an older nomenclature, pectic polysaccharides in situ were called protopectin. That terminology will not be retained here, but it emphasises the fact that irreversible changes often occur when pectins are extracted into solution and may indeed be necessary for their extraction. It seems likely that covalent bonds crosslink pectic molecules and keep many of them insoluble within the cell wall until either the crosslinks are cleaved (for example by alkali) or the pectic

BIOPHYSICAL PROPERTIES OF PECTINS

10!

Figure 4.1 Types of gel network, (a) Biopolymer gel network held together by regions of noncovalent association between chains, (b) Synthetic polymer gel network held together by single covalent crosslinks between chains. Note that network formation by some biopolymers, including native pectins in vivo, can involve covalent as well as noncovalent crosslinks.

chains themselves are cleaved between them (Jarvis, 1982; Kim and Carpita, 1992; Hwang et al., 1993; Renard and Thibault, 1993; Sakamoto et al, 1993). It is possible that a small number of crosslinks survive the extraction process and can be found linking pectic molecules in solution (Fishman et al, 2000), but if so their nature has not yet been established. It will emerge in this chapter that gaps in our understanding of the primary structure of pectins—especially of the covalent bonding between one chain and another—are at present one of the most important factors preventing us from constructing a detailed picture of how these molecules work in a mechanical sense in vivo. The pectic gel network is generally considered to interpenetrate the network of cellulose microfibrils crosslinked by xyloglucan (Carpita and Gibeaut, 1993), although cellulose is a more or less crystalline solid and the cellulose/xyloglucan network does not quite conform to the conventional picture of a gel. When polyelectrolytes like pectin are made into what are called co-gels with another polymer, the networks that they form sometimes differ radically from those formed by pectin alone. Phase incompatibility between what are in effect concentrated solutions of the two polymers can precipitate one or both of them to give an inhomogeneous pair of networks with unexpected properties (Chronakis et al., 1997; Dumay et al., 1999; Picout et al, 2000a,b). A theoretical description of two-component gel formation, incorporating the possibility of phase separation, has been given by Tanaka and Ishida (1999). There is some evidence for similar behaviour of pectin-cellulose composites (Chanliaud and Gidley, 1999), and at high concentrations phase separation is also possible in mixed solutions of pectins of contrasting structure (MacDougall et al, 1997). Thus, noncovalent as

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well as covalent network patterns may differ between gels of isolated pectins and the pectic gel in the cell wall. This is a problem even if identical pectins are being compared, but in practice the isolated pectins used for much of the published experimental work on pectin gels have been atypical polymers of commercial origin, usually dominated by the galacturonan component to a greater extent than is normal in native pectins. The exceptions (e.g. Hwang and Kokini, 1992: Kokini and Chou, 1993; MacDougall et al., 1996; Tibbits et al., 1998: Ryden el al., 2000) have been informative, particularly about the significance of the neutral side chains. The pectic group of polysaccharides comprise the following types of glycan chain: • • • • • •

galacturonans (homogalacturonans) alternating rhamnogalacturonan chains (RGI) based on a galacturonosylrhamnose repeating unit |3-D-(1—>-4)-galactans a-L-( 1 —>5)-arabinans and their branched derivatives xylogalacturonans the complex branched polymer fragment rhamnogalacturonan II (RGII)

For full details of structural elements present in pectic substances, see Chapter 1. It is often considered that chains of all these types, in varying proportions, are glycosidically linked together to form a single large pectic molecule. The evidence for and against this is complex. Where information is available it is discussed in chapters 1 and 2 of this book. The clearest statement that can be made is that the arabinans and ^-(1—»4)-galactans form side chains attached to rhamnosyl units of the alternating rhamnogalacturonan (e.g. Renard and Thibault, 1993) to make up what is often called rhamnogalacturonan I (RG-I). Otherwise, glycosidic links between chain types probably exist but the molecular topology—what is attached to what—is less clear (Round et al., 1997, 2001: Fransen et al., 2000; Huisman et al., 2001), and the picture is complicated by occasional covalent links to polymers that would not traditionally be considered pectic, particularly the f3-D-(l —*3/l-H»6)-galactans (type II arabinogalactans or arabinogalactan-proteins) (Andeme-Onzighi et al., 2000) but also proteins (Qi et al., 1995) and hemicelluloses such as xyloglucans (Femenia et al.. 1999: Thompson and Fry, 2000). This chapter is concerned with the strictly pectic chain types listed above. It deals first with the principles of gel formation by polysaccharides and the mechanical origins of gel cohesion. Then, for each of the principal types of pectic chain, what is known about the chain conformation will be used to explain both the elastic behaviour of isolated chains and their potential to form the junction zones of a pectic gel. What follows is focused as much as possible on the pectic polysaccharides in vivo, and not on the extracted pectins of commerce. It is not. however.

BIOPHYSICAL PROPERTIES OF PECTINS

! 03

irrelevant to the food industry because pectins in situ are centrally involved in the development of texture in fruit and cooked vegetable products (Kunzek el al., 1999). To understand how, for example, pectin structure controls the texture of a ripe apple or an apple puree, the best starting point is its properties within the living plant. 4.2

The mechanical properties of biopolymer gels

4.2.1 Gel structure Gels are not always considered to be a bonafide state of matter, but they ought to be. On changing the external conditions, it is commonly possibly to induce abrupt, clearly defined transformations from gels to the solid or solution state. These have the character of phase transitions, as shown for pectins in the phase diagrams of Garnier el al. (1993) and Axelos el al. (1994). A phase bounded by genuine phase transitions to recognisably different states of matter deserves to be called a state of matter in its own right; and if so then gels are one of the most important states of matter in the living world. Most biopolymer gels are held together by domains in which a small number of chains aggregate alongside one another (Figure 4.1). These regions of chain association were termed junction zones in the pioneering studies by Rees and co-workers (Rees, 1977). The gels formed by commercial pectins figured conspicuously in the development of ideas about noncovalent junction zones (Rees, 1977). Noncovalent gel networks must contain both polymer segments that are involved in junction zones and other, connecting, segments that are not. The associating and nonassociating polymer segments can be structurally different, or alternatively they may be structurally identical but differentiated by an equilibrium that leaves chain association incomplete under the prevailing conditions. Some of the underlying principles of noncovalent gel formation have been elegantly tested using recombinant proteins with controllable proportions of associating and nonassociating peptide segments (Petka etal, 1998). Polymer chemistry contains a well-established body of theory for gels held together by single covalent bonds (Figure 4.1), much of it established by Flory and his collaborators nearly half a century ago (Flory, 1953). Many of the Flory principles can be adapted for gels held together by noncovalent junction zones (Flory, 1975), although gels of that kind would be unfamiliar to the physical chemists who study synthetic polymers. Flory theory can explain many features of the mechanical behaviour of covalently networked gels. Adapting these concepts to the rather different structures of gels with noncovalent junction zones brings problems that have not been comprehensively tackled (Nossal, 1996; Ross-Murphy, 1998); this chapter will discuss some of these problems in a qualitative way, avoiding most of the mathematics and jargon of polymer physical chemistry. More rigorous accounts

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can be found elsewhere (Edwards, 1986; Clark and Ross-Murphy, 1987; Nossal, 1996; Ross-Murphy, 1998). A readable description of the principles may be found in Grosberg and Khokhov (1997). 4.2.2 Mechanisms for the deformation of gels under stress There are in principle several ways in which a polysaccharide gel may distort under mechanical stress, and may resist that stress. Only one of these, the rubberlike stretching of single chains between crosslinks, has a parallel in the classical Flory theory of polymer gels. Single chains of some polysaccharides can be stretched by another mechanism, quite unlike rubber elasticity (see below). Either single chains or larger aggregates may resist bending as well as stretching, although bending has been less investigated (Jarvis, 2000). It is also conceivable that noncovalent junction zones may come apart reversibly under lateral tensile stress (Nossal, 1996). Gel networks differ from conventional solids in that their volume is not fixed. Figure 4.2 shows that some forms of network distortion under tensile load will result in a reduction in volume, with water being squeezed out of the gel. In a polyelectrolyte gel, the osmotic potential associated with free charges and their counterions tends to draw water into the gel, and this swelling force is balanced by the volume modulus of the polymer network. The network is thus

Figure 4.2 Extension of a trellis type of gel network, shown here in two dimensions, may involve changes in volume with absorption or expression of water, (a) It the initial polymer orientation is close to normal to the applied force, the gel can extend with little sideways contraction and the volume may increase if water is available for absorption, (b) If the polymer orientation is close to parallel to the applied force, the gel contracts sideways more than it extends and water is squeezed out. In general, if the polymer orientation is at an angle 0 to the applied force, the longitudinal extension is given by d/dQ • sin 9 and the transverse contraction by d/d% • cos 9 for a two-dimensional network as shown, or d/dB • cos2 9 for a three-dimensional network. The transition from expansion to contraction in the volume of an extending three-dimensional trellis-like network occurs where d/dQ • sin 9 = d/dft • cos~ 0. which is at 9 = 30°.

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'pre-stressed' and will be more rigid as a result when subjected to an external tensile stress. These principles are derived in classical polymer chemistry (Flory, 1953). A quantitative treatment of the osmotic swelling effect was applied by Tibbits et al. (1998) and Ryden et al. (2000) to gels made from experimentally isolated pectins, although their interpretation of the results was hindered by the fact that calcium ions simultaneously stabilised the crosslinks and reduced the osmotic swelling potential by counterion condensation. This problem is also encountered with intact cell walls (Jarvis, 1992). However, the principle seems to be correct, and implies that pectic gels can function as simple molecular machines, converting chemical energy (in the form of changing apoplastic cation concentrations) into mechanical work against an external force. To summarise, the mechanical properties of gels depend on the frequency and stability of the crosslinks and the rigidity of the polymer segments between them. For polysaccharides, the conformation of the polymer chains is the key to all these properties, because the lateral association of the chains requires specific conformations and the elastic behaviour of an individual chain is also controlled by its conformation. 4.2.3 Single-chain mechanics The two fundamentally different mechanisms by which a single polymer molecule in solution can resist a stretching force are as follows; they apply also to chain segments between junction zones in a gel. (1) Any flexible polymer chain will not normally be straight: bombarded at every point by water molecules in thermal motion, it will take up a wandering, random-coil shape that constantly fluctuates (Figure 4.3). If the chain is straightened by an external force, this introduces order and carries an entropic penalty. Thus thermal motion generates elastic properties and restores the random-coil conformation whenever the external stress is relaxed. (2) If the polymer chain is already extended, the force may simply stretch it out of its most stable (lowest-enthalpy) conformation and thus make it longer. In the mechanism described by Marszalek et al. (1999a),

Figure 4.3 Pulling a random-coil chain conformation into a straight line is entropically unfavourable and will be resisted by thermal motion in the solution. This gives rise to what is called rubber elasticity or entropic spring behaviour.

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Figure 4.4 Mechanical tension on the diaxial a-( 1 —>4) linkage of a pectic galacturonan (the two vertical C-O bonds in the unstressed conformation on the left) creates two 'atomic levers' that tend to flip the pyranose rings out of their preferred 4 Ci chair (left) into the ' €4 chair conformation (right). The ' €4 chair conformation (right) is about 20% longer because the glycosidic linkage is now diequatonal. and aligned with the chain axis. However this conformation has higher energy owing to steric interactions between the other ring substituents, which are moved into the axial position. Cellulose and related polysaccharides cannot be stretched in this way because their glycosidic linkages are already diequatonal. Based on Marszalek el al. (1999a).

each monomer unit is pulled into a different ring conformation (Figure 4.4). Also, a related mechanism may be deduced from the experiments of Gilsenan et al. (2000); in many polysaccharides the most extended conformation that is sterically possible is not in fact a straight line, as suggested above, but a helix, and it is the axis of the helix that will be straightened during mechanism (1). But if two helical conformations are possible and these differ in projected length per monomer unit, then the transition between them will stretch the chain slightly. Polymers showing these two kinds of elastic behaviour under tension can be described respectively as (1) entropic springs and (2) enthalpic springs, following Marszalek et al. (1998). The term rubber elasticity is often used to describe the behaviour of entropic springs, because rubber is the classical example of a material with these properties—although in this case only the polymer molecules themselves are in thermal motion, and no solvent is present. The elastic restoring force that a rubber-like polysaccharide chain in water can generate depends on the length of the chain that is free, relative to the persistence length, which is a measure of how far it will stay approximately straight. The more flexible the chain, the shorter its persistence length and the more random coiling is possible within the length that is free between points of attachment (Figure 4.5). Rubber elasticity has long been recognised as the principal mechanism controlling the stiffness of the gels formed by covalent crosslinking of synthetic polymers (Flory, 1953). In contrast, the possibility of enthalpic molecular springs was recognised only recently with the discovery that single polymer molecules could be stretched by this mechanism in an atomic force microscope (Marszalek et al., 1998). For polysaccharides at least, enthalpic springs based on changes in monosaccharide ring conformation are so much stiffer than entropic springs that

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Figure 4.5 Effect of persistence length on entropic springs, (a) Highly flexible molecule with short persistence length takes up a rapidly fluctuating, tightly coiled chain conformation when not under tension, (b) Stiffer molecule with longer persistence length is not coiled so tightly and can be extended, against the entropic effect of thermal motion, by a smaller force. Very approximately to scale for galacturonans of low (a) and high (b) charge density with bar = 10 nm.

enthalpic stretching does not begin until entropic stretching is complete and has straightened out the chain, or at least has brought its regular helical axis into a straight line (Marszalek et al., 1998). Apart from that, one feature that can be used to distinguish between the entropic and enthalpic mechanisms of chain elasticity is their temperature dependences. Enthalpic springs become less stiff with increasing temperature, as might be expected. Entropic springs, however, become stiffer as the temperature rises. This behaviour is shown on the macroscopic scale by rubber and synthetic elastomers, which are softer and more readily deformed at low temperature and are cooled by stretching. Some caution is needed when applying the test of temperature dependence, however. A long-standing puzzle (Edwards, 1986) is that the stiffness of many biopolyrner gels is almost independent of temperature, within the limits set by phase transitions. If mechanical stress induces major changes on secondary structure during enthalpic stretching, for example the unfolding of a protein (Marszalek et al., 1999b), then there may be entropy changes in the associated water that complicate the temperature dependence observed. 4.2.4 Junction zones under mechanical stress Covalent crosslinks are not likely to be broken by mechanical forces large enough to disrupt most gels. Noncovalent junction zones, however, may disintegrate when the gel is stressed. Will this be a reversible process leading to elastic deformation on the macroscopic scale, or will it be irreversible and lead to plastic deformation or fracture? The mechanisms by which stress can dismantle junction zones have not been explored in current theories predicting the mechanical properties of polysaccharide gels, although the mechanical consequences have been discussed (Nossal, 1996; Ross-Murphy, 1998). A tentative qualitative treatment is presented here.

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Figure 4.6 Detachment of one chain from a junction zone by a force normal to the junction zone.

Consider a single polymer chain, under tension and having part of its length aggregated with other chains to form a junction zone that initially is normal to the tensile stress. The stress on the other end of the chain is increased until the chain starts to detach from the aggregate (Figure 4.6). What happens next will depend on the relative strength with which successive monomer units of the chain are attached. If the binding strength at the point of chain divergence increases as the chain begins to be stripped off, then it may stop at a point where the external force is matched by the binding strength. When the stress is removed, the aggregate will return to its original state. The existing bound region of the chain will direct the restoration of binding by the segment stripped off, so that it goes back to its original place. Under these conditions, therefore, the chain is stripped reversibly from the junction zone. If the external force is sufficiently large, of course, the chain may be removed completely and irreversibly. The energetic basis of this reversible detachment is as follows. If the segment of a chain involved in a junction zone is not delimited by structural features like the ends of a de-esterified galacturonan block, then its length will be statistically controlled around an optimum where the favourable enthalpy change on adding a further monomer unit to the bound segment is balanced by the unfavourable entropy term. In this case, when an external force starts to strip the chain segment from the aggregate to leave it shorter than the optimum length, there will be a rise in free energy that will result in resistance to the applied force and will return the system to its original state when the force is removed. From the simplified analysis above, it would be deduced that junction zones showing this 'statistical' resistance to stripping would be weakened by a rise in temperature and might melt if the temperature rise were sufficient. However, it may be misleading to describe the favourable and unfavourable terms in the free energy balance as 'enthalpy' and 'entropy', because there will be simultaneous changes in the ordering of associated water molecules and these may dominate the overall entropy changes involved, so that the ensuing temperature dependence of junction-zone stability is difficult to predict.

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If, on the other hand, starting to strip off a chain destabilises the junction zone, then the chain will immediately be pulled right out of the aggregate and removed to a new location where it may no longer be able to bind, or will bind into a different aggregate. The result then will be plastic deformation, creep or fracture. This irreversible mechanism may apply to pectic galacturonan chains that need minimum uninterrupted segments about 12 monomer units long to form junction zones (Powell et al., 1982). It will only apply, though, if the junction zones are initially of no more than this minimum length. Whether the stripping of a chain from a junction zone is reversible or not, the force required to initiate its detachment is what will determine the contribution of the junction zone to gel rigidity. This force does not depend on which scenario applies, but on the strength of binding of a single monomer unit into the aggregate and on some geometrical factors that are worth exploring. With an external stripping force normal to a completely flexible chain, only the bonding of one monomer unit to the junction zone is under stress. The free energy change per monomer unit will then simply determine the force required to initiate stripping of the chain. If the bound part of the chain is stiff, however, a segment longer than one monomer unit may come under stress and the free energy change for its detachment will be greater than that for one monomer unit. This effect is counterbalanced by the fact that the free part of a stiff chain will also bend through a relatively large radius, creating a molecular lever that will increase the local stress at the point where the chain diverges from the junction zone. The net effect will be small if the bound and free parts of the chain are equally stiff; but if the chain conformations are such that the bound part is stiff and the free part is flexible, then stripping of the chain will be more difficult. If the geometry of the point of detachment is examined more closely (Figure 4,7), it can be seen that the length of the bound chain segment over which the stripping load is distributed depends not only on chain stiffness but also on how far the restraining bonds can stretch before they break. This factor depends on what kind of bonds these are: more precisely, on their dissociation energy profile. For example, electrostatic forces will still be apparent at chain separations of several angstroms (unless they depend on the presence of small counterions and these escape). In contrast, dispersion forces diminish with the sixth power of the atom separation and will vanish at an early stage as the chain segment is detached. The strength of a hydrogen bond diminishes to near zero as it increases in O-O length from 2.5 A to about 3.5 A. Forces connected with ordering of water have complex energy profiles extending over several angstroms (Israelachvili and Pashley, 1983; Sorensen et al., 1999). It may be predicted, therefore, that junction zones consisting of stiff chains held together by electrostatic or hydrophobic forces may be more resistant to external stress than would be expected from their binding energy per monomer unit, because the stress is spread along a number of monomer units.

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Figure 4.7 The point of detachment from a junction zone, in more detail than in Figure 4.6. If the chain becoming detached is relatively rigid in bending mode, the detachment zone will be correspondingly long and the number of bonds being broken simultaneously will be greater than if the chain being detached is flexible.

When a junction zone remains intact under stress in the geometry shown in Figure 4.6, the bending of the free part of the chain through 90° will become tighter as the applied force increases. This may allow the bending stiffness of the free polymer chain to make a significant contribution to the elastic modulus of the gel. For pectic chains, it may be assumed that bending stiffness is reversible, is largely enthalpic and will decrease with rising temperature. Until now it has been assumed that the external force is normal to the junction zone. However if the junction zone holds together until the gel network has distorted considerably by other mechanisms, it will reorient so that the force is at an angle 0 to the junction zone (Figure 4.8). The minimum force required to detach the chain will then be increased by a factor of I/cos 0. Thus other mechanisms of distortion can in principle protect a gel network against damage to its junction zones. These other mechanisms do not include the tighter bending of the free

Figure 4.8 If other parts of the gel network are being distorted by the applied stress—for example, if chains between the junction zones are stretching—then the junction zones may become more aligned with the direction of the stress. This increases the stress required to detach a chain from a junction zone by the factor I/cos 9. where 9 is the angle shown.

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portion of a chain adjacent to a junction zone, because the junction zone would then reorient in the opposite direction: reorientation as shown in Figure 4.8 would reduce the potential for elasticity due to this type of chain bending. A quantitative treatment of these ideas will not be attempted here, but it can be seen that the incorporation of mechanical stress into the enthalpy terms of a conventional thermodynarnic treatment of junction zone stability is conceptually feasible and potentially has predictive power. In classical thermodynamics it is only heat that is allowed to disturb a macromolecular structure. To understand noncovalent networks under mechanical stress, on the other hand, we need to consider not only how they are held together but also the routes by which they come apart. We may begin to talk, therefore, of a kind of supramolecular fracture mechanics. 4.3

Mechanochemistry of the component chains of pectins

4.3,1 Chain conformation The conformations of the different chain types included in the pectic complex have been reviewed by Braccini et al. (1999) and Perez et al. (2000). The a-(l —>4)-linkage between galacturonic acid units has rather limited steric flexibility (Gouvion et al., 1995), although more than cellulose and its relatives. This linkage geometry, established by NMR measurements and modelling of oligosaccharides (Hricovini et al, 1991; Cros et al., 1993; DiNola et al., 1994; Gouvion etal., 1994;Bouchemal-Chibani, 1995;Ruggieroe/a/., 1995a) allows longer a-(l-»4)-galacturonan chains to form a number of regular helical conformations separated by relatively low energy barriers. These include twofold (2i) helices in which alternate monomer residues face in opposite directions. Cellulose also forms a twofold helix, but one that is almost flat, whereas the diaxial a-(l ->4)-galacturonosyl linkage in pectins forces the chain into a much less extended zig-zag shape. Also, the galacturonan chains can twist from this conformation in either direction to become right-handed or left-handed (3 \ or ^2) threefold helices with three monomer units per 360° turn. Scavetta et al. (1999) found glycosidic conformations similar to both 2\ and 3\ helical galacturonan segments in the crystal structure of a pectic lyase enzyme with its oligomeric galacturonan substrate attached. Galacturonan chains in solution explore all the conformational space delineated by these helical forms, but the classic circular dichroism study of Morris et al. (1982) suggested that conformations close to the twofold helical form predominated for low-methoxyl galacturonans in solution, at least at low concentrations, ambient temperature and neutral pH. Gilsenan etal. (2000) provided additional calorimetric and Theological evidence that the twofold helix predominated under neutral conditions but that the 3\ helix became more important in solutions below pH 3 or as the temperature was lowered, the transition

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between the two helical forms (or less regular conformations close to these) being followed by circular dichroism. Isolated galacturonan chains are relatively stiff. Modelling studies predict a persistence length in the range 50-100 A, equivalent to the extended length of 10-20 monosaccharide residues, provided that the flexibility of the monomer units themselves is taken into account (Boutherin et al., 1997): otherwise the chains are predicted to be stiffer (Ruggiero et al., 1995b). These predictions are in line with viscometric measurements (Axelos and Thibault, 1991; Chou et al, 1991; Hourdet and Muller, 199la; Axelos and Branger. 1993; Catoire et al., 1998), size-exclusion chromatography (Hourdet and Muller, 1991a,b) and neutron scattering (Cros et al., 1996). When the carboxyl group is ionised, repulsion between charges stiffens the polymer chain and increases the persistence length to the upper end of this range (Boutherin et al., 1997; Morris et al., 2000). The large-scale conformations of these stiff galacturonan chains have generally been represented as smooth, gently curving coils, but Catoire et al. (1997) suggest that straight 2\ helical segments 130 A long, joined by transient, thermally induced kinks, might be a more realistic model. Alternating rhamnogalacturonan chains appear to be rod-like in conformation, with comparable stiffness to galacturonans (Cros etal, 1996). It was once thought (Jarvis, 1984) that single rhamnose residues might introduce kinks in the conformation of a galacturonan chain, and these have been modelled (Ruggiero et al., 1995b), but there is now evidence that rhamnosyl residues generally occur within rhamnogalacturonans rather than as isolated residues of this kind (Zhan etal, 1998). The pectic side chains are less rigid. The pectic arabinans are branched molecules based on a-(l—»5)-linked chains of arabinofuranose units with (1,3) branch points. The a-(l—>5)-linked arabinan chain is exceptionally flexible because the monosaccharide rings are joined by a C1-O-C5-C4 linkage instead of the simple glycosidic oxygen that forms the bridging unit in other cell-wall polymers. This flexibility allows a wide range of disaccharide conformations (Cros et al, 1994). The conformation of arabinans in situ in the hydrated sugar beet cell wall was averaged by thermal motion within the NMR timescale (ns). to give direct-polarisation 13C NMR spectra (Renard and Jarvis, 1999a) very much like those observed in solution. The other polysaccharides of the beet cell wall are too rigid for their direct-polarisation 13C NMR spectra to be observed under these conditions. This implies that although each chain is covalently attached to a more rigid pectic segment at least at its inner end, most of the chain is not affected by this anchorage and can reorient and coil as freely as it does in solution, without any constraints imposed by aggregation. The ^-(l->4)-galactosidic linkage has limited but significant flexibility (Duda et al, 1991). Because the galactan chains are uncharged, they are less extended by electrostatic repulsion than are the galacturonans, as evidenced by NMR relaxation experiments on galactans in situ in cell walls and by the

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1 13

motional averaging of the I3 C chemical shifts to essentially their solution-state values on the NMR time scale (Foster et al., 1996; Jarvis and McCann, 2000). On this basis the conformation of the unstressed galactan chains is likely to be a tighter random coil than for the galacturonans, with shorter persistence length. 4.3.2 Single pectic chains under tension Pectic galacturonans are stiff molecules with a relatively limited capacity to act as entropic springs, because they do not coil up tightly when unstressed. The extent to which they coil depends on the density of free charges, which increases the persistence length, and on how far apart are the crosslinks. These factors correlated with gel rigidity, which increased with temperature, as expected for an entropy-driven effect, in the experiments of MacDougall et al. (1996). Galacturonan chains in solution might in principle also show another kind of entropy-driven elasticity due to the transition, under tension, from the 2\ helical chain conformation to the 3\ helical form. This will happen only if the projected length h of a monomer unit on the chain axis increases during the transition. It is an interesting characteristic of galacturonan chains that h changes relatively little across all the stable helical conformations (Perez et al, 2000). However, the data of Braccini et al. (1999) suggest a slight lengthening in h, by about 4%, on going from the 2\ to the 3\ helical form. This might be enough to permit a tension-driven conformational transition. The enthalpy change involved in the absence of mechanical stress is about 0.5kcal/mol (Gilsenan et al., 2000), but associated entropy changes, possibly connected with water structure, almost balance this. The two forms are therefore quite close to equilibrium even without tension, and the position of the equilibrium can be altered by manipulating pH or temperature (Gilsenan et al., 2000). Thus, if the temperature and ionic conditions are such that the 2\ helical form has slightly lower free energy in the absence of mechanical stress, the imposition of tension should stretch it into the 3\ helical form—provided that it is free to untwist. The ability of certain polysaccharides to behave as enthalpic springs has recently been demonstrated by the experiments of Marszalek et al. (1998, 1999a). When single galacturonan chains were tensioned by atomic force microscopy (AFM) (Marszalek et al., 1999a), first a small amount of entropic resistance was generated as the chains straightened out, and then much higher levels of enthalpic resistance appeared as each galacturonosyl ring was distorted from the most stable 4 C] chair conformation, first to a boat conformation and then to the inverse chair conformation. Each monosaccharide residue was stretched in length by about 20%, with a calculated energy input of 11 kcaUmol. The stretching of the molecule was completely reversible, with no hysteresis that might indicate destruction of intermolecular interactions.

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Whether a polysaccharide is capable of this kind of enthalpic elasticity depends on the configuration of the glycosidic linkages. In an a-(l—»4)-galacturonan the glycosidic link is diaxial; that is, both the C1-O1 and the C4-O1 bonds are at approximately right angles to the plane of the ring. When the chain is tensioned, each of these bonds acts as an 'atomic lever', generating torque to pull the ring out of its initial 4Q chair conformation (Marszalek el al., 1999a). Cellulose, in contrast, has both bonds to the glycosidic oxygen in the equatorial position so that they do not exert any torque at the pivot lines around which the ring must twist to change conformation, and substituted celluloses did not behave as enthalpic springs when placed under tension in AFM (Marszalek et al., 1998). The hemicelluloses all share the same diequatorially linked |3(1—>4)-pyranosyl structure and it may be assumed that like cellulose they will not show enthalpic elasticity under tension. So far as the potential for enthalpic elasticity in other kinds of pectic chain is concerned, there has been no similar experimentation, but the principles established by Marszalek et al. (1998) allow predictions to be made about the behaviour of these chains under tension once stretched into a linear conformation, lonisation or methyl-esterification of the carboxyl group on a galacturonan would make little difference. At most, the ionised form would have a slightly lower force constant for enthalpic extension due to electrostatic repulsion between the carboxyls on successive galacturonosyl residues (Boutherin et al., 1997). Acetylation or substitution with xylose on O-2 or O-3, likewise, would not prevent the galacturonan chain from behaving as an enthalpic spring, but might increase the force constant due to steric hindrance as the bulky subsitituent moved from an equatorial into an axial position. The 2-substituted ot-L-rhamnosyl residues of rhamnogalacturonan I are not capable of enthalpic extension but the 4-substituted a-D-galacturonosyl residues can be assumed to behave as enthalpic springs as in the galacturonan homopolymer (Marszalek etal., 1999a). The (3-D-O—»4)-galactan chains have a different glycosidic configuration. Only the bond from C-4 to the glycosidic oxygen is axial, but this should be sufficient to distort the ring into a boat conformation under tension, rather as in amylose (Marszalek et al., 1998), and allow the galactan chain to behave as an enthalpic spring up to about 10% extension. With rather tighter random coiling than the galacturonans owing to the lack of electrostatic repulsion, the galactan chains can be expected to behave as correspondingly stiffer entropic springs while being extended into the linear conformation for which enthalpic extension can commence. In the a-L-(l—*5)-arabinan chains, the arabinofuranose ring is much more flexible than the pyranose rings of the polysaccharides considered above, and the glycosyl linkage also allows a great deal of flexibility. In the more extended helical conformations suggested by Cros et al. (1994) the C1-O1 bond is correctly positioned to act as an atomic lever distorting the ring, but the possible

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11 5

ring conformations differ little in energy and a tensile force stretching the molecule in this way would meet little resistance. However, the flexibility of the arabinan chain conformation, manifested in still more solution-like NMR behaviour (Renard and Jarvis, 1999a) than for the p-(l-»4)-galactans, means that the arabinans are ideally configured to act as entropic springs. We arrive, therefore, at a generalisation: all the chain types that constitute the pectic complex are elastic when tensioned, although the mechanism of their elasticity can be entropic, enthalpic or both. In contrast, none of the other kinds of polysaccharide in the primary cell wall can act as enthalpic springs, and most of them are too stiff to be very effective as entropic springs. This distinction between pectic and other polysaccharides follows directly from fundamental features of their stereochemistry. At this point some caution is needed. A polymer can act as a spring only when it is stretched between two anchor points. Most pectic chains are glycosidically anchored at their reducing end, but we know very little about covalent anchorages elsewhere (Chapter 2). If pectic molecules form a gel by association of galacturonan chains to form junction zones, then there is an opportunity for the galacturonan segments between these junction zones to act as enthalpic springs. Classical polymer theory can lead to predictions of the macroscopic mechanical properties of gels based on the frequency of junction zones and the elastic properties of the chain segments between (e.g. MacDougall et al., 1996), although the elasticity is conventionally treated as entropic only. However the arabinan and galactan chains seem to have little potential for the formation of noncovalent junction zones (see Section 3.3) and, although covalently anchored at the inner end to rhamnogalacturonan, they are often assumed to be unattached to anything at their outer ends (except in beet and its relatives). If that is correct, then they cannot function as entropic springs. 4.3.3 Chain aggregation and the potential formation of junction zones Galacturonan chains can associate with one another in a number of ways depending on their structure. The most widely known type of aggregate is the calciumstabilised 'egg-box' dimer, first established in the crystal structure of the related polymer L-guluronan (Grant et al., 1973) and extended to calcium-galacturonan gels by Morris et al. (1982) using circular dichroism measurements. The 'eggbox' dimer structure has its galacturonan chains in the 2\ helical conformation but, as in solution, the 3\ helical form is also possible (Walkinshaw and Arnott, 1981b; Rigby et al., 2000) and there is little free energy difference between aggregates in these two conformations. 'Egg-box' junction zones appear to favoured in gels with low polymer concentration (Morris et al., 1982; Powell et al., 1982), while high polymer concentrations and low pH favour the formation of tetramers or similar-sized aggregates of chains in the 3\ helical form (Walkinshaw and Arnott, 1981b; Jarvis and Apperley, 1996; Gilsenan et al..

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Figure 4.9 Types of junction zones in calcium pectate gels, and the cable model (Goldberg et al. 1996) for their aggregation on two levels: dimerisation of galacturonan chains in the egg-box conformation, and grouping into larger aggregates in either the egg-box or the 3] helical chain conformation.

2000). A mixture of the two helical forms can be inferred from the solid-state NMR spectra of hydrated cell walls (Ha et al., 1997'; Tang et al., 1999). The 'cable' model of Goldberg et al. (1996) summarises these possible interactions (Figure 4.9). Calcium ions are bound more strongly by galacturonans in the gel state than in solution, and mechanically strong gels tend to bind calcium more tightly. Quantitatively, calcium binding has been described on the basis of a wide range of different physical models, some of which emphasise the relationship to the solution state (Gillet et al., 1998) and others that to solids (e.g. Dainty and Hope. 1961; Bush and McColl, 1987): this illustrates the Cinderella position of gels as a state of matter. The most difficult question is how to separate cation-specific binding interactions from the electrostatic contribution to the binding energy. That some ions are more strongly bound than others of the same valency is not in doubt: Pb2+ and Cu2+ are much more strongly bound than Ca2+, which is in turn more strongly bound than Mg2+ (Dronnet et al., 1996; Malovikova et al.. 1994). There is also variation in the strength of binding of monovalent cations (Ghagare et al., 1992; Abramovic and Klofutar, 1997). A selectivity coefficient is often introduced to describe the behaviour of the more strongly bound cations (Voue and Gillet, 1994; Dronnet et al., 1996). This assumes, however, that the electrostatic interaction is the same for all cations of the same valency, an assumption that may not be correct. If the Manning parameter ^ (Manning, 1978; but see Moss et al., 1994) is used to describe the linear charge density of the polymer chain in solution, then when on gelation a number of chains associate to form a junction zone, the linear charge density might be considered to include the negative charges on all the chains in the junction zone. The strength of purely electrostatic binding and the extent of

BIOPHYSICAL PROPERTIES OF PECTINS

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cation condensation would then be increased in proportion to the number of chains in the junction zone. This would be consistent with the greater observed affinity for cations of pectins in the gel state than in solution (e.g. Tibbits et al., 1998). If the number of chains in the junction zone varies with the size (steric fit) and hydration of the cation, this will affect the strength of cation binding independently of any selective effect of the chelation type. If chain packing is disturbed by acetylation there may be consequent effects on cation binding, even though a gel may still form (Oosterveld et al., 2000; Renard and Jarvis, 1999b). Such chelation effects do exist, however: modelling studies (Braccini et al, 1999) show that both helical forms of pectate have hydroxyl and carboxyl groups correctly positioned to match the coordination geometry of the Ca2+ ion, displacing up to four of its six associated water molecules. Using X~ray absorption fine structure analysis, Alagna et al. (1986) showed that calcium ions in pectate gels were indeed coordinated in this geometry. Features of the FTIR spectra of pectic films (Wellner et al., 1998) were also consistent with this type of coordination for a wider range of cations. The binding of cations other than calcium has been studied mainly in the context of utilising pectins as waste-water decontaminating agents (Dronnet et al., 1998; Harel et al., 2000). Some cations bind much more strongly than calcium, and although there is no doubt that calcium is a major pectic counterion within the native cell wall (Rihouey et al., 1995; Goldberg et al., 1996), it is uncertain whether other divalent ions, or cations of higher valency, might also be present in vivo. Binding of A13+ has been suggested (Blarney and Dowling, 1995; Chang et al., 1999), and features of the electron energy loss spectra of cell walls suggest the presence of Ti4+ (I.M. Huxham and M.C. Jarvis, unpublished). Polyamines are capable of electrostatic binding to pectic galacturonans, although their affinity for pectins is not very high (Messiaen and Van Cutsem, 1997, 1999). Strong peptide-galacturonan binding would be predicted for a cationic peptide if the charge spacing matched that of the negative charges in one of the preferred helical conformations of the galacturonan. Moderate interactions with polylysine have indeed been demonstrated (Bystricky et al., 1991), and were specific for one enantiomer of the peptide (Bystricky and Malovikova, 1992; Paradossi etal., 1999). There are a number of instances where noncovalent pectin-protein interactions appear to have biological significance (Penel and Greppin, 1996; Lord et al., 2000; Mollet et al., 2000; Rizk et al., 2000), although not all of these interactions are necessarily electrostatic and some that are may involve an anionic peptide moiety and a bridging inorganic cation, rather than a cationic peptide. The gels formed by high-methoxyl commercial pectins in the presence of sugar and acid are held together by junction zones containing about four galacturonan chains in the 3\ helical conformation (Walkinshaw and Arnott, 198la). The constituent chains are not twisted round one another but pack side-byside with the methoxyl groups, forming a hydrophobic column in the centre

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of the aggregate. The methyl groups, which are capable of rotation even in the absence of water, form an island of water-independent molecular motion within the hydrogen-bonded network of the gel (Ha etal., 1997; Williams et al., 1998). The need for hydrophobic association to hold the aggregate together is the reason why such gels require sugar, to lower the water activity, before they can form. Free negative charges appear to disperse aggregates of this type, but protonated carboxyl groups can be accomodated, and gels with both this and the calcium-mediated form of chain association have been described at low pH (Gidley et al, 1980; Fu and Rao, 1999,2001; Gilsenan et al., 2000). Dissolution at elevated temperature (Furuta et al., 2000) may be a characteristic of these mixed gels. It is not clear whether the water activity in cell walls is ever low enough to promote hydrophobic association. Water is certainly strongly bound by both cell walls and isolated pectins (lijima et al., 2000; Kerr and Wicker. 2000; Ryden etal., 2000). Regular associated forms of the P-( 1 —»4)-linked galactan side chains do not appear to form readily. No evidence of even limited crystallinity was found, for example, in cell walls of lupin seeds by solid-state NMR (M.C. Jarvis. D.C. Apperley and J.S.G. Reid, unpublished). Entanglement of the side chains is responsible for some features of the viscometric behaviour of branched pectins in solution (Hwang and Kokini, 1992) but this does not imply the capacity to resist sustained mechanical stress in the gel state. Linear a-( 1—>5)linked arabinans, however, can aggregate and precipitate from aqueous solution under some conditions. These precipitates can form a cloud in apple juice, but it seems that this occurs only after enzymatic debranching of the native, branched arabinans. The precipitates are microcrystalline, with the arabinan chains in a twofold helical conformation, which allows a number of possible arrangements of hydrogen bonding between adjacent chains (Radha and Chandrasekaran, 1997). Their CP-MAS 13C NMR spectrum, recorded under conditions suitable for rigid solids rather than the solution-like phases probed by the direct-polarisation I3C NMR experiments of Renard and Jarvis (1999a). showed relatively sharp lines superimposed on broader peaks (D.C. Apperley and M.C. Jarvis, unpublished). Narrow lines in this experiment are indicative of crystalline order. A doublet observed for C-l was consistent with a two-chain or two-residue unit cell. We have not recorded these distinctive features in the CP-MAS 13C NMR spectra of intact plant cell walls. It probably follows that this type of interaction between arabinan chains does not occur in situ, possibly because the native pectic arabinans are too branched. 4.3.4 Covalent crosslinks If pectins were retained in the cell wall only by noncovalent association of their galacturonan chains, it would be expected that the removal of divalent cations by chelating agents would lead to quantitative pectin solubilisation. as the other noncovalent mechanisms of chain association described above do

BIOPHYSICAL PROPERTIES OF PECTINS

! 19

not seem likely to keep pectins insoluble. However, only some of the pectic fraction is solubilised by chelators. The proportion is normally less than half (Jarvis et al., 1988; Renard and Thibault, 1993), unless either the cell walls are from ripe fruit in which there has been enzymatic depolymerisation of pectins, or the temperature of extraction is elevated into the range (>80°C) where galacturonan chains esterified on C-6 are depolymerised by the ^-elimination reaction. Much of the pectin that is not released by cation chelation becomes soluble on extraction with alkali under relatively mild conditions, comparable with the alkaline conditions needed to cleave methyl or acetyl esters. Pectins released on alkaline extraction normally contain more of the RG-I component and its associated side chains ('hairy regions') than those released by chelators (Jarvis, 1982; Redgwell and Selvendran, 1986). These observations are consistent with the idea that within the cell wall there are covalent, alkali-labile bonds crosslinking pectic chains either to one another or to insoluble, non-pectic polysaccharides. In the special case of sugar-beet and its relatives, feruloyl esters attached to the outer ends of the arabinan and galactan side chains (Rombouts and Thibault, 1986; Colquhoun et al., 1994) fulfil these criteria because they can be coupled by oxidative dimerisation of the ferulate portion. Extracted sugar-beet pectin can be made to form covalently crosslinked gels in this way in vitro. However, these pectic feruloyl esters appear to be scarce or absent outside the Centrospermae, and they can tentatively be discounted as the origin of alkali-extractable pectins in other dicot families. Also consistent with the idea of alkali-labile covalent crosslinking is the discovery by Kim and Carpita (1992) that 10-15% of the galacturonoyl carboxy 1 groups in maize pectin carry an unidentified substituent other than methanol that is released by similar alkaline conditions. We have confirmed their findings for dicot cell walls by methods independent of theirs (Mackinnon et al., 2002). The non-methyl substituents have generally been assumed to be esters, but amides are also possible (Perrone et al., 1998). The borodeuteride reduction method of Kim and Carpita (1992) unambiguously identifies the acyl moiety involved in an ester or amide linkage, but no corresponding procedure is available to identify the other moiety, because hydroxyl and amino protons are labile and the alcohol C-O bond remains intact during acid, alkaline and reductive cleavage of a galacturonoyl ester. It is possible, therefore, that pectic galacturonans are held within cell walls by galacturoyl ester or amide crosslinks identifiable with some of the non-methyl substituents found by Kim and Carpita (1992). But we do not know what is at the other end of these crosslinks. Until this is determined we cannot even guess at how external forces are distributed across the pectic gel network within the cell wall. It seems reasonable that an external force large enough to destroy noncovalent crosslinks between pectic molecules will then tension the sequence of chains connecting two covalent crosslinks. We need to know what this chain sequence is before we can evaluate how it will stretch when tensioned, and hence how the stressed gel will behave.

120

PECTINS AND THEIR MANIPULATION

Another form of covalent crosslinking has recently become well established. Borate forms acid-labile diester linkages (Kobayashi et al., 1996) with the apiosyl residues of the pectic constituent rhamnogalacturonan II (RG-II). The vicinyl dihydroxy system of apiofuranose is ideally configured to form either noncovalent or diester links to borate (Power and Woods, 1997), and a single borate moiety can form a tetrahedral complex that crosslinks apiosyl residues from two RG-II segments (Kobayashi et al, 1996; O'Neill et al, 1996). While some details remain to be elucidated, it is clear that the complex structure of RG-II is strongly conserved (Hirano et al, 1994; Ishii and Matsunaga. 1996: DocoetaL, 1997; Shine/ al, 1998; Shimokawae/a/., 1999; Edashige and Ishii. 1998; Vidal etal, 2000) in those higher plants that have been examined. It seems likely that the complexity of RG-II provides the conformation (Mazeau and Perez, 1998; du Penhoat et al, 1999) necessary to present the apiosyl residues in the correct orientation for diester crosslinking with borate. The borate-binding apiosyl residue in the A side chain of RG-II (Ishii et al. 1999) is located in a highly anionic environment flanked by the galacturonan core chain on one side, and on the other by a rhamnosyl residue doubly substituted with galacturonic acid. Two such apiosyl residues are crosslinked by the borate diester. Not surprisingly, the formation of the borate was found to be influenced by the nature of the cations present as well as by pH (O'Neill et al, 1996; Matoh and Kobayashi, 1998; Ishii et al. 1999; Kobayashi et al.. 1999). In these experiments the separation of kinetic and equilibrium factors was not entirely satisfactory, but it appears that (a) below pH 3-4 the equilibrium favours the monomer, while at high pH the dimer is stable (O'Neill et al.. 1996); (b) the rate of interconversion is slow above pH 5 (O'Neill et al.. 1996): (c) certain divalent cations (Ba 2+ , Sr2+, Pb 2+ ) with ionic radius > 1.1 A enhance dimerisation, apparently kinetically, although they bind preferentially to the dimer and their effect on the equilibrium and the rate of the reverse reaction is unclear (Ishii et al., 1999); (d) Ca2+ is claimed to stabilise the dimer in vitro (Matoh and Kobayashi, 1998; Kobayashi et al, 1999) and in vivo. The apiosyl residue in the B side chain of RG-II provides a further potential site for borate ester formation that must have been occupied in the RG-II dimer examined by Kobayashi et al (1999) as this contained two borate moieties. While a number of features of ester formation and hydrolysis remain to be clarified, it is clear from this that RG-II has characteristics that would normally be associated with enzymes and are unique for a carbohydrate: catalysis (or strictly autocatalysis) of the formation and cleavage of covalent bonds by a macromolecule in a tightly defined conformation with associated metal ions. Walls of suspension-cultured Chenopodium cells showed larger pore size under boron deficiency; when control cells reached the stationary phase, borondeficient cells continued to expand and eventually burst (Fleischer et al. 1998). The increase in pore size could alternatively be induced by removal of either borate or Ca 2+ , and was reversed within 1 min bv addition of borate (Fleischer

BIOPHYSICAL PROPERTIES OF PECTINS

12 1

et al, 1999). Control of pore size, and cohesion between layers within the cell wall, are recognised functions of the pectic polysaccharides (Jarvis, 1992; Fleischer el al., 1999). However, before we can describe in detail how RG-II dimers might participate in distributing stress within the cell wall, it will be necessary to know how they fit into the primary structure of pectic molecules; that is, how they are glycosidially attached to other pectic chains. Is it likely that covalent crosslinks between pectic molecules will be cleaved by mechanical stress? It is possible to give a partial answer to this question in qualitative terms. Carboxylic esters of pectin are technically unstable, in that free energy is released when they are hydrolysed, under the ambient conditions of the cell wall. The barrier to their hydrolysis at neutral pH is purely kinetic, and catalysis of the hydrolytic reaction by pectin methylesterase is evidence of this. If there is a role for mechanical stress, it is to reduce the activation energy for ester hydrolysis by pulling the ester linkage towards its transition state. The activation enthalpy will be reduced by the product of the force and the increase in length of the alcoholic C-O bond when the force is applied. We need, therefore, to know a good deal about both the reaction mechanism and the geometry of the molecular system under stress before any predictions can be made. In the case of carboxylic esters the free energy of activation for hydrolysis is of the order of tens of kcal/mol and it is clear that the forces needed to have any effect will be some orders of magnitude greater than those needed to disrupt noncovalent junction zones. Borate esters, being closer to equilibration, may be influenced by smaller forces, but less is known about the mechanism and geometry of their hydrolysis. For a more detailed discussion of links between pectic polysaccharides and other cell-wall components, see Chapter 2. 4.4 Conclusions: pectic gels under stress We have seen that an external force can deform a pectic gel by disrupting the crosslinks that hold it together or by stretching individual chains between crosslinks. Rough estimates are possible for the mechanical energy input needed to distort the gel structure each in these ways. With this information we can make informed guesses at the sequence of events as the magnitude of the external stress is increased. To do this it is necessary to reduce macroscopic stresses to forces at the molecular level. In a pectic gel of concentration 50 g/1 with half of the polymer incorporated in junction zones, the free chains are spaced so as to occupy a mean of about 10 nm2 of the gel in a plane normal to the chain axis. The internal swelling stress generated by osmotic pressure within the gel (Tibbits et al., 1998; Ryden et al, 2000) is of the order of 1 kN/m2 at the rather low ionic strengths typical of the apoplast (Goldberg et al., 1996; MacDougall et al., 1995). This stress is equivalent to 0.001 pN/nm 2 or a mean stress of 0.01 pN

122

PECTINS AND THEIR MANIPULATION

per chain, although some chains may be loaded more than others if the network geometry concentrates the stress. Based on the experimental data of Marszalek el al. (1999a), the mean stress per chain is not likely to be enough to straighten out any chain segments that are long enough, between crosslinks, to adopt a random-coil conformation. Any such chain segments will therefore be available to act as entropic springs under external stresses of greater magnitude than this internal swelling stress. Turgor-imposed stresses on cell walls are typically of the order of 10 MN/m 2 , both in the plane of the cell wall and normal to this plane at tricellular junctions (Jarvis, 1998). It is not clear to what extent the pectic fraction carries these stresses, but they correspond to a mean of about lOOpN per pectic chain. At single-chain stresses of this order, pectic chains will straighten out and behave as entropic springs if they are initially in a random-coil conformation: that is, if the length of chain that is free between crosslinks significantly exceeds the persistence length. This will be true for galacturonan chains with widely spaced crosslinks, at least some tens of monosaccharide residues apart. It will also be true for shorter arabinan and galactan side chains if—and only if—they carry a covalent crosslink at their outer extremity, as in feruloylated sugar-beet pectins. Single-chain stresses of this order should also be sufficient to induce a 2\3\ helical transition in galacturonan chains, if the ionic conditions are such that the 2\ helical form is only slightly favoured in the absence of mechanical stress. The data of Gilsenan et al. (2000) suggest that a typical free energy difference at ambient temperature would be of the order of 0.1 kcal/mol. two orders of magnitude less than is required for enthalpic stretching of the chain. This conformational transition is therefore possible under tensile stress of the same order as is required for entropic stretching. More importantly, a stressinduced change in helical conformation might facilitate the stripping of the chain from a junction zone in which the aggregated chains are in the 2\ helical form. It is not possible to predict either turgor-induced single-chain stresses or the stability of junction zones between galacturonans precisely enough to confirm whether damage to the junction zones would indeed occur; but the forces concerned are possibly of a similar order of magnitude. More extreme stresses, of the order of 100 MN/m 2 on the macroscopic scale, would be required to make pectic chains behave as enthalpic springs through change in ring conformation. Stresses on this scale, if applied to the pectic gel as a whole, would probably destroy noncovalent junction zones. However, a covalent gel network would survive under this level of mechanical stress and could be expected to behave elastically by the enthalpic mechanism. Polysaccharide chains can be cleaved by mechanical stress, but not easily (Corredig and Wicker. 2001). More realistically perhaps, localised enthalpic stretching might relieve the tension on particularly heavily loaded junction zones and equalise loads across the constituent chains of a nonuniform gel.

BIOPHYSICAL PROPERTIES OF PECTINS

1 23

Table 4.2 Types of distortion potentially induced by mechanical stress in pectic gels Mode of deformation Entropic stretching: galacturonans 2|/J| helical transition: galacturonans Entropic stretching: arabinans Detachment from junction zone: galacturonans Enthalpic stretching

Extension possible (%)

Tensile force/chain (pN)

Macroscopic stress (MPa)

<100 5 >100 <100 20

1-10 1-10 10-100 10-100 1000

0.1-1 0.1-2 I -10 1-10 100

This sequence of effects is summarised in Table 4.2. It must be noted that there is a considerable margin of uncertainty about the threshold stresses required for all these types of deformation, and the order in which the components of a pectic gel might come apart may differ depending on its structure. For purely noncovalent galacturonan gels, our knowledge or their internal mechanics is now sufficiently complete, and the gel structure is simple enough, for at least some aspects of their macroscopic mechanical behaviour to be understood at the molecular level. This is an encouraging precedent, as there are other biological systems, for example cartilage and other mammalian soft tissues, where this kind of understanding would be valuable. Real pectins in vivo are another matter, however. Our knowledge of their primary structure and the position of the possible covalent crosslinks does not allow us to say what sequence of pectic chains might be present between one crosslink and another. Without this knowledge we cannot say, for example, whether the RG-I side chains are in a position to fulfil their potential as entropic springs, nor what kind of chains—springy or otherwise—might lie between two RG-II units anchored by borate diester linkages. Nor can we construct hypotheses on how the structure of pectins might relate to their role in providing developmentally determined qualities of strength and resilience (Fleischer et al., 1998, 1999; McCartney et al,, 2000) to the walls of living plant cells. New research in these areas will be necessary before we can claim to understand the relationship between pectin structure and function.

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Ishii, T. and Matsunaga, T. (1996) Isolation and characterization of a boron-rhamnogalacturonan-II complex from cell walls of sugar beet pulp. Carbohydr. Res., 284, 1-9. Ishii, T, Matsunaga, T., Pellerin, P., O'Neill, M.A., Darvill, A. and Albersheim, P. (1999) The plant cell wall polysaccharide rhamnogalacturonan II self-assembles into a covalently crosslinked dinner, ,/. Biol. Chem., 274, 13098-13104. Israelachvili, J.N. and Pashley, R.M. (1983) Molecular layering of water at surfaces and origin of repulsive hydration. Nature, 306, 249-250. Jarvis, M.C. (1982) The proportion of calcium-bound pectin in plant cell walls. Planta, 154, 344-346. Jarvis, M.C. (1984) Structure and properties of pectin gels in plant cell walls. Plant Cell Environ., 7, 153-164. Jarvis, M.C. (1992) Control of thickness of collenchyma cell walls by pectins. Planta, 187, 218-220. Jarvis, M.C. (1998) Intercellular separation forces generated by intracellular pressure. Plant Cell Environ.,21, 1307-1310. Jarvis, M.C. (2000) Intercon version of the la and 1(3 forms of cellulose by bending.Carbohydr, Res., 325, 150-154. Jarvis, M.C. and Apperley, D.C. (1995) Chain conformation in concentrated pectic gels: evidence from 13 C NMR. Carbohydr. Res., 275, 131-145. Jarvis, M.C. and Apperley, D.C. (1996) Chain conformation in concentrated pectic gels: evidence from I3 C NMR. Carbohydr. Res., 275, 131-145. Jarvis, M.C., Forsyth, W. and Duncan, H.J. (1988) A survey of the pectic content of nonlignified monocot cell-walls. Plant Physiol, 88, 309-341. Jarvis, M.C., and McCann, M.C. (2000) Cell wall biophysics: concepts and methodology. Plant Biochem. Physiol., 38, 1-13. Kerr, W.L. and Wicker, L. (2000) NMR proton relaxation measurements of water associated with high methoxy and low methoxy pectins. Carbohydr. Polyrn., 42, 133-141. Kim, J.B. and Carpita, N.C. (1992) Changes in esterification of uronic acid groups of cell wall polysaccharides during elongation of maize coleoptiles. Plant Physiol., 98, 646—653. Kobayashi, M., Match, T. and Azuma, J. (1996) Two chains of rhamnogalacturonan II are crosslinked by borate-diol ester bonds in higher plant cell walls. Plant Physiol., 110, 1017-1020. Kobayashi, M., Ohno, K. and Matoh, T. (1997) Boron nutrition of cultured tobacco BY-2 cells. 2. Characterization of the boron-polysaccharide complex. Plant Cell Physiol., 38, 676-683. Kobayashi, M., Nakagawa, H., Asaka, T. and Matoh, T. (1999) Borate-rhamnogalacturonan II bonding reinforced by Cai+ retains pectic polysaccharides in higher-plant cell walls. Plant Physiol., 119, 199-203. Kokini, J.L. and Chou, T.C. (1993) Comparison of the conformation of tomato pectins with apple and citrus pectins. J. Texture Stud., 24, 117-137. Kunzek, H., Kabbert, R. and Gloyna, D. (1999) Aspects of material science in food processing: changes in plant cell walls of fruits and vegetables. Z. Lebensm. Unters. Forsch. A-FoodRes. Technol.,208, 233-250. Lord, E.M., Park, S.Y. and Mollet, J.C. (2000) Lily pollen tubes adhere to an in vitro stylar matrix of pectin and a 9kD cysteine-rich protein. Mol. Biol. Cell, 11, 2034. McCartney, L., Ormerod, A.P., Gidley, M.J. and Knox, J.P. (2000) Temporal and spatial regulation of pectic (1 -4)-(3-D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties. Plant J., 22, 105-113. MacDougall, A.J., Needs, P.W., Rigby, N.M. and Ring, S.G. (1996) Calcium gelation of pectic polysaccharides isolated from unripe tomato fruit. Carbohydr. Res., 293, 235-249. MacDougall, A.J., Parker, R. and Selvendran, R.R. (1995) Nonaqueous fractionation to assess the ioniccomposition of the apoplast during fruit ripening. Plant Physiol., 108, 1679-1689. MacDougall, A.J., Rigby, N.M. and Ring, S.G. (1997) Phase separation of plant cell wall polysaccharides and its implications for cell wall assembly. Plant Physiol., 114, 353-362. MacKinnon, I.M., Jardine, W.G., O'Kennedy, N., Renard, C.M.G.C. and Jarvis, M.C. (2002) Pectic methyl and non-methyl esters in potato cell walls../. Agric. Food Chem., 50, 342-346.

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Malovikova, A., Rinaudo, M. and Milas. M. (1994) Comparative interactions of magnesium and calcium counterions with polygalacturonic acid. Biopolymers, 34, 1059-1064. Manning, G.S. (1978) The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q. Rev. Biophys.. 11. 179-246. Marszalek. P.E., Oberhauser. A.F., Pang, Y.P. and Fernandez, J.M. (1998) Polysaccharide elasticity governed by chair-boat transitions of the glucopyranose ring. Nature, 396. 661-664. Marszalek. P.E., Pang, Y.P, Li, H.B., El Yazal, J.. Oberhauser, A.F. and Fernandez. J.M. (1999a) Atomic levers control pyranose ring conformations. Proc. Nail. Acad. Set. USA. 96, 7894-7898. Marszalek, P.E., Lu, H.. Li, et a/., (1999b) Mechanical unfolding intermediates in titin modules. Nature. 402, 100-103. Matoh, T. and Kobayashi. M. (1998) Boron and calcium, essential inorganic constituents of pectic polysaccharides in higher plant cell walls. J. Plant Res., Ill, 179-190. Mazeau, K. and Perez, S. (1998) The preferred conformations of the four oligomeric fragments of rhamnogalacturonan II. Carbohydr. Res.. 311, 203-217. Messiaen. J. and Van Cutsem, P. (1999) Polyamines and pectins. II. Modulation of pectic signal transduction. Planta. 208, 247-256. Messiaen. J.. Cambier. P. and Van Cutsem. P. (1997) Polyamines and pectins. 1. Ion exchange and selectivity. Plant Physio!.. 113, 387-395. Mollet. J.C., Park. S.Y., Nothnagel, E.A. and Lord, E.M. (2000) A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix. Plant Cell. 12, 1737-1749. Morris, E.R., Powell, D.A.. Gidley. M.J. and Rees, D.A. (1982) Conformations and interactions of pectins. I. Polymorphism between gel and solid states of calcium polygalacturonate. 7. Mol. Bioi. 155.507-516. Morris. G.A., Foster. T.J. and Harding. S.E. (2000) The effect of the degree of esterification on the hydrodynamic properties of citrus pectin. Food Hydrocolloids. 14. 227-235. Moss. W.F.. Lagu. S.G.. Bearden, D.W., Savitsky, G.B. and Spencer. H.G. (1994) Numerical and experimental studies of territorial binding of counterions in polyelectrolyte solutions including the added salt case. Polymer. 35, 3268-3271. Nossal, R. (1996) Mechanical properties of biological gels. PhysicaA. 231. 265-276. Oosterveld, A., Beldman. G., Searle-Van Leeuwen. M.J.F. and Voragen. A.G.J. (2000) Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium. Carbohydr. Polym.. 43. 249-256. Paradossi, G.. Chiessi. E. and Malovikova, A. (1999) Study of the interactions of D- and L-polylysine enantiomers with pectate in aqueous solutions. Biopolymers. 50. 201-209. Penel. C. and Greppin. H. (1996) Pectin binding proteins: characterization of the binding and comparison with heparin. Plant Physiol. Biochem.. 34. 479^188. Perez, S., Mazeau. K. and du Penhoat, C.H. (2000) The three-dimensional structures of the pectic polysaccharides. Plant Physiol. Biochem.. 38. 37-55. Perrone, P.. Hewage. C.M., Sadler. I.H. and Fry, S.C. (1998) Na and Ne-D-galacturonoyl-L-lysine amides: properties and possible occurrence in plant cell walls. Phytochemistry. 49. 1879-1890. Petka. W.A., Harden. J.L., McGrath, K.P., Wirtz, D. and Tirrell. D.A. (1998) Reversible hydrogels from self-assembling artificial proteins. Science. 281, 389-392. Picout, D.R., Richardson, R.K. and Morris. E.R. (2000b) Ca;+-induced gelation of low-methoxy pectin in the presence of oxidised starch. Part 2. Quantitative analysis of moduli. Carbohydr. Polym.. 43. 123-131. Picout, D.R.. Richardson. R.K., Rolin, C.. Abeysekera. R.M. and Morris. E.R. (2000a) Ca2*-induced gelation of low methoxy pectin in the presence of oxidised starch. Part 1. Collapse of network structure. Carbohydr. Polym.. 43. 113-122. Pilling. J.. Willmitzer. L. and Fisahn, J. (2000) Expression of a Petunia inflata. pectin methylesterase in Solanum tiiberosum L enhances stem elongation and modifies cation distribution. Planta. 210. 391-399.

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Powell, D.A., Morris, E.R., Gidley, MJ. and Rees, D.A. (1982) Conformations and interactions of pectins. 2. Influence of residue sequence on chain association in calcium pectate gels. J. Mol. Biol., 155,517-531. Power, P.P. and Woods, W.G. (1997) The chemistry of boron and its speciation in plants. Plant Soil, 193. 1-13. Qi, X.Y., Behrens, B.X., West, P.R. and Mort, A.J. (1995) Solubilization and partial characterization of extensin fragments from cell walls of cotton suspension cultures—evidence for a covalent crosslink between extensin and pectin. Plant Physiol., 108, 1691 -1701. Radha, A. and Chandrasekaran, R. (1997) X-ray and conformational analysis of arabinan. Carbohydr. Res., 298, 105-115. Redgwell, R.J. and Selvendran, R.R. (1986) Structural features of cell wall polysaccharides of onion Allium cepa. Carbohydr. Res., 157, 183-199. Rees, D.A. (1977) Polysaccharide Shapes, Chapman and Hall, London. Renard, C.M.G.C. and Jarvis, M.C. (1999a) A cross-polarization, magic-angle-spinning, l3 C-nuclear magnetic resonance study of polysaccharides in sugar beet cell walls. Plant Physiol.. 119. 1315-1322. Renard, C.M.G.C. and Jarvis, M.C. (1999b) Acetylation and methylation of homogalacturonans—2: Effect on ion-binding properties and conformations. Carbohydr. Polym., 39, 209-216. Renard, C.M.G.C. and Thibault, J.F. (1993) Structure and properties of apple and sugar-beet pectins extracted by chelating agents. Carbohydr. Res., 244, 99-114. Rigby, N.M., MacDougall, A.J., Ring, S.G., Cairns, P., Morris, V.J. and Gunning, PA. (2000) Observations on the crystallization of oligogalacturonates. Carbohydr. Res., 328, 235—239. Rihouey, C, Morvan, C, Jauneau, A. and Demarty, M. (1995) In vivo content of inorganic cations in cells and cell-walls of flax seedlings during their development.P/a«/ Physiol. Biochem., 33, 509-517. Rizk, S.E., Abdel-Massih, R.M., Baydoun, E.A.H. and Brett, C.T. (2000) Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea. Planta, 211, 423-429. Rombouts, F.M.. and Thibault J.-F. (1986) Feruloy lated pectic substances from sugar beet pulp, Carbohydr. Res., 154, 177-187. Ross-Murphy, S.B. (1998) Reversible and irreversible biopolymer gels—structure and mechanical properties. Ber. Bunsen-Ges. Phys. Chem. Chem. Phys., 102, 1534—1539. Round, A.N., MacDougall, A.J., Ring, S.G. and Morris, V.J. (1997) Unexpected branching in pectin observed by atomic force microscopy. Carbohydr. Res., 303, 251-253. Round, A.N., Rigby, N.M., MacDougall, A.J., Ring, S.G. and Morris, V.J. (2001) Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis. Carbohydr. Res., 331,337-342. Ruggiero, J.R., Urbani, R. and Cesaro, A. (1995a) Conformational features of galacturonans. I. Structure and energy minimisation of charged and uncharged galacturonan dimeric units. Int. J. Biol. Macromoi., 17, 205-212. Ruggiero, J.R., Urbani, R. and Cesaro, A. (1995b) Conformational features of galacturonans. 2. Configurational statistics of pectic polymers. Int. J. Biol. Macromoi., 17, 213-218. Ryden, P., MacDougall, A.J., Tibbits, C.W. and Ring, S.G. (2000) Hydration of pectic polysaccharides. Biopolymers, 54, 398-405. Sakamoto, T, Yoshinaga, J., Shogaki, T. and Sakai, T. (1993) Studies on protopectinase-C. Mode of action. Analysis of the chemical structure of the specific substrate in sugar-beet protopectin and characterization of the enzyme activity. Biosci. Biotechnol. Biochem., 57, 1832-1837. Scavetta, R.D., Herron, S.R., Hotchkiss, A.T. el al. (1999) Structure of a plant cell wall fragment complexed to pectate lyase C. Plant Cell, 11, 1081-1092. Shimokawa, T, Ishii, T. and Matsunaga, T. (1999) Isolation and structural characterization of rhamnogalacturonan II-borate complex from Pinus densiflora. J. Wood Sci., 45, 435-439.

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Shin, K.S., Kiyohara. H., Matsumoto. T. and Yamada, H. (1998) Rhamnogalacturonan II dimers crosslinked by borate diesters from the leaves of Panax ginseng C.A. Meyer are responsible for expression of their IL-6 production enhancing activities. Carbohydr. Res., 307. 97-106. Sorenson, J.M., Hura, G., Soper, A.K., Pertsemlidis, A. and Head-Gordon. T. (1999) Determining the role of hydration forces in protein folding. J. Phys. Chem. B. 103. 5413-5426. Sorensen, S.O.. Pauly, M., Bush, M., Skjot. M., et al. (2000) Pectin engineering: modification of potato pectin by in vivo expression of an endo-1.4-p-D-galactanase. Proc. Natl. Acad. Sci. USA. 97. 7639-7644. Tanaka. F. and Ishida. N. (1999) Thermoreversible gelation with two-component networks. Macromolecules,32. 1271-1283. Tang. H.R.. Belton, P.S.. Ng, A. and Ryden. P. (1999) I3 C MAS NMR studies of the effects of hydration on the cell walls of potatoes and Chinese water chestnuts. J. Agric. Food Chem.. 47. 510-517. Thompson. J.E. and Fry. S.C. (2000) Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells. Planta. 211, 275-286. Tibbits, C.W., MacDougall, A.J. and Ring. S.G. (1998) Calcium binding and swelling behaviour of a high methoxyl pectin gel. Carbohydr. Res.. 310. 101-107. Vidal, S., Doco. T, Williams, P., et al. (2000) Structural characterization of the pectic polysaccharide rhamnogalacturonan II: evidence for the backbone location of the aceric acid-containing oligoglycosyl side chain. Carbohydr. Res.. 326. 277-294. Voue, M. and Gillet, C. (1994) Site-specific counterion binding—application of the standard PoissonBoltzmann cell model to ionic polysaccharides of the plant cell wall. Biophys. Chem.. 51. 9-19. Walkinshaw. M.D. and Arnott, S. (198 la) Conformations and interactions of pectins. I. X-ray diffraction analyses of sodium pectate in neutral and acidified forms. 7. Mol. Biol.. 153. 1055-1073. Walkinshaw, M.D. and Arnott. S. (1981b) Conformations and interactions of pectins. 2. Models for junction zones in pectinic acid and calcium pectate gels. / Mol. Biol.. 153. 1075-1085. Wellner. N.. Kacurakova. M., Malovikova. A., Wilson. R.H. and Belton. PS. (1998) FT-IR study of pectate and pectinate gels formed by divalent cations. Carbohydr. Res.. 308. 123-131. Whitney. S.E.C., Gothard, M.G.E.. Mitchell. J.T. and Gidley. M.J. (1999) Roles of cellulose and xyloglucan in determining the mechanical properties of primary plant cell walls. Plant Physiol.. 121. 657-663. Williams, M.A.K.. Keenan, R.D., Halstead. T.K. (1998) A : H NMR lineshape study of -CH? group dynamics in pectin gels. Food Hydrocolloids, 12, 89-94. Wilson, R.H., Smith, A.C., Kacurakova, M.. Saunders, P.K.. Wellner. N. and Waldron. K.W. (2000) The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy. Plant Physiol.. 124. 397-405. Zhan. D.F.. Janssen, P. and Mort. A.J. (1998) Scarcity or complete lack of single rhamnose residues interspersed within the homogalacturonan regions of citrus pectin. Carbohydr. Res.. 308. 373-380.

5

Cell and developmental biology of pectins J. Paul Knox

5.1

Introduction to pectin biology

Pectic polysaccharides, which have long been isolated from citrus peel and apple pomace and exploited for their gelling properties, are thought to occur in the primary cell walls of all land plants, as well as in the walls of some green algae, such as Nitella and Chara. Some of the functions of pectins within plants are understood in broad terms, but much remains to be uncovered and it is highly likely that a range of properties and functions have yet to be identified. The long-held view that pectins are most abundant in the intercellular regions between cells, where they function in maintaining cell adhesion, is overly simplistic and far from being the only aspect of pectin biology. It is now clear that pectic polysaccharides are diverse in structure and vary in their location throughout cell walls and intercellular matrices. Certain structural features of pectic polysaccharides can be restricted to regions close to plasma membranes, to intercellular regions or to extracellular secretions. Pectins, therefore, are likely to impact upon a considerable range of cell functions and cell processes during plant growth and development. The chapter aims to give an overview of the cell and developmental biology of pectins, to provide pointers towards future studies and to highlight areas where considerable challenges remain. Detailed discussions of aspects of pectin biology have recently been covered elsewhere (Ridley et aL, 2001; Willats et aL, 200Ib). The structure of pectic polysaccharides, an understanding of which is essential for any consideration of pectin biology, is covered in this volume (Chapter 1) and elsewhere (O'Neill et al., 1990; Mohnen, 1999; Ridley et al., 2001). Pectin is a set of heterogeneous polysaccharides with considerable potential for covalent and noncovalent associations both within the pectic polymer grouping and with other cell wall polymers and molecules. Moreover, pectins have considerable potential to be modified in mum. Pectins therefore form one of the most structurally complex macromolecular networks in nature. They consist of three major galacturonic acid-rich polysaccharides that are thought to occur in most, if not all, higher plant organs and species. Homogalacturonan (HG) is the most abundant pectic polysaccharide. It is a homopolymer of galacturonic acid that appears to occur throughout all primary cell walls and intercellular matrices at middle lamellae. HG exists in the cell wall in various methyl-esterified, acetylated and substituted forms. HG appears to be

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functionally fine-tuned by the removal of methyl and acetyl groups by pectin methylesterases (PMEs) and pectin acetylesterases respectively. The extent of methyl-esterification determines the presence of free carboxyl groups along HG chains and this in turn influences factors such as cell wall pH and the capacity of HG to be crosslinked by calcium, leading to gel formation. Rhamnogalacturonan //(RG-II), which accounts for around 10% of pectin in cell walls, has an HG backbone and therefore appears to be a derived form of HG (thus raising questions about nomenclature). RG-II has four heteropolymeric side chains containing 11 different sugars in a range of linkages. This complex structure appears to be conserved within plants and across higher plant species. RG-II also appears to be a very stable structure that is not readily modified or degraded. Within cell walls, RG-II occurs predominantly as a borate-mediated dimer. Rhamnogalacturonan I (RG-I) comprises a highly diverse mixture of polymers grouped together by virtue of a backbone consisting of alternating rhamnose and galacturonic acid residues. The rhamnose residues can be sites of attachment of side chains rich in neutral sugars. Common components of side chains are (1—>4)-p-D-galactan and (l—»5)-a-L-arabinan. Polymers currently designated as RG-I appear to be highly regulated in relation to cell wall architecture and between cells, and evidence is emerging for a diversity of functions (Willats et al., 2001b and see below). Why are pectins so complex? This is a central question of pectin biology. The structure-function relationships of pectic polymers can be most reasonably approached by consideration of such questions as: What are the properties of the individual pectic polymers and their modified forms? How do these properties impact upon the higher-level properties of cell walls and of cells and tissues? How are pectic domains linked together and to other cell wall polymers to form a functional matrix? Currently, there are no comprehensive answers to these questions and this chapter reviews progress in understanding the biological basis and function of the structural complexity and heterogeneity of pectins.

5.2 Tools and approaches for the analysis of pectins in planta Pectins are complex, branched, acidic heteropolysaccharides occurring in the cell walls of dynamic multicellular plant organs and present many challenges with respect to understanding their cell and developmental biology. Our knowledge of pectin structure is derived from physicochemical analyses of polymers extracted from plant materials. These methods, such as compositional and linkage analyses of polysaccharides, continue to be of immense importance and the development of associated micro-methods of, for example, mass spectrometry, requiring smaller amounts of material will be of critical importance to the refinement of our understanding of pectin biology. However, these techniques

CELL AND DEVELOPMENTAL BIOLOGY OF PECTINS

11

generally require the homogenisation of cellular structures, resulting in the loss of most information concerning the spatial and developmental aspects of pectic polymer structure. Cell and developmental biology studies require knowledge of the occurrence of pectic polymers in relation to cell wall architecture and to cell development—pectins in context. Currently, anti-pectin antibodies are the best tools for generating this sort of information. The generation of anti-pectin antibodies is not a straightforward matter. Ideally, a good knowledge of the epitope (i.e. the oligosaccharide directly recognised by the antibody binding site) is critically important for analysis, although this is not always achievable. To obtain defined probes, defined oligosaccharides covalently attached to proteins should be used as immunogens wherever possible, but the availability of appropriate oligosaccharides can often be a problem. Table 5.1 shows the major anti-pectin monoclonal antibodies that have been developed and characterised to date. Full discussions of anti-pectin probes and the challenges inherent in the generation and characterisation of anti-carbohydrate antibodies can be found elsewhere (Knox, 1997; Jauneau et al., 1998; Willats et al., 2000a,b). Insights provided by anti-pectin monoclonal antibodies are discussed below. The analysis of the Arabidopsis genome has recently generated a vast resource for the elucidation of the mechanisms of plant growth and development (Arabidopisis Genome Initiative, 2000). Furthermore, it has also emphasised the major investment that this plant makes in modifying pectin in its cell walls. Table 5.1 Anti-pectin antibodies Antibody

Antigen/epitope

References

JIMS

HG/low/not metbyl-esterified

JIM7

HG/high methyl-esterified

2F4

HG/dimerised oligogalacturonides

PAM1 LM7

HG/blockwise pattern of de-esterification HG/nonblockwise pattern of de-esterification

Knox et al. (1990); Willats et al. (2000a) Knox et al. (1990); Willats et al (2000a) Liners et al. (1989): Liners et al (1992) Willats et al (1999a; 2000a) Willats et al (2001 c)

CCRC-RI antiserum

RG-II monomer RG-II borate-crosslinked dimer

Williams et al. (1996) Mat oh et al. (1998)

CCRC-M1 CCRC-M2 CCRC-M7

RG-I//-a-fucose-(l-»2Hinked to D-galactosyla RG-I/unknown b RG-I/arabinosylated (1 -> 6)-p-D-galactan

LM5

RG-I/( 1 ->4)-p-D-galactan

LM6

RG-I/(l-*5)-a-L-arabinanb

Puhlmann et al (1994) Puhlmann et al (1994) Puhlmann et al. (1994); Steffan etal. (1995) Jones et al (1997); Willats etal (1999b) Willats et al (1998; 1999b)

a

Epitope also occurs in xyloglucan. Epitope may also occur in arabinogalactan-protein proteoglycans.

b

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PECTINS AND THEIR MANIPULATION

For example, there are over 70 genes that appear to encode pectinesterases (PEs). In addition to the in muro modification of pectin, large numbers of genes are assumed to encode glycosyltransferases that are involved in the biosynthesis of the pectic polymers, with 21 estimated for the construction of RG-II alone and 53 for all pectic polymers. None of these pectin biosynthesis genes is yet identified, but progress in this area is discussed in Chapter 3. Moreover, this does not take into account other gene products that are likely to be involved in the transfer of pectic polymers from the Golgi bodies and their deposition and assembly at the cell surface. As indicated, pectic polysaccharide biosynthesis and associated genes are not easy to identify, but as the Ambidopsis genome becomes more extensively annotated it is providing a major genomic resource that can be applied to the understanding of pectin biology. Concomitant with the refinement of the Arabidopsis and other genomes will be the identification and appraisal of mutants with altered pectic structures derived by both forward and reverse genetic procedures. As pectic polymer sequences and substitutions are characterised, their locations within cell walls are determined and genes involved in their synthesis and modification are identified, another key area is the study of the properties of the pectic polymers themselves. Conformational studies and modelling of pectic polymers are an important aspect of this (Chapter 4; Perez et al. 2000). How do pectic polysaccharide structures relate to their properties and functions in the context of cell wall architecture and how do they impact on cell wall properties? For example, how do substitutions of pectic backbones, modified chain lengths and the presence or absence of side chains change the properties of pectic polymers? One useful approach that can provide important insights involves in vitro studies on the gelling and Theological properties of polymers isolated from a range of biological sources (for example, see Tibbits et al.. 1998: MacDougall et al. 2001: Willats et al. 200Ic). 5.3 Pectins and the cell wall 5.3.1 Pectic polysaccharides and matrix properties Plant cell walls are fibrous composites. Pectin comprises between 30% and 40% of the polysaccharide polymers of primary cell walls. Current models indicate that the cellulose microfibrils, which provide the tensile strength of cell walls, are tethered together by single-chain hemicellulose crosslinks. In primary cell walls these crosslinks appear to be the targets for protein-driven modulation that appears to be a crucial component of cell expansion and hence plant growth (Cosgrove, 1999, 2000; Darley et al. 2001). The load-bearing cellulose/hemicellulose network is embedded in pectic polymers that form a highly hydrated and crosslinked three-dimensional network. There is some evidence that pectic components are attached to the hemicellulose xyloglucan

CELL AND DEVELOPMENTAL BIOLOGY OF PECTINS

1 35

and hydroxyproline-rich glycoproteins and links to these molecules may account for the occurrence of pectic polymers in cellulose residues after alkali extractions (Chapter 2; Ridley et al, 2001). A series of observations suggest the occurrence of regulatory links during the assembly and function of the co-extensive pectic and cellulose/hemicellulose networks. The downregulation of an expansin in tomato fruit has resulted in reduced pectin degradation (Brummell et al., 1999) and the downregulation of cellulose synthase genes or application of inhibitors of cellulose biosynthesis has resulted in increased levels of pectic polymers (Shedletzky et al, 1992; Burton etal, 2000). The pectic gel/network is the matrix through which cellulose microlibrils move or slide apart during cell expansion and it is the environment in which factors that act upon the cellulose/hemicellulose network move and operate. Pectin is therefore a major contributor to a dynamic operating environment for cell wall proteins and other factors that act to modulate cell wall properties. Pectin-dependent factors of this operating environment will include ionic status, pH, porosity, etc. The complexity of pectic polymers and their extensive capacity for structural modulation and interaction with a range of other cell wall molecules (from other cell wall polymers to cations) therefore provides considerable potential for the local modulation or remodelling of cell wall matrix properties. Porosity is likely to be an important factor of the matrix environment and may influence the access of cell wall-modifying proteins such as expansins to sites of action within the wall. See Read and Bacic (1996) for an excellent review of cell wall porosity and its measurement. The extent of HG gel formation will influence matrix porosity and recent work has also suggested that borate diester-mediated RG-II dimerisation may also be involved in the regulation of cell wall porosity (Fleischer et al, 1999). The reversible cationinduced effects on the hydraulic resistance of pit membranes of xylem vessels provides a good example of the reversible modulation of the structure of a pectic polymer and hence cell wall properties (Zwieniecki et al, 2001). Moreover, the possible ionic or covalent association of proteins with the pectic network (e.g. Mollet et al, 2000; Rizk et al, 2000; Carpin et al, 2001; Wagner and Kohorn, 2001) is also likely to modulate pectic polymer and matrix properties and may influence pectin-plasma membrane links. Pectin-protein interactions are not well understood, but this is likely to be an important area for future studies. 5.3.2 Pectin and cell wall architecture Recent years have seen an increased understanding of the spatial regulation of pectin structure at the level of cell walls. Observations have generally indicated that HG is distributed throughout primary cell walls but that the extent of its methyl-esterification is highly variable (Knox et al, 1990; Knox, 1997; Liners et al, 1992; Willats et al., 200Ic). There are three major regions where pectic

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components appear to vary in relation to primary cell wall architecture. These are the intercellular matrix of the middle lamellae (and where separated at intercellular spaces), in the region of pit fields and in the inner region of cell walls close to plasma membranes. Examples of the occurrence of pectic domains in relation to these features of cell walls are shown in Figures 5.1 and 5.2. In

Figure 5.1 Spatial regulation of pectic polysaccharide epitopes in relation to intercellular spaces of parenchyma systems as indicated by immunofluorescent labelling. Hotnogalacturonan (HG) occurs through all primary cell walls (PCW). middle lamellae (ML) and the expanded ML at corners of intercellular spaces as shown by binding of JIM7. The schematic of an intercellular space at the junction of three cells (c), is used to indicate examples of the restricted patterns of occurrence of the PAM1 and LM7 HG epitopes in regions of cell wall lining intercellular spaces in pea stem parenchyma (see Willats et al.. 2001 c), and the occurrence of the LM5 (1 —»• 4 )-p-D-galactan and LM6 (1 —* 5 )-a-L-arabinan epitopes, associated with RG-I, in legume cotyledon parenchyma (see McCartney et al.. 2000). In this case, the LM5 (1 —>4)-p-D-galactan epitope is restricted to a narrow region of cell wall close to the plasma membrane. PM = plasma membrane. IS = intercellular space. Scale bars = 1 |im.

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Figure 5.2 Spatial regulation of pectic polysaccharide epitopes in relation to pit fields in cell walls of tomato fruit pericarp as indicated by immunofluorescent labelling. A radial section of tomato fruit pericarp labelled with LM5 (l->4)-|3-D-galactan indicates regions of cell walls that do not contain the epitope. These are pit fields as indicated by co-localisation with the callose-binding fluorochrome aniline blue. JIM5 binding indicates that HG occurs throughout the cell walls in pit field regions. The schematic indicates the pattern of JIMS, LM5 and LM6 epitopes on the inner face of pericarp cell walls and in cell walls at pit fields in a three-dimensional cutaway format. For full details see Orfila and Knox (2000). Scale bars = 10 (im.

summary, it can be stated that HG occurs throughout primary cell walls and middle lamellae and that its methyl-esterification shows extensive regulation in both extent and pattern. The modulation of methyl-esterification is generally most dynamic in regions of cell walls that surround intercellular spaces and in the expanded middle lamellae at the corners of intercellular spaces (Figure 5.1). The methyl-esterification of HG will be discussed below in the context of cell adhesion and its loss. Although RG-II has a HG backbone, and can be viewed as a derived form of HG, it appears to be restricted to primary cell walls and not to occur at middle lamella regions (Williams et al., 1996; Matoh et al., 1998).

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Polymers of the RG-I group have been located with antibodies to what are thought to be components of side chains. These studies show that the polymers are highly spatially and developmentally regulated and this may be indicative of a wide range of functions (Freshour etal.,\ 996; Willats et al., 1999b; McCartney et al., 2000). RG-I side chain epitopes often appear to be absent from middle lamellae between adherent cells and expanded middle lamellae in the region of intercellular spaces. Furthermore, the RG-I epitopes are spatially regulated in inner regions of cell walls and at cell walls in regions of pit fields as shown in Figures 5.1 and 5.2. Pit fields are regions of primary cell walls that contain numerous plasmodesmata that link the plasma membrane and ER of adjacent cells. HG components have been observed to be regulated in relation to pit fields in apple fruit (Roy et a/., 1997) and the LM5 (1—»4)-fi-D-galactan epitope is generally absent from pit fields (Bush and McCann, 1999; Orfila and Knox. 2000), perhaps reflecting distinct mechanical properties in these regions. The occurrence of pectic polymers at pit fields has been most extensively studied in the pericarp of tomato fruit (Casero and Knox, 1995; Orfila and Knox, 2000). These studies have indicated complex arrangements of HG- and RG-I-related epitopes in relation to pit fields at the inner surface of pericarp cell walls (Orfila and Knox, 2000). The significance of such patterns of pectic polymers at the plasma membrane face of cell walls is far from clear, but may reflect aspects of polymer deposition or the occurrence of microdomains of the matrix with distinct properties. In these studies, the spatial relationship of RG-I side chain epitopes to other domains within RG-I polymers is also uncertain. Antibody probes for the backbone of RG-I would be useful in this regard.

5.4

Pectins and cell processes

5.4.1 Pectins, metabolism and signalling An important aspect of the biology of certain plant polysaccharides is that oligomers enzymatically cleaved from larger polymers have biological activity. These oligosaccharides, termed oligosaccharins, exhibit structural specificity in relation to diverse cell processes and aspects of cell physiology. Of the pectic polysaccharides, only oligomers derived from unesterified HG (resulting from the combined action of PMEs and polygalacturonases (PGs)) have as yet been shown to possess biological activity. Oligogalacturonides function in development, growth and defence. This important aspect of HG polymers has been covered extensively elsewhere (Prade et al., 1999; Dumville and Fry, 2000; Ridley et al., 2001). It is important to bear in mind the potential for cleavage and solubilisation of HG components when dynamic aspects of the modulation of HG polymers or the occurrence of HG epitopes are considered in relation to cell development.

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5.4.2 Cell proliferation There is currently no evidence that pectic polysaccharides function directly in the process of cell proliferation. However, in developing plant systems cell proliferation is in complex interplay with other processes such as cell expansion and cell adhesion. The observation that the LM6 (l->5)-a-L-arabinan epitope is most abundant in cell walls of meristematic cells at the carrot root apex and the LM5 (l->4)-|3-D-galactan epitope is absent appears to indicate some correlation of RG-I-associated polymers with cell proliferation (Willats et al., 1999b). A similar correlation was observed in a carrot suspension cell culture (Willats et al,, 1999b). The developmental^ regulated occurrence of the LM5 galactan and LM6 arabinan epitopes in the carrot root and cell culture systems is shown in Figure 5.3. Compositional analyses of carrot suspension cell wall polymers has indicated correlations between the presence of arabinose- and galactose-containing pectic polymers and the size of cell clusters that may related to aspects of cell proliferation in cell culture systems (Kikuchi et al., 1996a,b; Iwai et al., 2001). Of course, these observations may reflect factors such as the switch from cell proliferation to cell expansion or to increased cell separation (see below). More work needs to be done in this area to determine the function of altered pectic structures in these cell culture systems and within meri stems. 5.4.3 Cell expansion The observation that pectins are generally restricted to primary cell walls and absent from most secondary cell walls has suggested that pectins function in cell expansion and that they are key components allowing primary cell walls to be extensible. Clearly, pectic polymers are major components of the matrix environment that allows cellulose microfibrils to slide apart due to forces of turgor pressure and the action of cellulose/hemicellulose-modifying factors such as expansins and xyloglucan endotransglycosylases (Cosgrove, 2000; Darley et a/., 2001). What is not known in any precise way is how the pectic matrix contributes to or regulates cell wall extensibility. An important aspect of plant cell expansion is that cell walls are maintained at an even thickness and that new cell wall material is continuously being assembled during cell expansion. Pectins may function during this assembly rather than during wall stretching per se. Some evidence supporting the idea that pectins can influence mechanical properties during cellulose microfibril synthesis has been obtained from in vitro studies on the formation of synthetic wall composites (Chanliaud and Gidley. 1999). It has generally been proposed that HG polymers are deposited in cell walls in highly methyl-esterified forms (see Chapter 2 for a discussion of this issue) and that in this form they have reduced capacity to form calcium crosslinks. The

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Figure 5.3 Developmental regulation of LM5 (1—»-4)-p-D-galactan and LM6 (1—»5)-a-L-arabinan epitopes in relation to cell development at the carrot root apex and at the surface of intact carrot suspension-cultured cells by immunofluorescent labelling. Labelling of medial longitudinal cryosections of the carrot root apex indicates that the LM5 galactan epitope does not occur in cell walls at the centre of the root meristem but appears during development of both the root cap and the root body. In contrast, the LM6 arabinan epitope is most abundant in at the centre of the meristem (arrow). In a cell culture system proliferating cells can be induced to switch from proliferation to elongation by removal of 2.4dichlorophenoxyacetic acid (2.4-D) from the growth medium. Immunolabelling of intact cells indicates that the galactan epitope is abundant at the surface of elongated cells (arrow) and the LM6 arabinan epitope is absent. For full details see Willats et al., 1999b. Scale bars = 100 |im.

subsequent action of PMEs on HG in the cell wall can have a range of effects that may both increase or decrease cell wall extensibility. For example, PME action can increase the extent of acidic sites of HG chains and the resulting lowered pH may promote the activity of expansins and increase cell wall extension. However, the capacity of HG polymers to form calcium crosslinks may lead to cell wall stiffening that may contribute to reduced extensibility. Increases in PME activities and increased levels of unesterified Gal A residues are associated with cell expansion in legume hypocotyls (Liberman etai, 1999). An increase in the level of esterification of pectin during cell elongation followed by a decline has been reported for suspension-cultured cells (McCann and Roberts. 1994). The use of anti-HG JIM5 and JIM7 monoclonal antibodies has not indicated any clear relationship of HG methy 1-esterification and cell expansion/elongation

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14!

(e.g. McCann et al, 1993; McCann and Roberts, 1994; Fujino and Itoh, 1998; Liberman et al., 1999). However, it is now clear that these two monoclonal antibodies cannot clearly indicate fully methyl-esterified or fully de-esterified domains of HG in cell walls and studies using these antibodies need to be interpreted with caution (Willats etal, 2000a). Currently, it is not easy to define relationships between HG structure and cell wall extension using current probes and approaches. Homogalacturonan is likely to function in a range of cell processes in addition to cell expansion, and it not ease to dissect these roles. There are also likely to be complex relationships between HG polymer structure and polymer length (and properties) resulting from the interplay of a range of HG-modifying enzymes such as PMEs, PGs and pectate lyases. PMEs are encoded by large multigene families and have complex action patterns (Catoire et al., 1998; Goldberg et al., 2001; Micheli, 2001) and are likely to participate in several processes. The same is likely to be the case for PGs (Hadfield and Bennett, 1998). The functions of HG-modifying enzymes are now being dissected using the techniques of reverse genetics. The inhibition of PME activity in the pea root has resulted in reduced root elongation and also altered root structure and reduced separation of root cap cells (Wen et al., 1999). The overexpression of a Petunia PME in potato resulted in complex effects and these included a reduction of PME activity in the shoot apical tissues that was associated with increased elongation (Pilling et al., 2000). Our understanding of the role of HG in cell wall architecture and cell wall extension requires refinement, and antibody probes to defined methyl-esterification (and acetylation?) states of HG will be important for this. There is, as yet, no evidence for the direct involvement of RG-II in cell expansion, although its possible functions in the regulation of cell wall porosity (Fleischer etal.,\ 999) or cell wall thickness (Ishii et al., 2001) may have indirect effects on the capacity of cell walls to expand. Recent, elegant experiments with the murl mutant of Arabidopsis indicate that the dimerisation of RG-II with boron is required for growth (O'Neill et al, 2001), but how this relates to cell expansion is not yet clear. As indicated in Section 5.4.2 on cell proliferation, LM5 pectic galactan and LM6 pectic arabinan epitopes are developmentally regulated at the carrot root apex and in suspension-cultured cells and this can be interpreted in relation to cell processes such as cell proliferation or cell expansion. But how the presence or absence of these cell wall components impacts on cell wall properties, if at all directly, is unknown. Carrot cells in culture that can be induced to elongate by hormonal manipulation have been used to demonstrate that pectic galactan is upregulated in cells prior to any change in cell size (Figure 5.3; Willats et al., 1999b). This may indicate that the galactan epitope is required in cell walls for cell elongation. However, we currently have no understanding of how it may function in this.

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Most studies have considered pectin in relation to the axial extension of multicellular organs such as roots or hypocotyls. Tip growing systems with rapid cell elongation of unadhered cells make a good system for study of cell wall components. In pollen tubes pectic epitopes have been reported to occur at the pollen tube surface in periodic rings, but these do not appear to reflect growth rates (Stepka et al., 2000). It is of interest that pectic epitopes appear to be largely absent from the surface of root hairs of Arabidopsis seedling roots even although epitopes are present at the root surface (Willats er al.. 2001 a). 5.4.4 Cell differentiation The formation of specific cell wall structures is an important aspect of plant cell differentiation. However, the interpretation of the patterns of developmental regulation of cell wall molecules during cell differentiation and the elucidation of the function of these molecules is not easy, as discussed above, owing to the complex spatial relationships between cell proliferation, cell expansion and cell differentiation in developing plant organs. Homogalacturonan appears to be distributed throughout all cell walls at meristems, although there is some indication that this may not be the case in root apices of graminaceous monocotyledons (Knox et al., 1990). (The exploration of the taxonomic aspects of pectin using anti-pectin probes—in algae, moss through to angiosperms—is an area that currently does not receive much attention but would seem to have considerable potential for increasing our understanding of the functional significance of pectin complexity.) It is clear that the fine structure of HG is modified due to the action of PMEs and related enzymes during cell development, but the significance of this, if any. in relation to the differentiation of particular cell types is not clear. RG-I polymer epitopes are currently known to be extensively regulated in occurrence in developing systems. This has so far been studied using probes for epitopes occurring in side chains of RG-I and the focus has mostly been on roots (Freshour et al., 1996; Willats et al., 1999b; Vicre et al, 1998; Willats et al. 2001 a). The LM5 pectic galactan and CCRC-M2 arabinogalactan epitopes are absent from proliferating cells of meristems and appear during differentiation, although not equally in all cells. In contrast, the LM6 arabinan epitope occurs most abundantly in the cell walls in the centre of the meristem at the carrot root apex and the levels of this epitope appears to decline during development, as seen in Figure 5.3. We do not know whether these dynamics of epitope occurrence reflect the expression and turnover of the antigens or an alteration/unmasking of epitopes in the cell wall. Shoot apices have been less extensively studied, but examination of longitudinal sections of the milkwort shoot apex (Serpe et al. 2001) and potato stolon apices (Bush etal, 2001) with LM5 and LM6 indicates that the LM5 galactan epitope is absent from the meristematic cells and appears during development.

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The developmental appearance of the LM5 galactan epitope is therefore emerging as a key aspect of pectic structure modulation during cell development in a range of systems. During pea cotyledon development the LM5 epitope appears relatively late in development (around 30 days after anthesis, just prior to seed drying) in regions of the cell wall close to the plasma membrane, and its appearance correlates with an increase in firmness of the cotyledon parenchyma (McCartney et al, 2000). The function of pectic galactan in cell walls of potato tubers has been explored by a transgenic approach with the expression of a microbial galactanase that removes most of the pectic galactan (S0rensen et al., 2000). The resulting plants develop as normal, suggesting that the pectic galactan of potato tuber cell walls is not required for developmental events but that it may function in other aspects of cell physiology such as mechanical properties (S0rensen et al., 2000). No consistent picture of the LM6 arabinan epitope in relation to meristem or organ development has yet emerged. Antibody probes allow the ready determination of pectic components in cell walls of specific cell types. Examples include the development of phloem sclerenchyma fibres in flax (Andeme-Onzighi et al., 2000) and tomato petiole collenchyma primary cell walls (Jones et al., 1997) where the LM5 galactan epitope occurs mostly in the regions close to the plasma membrane. A particular abundance of the LM6 pectic arabinan epitope has been reported in the intrusive tip-growing latex-containing laticifers in Asclepias speciosa (Serpe et al, 2001). As the number of such observations on a range of cell types in a range of species increases, they may provide insights into specific pectic polymers and their contribution to the specific morphology and properties of cell walls of diverse cell types.

5.4.5 Pectins and the intercellular matrix: cell adhesion Pectin is widely considered to be the intercellular glue, but surprisingly little is understood about the molecular details of pectic polymers in cell adhesion. It is now recognised that pectins are distributed throughout primary cell walls and the intercellular matrices at middle lamellae but that not all pectic polymers are equally distributed throughout these regions. Our current understanding is that HG and related components are the major pectins in middle lamellae and cell walls at regions of intercellular spaces and that RG-II and RG-I side chains are generally absent from these regions. In certain systems the application of a calcium chelator to adhered cells results in cell separation, the implication being that removal of calcium allows solubilisation of crosslinked HG polymers that were holding the cells together. This only occurs in a few instances, such as ripe tomato fruit periciarp (Orfila et al., 2001). In other cases, treatment with pectinases or heat treatment is required for cell separation, indicating that bonds, other than calcium-mediated ones, link adjacent cells. A key point is that the

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pectin at the middle lamellae that links adjacent cells must also be linked to the primary cell walls in some way. Therefore, key links that are broken to cause cell separation may occur at the middle lamella or within the primary cell wall. A study of potato tuber parenchyma has indicated that the edges of cell faces are key points for cell-to-cell attachment and that HG epitopes can occur specifically in these regions (Parker et al., 2001), but key molecular links have not yet been identified. In general, plant cell adhesion is a non-active process that occurs as a result of cytokinesis. Cell separation at cell corners occurs to a limited extent in most tissues and complete cell separation occurs as a regulated developmental event, for example during the sloughing off of root cap cells or during organ abscission (Knox, 1992). The limited cell separation of adjacent plant cells is seen most readily in transverse sections of parenchyma tissues. Some insight into the modulations of HG structure in parenchyma has recently been obtained with monoclonal antibodies to different methyl-esterification states of HG. LM7 binds to a non-blockwise de-esterified epitope that contains both methylesterified and unesterified Gal A residues (Willats et al.. 200 Ic). The LM7 epitope is at located points of adhesion and separation throughout the development of intercellular spaces, that is at the corners of developing intercellular spaces (Figure 5.1). The LM7 epitope has been observed in this location in all parenchyma systems in all higher plant species so far examined. In vitro studies have indicated that the non-blockwise pattern of methyl-esters recognised by LM7 may confer specific mechanical and porosity properties to the expanded middle lamella at the corners. The precise properties and role of this structure are unknown. In contrast, a phage display antibody PAM1 that binds to long de-esterified blocks of GalA is most abundant in the regions of cell walls lining intercellular spaces (Figure 5.1). The function of extensively de-esterified HG at this location is not clear but, again, is likely to involve the generation of local matrix properties. Leaf and organ abscission and pod dehiscence are specialised cases of complete cell separation of specific files of cells at a determined point in development. These processes have been studied extensively, largely from the molecular genetic angle, focusing on the identification of the enzymes involved. In these cases, cell separation is a highly developmentally regulated process and occurs subsequently to differentiation of defined abscission layers. PGs are the enzymes that are most directly implicated in breaking bonds leading to cell separation during abscission and dehiscence and no clear evidence of a prior requirement for PMEs appears to have been reported (Roberts et al., 2000: Patterson, 2001: Sander etal., 2001; Taylor and Whitelaw, 2001). The nature of the HG domains (i.e. extent of methyl-esterification) in the cleaved intercellular regions does not appear to have been directly examined in most systems. The JIM5 and JIM7 anti-HG probes have been used in a study of ethylene-induced abscission of Azolla branches, indicating a persistence of methyl-esterified HG at the

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middle lamella fracture surface throughout the abscission process (Uheda and Nakamura, 2000). A recent, detailed study of the mechanism of pollen tube adhesion to the stylar transmitting tract epidermis in lily has made significant advances in the understanding of the role of a pectic polymer in adhesion events (Mollet et al., 2000; Park et al., 2000). Two molecular components have been identified as being crucial for this adhesion. The first is a small cysteine-rich protein, with some similarities to lipid transfer proteins, now known as stigma/stylar cysteine-rich adhesin (SCA) (Park etai, 2000). The second is an HG-rich pectic polysaccharide in the style that binds to SCA in a pH-dependent manner (Mollet eta!., 2000). Most studies of pectin in relation to cell adhesion have implicitly considered the HG domain, and it is the HG domain that shows extensive spatial regulation of structure at middle lamellae and intercellular spaces. However, it will also be important to maintain an open mind about a role for the other pectic domains in cell adhesion. Several recent observations have provided circumstantial evidence that pectic arabinan may be involved in in cell adhesion events in some way. As discussed above, and shown in Figure 5.3, the LM6 arabinan epitope occurs abundantly in the tightly adhered cells at the centre of the carrot root meristem. Furthermore, analysis of suspension-cultured Nicotiana cells has indicated a correlation between cell adhesion and the presence of (1—>5)-aL-arabinan (Iwai et al., 2001). Although analysis of the Cnr tomato mutant, in which pericarp cells show reduced cell-cell adhesion, has indicated altered middle lamella HG with reduced capacity for calcium binding, the deposition of (1 ~>5)-a-L-arabinan is also disrupted in this system (Orfila et al, 2001). Even though (l-»5)-a-L-arabinan is generally absent from middle lamellae and cell walls lining intercellular spaces, it may be involved in linking primary cell wall pectic polymers to intercellular matrix HG domains. The ripening of fleshy fruits involves changes to cell walls that often result in reduced cell adhesion in addition to cell wall swelling and the softening of fruit tissues. Modifications, involving degradation of fruit pectic polymers appear to be key components in the development of the properties of ripe fruit and these aspects are covered in Chapter 6. 5.5 Prospects Clearly, there is still much to uncover about the cell and developmental biology of pectin. The subtle modulation of pectic domain structure and pectic network interactions are likely to have considerable capacity to influence matrix properties and these in turn may impact upon a diverse range of cell functions and developmental events. The extent or significance of interconnectedness of the components of the pectic network throughout the plant body is not clearly

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understood, but it seems possible that a network of attached pectic molecules ramifies throughout groups of adhered cells. Defining the complete array and spatial relationships of pectic polymers in a developing system or within a cell wall still presents major challenges. Furthermore, refining understanding so that we know the consequences of a particular grouping of pectic polysaccharides for local cell wall properties and tissue properties will not be an easy task. However, the combination of new probes and approaches with the powerful resource of the Arabidopsis genome should lead to rapid progress in the coming decade. References Andeme-Onzighi, C., Girault, R., His, I., Morvan, C. and Driouich, A, (2000) Immunocytochemical characterization of early-developing flax fiber cell walls. Pmtoplasma, 213. 235-245. Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408, 796-815. Brummell, D.A., Harpster, M.H., Civello, P.M.. Palys, J.M., Bennett, A.B. and Dunsmuir. P. (1999) Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism during ripening. Plant Cell, 11. 2203-2216. Burton, R. A., Gibeaut, D.M., Bacic. A., et al. (2000) Virus-induced silencing of a plant cellulose synthase gene. Plant Cell, 12, 691-705. Bush. M.S. and McCann, M.C. (1999) Pectic epitopes are differentially distributed in the cell walls of potato (Solanum tuberosum) tubers. Physiol. Plant.. 107, 201-213. Bush. M.S., Marry, M., Huxham, I.M., Jarvis. M.J. and McCann, M.C. (2001) Developmental regulation of pectic epitopes during potato tuberisation. Planta. 213, 869-880. Carpin, S., Crevecoeur, M., de Meyer, M.. Simon. P.. Greppin. H. and Penel. C. (2001) Identification of a Ca2+-pectate binding site on an apoplastic peroxidase. Plant Cell. 13. 511-520. Casero, P.J. andKnox, J.P. (1995) The monoclonal antibody JIM5 indicates patterns of pectin deposition in relation to pit fields at the plasma-membrane-face of tomato pericarp cell walls. Protoplasma. 188.133-137. Catoire. L., Pierron, M.. Morvan. C., Herve du Penhoat, C. and Goldberg. R. (1998) Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications in cell wall expansion. J. Biol. Chem., 273. 33150-33156. Chanliaud, E. and Gidley, M.J. (1999) In vitro synthesis and properties of peclin/Acetobacter xylinus cellulose composites. Plant J., 20, 25-35. Cosgrove. D.J. (1999) Enzymes and other agents that enhance cell wall extensibility. Anna. Rev. Plant Physiol. Plant Mol. Biol.. 50. 391-417. Cosgrove. D.J. (2000) Expansive growth of plant cell walls. Plant Physiol. Biochem.. 38. 109-124. Darley. C.P.. Forrester. A.M. and McQueen-Mason. S.J. (2001) The molecular basis of plant cell wall extension. Plant Mol. Biol.. 47, 179-195. Dumville. J.C. and Fry, S.C. (2000) Uronic acid-containing oligosaccharins: their biosynthesis. degradation and signalling roles in non-diseased plant tissues. Plant Physiol. Biochem.. 38. 125-140. Fleischer. A.. O'Neill. M.A. and Ehwald, R. (1999) The pore size of non-graminaceous plant cell walls is rapidly decreased by borate ester crosslinking of the pectic polysaccharide rhamnogalacturonan II. Plant Physiol.. 121. 829-838. Freshour. G., Clay. R.P.. Fuller. M.S.. Albersheim, P.. Darvill, A.G. and Hahn. M.G. (1996) Developmental and tissue-specific structural alterations of the cell wall polysaccharides of Arabidopsis thaliana roots. Plant Physiol.. 110. 1413-1429.

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Fujino, T. and Itoh, T. (1998) Changes in pectin structure during epidermal cell elongation in pea (Pisum xativum) and its implications for cell wall architecture. Plant Cell Physiol, 39, 1315-1323. Goldberg, R., Pierron, M., Bordenave, M., Breton, C., Morvan, C. and Herve du Penhoat, C. (2001) Control ofmung bean pectinmethylesterase isoform activities. Influence of pH and carboxyl group distribution along the pectic chains. J. Biol. Chem., 276, 8841-8847. Hadfield, K.A. and Bennett, A.B. (1998) Polygalacturonases: many genes in search of a function. Plant Physiol., 117, 337-343. Ishii, T., Matsunaga, T. and Hayashi, N. (2001) Formation of rhamnogalacturonan H-borate dimer in pectin determines cell wall thickness of pumpkin tissue. Plant Physiol., 126, 1698-1705. Iwai, H., Ishii, T. and Satoh, S. (2001) Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells. Planta, 213, 907-915. Jauneau, A., Roy, S., Reis, D. and Vian, B. (1998) Probes and microscopical methods for the localization of pectins in plant cells. Int. J. Plant Sci., 159, 1—13. Jones, L., Seymour, G.B. and Knox, J.P. (1997) Localization of pectic galactan in tomato cell walls using a monoclonal antibody specific to (1 —>4)-p"-D-galactan. Plant Physiol., 113, 1405-1412. Kikuchi, A., Edashige, Y., Ishii, T., Fujii, T. and Satoh, S. (1996a) Variations in the structure of neutral sugar chains in the pectic polysaccharides of morphologically different carrot calli and correlations with the size of cell clusters. Planta, 198, 634-639. Kikuchi, A., Edashige, Y, Ishii, T. and Satoh, S. (1996b) A xylogalacturonan whose level is dependent on the size of cell clusters is present in the pectin from cultured carrot cells. Planta, 200, 369-372. Knox, J.P. (1992) Cell adhesion, cell separation and plant morphogenesis. Plant J., 2, 137-141. Knox, J.P. (1997) The use of antibodies to study the architecture and developmental regulation of plant cell walls. Int. Rev. Cytoi, 171, 79-120. Knox, J.P., Linstead, P.J., King, J., Cooper, C. and Roberts, K. (1990) Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices. Planta, 181, 512-521. Liberman, M., Mutaftschiev, S., Jauneau, A., Vian, B., Catesson, A.M. and Goldberg, R. (1999) Mung bean hypocotyl homogalacturonan: localization, organization and origin. Ann. Bot., 84, 225-233. Liners, F, Letesson, J.-J., Didembourg, C. and van Cutsem, P. (1989) Monoclonal antibodies against pectin. Recognition of a conformation induced by calcium. Plant Physiol, 91, 1419-1424. Liners, F, Thibault, J.-F. and van Cutsem, P. (1992) Influence of the degree of polymerization of oligogalacturonates and of esterification pattern on pectin on their recognition by monoclonal antibodies. Plant Physiol., 99, 1099-1104. McCann, M.C. and Roberts, K. (1994) Changes in cell wall architecture during cell elongation. J. Exp. Bot., 45, 1683-1691. McCann, M.C., Stacey, N.J., Wilson, R. and Roberts, K. (1993) Orientation of macromolecules in the walls of elongating carrot cells.J. Cell Sci., 106, 1347-1356. McCartney, L., Ormerod, A.P., Gidley, M.J. and Knox, J.P. (2000) Temporal and spatial regulation of pectic (1 —>4)-|3-D-galactan in cell walls of developing pea cotyledons: implications for mechanical properties. Plant J., 22, 105-113. MacDougall. A.J., Brett, G.M., Morris, V.J., Rigby, N.M., Ridout, M.J. and Ring, S.G. (2001) The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin. Carbohvdr. Rex,, 335, 115-126. Matoh, T., Takasaki, M., Takabe, K. and Kobayashi, M. (1998) Immunocytochemistry of rhamnogalacturonan II in cell walls of higher plants. Plant Cell Physiol., 39, 483—491. Micheli, F. (2001) Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci., 6, 414-419. Mohnen, D. (1999) Biosynthesis of pectins and galactomannans, in Comprehensive Natural Products Chemistry, vol. 3 (eds D. Barton, K. Nakanishi and O. Meth-Cohn), Elsevier Science, Amsterdam. pp.497-527.

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Mollet. J.-C., Park, S.-Y., Nothagel, E.A. and Lord, E.M. (2000) A lily stylar pectin is necessary for pollen tube adhesion to an in vitro stylar matrix. Plant Cell, 12, 1737-1749. O'Neill, M.A., Albersheim, P. and Darvill, A. (1990) The pectic polysaccharides of primary cell walls, in Methods in Plant Biochemistry, vol. 2 (ed. P.M. Dey), Academic Press, London, pp. 415-441. O'Neill, M.A., Eberhard, S.. Albersheim, P. and Darvill, A.G. (2001) Requirement of borate crosslinking of cell wall rhamnogalacturonan II for Arabidopsis growth. Science, 294, 846-849. Orfila. C. and Knox, J.P. (2000) Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp. Plant Physiol., 122, 775-781. Orfila, C., Seymour, G.B.. Willats, W.G.T.. el al. (2001) Altered middle lamella homogalacturonan and disrupted deposition of (1 —»-5)-a-L-arabinan in the pericarp of Cnr. a ripening mutant of tomato. Plant Physiol.. 126. 210-221. Park, S.Y., Jauh, G.Y., Mollet. J.C.. et al. (2000) A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix. Plant Cell. 12. 151-163. Parker. C.C., Parker. M.L., Smith, A.C. and Waldron. K.W. (2001) Pectin distribution at the surface of potato parenchyma cells in relation to cell-cell adhesion. / Agric. Food Chem.. 49. 4364-4371. Patterson, S.E. (2001) Cutting loose: abscission and dehiscence in Arabidopsis. Plant Physiol.. 126. 494-500. Perez. S., Mazeau, K. and Herve du Penhoat. C. (2000) The three-dimensional structures of the pectic polysaccharides. Plant Physiol. Biochem.. 38. 37-55. Pilling. J.. Willmitzer. L. and Fisahn. J. (2000) Expression of a Petunia inflata pectin methylesterase in Solanum tuberosum L. enhances stem elongation and modifies cation distribution. Planta. 210. 391-399. Prade, R.A.. Zhan, D.F., Ayoubi, P. and Mort, A.J. (1999) Pectins, pectinases and plant-microbe interactions. Biotechnol. Genet. Eng. Rev.. 16, 361-391. Puhlmann. J., Bucheli. E., Swain. M.J.. et al. (1994) Generation of monoclonal antibodies against plant cell wall polysaccharides. I. Characterization of a monoclonal antibody to a terminal a-(l-»2)linked fucosyl-containing epitope. Plant Physiol., 104. 699-710. Read. S.M. and Bacic. A. (1996) Cell wall porosity and its determination in Modern Methods of Plant Cell Wall Analysis, vol. 17 (eds H.F. Linskens and J.F. Jasckson). Springer-Verlag. Berlin, pp. 63-80. Ridley. B.L.. O'Neill, M.A. and Mohnen, D. (2001) Pectins: structure, biosynthesis, and oligogalacturonide-related signaling. Phvtochemistry, 57. 929-967. Rizk. S.E.. Abdel-Massih, R.M., Baydoun. E.A.H. and Brett. C.T. (2000) Protein- and pH-dependent binding of nascent pectin and glucuronoarabinoxylan to xyloglucan in pea. Planta, 211,423^429. Roberts. J.A.. Whitelaw. C.A.. Gonzalez-Carranza. Z.H. and McManus. M.T. (2000) Cell separation processes in plants—models, mechanisms and manipulation. Ann. Bot., 86. 223-235. Roy, S.. Watada, A.E. and Wergin. W.P. (1997) Characterization of the cell wall microdomain surrounding plasmodesmata in apple fruit. Plant Physiol., 114, 539-547. Sander. L.. Child, R.. Ulvskov. P.. Albrechtsen. M. and Borkhardt. B. (2001) Analysis of dehiscence zone endopolygalacturonase in oilseed rape (Brassica napus) and Arabidopsis thaliana: evidence for role in cell separation in dehiscence and abscission zones, and in stylar tissues during pollen tube growth. Plant Mol. Biol.. 46, 469-479. Serpe, M.D.. Muir. A.J. and Keidel. A.M. (2001) Localization of cell wall polysaccharides in nonarticulated laticifers of Asclepias speciosa Torn Protoplasma, 216, 215-226. Shedletzky. E.. Shmuel. M.. Trainin, T. Kalman. S. and Delmer, D. (1992) Cell wall structure in cells adapted to growth on the cellulose-synthesis inhibitor 2.6-dichlorobenzonitrile. Plant Physiol.. 100. 120-130. S0rensen. S.O., Pauly. M., Bush. M.. et al. (2000) Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-p-D-galactanase. Proc. Nat. Acad. Sci. USA. 97. 7639-7644. Steffan, W.. Kovac, P.. Albersheim. P.. Dan-ill. A.G. and Hahn. M.G. (1995) Characterization of a monoclonal antibody that recognizes an arabinosylated (1—»6)-p-D-galactan epitope in plant complex carbohydrates. Carbohydr. Res.. 275. 295-307.

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Stepka, M., Ciampolini, F., Charzynska, M. and Cresti, M. (2000) Localization of pectins in the pollen tube wall of Ornithogalum virens L. Does the pattern of pectin distribution depend on the growth rate of the pollen tube? Planta, 210, 630-635. Taylor, J.E. and Whitelaw, C.A. (2001) Signals in abscission. New Phytologist, 151, 323-339. Tibbits, C.W., MacDougall, A.J. and Ring, S.G. (1998) Calcium binding and swelling behaviour of a high methoxyl pectin gel. Carbohydr. Res., 310, 101-107. Uheda, EL and Nakamura, S. (2000) Abscission of Azolla branches induced by ethylene and sodium azide. Plant Cell Physioi, 41, 1365-1372. Vicre,M., Jauneau, A., Knox, J.P. andDriouich, A. (1998)Immunolocalizationof |J(l-»-4)- andP(.]—>6)D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues. Protoplasma, 203,26-34. Wagner, T.A. and Kohorn, B.D. (2001) Wall-associated kinases are expressed throughout plant development and are required for cell expansion. Plant Cell, 13, 303-318. Wen, F., Zhu, Y. and Hawes, M.C. (1999) Effect of pectin methylesterase gene expression on pea root development. Plant Cell, 11, 1129-1140. Willats, W.G.T., Marcus, S.E. and Knox, J.P. (1998) Generation of a monoclonal antibody specific to (l-*5)-a-L-arabinan. Carbohydr. /to., 308, 149-152. Willats, W.G.T., Gilmartin, P.M., Mikkelsen, J.D. and Knox, J.P. (1999a) Cell wall antibodies without immunization: generation and use of de-esterified homogalacturonan block-specific antibodies from a naive phage display library. Plant J., 18, 57-65. Willats, W.G.T., Steele-King, C.G., Marcus, S.E. and Knox, J.P. (1999b) Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation. Plant J., 20,619-628. Willats, W.G.T., Limberg, G., Bucholt, H.C., et al. (2000a) Analysis of pectic epitopes recognised by hybridoma and phage display monoclonal antibodies using defined oligosaccharides, polysaccharides and en/ymatic degradation. Carbohydr. Res., 327, 309-320. Willats, W.G.T., Steele-King, C.G., McCartney, L., Orfila, C, Marcus, S.E. and Knox, J.P. (2000b) Making and using antibody probes to study plant cell walls. Plant Physiol. Biochem., 38, 27-36. Willats, W.G.T., McCartney, L. and Knox, J.P. (2001a) In-situ analysis of pectic polysaccharides in seed mucilage and at the root surface of Arabidopsis thaliana. Planta, 213, 37-44. Willats, W.G.T., McCartney, L., Mackie, W. and Knox, J.P. (2001 b) Pectin: cell biology and prospects for functional analysis. Plant Mol. Biol., 47, 9-27. Willats, W.G.T., Orfila, C., Limberg, G., et al. (2001c) Modulation of the degree and pattern of methylesterification of pectic homogalacturonan in plant cell walls: implications for pectin methylesterase action, matrix properties and cell adhesion. /. Biol. Chem., 276, 19404-19413. Williams, M.N.V., Freshour, G., Darvill, A.G., Albersheim, P. and Hahn, M.G. (1996) An antibody Fab selected from a recombinant phage display library detects de-esterified pectic polysaccharide rhamnogalacturonan II in plant cells. Plant Cell, 8, 673-685. Zwieniecki, M.A., Melcher, P.J. and Holbrook, N.M. (2001) Hydrogel control of xylem hydraulic resistance in plants. Science, 291, 1059-1062.

6

Modification and degradation of pectins Gregory A. Tucker and Graham B. Seymour

6.1 Introduction Cell wall pectins are modified or degraded during major developmental events such as growth, abscission and fruit ripening. All these events involve alterations in the degree of cell-to-cell adhesion and it is likely that changes in pectin structure play a major role in cell separation. Indeed, pectins are appropriately localised to take this role (Chapter 5) and cell separation in some tissues can be induced using pectin-degrading enzymes or in certain cases calcium chelators alone (Vennigerholz and Walles, 1987; Van Buren, 1991). The structure of pectic polysaccharides is complex (Chapter 1) and the function of the various elements (e.g. rhamnogalacturonan II, RG-II) is still quite poorly understood. However, modifications of pectin structure are common and are likely to have a significant impact on the physical properties of the cell wall. These changes can include alteration in the pore size of the network, which affects the passage of both macromolecules and water; for example, flow of water through xylem may also be regulated by alteration in the size of pores in pectin in pit membranes (Zwieniecki et ai, 2001). The removal of methyl groups is likely to enhance the degree of calcium binding between adjacent polyuronide chains. Furthermore, degradation of side chains may reduce opportunities for entanglement. The aim of this chapter is to clarify the functional role of these, and other, modifications in pectin structure. An area that is not covered in the present chapter is the role of biologically active oligogalacturonides. This topic is covered in detail in a recent review (Ridley et al.. 2001).

6.2 Pectin-degrading enzymes Given the complex structure of pectin, it is perhaps not surprising that there is a wide range of enzymes capable of degrading this polymer. These pectindegrading enzymes, orpectinases, are ubiquitous in pathogenic and saprophytic bacteria and fungi, where they play an important role in the pathogenicity of the organisms (Prade et al., 1999). Pectinases are also found in all plants, where they are closely associated with many aspects of plant development such as pollen tube growth, cell expansion, abscission (Sander et al., 2001) and fruit ripening (Tucker and Grierson, 1987; Fischer and Bennett. 1991).

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Table 6.1 Some commonly occurring pectinases and estimate of gene copy number in Ambidopsis (based on searches of the Arabidopsis database at www.tigr.org, July 2001) Enzyme Pectinesterase Polygalacturona.se Pectate lyase Pectin acetylesterase |3-Galactosidase Arabinosidase

Arabidopsis gene copy number 59 65 24 11 15 1

Table 6.1 lists common pectin-degrading enzymes and an estimate of the potential gene copy number in Arabidopsis (a more comprehensive survey can be found in Henrissat et al., 2001; see also http://afrnb.cnrs-mrs.fr/CAZY/ index.html). This list is by no means exhaustive but does include the major enzymes thought to be involved in the degradation of pectin. Figure 6.1 provides a summary of the mode of action and possible interaction of these enzymes. The enzymes fall generally into two groups. In the first group there are the enzymes responsible for the depolymerisation of the homogalacturonan backbone of the pectin. These include pectate lyase, which is perhaps the most important microbial enzyme activity in this respect, and polygalacturonase, which may be more significant in plant tissues. This group also includes pectinesterase (or pectin methylesterase as it is sometimes called) which may act in a synergystic manner with either pectate lyase or polygalacturonase. The second group of enzyme activities is directed towards the degradation of the highly branched rhamnogalacturonan regions of the pectin polymer. These include rhamnogalacturonase (RGase) and rhamnogalacturonanan lyase, which again act to depolymerise the polymer, and |3-galactosidases and a-arabinosidases, which act to degrade the galactan/arabinan or arabinogalactan side chains found associated with the rhamnosyl residues. The distribution of these specific pectinases varies both between species and within plant tissues. Some, such as pectinesterase, are found in plants, bacteria and fungi, while others, such as the rhamnogalacturonases may be more restricted in their occurrence, being found either only in microorganisms or within certain plant organs. It is also generally true that the enzyme activities within a given species or tissue often occur as several isoforms. The significance of these isoforms is often very unclear, but may reflect subtle differences in substrate specificity. Gene sequences for many of these enzymes have been reported and in many cases they exist as multigene families. Table 6.1 illustrates this point by listing the known pectinase genes so far identified within the Arabidopsis genome sequence. This diversity of genes within organisms also reflects the isoform nature of the enzyme activities.

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Figure 6.1 Stylised mode of action of the major pectinases found in microorganisms and plants. A Gal: O. GalA; •, Rha: ®. Ara: •. Ac: o. Me: 9. 4-O-Me. (Adapted from Schols and Voragen. Chapter 1.)

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It is beyond the scope of this review to cover all of the known pectinases; instead emphasis will be placed on those most commonly found in plants or microbes. For a review of microbial pectinases see Chapter 7. 6.2.7 Pectinesterase Pectinesterase (PE) (EC 3.1.1.11) removes the methyl esters from esterified galacturonic acid residues, and in so doing converting the C-6 carbon to the carboxylic acid. The enzyme is found in many microorganisms and most, if not all, plant tissues, where it tends to be expressed throughout development. In most plant tissues PE activity is known to be associated with several isoforms, although in many instances their mode of action and relative roles are not known. The mode of action is a particularly important property since this dictates the extent and nature of the blocks of unesterified regions within the polygalacturonic backbone of the pectin. There are three generally recognised modes of action for PE (see Grasdalen et al., 1996): •

•

•

The 'multiple-chain' mechanism, in which the enzyme-pectin complex dissociates after the de-esterification of a single galacturonic acid residue, which results in random de-esterification of the pectin backbone. The 'single-chain' mechanism, where the PE de-esterifies all the residues in an esterified block. Thus GEEEEEEG would be converted to GGGGGG GG, where G represents galacturonic acid, and E represents methylgalacturonic acid residues. The 'multiple attack' mechanism in which the PE de-esterifies a fixed number of residues within an esterified block (known as the degree of multiple attack) with each enzyme substrate encounter. Thus for a degree of multiple attack of 3, GEEEEEEG would be converted to GGGGEEEG.

More recently it has been suggested that these mechanisms be modified to take into account both intra- and inter-chain patterns of de-esterification (Denes et al., 2000). Studies of PE action in vitro have suggested that acidic PEs, such as those found in fungi, tend to follow random patterns of de-esterification, while PEs with alkaline pH optima, such as found in plants, result in blockwise deesterification of the pectin. Catoire et al. (1998) examined the mode of action of three isoforms of PE isolated from mung bean and found that they exhibited different mechanisms. This group also demonstrated that the mechanism differed depending on whether the PE being characterised was soluble or in the cell wall-bound form. Denes et al. (2000) have determined the mode of action of apple fruit PE and have shown that this can vary depending on the pH of the assay. In addition, Limberg et al. (2000) have observed variations in the mode of action of PE from orange peel depending on the degree of methyl-esterification (DM) of the pectin. These observations clearly demonstrate that the mode of action of PE as determined in vitro may not reflect its actual action in situ.

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The three-dimensional structure of a PE from Erwinia chrysanthemi has recently been described (Jenkins et al, 2001). The PE enzyme has been shown to comprise a right-handed parallel {3-helix similar to that seen in polygalacturonase, pectin lyase, pectate lyase and rhamnogalacturonase enzymes. Interestingly, another pecteolytic esterase—rhamnogalacturonan acetylesterase—has a very different structure, having a a/P hydrolase fold. The active site of the PE appears to contain a pair each of aspartate and arginine residues. 6.2.2

Polygalacturonase

Polygalacturonase (PG) (EC 3.2.1.15) is another pectinase often associated with both microbial and plant tissues. PG hydrolyses the a-(l—»4) linkage between two adjacent galacturonic acid residues within the pectin backbone. As such, this is a key enzyme responsible for the depolymerisation of pectin. The exact substrate requirement for PG action is still subject to debate, but it is generally acknowledged that the enzyme will cleave only between de-esterified galacturonic residues. This raises the possibility of synergy between PG and PE in the degradation of pectin, the action of PE generating blocks of de-esterified galacturonic acid residues that in turn act as sites for PG action. PGs normally have pH optima in the acid range (pH 4-6) and can be either exo- or endo-acting. Endo-PG is perhaps the more common activity. Polygalacturonase has been shown to be expressed at high levels in many plant tissues including fruit, flowers, pollen and leaf abscission zones (Brown and Crouch, 1990; Fischer and Bennett, 1991; Kalaitzis et al., 1997) and is secreted by a wide range of pathogenic microorganisms (Prade et al., 1999). Indeed, these enzymes can be grouped according to their amino acid sequences into 'fruit', 'pollen' or 'abscission' type enzymes. However, low levels of PG activity are often associated with other plant organs such as young seedlings (Pressey and Avants, 1977) and roots (Hawes and Lin, 1990). Polygalacturonase activities have also been characterised from a wide range of microorganisms; indeed the endo-PG from Erwinia carotovora was the first PG to have its crystal structure determined (Pickersgill et al, 1998). Since then the structure of the endo-PG from Aspergillus niger has also been determined (Van Santen et al., 1999). These structures have shown that the protein folds to give a 'tunnel-like' cleft in which the active site is located. Structural analysis and site-directed mutagenesis have localised tryptophan, histidine, aspartate and arginine residues within the active site and indicated that these may be important for catalytic activity (Niture et al., 2001). Genes encoding PG have also been identified in many plants and microorganisms. Numerous PG genes have been described in the model plant Arabidopsis thaliana. A recent report discusses the analysis of \9Arabidopsis PG genes. The existence of five classes was suggested: clade A containing those involved in leaf and flower abscission; clade B. those expressed in the fruit dehiscence zone:

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clade C, mainly expressed in flower buds and flowers; clade D, containing genes whose expression pattern has yet to be determined; and clade E, comprising genes expressed in young seedlings and roots (Torki et al, 2000). Polygalacturonase is a key enzyme in the degradation of plant cell walls by microorganisms such as Erwinia, and several plants have evolved a resistance mechanism. This relies on the presence within the cell wall of PG-inhibiting proteins (PGIP) that can inhibit the microbial enzyme and favour the accumulation of elicitor-active oligogalacturonides (De Lorenzo and Cervone, 1997). These PGIP proteins belong to a super-family of leucine-rich repeat proteins that are specialised for the recognition of non-self molecules and the proteins themselves are structurally related to the products of several recently cloned resistance genes in plants (Jones and Jones, 1997). A detailed analysis of the secondary structure of a PGIP isolated from Phaseolus vulgaris has been reported (Mattei et al., 2001). There are numerous PGIPs found in plants, each with a slightly modified amino acid sequence. These modifications are thought to influence the specificity and affinity of the PGIPs for fungal endo-PGs (Leckie et al,, 1999). 6.2.3 Pectate lyase Pectate lyase (PL) (EC 4.2.2.2), also known as pectate transeliminase, carries out a reaction very similar to that performed by PG. In this case the a-(l -»4) linkage between galacturonic acids is broken, not by a hydrolytic reaction as with PG, but by a ^-elimination reaction that causes the formation of a double bond between carbons 4 and 5 of one of the galacturonic acids. This again results in the depolymerisation of the pectin backbone. Unlike PGs, which tend to have acidic pH optima, PLs seem to be most active at alkaline pH. Some lyases can cleave the a-(l->4) linkage when either, or both, of the adjacent galacturonic acid residues are esterified. In this case the enzyme may be termed a pectin lyase. Other PL enzymes may require both of the galacturonic acid residues to be de-esterified. In addition to this variable requirement for methylesterification, the PL enzymes can be either endo- or exo-acting and may have their activity restricted to short oligosaccharides. The exo-acting forms of the enzyme can result in the production of di- or trioligomers. Pectate lyase activity is commonly associated with fungi and bacteria (Collmer and Keen, 1986) and it was originally thought that this enzyme rarely occurred in plant tissues. However, with the advent of molecular cloning techniques, genes or cDNAs with homology to microbial PL have been clearly identified in several plant tissues. Thus two genes expressed in tomato pollen (Wing et al., 1989) showed homology to PL from Erwinia. While many PL-like genes in plants seem to be associated with pollen, they have also been shown to be active in other tissues such as ripening fruits (Medina-Suarez et al,, 1997), vascular bundles and shoot primordia (Domingo et al., 1998). The presence of actual PL activity within a plant tissue has been more difficult to establish; however.

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Domingo et al. (1998) expressed a PL cDNA sequence from Zinnia elegans in E. coli and demonstrated that this possessed calcium-dependent PL activity. More recently, PL gene expression and enzyme activity have been reported in the latex of opium poppy (Pilatzke-Wunderlich and Nessler, 2001) and banana fruit pulp (C. Marin, K. Manning, J. Orchard and G. B. Seymour, unpublished). Pectate lyase has been most extensively investigated in microorganisms such as the Erwinia spp. and a wide range of isoforms have been characterised (Hugouviex-Cotte-Pattat et al., 1996). In most cases the PL enzymes, like many other microbial pectinases, are secreted into the extracellular medium, this secretion being predominantly via the type II secretory machinery. However, there are some cell-bound isoforms, these being either periplasmic or cytoplasmic in localisation (Shevchik et al., 1999). The PLs have been classified into five families, based on their relative amino acid sequences. Family 1 is perhaps the largest group and comprises many bacterial and fungal lyases. In Erwinia chrysanthemi, for example, there are at least five secreted endo pectate lyases (PelA to PelE) classified as being within family 1 (Shevchik et ai, 1999). The PL proteins predicted from the plant genome sequences also fall within the family 1 grouping. The other families also contain examples of endo pectate lyases, for instance Pell, PelL and PelZ from Erwinia chrysanthemi are all endo pectate lyases but are grouped in families 3, 4 and 5, respectively (Shevchik et al, 1999). The periplasmic exopolygalacturonase lyase (PelX) from Erwinia chrysanthemi is classified in family 4 and this bacterium also possesses a cytoplasmic oligogalacturonate lyase (Ogl) the activity of which is restricted to short oligogalacturonates (Shevchik et al., 1999). The PelX isoform actually catalyses the formation of unsaturated digalacturonates by attack from the reducing end of the polymer. The threedimensional structures of several pectate and pectin lyases have been elucidated (Yoder et al, 1993; Lietzke et al, 1994, 1996; Pickersgill et al, 1994). 6.2.4 Pectin acetylesterase Several acetylesterase activities have been described in either microorganisms or plants. Some of these have the potential to degrade pectin polymers. Pectin acetylesterase (PAE) is one such enzyme. This enzyme acts in a fashion similar to PE in that it hydrolyses the ester bond between a glycosyl carbon and an acetyl group. Pectin acetylesterase activity has been purified from orange fruit (Williamson, 1991) and shown to have an MT of 29000. The enzyme did not possess any pectinesterase activity itself but its activity against sugar beet pectin was enhanced by pretreatment with PE. The pH optimum for the enzyme was 5.5. Acetylesterases have also been reported in, and purified from, microorganisms. Searle-van Leeuwen et al (1996) reported the characterisation of three acetylesterases from Aspergillus niger. Only one of these, which was classed as a PAE, was capable of releasing acetyl groups from the homogalacturonan

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region of sugar beet pectin. Again this enzyme was shown to work cooperatively with other pectinases. A second acetylesterase was shown to remove at random the acetyl esters from the hairy ramified regions of apple pectin and this enzyme was termed a rhamnogalacturonan acetylesterase. The third acetylesterase was found to be specific for the removal of feruloyl esters from xylan oligomers, but no activity was shown against pectins. Kroon etal. (1996) reported the isolation of an acetylesterase, again from Aspergillus niger, which was marginally active against sugar beet pectin, releasing only 0.9% of the alkali-extractable ferulic acid. However, when the sugar beet pectin was incubated with a combination of endo-arabinase and the acetylesterase, this resulted in a 14-fold increase in the release of ferulic acid. This, along with the fact that the enzyme showed activity against a range of soluble feruloylated oligosaccharides derived from sugar beet pectin, suggests that it is indeed a feroulyl acetylesterase that can degrade pectin. The three-dimensional structure of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus has been described by Molgaard et al. (2000). 6.2.5 $-Galactosidase and a-arabinosidase All the enzymes discussed so far are active primarily against the homogalacturonan backbone region of the pectin. Other enzyme activities are directed at the degradation of the galactan and arabinan side chains. These include Pgalactosidase and ot-arabinosidases. Again these enzymes are commonly associated with both microorganisms and plants (Chin et al., 1999), It is common to assay these enzymes through degradation of/?-nitrophenol glycosides. However, the results obtained with these artificial substrates must be treated with caution. For instance, tomato fruit has been shown to contain at least three isoforms of ^-galactosidase activity (Pressey, 1983), but only one of these enzymes actually possesses the ability to degrade a j3-(l-*4) galactan (Pressey, 1983; Carey et al., 1995). In all instances so far reported the p-galactosidases associated with plant tissue have been found to have exoacting activities. In contrast, microorganisms have been shown to possess both exo- and endo- acting isoforms. In addition to p-galactosidase activity, many microorganisms and plants also possess a-galactosidase activity (deVries et al., 1999). The significance of this for pectin degradation is unclear as the occurrence of this type of bond within pectic polymers is rare. 6.2.6 Rhamnogalacturonase and minor pectinases In addition to the commonly found, and highly expressed, enzyme activities discussed above there are many other 'minor' pectinases. However, although these enzymes may be more restricted in their distribution and/or level of expression, this does not imply that they are any less important for the modification of pectin. One such pectinase is rhamnogalacturonase (RGase) (Schols et al.,

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1990). This enzyme, which cleaves the alternating a-(l->2) links between rhamnose and galacturonic acid is thought to attack the backbone structure in rhamnogalacturonan I regions of the pectin polymers. An RGase has been purified fromAspergillus aculeatus and its structure has been completely solved (Petersen et al, 1997). The primary amino acid sequence of this RGase showed 20% homology with that of the endo-PG from Erwinia carotovora. RGase activity has also been detected in tomato, apple and grape (Gross et al., 1995). Several other similar enzymes have been characterised (Mutter et al., 1998) again from Aspergillus aculeatus. These include rhamnogalacturonan lyase, rhamnogalacturonan-rhamnohydrolase (Mutter et al., 1994) and rhamnogalacturonan-galactohydrolase (Mutter et al.. 1996). For further details, see Chapter 7. 6.2.7 Peroxidase It is well recognised within the food industry that free radicals produced during food processing can result in the nonenzymatic depolymerisation of polysaccharide polymers. Hydrogen peroxide is a common cause of such nonenzymatic degradation and Miller (1986) showed that several plant cell wall polymers (including galacturonan) could undergo scission when incubated with 0.1lOmM H2C»2. The much more reactive hydroxyl radical has also been shown to cause scission in a wide range of polymers including polysaccharides. Fry (1998) demonstrated in vitro the nonenzymatic degradation of several cell wall polymers (again including pectin) by a system producing hydroxyl radicals under conditions likely to be encountered within the cell wall. Hydroxyl radicals can be produced by a Fenton reaction involving ascorbate and copper ions, as used by Fry (1998). However, hydroxyl radicals can also be produced from oxygen by the action of horseradish peroxidase (Chen and Schopfer, 1999) and this system has also been demonstrated to cause scissions in wall polymers such as pectin (Schweikert et al., 2000). It has thus been postulated that peroxidase action may be able to generate these radicals in situ within the cell wall. Peroxidases are present in plant cell walls and are often found associated with polysaccharides (Everse et al, 1991). While the hydroxyl radicals are highly reactive, they are also very short lived. This raises the possibility that localised peroxidase activity in the vicinity of specific cell wall polymers may result in their nonenzymatic cleavage. However, the occurrence of such a mechanism in the wall and its significance for development, if any. remain to be demonstrated. 6.3 Pectin modification during plant development 6.3.7 Fruit ripening The ripening of many fruits is accompanied by softening of the tissue. While this softening may be partially attributed to changes in turgor. it is likely that it is

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the degradation of the cell wall that is of primary importance. This degradation is thought to result in either wall weakening or loss of cell-to-cell adhesion, or both. Light microscopy and electron microscopy reveal that ripening is accompanied by changes in the ultrastructure of the fruit cell wall, and the pectinrich middle lamella in particular seems to undergo significant alteration (Ben Arie etal, 1979). These changes in ultrastructure are generally accompanied by characteristic modifications in the composition and properties of the cell wall polymers. Thus it has been shown that the pectin becomes progressively more soluble, the degree of methyl-esterification is reduced and depolymerisation occurs during ripening. There is also a significant loss of neutral sugars from the pectin fraction, primarily galactosyl, and in some instances arabinosyl residues. While these changes occur in a wide range of fruit tissues, they are by no means associated with the softening of all fruit types. Strawberry, for example, while exhibiting an increase in pectin solubility, does not demonstrate either the depoiymerisation of the pectin or the loss of methyl esters commonly associated with ripening (Knee et al., 1977). Tomato, on the other hand, exhibits all of these cell wall changes. Tomato ripening has also been extensively investigated and serves as the model in this case to illustrate the role of pectinases in fruit ripening. The changes in the pectin polymers outlined above suggest key roles for PG, PE and |3-galactosidase and it is these three classes of enzyme that will be discussed in more detail. PE activity is present throughout both the development and ripening of the tomato fruit (Hobson, 1963). This suggests a function in both cell expansion and ripening. However, it is apparent that this activity resides in several isoforms and that the pattern of expression of these may vary during development and ripening. Gaffe et al. (1994), using isoelectric focusing gels, demonstrated at least five PE isoforms in the tomato plant. Two of these represented a basal activity found in all tissues including fruit, while the remaining forms appeared to be fruit specific. Tucker et al. (1982, 1999) and Warrilow et al (1994) have both reported the characterisation of three PE isoforms from ripe tomato fruit (PE1, PE2 and PE3 using the nomenclature of Tucker). The fruit-specific isoform (PE2) accounted for the majority (80%) of the activity in ripe fruit, while PE1 was the next major isoform and PE3 the minor isoform. The PE1 and PE3 isoforms may also be found in vegetative tissues. In addition to this tissue distribution of the isoforms, there was also a developmentally regulated expression of PE2 in the fruit, with levels of this isoform increasing dramatically during fruit development, commencing at around 15-20 days after anthesis, which is prior to the onset of ripening (Tucker and Zhang, 1996). Levels of PE 1 activity also seem to increase during ripening, while those of PE3 seem to remain fairly constant throughout development. The PE2 isoform has been completely sequenced (Markovic and Journval, 1986) and corresponding cDNAs have been identified (Ray et al., 1988; Hall et al., 1994). Analysis of the tomato genome has identified three genes, organised in tandem, with homology to PE2. One of the genes appears to have no

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corresponding mRNA and as such may represent an inactive pseudogene. Hall et al. (1994) described two cDNA clones for PE2—pB8 and pB16. These had 93.7% homology to each other in the coding regions but the sequence for pB8 showed greater homology to the actual amino acid sequence of the PE2 protein. Analysis of mRNA levels equivalent to either pB8 or pB16 have demonstrated a 40-fold higher accumulation of the former. Thus pB8 may represent the gene responsible for the bulk of PE2 production in the fruit. The pB8 has an ORF encoding a protein of 546 amino acids. Comparison with the N-terminus of the actual PE2 enzyme (which contains only 317 amino acids) suggests the presence of an N-terminal extension of 229 amino acids, the function of which is not clear. The PE1 and PE3 isoforms have both been purified (Warrilow et al., 1994; Zhang, 1994) and N-terminal amino acid sequences have been determined. It is apparent from these sequences that these other two isoforms derive from the action of a different gene or gene family from that responsible for PE2. However, the precise nature of these genes remains to be elucidated. Gaffe et al. (1997) characterised a ubiquitously expressed PE sequence isolated from tomato fruit. This gene was expressed at high levels in young root, leaf and fruit tissues, but at a lower level in older tissues. This gene may well correspond to one of the other isoforms of the enzyme. Polygalacturonase activity unlike that of PE, increases dramatically in tomato fruit commensurately with ripening (Hobson, 1964). There is a low level of PG activity detectable in green immature fruit, but this appears to reside in an exo-acting isoform. There are at least two major isoforms of endo-PG (PG1 and PG2) synthesised during ripening (Tucker et a/., 1980). Polygalacturonase 1 is the first isoform to appear and this has an Mr of about lOOkDa and on analysis by SDS-PAGE has been shown to be a dimer with subunits of 45 and 38kDa, respectively (Moshrefi and Lu, 1984). However, PG1 represents only a minor form of the enzyme in fully ripe fruit since PG2 appears fairly soon after the onset of ripening and accumulates rapidly. This isoform has again been characterised and shown to have an Mr of about 43 kDa and to run as a single polypeptide on SDS-PAGE. Comparison of the two 43 kDa polypeptides in both PG 1 and PG2 has shown them to be identical and as such this appears to represent the catalytic subunit of the enzyme. Thus it would appear that there is a single active polypeptide (PG2) and that formation of PG1 results from the association of this with a second polypeptide, which has been termed the (3-subunit. This p-subunit is synthesised before the catalytic subunit and has been shown by immunolocalisation to be deposited in the cell wall (Pogson et al, 1991). The (3-subunit may thus act in some way as a target for PG action within the wall and be responsible for controlling the activity of this enzyme to some extent, perhaps by limiting mobility. Removal of the ^-subunit by gene silencing does result in an increased depolymerisation of the pectin during ripening (Watson et al., 1994) and an increased rate of softening, suggesting that this is indeed the case (Chun and Huber, 2000).

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A single gene encoding the ripening specific endo-PG has been identified in tomato fruit (Bird et al., 1988). This gene gives rise to a polypeptide that is then subjected to post-translational glycosylation to give rise to the 43kDa PG2, In fact, there seem to be two isoforms within the PG2 group (Mohd and Brady, 1982), which may arise from differential levels of glycosylation. The 5; and 3' flanking regions of the endo-PG gene have been used in a DNA construct to drive the expression of a CAT (chloramphenicol acetyltransferase) reporter gene. This reporter gene construct has been used to generate transgenic tomato plants. The CAT was expressed in a fruit- and ripening-specific manner (Grierson et al., 1990). This expression corresponds to that of the endogenous PG gene in normal fruit. The levels of PE and PG found in ripe tomato fruit are so high that it has been estimated that they could degrade the cell wall pectin completely within 4 minutes. It is thus clear that the activity of both enzymes must be severely restricted in vivo and this was readily demonstrated by observing the extent to which cell wall pectins were degraded by exogenous PG and PE in comparison with changes in vivo (Seymour et al., 1987a,b). Use of silver ions to arrest further synthesis of PG has shown that the enzyme already present in the wall prior to treatment is incapable of any further degradation of the pectin (Smith et aL, 1989), suggesting that it is in some way sequestered within the wall. How this may occur is not clear. It may involve attachment to target proteins, such as the |3-subunit (Chun and Huber, 2000), or, given that both PG and PE are very basic proteins, may involve electrostatic attraction between the positively charged enzymes and the negatively charged regions of de-esterified homogalacturonans. While endo-PG is the pectin-depolymerising activity most commonly associated with ripening, there are several fruits in which PG activity is either low or undetectable; these include banana and strawberry. Genes for PL have been identified and mRNA transcripts have been demonstrated to be present during the ripening of strawberry (Medina-Escobar et al., 1997) and banana (Domingues-Puigjaner et al., 1997; Medina-Suarez et al., 1997) fruit. It is possible that PL is more widespread and may occur in association with PG. The significance of the production of two different enzymes with essentially the same biological consequence in terms of pectin modification is unclear. Cell wall galactan-degrading activity is present in ripening tomato fruits. Pressey (1983) showed that in tomato, as in many other fruit, the (3-galactosidase activity could be resolved into isoforms. There were three in the case of the tomato and only one of these (|3-gal II) was capable of degrading a j3-(l-»4) galactan substrate. This fi-gal II isoform has been purified and shown to be able to degrade a natural fH 1 ->4) galactan with the release of free galactose, suggesting that this enzyme, as usual for plants, is an exo-acting P~( l->4) galactanase (Carey et a/., 1995). The (3-galactosidase activity in tomato is encoded for by a rnultigene family with at least seven members (Smith and Gross, 2000).

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One of these (TBG4) appears to represent the major transcript in ripe fruit and encodes for the ^-gal II galactanase protein previously purified (Smith et al., 1998; Smith and Gross, 2000). The other members of the family show variable patterns of expression. Some are highly expressed early in fruit development and then decline, while others show transient expression at certain times during development. 6.3.2

Abscission

The primary site of cell wall breakdown in abscission zones is the pectinrich middle lamella. Increases in the activity of pectolytic enzymes have been reported in the abscission zones of leaves, flowers and fruit (Roberts et al.. 2000). It is known that tomato tissues other than fruit express PG activity and the abscission zone is one of these (Taylor et al., 1990). Low levels of PG activity are detectable in abscission zone tissue prior to separation. However, in response to ethylene treatment this activity increases dramatically as the separation zone develops. This occurs both in the leaf abscission zone and also in the zone associated with flower and fruit abscission. Two PG genes have been characterised from tomato abscission zones and their promoters have been shown to direct the tissue specific expression (Hong et al., 2000). One of these PG genes is also expressed in the tomato flower along with another member of the tomato PG gene family (Hong and Tucker, 2000). PG activity has also been observed in the abscission zone of S. nigra and is endo acting (Taylor et al., 1993). There is also evidence for the action of PG in the dehiscence zone of pods of B. napus (Peterson et al., 1996) and those of Arabidopsis siliques. anthers and floral abscission zones (Sander et al.. 2001). Studies on changes in the structure of pectic polysaccharides in abscission zones have been very limited (see Chapter 5). 6.3.3 Growth Pectin-degrading enzymes are active in numerous growth-related processes. For instance in Arabidopsis, PG promoter GUS fusions revealed expression in stylar tissues during pollen growth, branch points between stems and pedicel, and expression associated with the apical meristem of seedlings (Sander et al., 2001). Pollen tubes extend by tip growth and require a rapid turnover of the tip wall materials. This is achieved by delivery of pectin-containing vesicles to the growing tip (Steer and Steer, 1989). Alterations in the extensibility of pollen cell walls are correlated with pectin esterification and de-esterification (Li etal. 1996). Esterified pectin is located predominantly at the apex of the pollen tube, indicating that it is related to tip wall loosening. De-esterification of pectin leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall (Li etal.. 1994).

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Developing roots show similarities to growing pollen tubes. At the growing root tips of Arabidiopsis, dividing cells have more esterified pectin and the nondividing cells have more de-esterified pectin (Dolan et at., 1997). In leek seedlings, a steady increase in PG activity was associated with the emergence of lateral roots and in situ localisation with PG antibodies showed its presence over the meristems of the lateral root primordia (Peretto et al., 1992). A similar pattern of events is found in elongating epidermal cells, with de-esterified pectins being much more abundant in the cells of the nonelongating region. Furthermore, the pore size was larger in the nonelongating region than in the elongating region (Fujino and Itoh, 1998). The pectins were observed to become granular in elongating regions of the wall and then this material almost disappeared in nonelongating regions. The authors suggest that this represents a modification in the molecular form of the pectins during elongation, possibly associated with changes in the structure of the neutral sugar side chains. The role of neutral sugar side chains in pectin structure is still poorly understood. However, extension growth is associated with changes in the levels of galactosyl residues (Tanimoto, 1988). S0rensen et al. (2000) reported the effects of overexpressing a fungal endo-galactanase in transgenic potato tubers. There were substantial reductions in the level of galactosyl residues associated with isolated rhamnogalacturonan fragments compared with wild type. Furthermore, pectin solubility increased in the transgenic cell walls challenged with endoPG/PE. These data indicate that the polyuronides in the transgenic walls were more accessible to endo-PG/PE compared to wild-type walls. Thus, removal of galactan side chains in mum may result in a more porous cell wall architecture. Galactan and arabinan side chains appear to participate in the formation of cell clustering and intercellular attachment in carrot calli (Kikuchi et al., 1996). Indeed, a recent study of a mutant line of callus with very loosely attached cells showed that the pectic polysaccharides are not retained in the walls. Furthermore, the arabinans associated with the hemicellulosic fraction were absent in the mutant. These arabinose-rich pectins, which are strongly associated with cellulose-hemicellulose complexes, may therefore play an important role in intercellular attachment in the architecture of the wall (Iwai et al., 2001).

6.4

Functional analysis of pectinases

The function of pectinases in microorganisms is fairly obvious. Pectin accounts for a significant proportion of the total sugar in plant cell walls. Thus, for pathogenic and saprophytic organisms this pectin is a potential carbon source. They respond to the presence of extracellular pectin by the synthesis and secretion of pectinases (Hugouviex-Cotte-Pattat et al., 1996). The primary aim of these secreted enzymes is the depolymerisation of the pectin into a form that can be readily transported into the microorganism. Other periplasmic and

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cytoplasmic enzymes, such as the oligogalacturonate lyase, are then used to further metabolise and assimilate the sugars (Prade et al., 1999). The precise function of these pectinases in planta, however, is often unclear. For instance, the action of PE in producing long regions of de-esterified homogalacturonan could function to weaken the cell wall by synergy with either PG or pectate lyase, resulting in depolymerisation of the pectin. Similarly, the generation of immobilised charges on the pectin polymers may result in Donnan forces, resulting in increased hydration and swelling of the wall. Alternatively, interactions of these de-esterified blocks with calcium could serve to strengthen the wall by the formation of the so-called 'egg-box' structures (Grant et al., 1973). Function is often implied by the spatial and temporal expression of these pectinases in plant tissue. Thus the fruit-ripening-specific endo-PG may be implicated in the process of fruit softening. However, more direct evidence for the role of these enzyme activities is being accumulated by the study of naturally occurring mutants and by the application of gene silencing technology.

6.5 Application of gene silencing techniques The cDNA encoding the fruit-specific PE2 (Ray et al., 1988) has been used to produce genetically modified tomato plants in which the activity of PE has been downregulated to levels about 10% of that found in wild-type fruit (Hall et al., 1993). Fruit of the transgenic plants showed little or no PE2 activity, while the activities of the other two isoforms (PE1 and PE3) were unaffected (Tucker and Zhang, 1996). Similarly, levels of PE activity in the leaves of these transgenic plants was also unaffected, showing that expression of the basal PE isoforms had not been modified. Pectin metabolism in these transgenic fruit was investigated and it was found that while the extent of pectin de-esterification was reduced in the transgenic fruit, it was by no means eliminated completely. Wild-type fruit showed a decline in methyl ester content from about 75% to 50% in green and ripe fruit, respectively. In contrast, the reduced PE fruit showed a decline from about 85% to 65% at comparable stages of ripening (Tucker, 1993). The solubility and molecular weight of the pectin in the transgenic fruit both seemed to be unaffected by the reduction in PE activity, and fruit texture during ripening was also unaltered. The original transformation was achieved using only a fragment of the cDNA encoding PE2. More recently the entire coding sequence for the mature PE2 protein has been used to generate transgenic tomato plants. In this case both PE2 and PE3 activities were downregulated, resulting in fruit with only one remaining PE isoform (PE1) (Simons and Tucker, 1999) but again with little or no evident effect on fruit texture. In a similar manner, antisense PE plants have also been produced by Tieman et al. (1992). Here the reduction of PE activity in the fruit again resulted in an

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increase in the degree of esterification of the pectin, but depolymerisation of the pectin was also reduced. Furthermore, the transgenic fruit exhibited a decrease in the levels of total and chelator-soluble pectins, compared to wild-type controls. The reduction of PE was accompanied by a reduction in the level of calcium associated with the cell wall (Tieman and Handa, 1994). Although there was no apparent effect on the firmness of fruit during ripening, the reduction of PE did lead to a complete loss of integrity as the fruit entered senescence. In this instance the reduction in PE had commercial significance as it has been shown to improve the quality of the tomato juice produced from the transgenic fruit (ThakmetaL, 1996). Two groups have downregulated tomato fruit endo-PG expression (Sheeny et al, 1988; Smith et al., 1988). In both instances PG expression in transgenic fruit was reduced to less than 0.5% of normal activity. As expected, all the isoforms of PG were effected by this transformation since only a single gene is involved. During the ripening of these transgenic fruits, pectin solubility and degree of esterification were unaltered, while the depolymerisation of pectin normally associated with ripening was markedly reduced (Smith et al., 1990). However, this modification to pectin metabolism did not result in any significant alteration to the softening of the fruit during ripening. Despite this failure of reduced PG to prevent fruit softening, the genetic modification still had commercial significance as the paste produced from these fruits had higher solid content and viscosity (Schuch et al., 1991). The fact that PG activity alone cannot bring about fruit softening was further demonstrated by the work of Giovannoni et al. (1989). This group transformed a tomato nonripening mutant (rm), which is deficient in endo-PG activity and which does not soften, with a full-length sense cDNA encoding the tomato PG2 enzyme. The resultant transgenic fruit now expressed significant levels of endo-PG activity but failed to show an increase in softening during ripening. The effect on fruit softening of downregulating two tomato p-galactosidase genes (TBG1 and TBG4) has been investigated. The protein product of TBGI was shown to have galactan-degrading activity, but reduction of TBGI gene expression to 10% of normal levels failed to affect fruit softening (Carey et a/., 2001). However, downregulation of [3-gal II (TBG4) resulted in a reduction of galactanase activity to between 5% and 30% of normal, and yielded fruit that showed a 40% increase in firmness (David Smith and Kenneth Gross, USDAARS, Beltsville, personal communication). It is very likely that these pectinases act in concert with other wall-degrading enzymes such as cellulase, to bring about the changes in the wall that are required for softening. Indeed such an interaction has been suggested for the action of pectinases and expansins during tomato ripening (Bmmmell et al., 1999). Gene silencing has also been employed to examine these interactions. A technique has been developed using chimeric constructs containing DNA sequences for two target genes that results in the silencing of both target enzymes simultaneously

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(Seymour et al., 1993). This has been used to silence both PE2 and PG activities in tomato fruit (Seymour et al., 1993; Simons and Tucker, 1999). Gene silencing techniques have also been employed to study the transport of these cell wall hydrolases into the cell wall. A gene encoding a small GTPase (rabll), which may be involved in such trafficking, has been identified in mango (Zainal et al.. 1996) and tomato (Lu et al., 2001) fruit. This gene is expressed in a ripeningspecific manner and when silenced in tomato plants, using antisense technology, resulted in much firmer fruit in which the levels of both PG and PE activities were reduced (Lu et al., 2001). The role of PE in border cell separation in pea root caps has been tested using transgenic plants. Expression of a PE was found to be tightly correlated both spatially and temporally with border cell separation. Partial inhibition of the gene's expression with antisense RNA prevented normal separation of the root border cells from the root tip into the external environment (Wen et al., 1999). Reduced PE expression resulted in a higher extracellular pH. Furthermore, the antisense construct resulted in the growth of emerging hairy roots being stunted by 50%. Also, cells in the root tip were deformed. In the antisense plants, border cells were made but they accumulated in a ball instead of being dispersed into the medium. It seems possible that PE functions to reduce the extracellular pH: this promotes acid growth, induces the action of PGs and results in cell separation (Wenerfl/., 1999). 6.6

Use of mutants

Mutants have been characterised that show aberrant cell adhesion phenotypes or altered growth patterns, whose phenotype might be attributed to altered pectin structure. During pollen development several tissues in the anther show cell separation. The microspores separate from their meiotic siblings during pollen development. Finally, once the pollen grains reach maturity, the septum tissue that separates the two locules of the anther undergoes cell separation. In the quartet mutants (qrt) of Arabidopsis, the microspores do not separate from each other and the exine is deposited on the cluster of four meiotic products, resulting in tetrad pollen (Rhee and Somerville, 1998). The tetrad of microspores is surrounded by a callose wall and then by the pollen mother cell (PMC) wall. In the wild type the callose and PMC cell wall are eventually degraded and the pollen is released into the locule. In the qrt mutants the callose is degraded, but the PMC remains intact. These qrt mutants show varying wall modifications including, in qrtl, the absence of de-esterified pectin. The mutations may be in genes encoding cell wall-degrading enzymes or regulatory components and further characterisation of the qrt mutants is awaited. There are often difficulties in associating phenotypes with specific changes in enzymes or pectin modifications. Mutants defective in fi-galactosidase isolated

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from Brassica campestris have impaired fertility (Singh and Knox, 1985), suggesting an important role for this enzyme in pollen tube growth or fertilization. However, in the pleiotropic tomato ripening mutations nor, rin and Cnr, pectin degradation is substantially altered along with fruit softening, but these mutations affect numerous aspects of the softening process and not just pectin degradation. The cell wall changes in Cnr have been particularly well characterised (Thompson et al., 1999; Orfila et a/., 2001) and some tentative conclusions can be drawn about the role of pectins in cell adhesion in this mutant. Ripe Cnr fruits show very reduced cell adhesion and the pericarp tissue has a mealy appearance. The cell walls of the fruit show many of the characteristics so clearly associated with cell separation that have been described earlier (see also Chapter 5). The cell walls of Cnr have significantly less calcium-binding capacity in comparison with wild type. Antibody studies show that in Cnr levels of calcium cross-linkable homogalacturonan capable of maintaining cell adhesion are severely reduced. Additionally, levels of «-(!->5) arabinan are also low in Cnr fruit cell walls.

6.7

Conclusion and prospects

Pectin degradation and modification are important throughout plant development and are involved in the growth of plant organs such as roots, shoots and pollen tubes. Pectins are also modified and degraded in abscission zones and the tissues of ripening fruits. The role that pectins are thought to play in these processes is either to maintain cell-to-cell adhesion or to allow controlled cell separation. The alterations in pectin structure that are critical for these events are not precisely understood at present. The large number of the genes in the Arabidopsis genome that are likely to encode pectinases almost certainly reflects the many different tissues in which these enzymes are required and must also indicate a wide variety of subtly different isoforms and substrate requirements. PE and PG activities are very widespread and this is consistent with the likely large number of PG- and PE- related sequences in plant genomes. Pectate lyases are also well represented in the Arabidopsis genome, but have only recently become the focus of research in some areas of plant development. Immunocytological studies and more recently transgenic plant experiments indicate that PG and PE have important roles in cell adhesion in root caps, pollen tubes and ripening fruits, but a full picture is yet to emerge of the relationship between pectin modifications and changes in function. It has long been known that degradation of pectic galactans is a common feature in ripening fruits and the findings from studies with cell cultures and ripening mutants are consistent with a role for neutral sugar side chains in cell-to-cell adhesion. Recent work on genes that may be involved in transport of hydrolases to the cell wall indicates the importance of these processes for modulating events

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such as fruit softening. However, the identity of genetic factors that regulate the transcription of genes encoding the cell wall hydrolases or the control of their action within the wall are areas that represent serious gaps in our knowledge. References Ben-Arie. R., Kislev, N. and Frenkel, C. (1979) Ultrastructural changes in the cell walls of ripening apple and pear fruit. Plant Physiol., 64, 197-202. Bird. C.R., Smith, C.J.S., Ray, J.A. etal. (1988) The tomato polygalacturonase gene and ripening specific expression in transgenic plants. Plant Mol. Bioi. 11, 651-662. Brown, S.M. and Crouch, M.L. (1990) Characterisation of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase. Plant Cell. 2, 263-274. Brummell, D.A., Harpster, M.H., Civello, P.M., Palys, J.M.. Bennett. A.B. and Dunsmuir. P. (1999) Modification of expansin protein abundance in tomato fruit alters softening and cell wall polymer metabolism. Plant Cell. 11, 2203-2216. Carey, A., Holt, K., Picard, S., Wilde, R., et al. (1995) Tomato exo-( 1 -4)-p-D-galactanase: isolation and changes during ripening in normal and mutant tomato fruit and characterisation of a related cDNA clone. Plant Physiol. 108. 1099-1107. Carey, A.T., Smith, D.L., Harrison, E., etal. (2001) Downregulation of a ripening-related p-galactosidase gene (TBGI) in transgenic tomato fruits. J. Exp. Bot.. 52. 663-668. Catoire, L., Pierron, M., Morvan, C.. du Penhoat. C.H. and Goldberg (1998) Investigation of the action patterns of pectinmethylesterase isoforms through kinetic analyses and NMR spectroscopy. Implications in cell wall expansion. J. Biol. Chem.. 273. 33150-33156. Chen. S.-X. and Schopfer. P. (1999) Hydroxyl-radical production in physiological reactions. A novel function of peroxidase. Eur. J. Biochem., 260. 726-735. Chin, L.H., AH, Z.M. and Lazan, H. (1999) Cell wall modifications, degrading enzymes and softening of carambola fruit during ripening. J. Exp. Bot.. 50, 767-775. Chun, J.P. and Huber, D.J. (2000) Reduced levels of beta-subunit protein influence tomato fruit firmness. cell-wall ultrastructure, and PG2-mediated pectin hydrolysis in excised pericarp tissue. / Plant Physiol.. 157, 153-160. Collmer, A. and Keen, N.T. (1986) The role of pectic enzymes in plant pathogenesis. Annu. Rev. Phytopathol.. 24, 383^09. De Lorenzo. G. and Cervone., F. (1997) in Plant-Microbe Interactions, vol. 3 (eds G. Stacey and N.T. Keen), Chapman and Hall, New York, pp. 76-93. Denes, J.-M., Baron, A., Renard, C.M.G.C., Pean, C. and Drilleau. J.-F. (2000) Different action patterns for apple pectin methylesterases at pH 7.0 and 4.5. Carbohydr. Res., 327, 385-393. de Vries. R.P., van den Broeck, H.C., Dekkers. E.. Manzanares. P.. de Graaff. L.H. and Visser. J. (1999) Differential expression of three alpha-galactosidase genes and a single beta-galactosidase gene from Aspergillus niger. Appl. Environ. Microbiol.. 65, 2453-2460. Dolan, L. Linstead, P. and Roberts. K. (1997) Developmental regulation of pectic polysaccharides in the root meristem of Arabidopsis. J. Exp. Bot.. 48, 713-720. Domingo. C., Roberts. K., Stacey, N.J.. Connerton, L. Ruiz-Teran. F. and McCann. M. (1998) A pectate lyase from Zinnia elegans is auxin inducible. Plant J., 13. 17-28. Dominguez Puigjaner. E.. Llop, I.. Vendrell, M. and Prat. S. (1997) A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases. Plant Physiol.. 114. 1071-1076. Everse. J., Everse, K.E. and Grisham. M.B. (1991) Peroxidases in Chemistry and Biology, vols 1 and 2. CRC Press. Boca Raton. FL.

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7

Microbial pectinases Jacques A. E. Benen, Jean-Paul Vincken and Gert-Jan W. M. van Alebeek

7.1

Introduction

The highly complex structure of pectin is discussed in detail in chapter 1. In addition to homogalacturonans, known as the smooth regions of pectin, branched or hairy regions also occur (Schols and Voragen, 1996). These hairy regions are composed of the heterogalacturonans rhamnogalacturonan (RG) and xylogalacturonan (XGA), together with arabinan and (arabino)galactan. This chapter deals with carbohydrases having activity towards the homogalacturonans, RG and XGA. A third highly complex heterogalacturonan present in plant cell walls is rhamnogalacturonan II. No enzymes have yet been characterized with activity towards this polysaccharide. Given the complexity of pectin, it can easily be envisaged that microorganisms decomposing pectin require a large set of enzymes to fully degrade the polymer. An overview of known microbial pectinases is presented in Table 7.1. The assignment into families by Coutinho and Henrissat (1999) is based on amino acid similarities rather then biochemical properties. The hydrolytic pectinases for which sequences are available are all grouped into family GH28. The pectic lyases are apparently more diverse and so far six different families are known. Although the GH28 members and the pectic lyases catalyze different reactions, hydrolysis versus ^-elimination, and their sequence identity is marginal (less than 18%), there is a striking similarity in structural topology: a righthanded parallel ^-helix, first described for pectate lyase C from Erwinia chrysanthemi (Yoder et al., 1993). As an example of this topology, the Ca-trace of pectate lyase C is presented in Figure 7.1. In addition to the pectic lyases and hydrolases, this topology was also reported for pectin methylesterase (PME) from E. carotovora (Jenkins et al., 2001). The major differences in topology are found in the number of turns of the (3-helix (between 7 and 13) and the length of the loops extending from the helix barrel. Very recently, the structure of a family-10 polysaccharide lyase (pectate lyase) has been solved and is completely different from the other pectic lyases (G.J. Davies and G.W. Black, personal communication). In the following sections an overview of the biochemical properties of pectinases will be presented. Emphasis will be on substrate specificity and mode of action.

175

MICROBIAL PECTINASES Table 7.1 Overview of microbial pectinolytic enzymes Enzyme

Abbreviation

Family3

Comments

Endopolygalacturonase Exopolygalacturonase Endoxylogalacturonase

EndoPG ExoPG EndoXGH

GH28 GH28 GH28

Endorhamnogalacturonase RG rhamnohydrolase RG galacturonohydrolase Endopectate lyase Endopectin lyase Exopectate lyase Endopectate lyase Endorhamnogalacturonan lyase Endopectate lyase Exopectate lyase Endopectate lyase Endorhamnogalacturonan lyase Pectin methylesterase Pectin acetylesterase RG acetylesterase

EndoRGH ExoRGR ExoRGG EndoPAL EndoPL ExoPAL EndoPAL EndoRGL EndoPAL ExoPAL EndoPAL EndoRGL PME PAE RGAE

GH28

3D structure, ^-helical protein Also active on xylogalacturonan Presumably requires xylosyl side chains for activity 3D structure, p-helical protein No sequence information No sequence information 3D structures, |3-helical protein 3D structures, {3-helical protein Cytosolic enzyme

a

9 •7

LI LI L2 L3 L4 L9 L9 L10 Lll CE8 CE12 CE12

Periplasmic enzyme 3D structure, modular protein Modular protein 3D structure, P-helical protein 3D structure, modular protein

GH, glycosy! hydrolase; L, lyase; CE, carbohydrate esterase; RG, rhamnogalacturonan.

Figure 7.1 Ca trace of Erwinia chrysanthemi B16 pectate lyase C.

7.2

Polygalacturonases

Polygalacturonases (PGs) cleave the a-1,4-D-galacturonosidic linkage in homogalacturonan by hydrolysis. During hydrolysis, the anomeric configuration changes from a to ^ and thus these enzymes are inverting enzymes (Biely et al., 1996). The endopolygalacturonases (EC 3.2.1.15) randomly attack the substrate, whereas the exopolygalacturonases release galacturonic acid monomers (Gal/?A) (EC 3.2.1.67) or (Gal/?A)2 (EC 3.2.1.82) from the nonreducing end

MICROBIAL PECTINASES

177

(see Figure 7.2). PGs generally prefer the nonesterified substrate, polygalacturonic acid, and show decreasing activities with increasing degree of methyl-esterification (DM), although some enzymes are most active on partially (low-) esterified pectin (Pafenicova et al., 2000a). The existence of polymethylgalacturonases (PMG), enzymes that, according to their name, should prefer high-DM pectins, has been reported (Sakai et al, 1993; Pashova et al, 1999). However, there are no detailed studies with respect to biochemical characterization of such PMGs. Microorganisms normally use the PGs to mobilize pectin to be used as food. However, in some yeasts like Kluyveromyces marxianus (Schwan and Rose, 1994) and Saccharomyces cerevisiae (Blanco et al, 1997), which can not utilize galacturonic acid, PG production has been demonstrated and genes encoding PGs have been cloned. Recently it was shown that in Saccharomyces one such gene, PGUI, is involved in the formation of pseudohyphae and as such in the complex process of invasion of the host plant (Cognies et al, 2001). Involvement of pectinases in the infection process of plants by phytopathogenic microorganisms has been well documented. However, this is beyond the scope of this book. In the past many PGs purified from commercial pectinase preparations have been characterized (Kester and Visser, 1990; Pasculli et al, 1991). However, often no clues to the identity of the enzyme are available. During the last decade, many genes encoding PGs have been cloned and the number of genes in the databases is continuously expanding. Unfortunately, only a fraction of these genes have been overexpressed with subsequent in-depth biochemical characterization of the corresponding PGs. Among the best-characterized pectinases for which corresponding genes are known are those from Erwinia chrysanthemi, E. carotovora, Aspergillus niger, A, tubingensis and A. aculeatus. Recently, a detailed analysis of the Fusarium moniliforme endoPG has been reported (Bonnin et al, 2001). The description of enzymatic properties of pectinases presented here will focus on enzymes obtained from these species. Whenever appropriate, data for enzymes from other species will be presented as well. To provide the necessary background to understand the mode-of-action analyses of pectinases that will be presented, a short introduction to the concept of subsites will be given. A subsite is a spot on or in the enzyme where one of the building blocks of a carbohydrate substrate binds. In general all depolymerizing enzymes have multiple subsites and the number of subsites can vary from 2 to 1.4 (Thoma et al, 1971). The subsites are aligned in a linear array and the active site is located somewhere in the array. Subsites are numbered from the catalytic site that is located between/at subsites —1 and +1 (see Figure 7.3) and the subsites with a negative sign extend to the left. By convention oligoand polysaccharides are depicted with the nonreducing end to the left. As a consequence, the nonreducing end always binds to a subsite with a negative sign. As a result of the presence of multiple subsites, the substrate can bind in a

M1CROBIAL PECTINASES

1 79

productive or an unproductive way. Catalysis only takes place when the substrate covers subsites —1 and 4-1. Any binding to other subsites not covering — 1 and -f 1 is unproductive and in fact constitutes an inhibitory (competitive) complex. The specificity of a polysaccharidase is determined by the actual total number of subsites, the affinity of each subsite for a sugar residue and the location of the active site. These features can all be presented as a subsite map. In the past, methods have been developed for subsite mapping by Thoma et al. (1971) and Hiromi et al. (1973) with subsequent alterations and additions (Allen and Thoma, 1976; Suganuma et al, 1978). By determination of a subsite map, insight is gained into which contribution each subsite makes in binding the substrate. In combination with 3D structural data this opens the way to change substrate specificity or other particular properties of an enzyme by mutagenesis techniques. Subsite mapping is based on the mode-of-action analysis using defined oligomeric substrates of increasing degree of polymerization (DP), viz. the determination at which position(s) such oligomers are cleaved (so-called bond cleavage frequencies (BCFs)) and on the kinetic parameters VmaK app and ^m app (Thoma et al., 1971). Even if calculation of a complete subsite map cannot be achieved, mode-of-action analysis and reaction rate determinations give valuable information about relative subsite affinities. An in-depth analysis of a pectinase ideally includes such a mode-of-action analysis. 7.2.7

Exopolygalacturonases

With respect to mode of action, exoPGs are much simpler enzymes than endoPGs because always only one product is released, either a monomer or a dimer. Typically, all bacterial exoPGs analyzed so far—Erwinia chrysanthemi (Collmer et al., 1982), Ralstonia solanacearum (Huang and Allen, 1997), Yersinia enterocolitica (Liao et al., 1999) and Bacillus strain KSM-P567 (Kobayashi et al., 2001)—only released the dimer, whereas in fungi like Aspergillus and Fusarium oxisporum (Garcia-Maceira etal, 2000) only the monomer-releasing activity was reported. From Clostridium thermosaccharolyticum (van Rijsel et al., 1993), an exoPG activity was purified as part of a complex with PME activity that had a low degree of trimer-releasing activity in addition to dimer release. All exoPGs studied so far attack the substrate chain from the nonreducing end. For the E. chrysanthemi PEHX this was inferred from the analysis of products obtained after incubation of a mixture of A4,5-unsaturated Gal/?A oligomers by Collmer et al. (1982). This was later confirmed by Shevchik et al. (1999a) using reduced (Gal/?A>6. Furthermore, Collmer et al. (1982) deduced that the enzyme degraded the substrate by multichain attack and that PEHX preferred A4,5-unsaturated substrate over saturated substrate. The latter was confirmed by Kester et al. (1999) using saturated and A4,5-unsaturated trimers. Kester et al. (1999) also showed that the enzyme requires an nonesterified GalpA unit at subsites —2, — 1 and +1.

180

PECTINS AND THEIR MANIPULATION

Although PGs are generally active between pH 3 and 7, with a more acidic pH optimum for the fungal enzymes, some remarkable PGs, both endo- and exoacting, have recently been purified from alkaliphilic Bacillus species (Kapoor et al., 2000; Kobayashi et ai, 2001). From Bacillus KSM-P567 an exoPG with an optimum at pH 10 was purified that was shown to be devoid of lyase activity (Kobayashi etal., 2001). Kapoor et al. (2000) partially purified a PG (it was not shown whether this enzyme was endo- or exo-acting) from Bacillus MG-cp-2 that not only showed the same high pH optimum but that, in addition, was quite thermostable with a half-life of 120 min at 60CC. The most detailed characterizations have been carried out for the exoPG from A. tubingensis and A. aculeatus, most likely as a result of the industrial importance of the pectinases of these fungi. Kester et al. (1996) showed that the A. tubingensis exoPG hydrolyzes monomers from the nonreducing end. Using oligoGalpA of various DP, a subsite map was calculated that comprised four subsites, —1 to +3. Subsite -1-1 was shown to have a very high affinity for GalpA units (24.5 kJ/mol) whereas subsite — 1 even had some repelling force (— 1.6 kJ/mol). The contribution of subsites +2 and -1-3 to binding of the substrate was only small. The calculated potential intrinsic rate (716s~') was more than threefold higher than the actual turnover (220 s~'). This discrepancy was explained by the formation of unproductive complexes, binding to the enzyme from subsites +1 to +3 (not covering subsite — 1) as a result of the high affinity at subsite -I-1 and negative affinity at subsite — 1. This is supported by the fact that GalpA is quite a strong inhibitor (K t = 0.3 mM) (Kester et al. 1996). During their characterization of the A. aculeatus exoPG, Beldman et al. (1996) discovered the ability of this enzyme to release xylogalacturonic acid dimers (p*-xylose-l,3-galacturonic acid) from the xylogalacturonan part of soy bean pectin (see Figure 7.2). This enzyme also possessed the 'ordinary' exoPG activity towards homogalacturonan. Kester et al. (1999) were able to demonstrate a similar exo-xylogalacturonase activity for the A. tubingensis exoPG. However, this required the continuous removal of the inhibitory GalpA by dialysis. In a subsequent study on the effect of methyl-esterification, using defined monomethyl-esterified di- and triGalpA, Kester et al. (1999) demonstrated that the A. tubingensis exoPG can accommodate methyl-esterified GalpA at subsites — 1 and +1, although this is accompanied by a reduced efficiency. By analyzing A. tubingensis exoPG digests of partially methyl-esterified pectins by mass spectrometry, Korner et al. (1999) concluded that this enzyme does not hydrolyze a non-methyl-esterified GalpA from the reducing end when the following residue is methyl-esterified. Kester et al. (1999) explained the difference between these two studies by the presence of the inhibitor GalpA in the oligomer mixture used by Korner et al. (1999) and concluded that, during pectin hydrolysis. exoPG will leave a nonmethylated residue at the nonreducing end.

MICROBIAL PECTINASES

181

7.2.2 Endo-xylogalacturonan hydrolase In the previous section, the exo-xylogalacturonase activity of exoPG was described, albeit that this activity is much lower than the exoPG activity. Since continuous stretches of xylogalacturonan are present in some pectins, an enzyme specific for this substrate was searched for (van der Vlugt-Bergmans et al, 2000). To this end, an A. tubingensis expression library in Kluyveromyces lactis was screened using modified gum tragacanth (saponified and treated with dilute acid; XyhGalpA ratio of 1:2), a mimic of xylogalacturonan, as a substrate and a new sensitive reducing end group assay for detection (Meeuwsen et al., 2000). Following nucleotide sequencing of clones showing degradation of the substrate it was established that the newly identified enzyme belongs to GH family 28 and shares highest sequence identity with exoPGs (van der Vlugt-Bergmans et al, 2000). Further characterization of the purified enzyme revealed that it can degrade saponified apple hairy regions (containing xylogalacturonan). The major products identified were the disaccharide Xyl-GalpA and an unknown oligosaccharide. Digestion of the modified gum tragacanth resulted in a complex mixture of many xylosylated oligogalacturonides, demonstrating its endolytic action. Furthermore, it was shown that the enzyme is less active on nonesterified homogalacturonan than on non-methyl-esterified xylogalacturonan, suggesting that the enzyme has a requirement for xylosyl side chains and it is therefore believed to cleave between two xylosylated Gal/?A residues (see Figure 7.2) (van der Vlugt-Bergmans et al., 2000).

7.2.3

Endopolygalacturonases

EndoPGs have been known for a long time and as early as 1973 Rexova-Benkova described the first mode of action study of an A. niger endoPG. Kester and Visser (1990) extended this study to six endoPGs purified from a commercial A. niger pectinase preparation. However, not until a family of seven genes encoding endoPGs from A. niger was cloned and individually overexpressed, and analysis techniques had improved, did in depth analysis become possible (Bussink et al., 1992; Pafenicova et al, 1998, 2000a,b; Benen et al, 1999). The elucidation of 3D structures of endoPG from E. carotovora (Pickersgill et al, 1998) and endoPGII from A. niger (van Santen et al, 1999) has also added greatly to the insight on the mode of action of these enzymes. Furthermore, site-directed mutagenesis studies on A. niger endoPGII, now considered a model enzyme, have further improved our understanding of substrate specificity and catalytic properties (Armand et al, 2000; Pages et al, 2000, 2001). An overview of the research carried out for the A. niger endoPGs, and other enzymes where appropriate, will give a good impression of the current status of the knowledge gathered about endoPGs.

182

PECTINS AND THEIR MANIPULATION

The pectinase preparation studied by Kester and Visser (1990) contained two major endoPG activities designated PGI and PGII. By a reverse genetics approach, the corresponding genes pgal and pgall were cloned (Bussink et ai, 1991a,b); and by library screening using the pgall gene as a probe, five additional classes, A to E (based on restriction enzyme mapping), of hybridizing phages were obtained (Bussink et ai, 1992). The endoPG-encoding genes were subsequently cloned and designated pgaA to pgaE and the enzymes PGA to PGE (Parenicova et al., 1998,2000a,b). Basic characterization revealed that the enzymes exhibited a large variation in activity on the model substrate polygalacturonic acid. Whereas PGC, PGD and PGE were reasonably active (Vm^ of 25, 96 and 80U/mg, respectively), PGI, PGB and PGA showed good activity (Vmax of 800, 900 and 1200U/mg, respectively) and PGII was most active with Vmax of 4000 U/mg. Using partially methyl-esterified substrates with various DM, it was established that except for PGA and PGB the enzymes gradually became less active upon increasing DM. PGA and PGB were most active on moderately esterified pectin (DM 22^5%) (Parenicova et al, 2000a). For PGC and PGE it was concluded that the natural substrate is most likely different from polygalacturonic acid. For PGD, which has some unique properties, such as a limited number of subsites (4), digalacturonate-hydrolyzing capacity and extreme processive behavior on oligogalacturonides exceeding degree of polymerization (DP) 4, it was proposed that this enzyme is in fact an oligogalacturonase (Parenicova etal., 2000b). EndoPGs are considered to be random-acting enzymes on polymer substrates. In a strict sense this is indeed true for all enzymes characterized so far. However, for some enzymes it turned out that after the first random encounter with the substrate the enzyme remains bound to the substrate to further degrade this in a multiple-attack fashion. For the enzymes characterized in this respect, the multiple attack or processivity releases monomers from the reducing end, in contrast to exoPGs that release monomers from the nonreducing end, and thus the enzymes appear to act in an exolytic way. Even for the truly randomacting enzymes nonrandom hydrolysis occurs when the DP of the substrate is smaller than the number of subsites, as then the affinities of the individual subsites become important. As a result, the appearance/disappearance and amounts of individual oligogalacturonides during hydrolysis of polymer substrate is typical for each endoPG and can be presented as a product progression profile. Product progression profiles for PGI and PGII from A. niger are presented in Figure 7.4 (from Benen et al., 1999). The product progression of PGII is typical for random endo-acting enzymes and is therefore easier to understand than the one for PGI. Random hydrolysis of a polymer substrate will result in large products at the onset of the reaction. These larger products will then be hydrolyzed into smaller products until the smallest substrate that can efficiently be hydrolyzed is depleted. The product progression for PGII closely follows this theoretical profile. Since the trimer is only very slowly hydrolyzed, it will only be degraded after prolonged incubation. A similar profile as found for PGII

Image Not Available

Figure 7.4 Progression of products formed by endopolygalacturonases I and II acting on polygalacturonate. Compilation of products produced during the first 4 h of hydrolysis of 1 % (mass/vol) polygalacturonate in 1 ml 50 mM Na-acetate pH 4.2 at 30°C. 50jxl samples were analyzed by HPAEC-PAD. (a) Incubation with 180ng endopolygalacturonase I; (b) Incubation with 40 ng endopolygalacturonase II. •, (Gal/?A)i; O, (GalpA)2; •. (GalpA)3; D, (GalpA)4; A, (GalpAJs; A, (GalpA)6_g. Taken from Benen er al. (1999) and reprinted with permission from the European Journal of Biochemistry.

184

PECTINS AND THEIR MANIPULATION

was also recorded for A. niger PGB and PGE (Pafenicova et al, 1998, 2000a) and for F. moniliforme PG (Bonnin et al., 2001). The product progression of PGI (Figure 7.4a) is characterized by a rapid increase of the monomer from the onset of the reaction and, moreover, no substantial amounts of oligoGal/?A of DP > 5 were detected. This demonstrates that PGI is not a fully random-acting enzyme. Similar profiles to that described for PGI were found for PGA, PGC and PGD (Pafenicova el al., 1998, 2000a; Benen etal, 1999). Mode-of-action analyses on oligoGal/?A of defined DP were carried out to elucidate the differences in product progression. On oligoGal/?A, PGs form products for which it can not be established directly whether they originate from the nonreducing or the reducing end. Therefore, borohydride-reduced oligoGalpAs were prepared and included in the mode of action analysis. The reduced end at one of the two products generated allowed the orientation of binding to be established (Pafenicova et al, 1998; Benen et al., 1999). Bonnin et al. (2001) applied 18O labeling of the trimer with subsequent mass spectrometric analysis to establish the direction of binding. The latter method leaves the substrate unchanged and avoids artifacts. For the A. niger enzymes as well as for the F. moniliforme enzyme, the trimer binds from subsites — 2 to -t-1. The data for the mode-of-action analyses of PGI and PGII, two representative enzymes, are presented in Table 7.2. The smallest substrate hydrolyzable by the A. niger and F. moniliforme PGs is the trimer. Only PGD was capable of hydrolyzing dimers. The hydrolysis rate of the trimer is low for all enzymes. Like the trimer, the tetramer is hydrolyzed exclusively or with high preference at the first glycosidic bond from the reducing end, although the rate is increased dramatically compared to trimer hydrolysis. This demonstrates that the substrate binds with very high affinity at subsite —3. The BCFs become specific for each enzyme when DP exceeds 4 (Pafenicova et al, 1998, 2000a,b; Benen et al, 1999). For the processive enzymes the amounts of each of the products originating from a certain binding mode were not equal. For PGI this was observed when DP > 5, for PGA and PGC when DP > 6, and for PGD when DP > 4. For PGI the BCFs for hexamer could not be determined as the product pairs deviated from stoichiometry. For PGI, the data shown are those obtained for reduced heptamer. This nonstoichiometric product distribution for PGI, PGA, PGC and PGD was shown to originate from processive behavior rather than secondary attack (Benen et al, 1999; Pafenicova et al, 2000a,b). Secondary attack means that both products are released from the enzyme and one of the products (usually the larger) serves again as a substrate. During processive attack only one product, usually the smaller, is released, whereas the larger remains bound to the enzyme and then shifts over the active site for another catalytic event. The basic principle of processivity, retainment of the product at the enzyme, has as a consequence that during steady state turnover a fraction of the enzyme population is tied up in an inactive/unproductive EP complex. In the

MICROBIAL PECTINASES

1 85

Table 7.2 Bond cleavage frequencies and hydrolysis rates for endopolygalacturonase I and II. Assay conditions: 500 (iM oligogalacturonides in 0.5 ml 50 mM Na-acetate pH 4.2. At timed intervals 50j.il aliqouts were withdrawn and mixed with 50 nJ stopmix to raise the pH to 8.3-8.5. Products were analyzed and quantitated by HPLC. Bold type indicates the reducing end. Bond cleavage frequencies are given in percentages; n = degree of polymerization n

Rate (U/mg)

Enzyme

PGI

G

3

—

G

—

G

1.6

100

G

4 5

G

6

_

G

— 2 G — G — G — 46 — Image G — Not G Available — G — 3 15

G

G

155

G

96

G

244

98

G G

54 — 82

PGII

3

G

4

G

5 6

G

—

G

_

G —

G

G

—

G

G

—

G

G — 8

G

— 100 — 100

G 37 — 57

G

63 — 35

G

0.4

G

155

G

663

G

552

These data were taken from Benen et al. (1999) and are reprinted with permission from the European Journal of Biochemistry.

mode-of-action analysis, for PGI as an example, this is reflected as a decreased rate of turnover of the pentamer compared to the tetrarner. In general, with every increase of DP an increase in turnover is associated until all subsites on the enzyme are covered. When the hexamer is hydrolyzed in the preferred binding mode, from subsites —5 to +1, the pentamer product remains bound to subsites —5 to —1. Thus, subsites —5 to —1 have high affinity for a pentamer. When pentamer is used as a substrate, in addition to productive complexes covering subsites —4 to 4-1 and —3 to +2, the unproductive complex covering subsites —5 to — 1 will also occur. Hence, pentamer substrate is a competitive inhibitor of PGI. Based on the BCF analyses for PGI and PGII, a provisional subsite map was calculated, shown in Figure 7.5. The major difference between these enzymes is the relatively high affinity at subsite —5 of PGI. This high affinity is the underlying principle of the processive behavior in endoPGs from A. niger. This applies not only to PGI but also to PGA and PGC, as will be discussed in the section on structure-function relationships in PGs.

186

PECTINS AND THEIR MANIPULATION

Figure 7.5 Provisional subsite maps of PGI and PGII. The number of the subsite is indicated. The binding energies (kJ/mol) are indicated above the corresponding subsites.

7.2.3.1 Structure-Junction relationships in A. niger endopolygalacturonases With the 3D structure for the well-characterized PGII available (van Santen et al., 1999), this enzyme provides an excellent candidate to further insight into the mechanism of the PGs and to assess determinants of specificity in a site-directed mutagenesis approach. Identification of the active-site residues. An elaborate sequence alignment of GH28 members revealed the strict conservation of only a small number of amino acids among PGs. For catalysis only charged residues were taken into account since generally only Asp and Glu residues are involved in catalysis in glycosyl hydrolases. In addition, positively charged residues were considered to be involved in binding of the negatively charged substrate close to the active site. The set of mutated enzymes prepared and studied by Armand et al. (2000) is listed in Table 7.3. Although all mutated enzymes were affected in catalysis, the strongest effect was observed upon mutagenesis of D180 or D201. For D202 and H223 reasonable activities were still obtained, depending on the side chain engineered. Enzyme R256Q was moderately active. Residues D202, H223 and R256 are not conserved in GH28, thus those three residues can stricto senso never be part of the catalytic machinery. Armand et al. (2000) suggested the following roles for the amino acids mutagenized. R256 and K258 are involved in the binding of the substrate at subsites +1 and -1 respectively. D180, assisted by D202, activates H2O, which acts as a nucleophile. D201 serves as the acid that protonates the leaving group. H223 most likely shares a proton with D201 and thus helps maintain the proper ionization state of D201. In the 3D structure the arrangement of catalytic residues is completely different from the arrangement found in inverting enzymes. Generally, a distance of 9-9.5 A between acid and base is found in inverting hydrolases, whereas in PGs this distance is on the order of 4-5 A, indicating that the nucleophilic attack and the protonation occur from the same side instead from opposite sides of the glycosidic bond (van Santen et al., 1999; Armand et al., 2000).

MICROBIAL PECTINASES

1 87

Table 7.3 Kinetic parameters of endopolygalacturonase II mutated at active-site residues. Kinetic parameters were determined in 50 mM Na-acetate (pH 4.2) at 30°C using polygalacturonic acid a substrate Kinetic parameters

Enzyme Wild type D180A D180E D180N D201E D201N D202E D202N D180E/D201E H223A H223C H223Q H223S R256Q K258N

'max

^m

(U/mg)

(mg/ml)

2000 0.17 0.24

1.4

0.04 Image Not Available 0.19 12.7

0.3 0.04 10.0 21.5 0.36

1.7 278 16.2

< 0.15 0.15

0.3 1.5 0.3 0.3 0.7 1.5 <0.15 0.15 0.80

1.1 1.5 1.7 2.8

These data were taken from Armand et al. (2000) and are reprinted with permission from the Journal of Biological Chemistry.

Localizing subsites by site-directed mutagenesis. In the absence of a 3D structure of a PGII-substrate complex, Pages et al. (2000) carried out site-directed mutagenesis studies at selected amino acids to reveal the position with respect to subsite and role of selected amino acids. Figure 7.6 depicts the actual position in the 3D structure of the amino acids mutagenized and Table 7.4 lists the kinetic parameters of the mutant enzymes. Table 7.4 shows that all mutated enzymes are kinetically competent except for enzymes Y291L,Y291FandD183N. Since Y291 is one of the strictly conserved amino acids in all PGs, a significant effect of mutagenesis of Y291 was expected. From its aromatic character and its location in the structure, it is likely that Y291 stacks with the sugar ring of GalpA at subsite 4-1. Mutant Y291F shows that, in addition to a stacking effect of the ring, a strong interaction with the hydroxylic function of Y291 also occurs. The close proximity of the carboxylate oxygens of D183 to those from D180 (activating the water) at 3.6 A strongly indicates the sharing of a proton. It can therefore be expected that D183 helps to maintain the appropriate ionization state of D180 (Pages et al, 2000). Mutagenesis of amino acids more remotely from the catalytic site resulted in small effects on overall catalysis, although some quite profound changes in product progression and mode of action were recorded (see Table 7.5). These data were used to assign the amino acids mutagenized to individual subsites (see Figure 7.7) (Pages et al, 2000).

188

PECTINS AND THEIR MANIPULATION

Image Not Available

Figure 7.6 Stereo figure showing the location of the mutated residues in the substrate-binding cleft. Residues D180, D201, D202, H223, R256 and K258 are strictly conserved residues and are involved in catalysis. Residues Ml50 and N186 are involved in substrate binding. Residue S91 is important for processive/nonprocessive behavior. From Pages el al. (2001) and reprinted with permission from the Journal of Biological Chemistn-. Table 7.4 Kinetic parameters of subsite mutants of endopolygalacturonase II. Kinetic parameters were determined in 50 mM Na-acetate (pH 4.2) at 30°C using polygalacturonic acid a substrate Kinetic f )arameters 'max. app

^m. app

Enzyme

U/mg

mg/ml

Wild type M150Q D183N N186E E252A D282K Q288E Y291L Y291F Y326L

4000 1700 500 3000 3000 2500 2600 35 250 4200

0.15 0.4 0.5 0.5 0.6 0.3 1 1.9 1.8 0.2

Image Not Available

These data were taken from Pages et al. (2000) and are reprinted with permission from the Journal of Biological Chemistry.

The origin of processivity. From the provisional subsite maps calculated for PGI and PGII it was deduced that the high affinity at subsite —5 in PGI might account for the processive behavior (Benen et al., 1999). The strong similarities in mode of action of PGI, PGA and PGC indicate a similar origin for processivity in all three enzymes. Since the six A. niger share high sequence identity. Pages

MICROBIAL PECTINASES

1 89

Table 7.5 Bond cleavage frequencies on (GalpA>6 and (GalpA)s for wild-type and subsite mutants of PGII. Assay conditions: 500 M-M oligogalacturonides were incubated with wild-type or mutated PGII in 0.5 ml 50 mM Na-acetate buffer, pH 4.2, at 30°C. At timed intervals 50 (xl aliquots were withdrawn and mixed with 20 |xl stopmix to raise the pH to 8.3-8.5. Products were analyzed and quantified by HPLC. Bold type indicates the reducing end. Bond cleavage frequencies are given in percentages; n = degree of polymerization Enzyme

6

G

—

G

-_

G

—

G

8 8 11 31 10 16 9 10 10 11

Wild type M150Q D183N N186E E252A D282K Q288E Y291L Y291F Y326L

—

G

G

-

G

—

G

— 32 42 51 69 30 16 37 43 44 30

—

G 552 258 250 770 700 500 561 5 78 650

35 14 14 16 30 42 23 15 12 32

57 78 75 53 60 42 68 75 78 57

Image Not Available 5

Wild type M150Q DI83N N186E E252A D282K Q288E Y291L Y291F Y326L

Rate (U/mg)

n

G

— 68 58 49 31 70 84 63 57 56 70

G 663 134 80 611 718 380 502 1.3 29 702

These data were taken from Pages et al. (2000) and are reprinted with permission from the Journal of Biological Chemistry.

et al. (2001) explored both the primary sequence alignment and PGII structure for ammo acids that can explain the observed behavior. With knowledge of the position of subsites —1 and +1 combined with the distance of approximately 4.4 A that each GalpA residue will span, the potential location of subsite —5 was identified in the structure of PGII (Pages et al., 2001). Amino acid residues at subsite —5 were identified and compared to the other five PGs. This revealed the presence of a Ser in the nonprocessive enzymes (see Figure 7.6) or an Arg at the equivalent position in the processive enzymes. The high affinity at subsite —5 in the processive enzymes might well be caused by the interaction of the positively charged arginine with the uronate of the substrate. This hypothesis was tested by preparing and characterizing mutant enzymes PGII-S91R and PGI-R95S with respect to product progression and bond cleavage frequencies (Pages etal., 2001).

190

PECTINS AND THEIR MANIPULATION

Image Not Available

Figure 7.7 Schematic overview of subsites of PGII and amino acids assigned based on site-directed mutagenesis. Modified from Pages el al. (2000) and reprinted with permission from the Journal of Biological Chemistry.

The product progression of enzyme PGII-S91R resembled the product progression of wild-type PGI, whereas for mutant PGI-R95S the profile resembled that for wild-type PGII (see Figure 7.8). This strongly indicated that the processive behavior was interchanged between the two enzymes. Proof was obtained from the mode-of-action analysis, which revealed dramatic changes in BCFs that comply with a very high affinity at subsite —5 in PGII-S91R and a reduced affinity in mutant PGI-R95S (not shown). Furthermore, the pentamer substrate now appeared a very strong inhibitor of PGII-S91R (20-fold decreased hydrolysis rate compared to the wild type), again demonstrating the high affinity at subsite —5 and, moreover, also demonstrating the ability of this enzyme to retain the substrate as an unproductive complex. The latter is a prerequisite for processive behavior. These data show that a single amino acid at a subsite remote from the active site can cause dramatic changes in kinetic behavior.

7.3 Pectate and pectin lyases Pectate lyases (endo-acting EC 4.2.2.2 and exo acting EC 4.2.2.9) and pectin lyases (EC 4.2.2.10) cleave the a-(l—»4)-D galacturonosidic linkage by Pelimination, resulting in the formation of a A4,5-unsaturated bond at the newly formed nonreducing end. As their names indicate, pectate lyases are generally most active towards polygalacturonic acid or low-DM pectin, whereas pectin lyases prefer pectins with a high DM. For the pectin lyases characterized, the specific activity increases with increasing degree of methyl-esterification (DM), whereas for many pectate lyases the highest specific activity was observed when the substrate was moderately methyl-esterified. In a recent report by Soriano et al. (2000) a pectate lyase from Bacillus sp. is described that displays highest activity on both low- and high-DM pectin with a low activity on partially methylesterified pectins. Also, two pectate lyases, from Clostridium cellulovorans (Tamaru and Doi, 2001) and Pseudomonas cellulosa (Brown et al., 2001) have recently been described that have cellulose-binding domains. Even a dockerin domain to be incorporated into the cellulosome is present in the C. cellulovorans

Image Not Available

Figure 7.8 Progression of products formed by the mutated enzymes endoPGI-R95S and endoPGIIS91R. Products produced during the first hours of hydrolysis of 0.8% (mass/vol) polygalacturonic acid in 1 ml of 50 mM Na-acetate, pH 4.2 at 30°C. Samples (50 (xl) were analysed by HPLC. (a) Products formed by endoPGl-R95S; (b) Products formed by endoPGII-S91R. •, (GalpA),; O, (Gal/?A)2; •, (Gal/?A)3; D, (Gal/>A)4; A, (GalpA)5; A, (GalpA.)^. From Pages et al. (2001) and reprinted with permission from the Journal of Biological Chemistry.

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PECTINS AND THEIR MANIPULATION

enzyme (Tamaru and Doi, 2001). Thus, the more pectic lyases that are characterized the more diverse the spectrum of properties becomes. So far the only discriminating factor between pectate lyases and pectin lyases that remains is the requirement for Ca2+ ions for catalysis by pectate lyases. However, it may turn out that this dogma can no longer hold as the cytosolic E. chrysanthemi 3937 PelW Ca2+ is substituted by Mn 2+ (Shevchik et al, 1999b). The diversity found among the pectic lyases may be reflected by the fact that these enzymes have been classified into six different polysaccharide lyase families (Coutinho and Henrissat, 1999). Currently quite a number of 3D structures of pectate and pectin lyases are known. These include the family L1 pectate lyases PelC (Yoder et al., 1993) and PelE (Lietzke et al, 1994) from E. chrysanthemi EC 16, BsPel from Bacillus subtilis (Pickersgill et al., 1994), and the pectin lyases PLA (Mayans et al., 1997) and PLB (Vitali et al., 1998) from A. niger. Structures have also been solved for the family L3 pectate lyases from Bacillus sp. KSM-P15 pectate lyase (Akita et al., 2000) and PelL from E. chrysanthemi 3937 (J. Jenkins, personal communication). Very recently the structure for the family L10 pectate lyase from Pseudomonas cellulosa has been solved, but the coordinates are not yet publicly available (G.J. Davies and G.W. Black, personal communication). The E. chrysanthemi EC 16 PelC structure was the first to be described (Yoder et al. 1993) and turned out to constitute a new structure with a unique topology of a right-handed parallel ^-helix. All subsequently reported pectate and pectin lyase, rhamnogalacturonase and polygalacturonase structures have revealed the same topology. However, the P. cellulosa pectate lyase is not a (3-helical enzyme (G.J. Davies and G.W. Black, personal communication). The main differences between the pectate lyase and pectin lyase structures occur in the substrate-binding cleft as a result of the differences in charge of the preferred substrates. As a result, the substrate-binding cleft of the pectate lyases contains charged and polar residues, whereas the pectin lyases have a very high number of aromatic and apolar residues in their substrate-binding cleft. Of great help for the understanding of the catalytic properties (to be described below) was the recent elucidation by Scavetta et al (1999) of the 3D structure of pectate lyase C from E. chrysanthemi EC 16 in complex with a pentagalacturonide. This is the first structure of a pectic enzyme-substrate complex, which confirms the initially proposed substrate binding cleft and provides insight into the necessity of Ca2+ for catalysis. Initially, pectate lyases were mainly isolated from phytopathogenic microorganisms, though in recent years pectate lyases have also been reported in many other nonphytopathogenic microorganisms. The best-studied bacteria with respect to pectate lyases are the phytopathogenic E. chrysanthemi and E. carotovora (Barras etal., 1994; Hugouvieux-Cotte-Pattat etal, 1996). Mutagenesis programs have revealed that some pectate lyases are important for

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effective infection, whereas other pectate lyases secreted by the same organism have only limited impact on infection. In E. chrysanthemi 3937 eight endo-acting pectate lyases (PelA-PelE, Pell, PelLandPelZ(Lojkowska
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PECTINS AND THEIR MANIPULATION

Table 7.6 Biochemical properties off. chrysanthemi 3937 endo-pectate lyases. Kinetic parameters were determined using polygalacturonic acid. %DM signifies the preferred degree of methyl-esterification Enzyme PelA PelB PelC PelD PelE Pell PelL PelZ

Family

pH optimum

%DM (range)

1 1 1 1 1 3 9 1

8.5 9.3 9.2 8.8 8.0 9.2 8.5 8.5

0 (0-30) 31 (0-50) 31 (0-45) 0(0-25) 0 (0-25) 31 (10-55) 31 (0-70) 7(0-45)

^m

Vmax

mg/ml

U/mg

Reference

0.3 0.03 0.02 0.28 0.42 0.12

46 2448 762 670 3803 230 5 9

Tardy el al. (1997) Tardy et al. (1997) Tardy et al. (1997) Tardy et al. (1997) Tardy et al. (1997) Shevchik et al. (1997a) Lojkowska et al. ( 1 995 ) Pissavin et al. (1998)

0.15

In addition to the biochemical studies, mode-of-action analyses have been carried out for pectate lyases as well. A first study was reported by Preston et al. (1992). Each of the four enzymes studied showed a distinctive product progression during polygalacturonic acid degradation. These profiles are compatible with the endolytic character of these enzymes as initially larger oligomers are formed that are further degraded into typical smaller oligomers such as unsaturated dimer and/or trimers. In addition to product progression. Roy et al. (1999) also analysed the mode of action and cleavage rate of five E. chrysanthemi 3937 pectate lyases on oligoGalpA of defined DP (2-8). These studies have resulted in the estimation of the number of subsites and relative affinities of PelA, PelB, PelD, Pell and PelL. 7.3.2.1 Identification of the catalytic machineryIt has long been known that pectate lyases require Ca2+ for catalysis. A Ca2^ ion was indeed observed in the 3D structure of BsPel (Pickersgill et al., 1994). coordinated by three acidic amino acid residues. However, no direct clue about catalytic residues could be derived from the structure. A first indication of such residues was obtained from mutagenesis studies on E. chrysanthemi B16 PelC carried out by Kita et al. (1996). Strong effects on catalysis were observed upon mutagenesis of the Ca2+-coordinating acidic residues. In addition to this, it surprisingly turned out that mutagenesis of an arginine (R218) almost completely abolished catalysis (Kita et al, 1996). No direct role of an arginine in catalysis could be envisaged at that time. However, the R218K mutant proved crucial in obtaining an enzyme-substrate complex (Scavetta et al. 1999). By superimposing the wild-type structure onto the structure of the enzyme-substrate complex, it was shown that this arginine is ideally positioned in relation to the C-5 proton of the substrate that is to be extracted to initiate the ^-elimination reaction (Scavetta etal, 1999). Mutation of the corresponding arginine in pectin lyase B (Herron et al, 2000) or pectin lyase A (P. Sanchez-Torres, J. Visser and J. Benen, unpublished) also completely inactivates the enzymes. Furthermore, a (GalpA)4 molecule modeled into the pectin lyase B structure results in a similar

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geometry of the arginine with respect to the C-5 of the substrate (Herron et al., 2000). A similar observation was made for modeling a decamer substrate into the PLA structure (P. Swaren, personal communication). It is therefore now accepted that an arginine is the critical amino acid for catalysis in pectate and pectin lyases (it is not known yet whether the same holds for family L10 lyases). 7.3.2.2 The role of calcium ions in catalysis Polygalacturonic acid and low-DM pectins have high affinity for Ca2+ ions and form gels quite easily. Kinetic studies with pectate lyases and these substrates are therefore a hazardous enterprise. Furthermore, the ionic strength is important as is the presence of other salts like NaCl, which can have a strong impact on catalysis via the so-called 'swamping effect' (Morris et al., 1982; Benen et al, 2000). Ideally, kinetic studies are carried out using oligoGalpAs, which are insensitive to the swamping effect (Kohn, 1971) and do not form gels or precipitate until at high Ca2+ concentration. It is known that Ca2+ is tightly bound to the pectate lyases (Kn of 2.3 jxM in case of BsPel; R. Pickersgill, personal communication). It is also known that Ca2+ behaves like a substrate, which it should not do when only being bound tightly to the enzyme. Atallah and Nagel (1979) studied the role of Ca2+ ions with a pectate lyase from a Cephalosporium sp. in a two-substrate kinetic analysis using (GalpA)4 and Ca2+. This detailed analysis showed clear upward curvature in Line weaver-Burke plots (sigmoidal Michaelis-Menten plots), from which further replotting of intercepts and slopes allowed the conclusion that the substrate binds as a Ca2+-tetragalacturonide complex and that Ca2+ might act as an activator of the enzyme. Such an analysis has recently also been carried out for the A. niger pectate lyase (PLYA) and included (Gal/?A)6 in addition to (Gal/?A)4 in the kinetic analysis. A Ca2+-dependent mode-of-action analysis using oligoGal/?A of DP 2-8 was also carried out (Benen et al, 2000). The mode-of-action analysis is presented in Table 7.7. (GalpA)2 and (GalpA)3 were not cleaved by the enzyme. For (Gal/?A)4 only one productive binding mode was detected, irrespective of the [CaCy, resulting in A4,5 unsaturated (Gal/?A)3 and GalpA. However, a strong rate increase was recorded upon increasing [CaCy. For (Gal/?A)5 and (Gal/?A)6 two and three productive binding modes were found, respectively. In addition to the strong increase in cleavage rate as a function of [CaC^], a change in the mode of action occurred. At higher [CaCh] a shift was observed towards cleaving the substrate at the ultimate glycosidic linkage, counting from the nonreducing end (Benen et al, 2000). Such a shift can only be explained by a direct role of Ca2+ ions in the binding of the substrate at a particular subsite or particular subsites. In the 3D structure of the PelC-substrate complex, Ca2+ ions were found that form bridges between acidic residues of the enzyme and the carboxylic functions of the substrate (Scavetta et al, 1999). The Ca2+ ions in this structure appeared to be in different positions from those normally found in interstrand

Image Not Available

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contacts. Furthermore, the substrate appeared to have adopted a partial 3\-2\ helix that deviates from the 2\ helix in solution (Scavetta et al., 1999). These data combined with the mode-of-action analysis and kinetic studies allowed the conclusion that the substrate enters the enzyme as a substrate-Ca2+ complex. The rate-limiting step is most likely the distortion of the helix upon binding of the substrate with concomitant rearrangement of the Ca2+ ions (Benen et al., 2000). 7.3.3 Pectin lyases Little attention has been paid to this group of enzymes in recent years. Although the number of nucleotide sequences in the databases is increasing steadily, hardly any biochemical data have become available. Pectin lyases have mostly been identified in fungi such as Neurospora, Botrytis, penicillia and aspergilli, in contrast to pectate lyases, which have predominantly been isolated from bacteria. Currently, only endo-acting pectin lyases are known, which have all been grouped into lyase family LI. The best-characterized pectin lyases are most likely those from aspergilli. These enzymes were subject of the PhD research carried out by Voragen (1972) and van Houdenhoven (1975). The resulting PhD dissertations are hardly available anymore and it is beyond the scope of this chapter to reproduce all data. None the less, some interesting aspects from these investigations will be presented here. Voragen (1972) mainly focused on substrate specificity and the influence of DM on kinetic parameters. It was shown that in addition to a lowering of the activity, a lowering of the pH optimum also occurred when the DM of the pectins was decreased. This shift of the pH optimum and the decrease in activity correlated with the distribution of the methyl groups. Kinetic studies showed that Vmax remained constant whereas Km increased upon lowering DM. In contrast, on lowering the pH, the Km for the lower-DM pectins increased. Voragen (1972) also observed that, using pectins of the same DM, lyase activity was higher when the pectin was de-esterified by a plant pectin methylesterase instead of by alkaline treatment. Enzymatic de-esterification results in a blockwise removal of methyl groups when plant enzymes are used, whereas alkaline treatment results in a random removal of methyl groups (see also Section 7.5.1 on the pectin methylesterases). Although pectin lyases do not require Ca2+ for activity, Ca2+ appeared to influence the activity of the enzymes. Above the pH optimum, Ca2+ stimulated the activity, notably on partially alkaline de-esterified substrates, and addition of Ca2+ to the reaction mixtures also resulted in a lowering of the pH optima (Voragen, 1972). With current knowledge on pectins and pectinases some aspects of Voragen's research can now be explained. The fact that Vmax remains constant and Km

198

PECTINS AND THEIR MANIPULATION

increases using blockwise de-esterified pectins can be explained by the preference of high DM by pectin lyases. When blocks of methyl groups are removed, only the concentration of 'proper' substrate changes and thus Km increases and Vmax remains constant. However, alkaline treatment of pectins changes the nature of the substrate as the distribution of methyl groups becomes more random, and therefore Vmax decreases. When the pH of the reaction mixture is lowered, more of the de-esterified groups become protonated and the net charge of the substrate decreases, which results in an increased affinity (pectin lyases prefer uncharged substrates). A similar effect can be expected from Ca2^ ions that compensate the charge of the carboxylates of the substrate. This effect will be stronger at higher pH. The work carried out by van Houdenhoven (1975) focused on enzymology rather than substrate specificity. The research involving pectin lyase I (PLI) and PLII revealed that the pH optima of these enzymes are quite low (pH 6) compared to pectate lyases. However, potentially these enzymes are more active at higher pH. The fact that this is not observed is a result of reversible inactivation at higher pH, as was shown by circular dichroism and fluorescence studies (van Houdenhoven, 1975). Addition of relatively high concentrations of NaCl prevented the inactivation. Detailed pH- and temperature-dependent kinetic studies allowed calculation of reaction enthalpies and identification of potential amino acids involved in binding/catalysis. Using oligoGal/?A of defined DP, van Houdenhoven (1975) was able to estimate the least number of subsites for PLI (8 subsites) and PLII (9 or 10 subsites). Recently, these determinations proved quite accurate when the structure of PL A (identical to PLII) was solved and used to model a substrate molecule (decamer) into the enzyme (P. Swaren, personal communication). In a similar way to that described for the polygalacturonases. in a reverse genetics approach (PLI and PLII as input) and subsequent screening of an A. niger genomic library a family of six pectin lyase genes was identified, cloned and overexpressed (Gysler el al.. 1990: Harmsen el al. 1990; Kester and Visser, 1994). PLI and PLII correspond to PLD and PL A. respectively. PLB was characterized by Kester and Visser (1994). This enzyme differs from PLI and PLII as the pH optimum is 8.5 and the activity is much higher: Vmax 1288U/mg compared to Vmax 125U/mg for PLI and Vmax 44U/mg for PLII. The high activity could only be achieved in the presence of 0.5 M NaCl. PLB was the second pectin lyase for which the 3D structure was solved (Vitali etal., 1998). This structure also served to model a substrate (tertramer) molecule and PLB was the first pectin lyase subjected to site-directed mutagenesis to identify catalytic residues (Herron et al., 2000). Van Alebeek and Benen have embarked on a detailed study of PLA that comprises detailed mode-of-action analysis on oligoGal/?A. performance on synthetic substrates and site-directed mutagenesis studies to change substrate specificity. Parts of this work will be presented in the following sections.

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Figure 7.9 Structures of (un)derivatized galacturonic acid. R2 = OH combined with R 1 = OH, 6-0methyl, 6-O-ethyl, NHa, 6-W-methyl. R 2 = 1-0-methyl combined with R 1 = 6-0-methyl-GalpA.

7.3.3.1 A. niger pectin lyase A As was demonstrated by Voragen (1972), pectin lyase activity is highly dependent on the distribution of the methyl esters. To assess the importance of the nature of the modification of the uronate functionality, oligoGal/?As of defined DP were purified and derivatized under controlled conditions. Generation of model substrates of defined degree of polymerization. Purified saturated and unsaturated oligoGal/?As (van Alebeek et al., 2000) were derivatized at the C-6 position and/or the C-l position (Figure 7.9). Methyl- and ethyl-esterification was carried out in acid methanol (0.02 N H^SCU) at 4°C. The purity and molecular weight were checked by MALDI-TOF MS (van Alebeek et al., 2000). Amidated oligoGal/?As were synthesized at a yield of 95-97% by amidation of fully methyl-esterified oligoGal/?A using methylamine, whereas a yield up to 70% was reached using ammonia (van Alebeek et al., 200la). Glycosidation at the C-l position (reducing end) was performed in acid methanol (0.1 N H^SCU) at room temperature. At this high acidity both methyl-esterification and methyl-glycosidation occurred. All reactions were followed using MALDI-TOF MS and the purity (degree of amidation, degree of esterification) was determined from mass spectra (van Alebeek et al., 2001b). Mode of action of A. niger pectin lyase A. The mode of action of PLA was studied using the model oligoGalpA generated. Since PLA is known to prefer high-DM pectin, the mode of action of PLA was first studied using fully methylesterified oligoGalpA. The reactions were monitored by high-performance anion-exchange chromatography (HPAEC) using pulsed amperometric detection (PAD) showing all compounds and UV detection that reveals only the unsaturated products, as shown in Figure 7.10a for PLA digestion of (6-0CH3-Gal/?A)6. Figure 7.10 shows that PLA binds GalpA6-6CH3 in three different productive modes, resulting in saturated (Gal/?A)2 and A4,5-unsaturated (Gal/?A)4, (GalpA)3 and A4,5-unsaturated (Gal/>A)3, and (Gal/?A)4 and A4,5-unsaturated

200

PECTINS AND THEIR MANIPULATION

Figure 7.10 (a) HPAEC elution patterns of a PLA digest of (6-O-CH^-Ga\pA)f, after 0 and 4 h of incubation detected by either PAD or UV (235 nm) and (b) corresponding progression profile. Saturated (open symbols) and unsaturated (solid symbols) products are indicated as follows: (GalpA>2 (A. A). (GalpAb (O, •), and (GalpA)4 (D. •).

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201

Table 7.8 Effect of the C-l and C-6 substituent on the PLA activity Substrate (GalpA)6 (6-<9-CH3-Gal/7A)6 (6-0-C2H6-GalpA)6 (6-NH2-Gal/?A)6 (6-Af-CH3-GalpA)6 l-0-CH3-(6-0-CH3-GalpA)6

PLA reaction rate (mU/mg) 0 538 3 0 0 107

(Gal/?A)2 with a strong preference for binding from subsites —3 to +3. Following quantification of the products, progression profiles were constructed (Figure 7.1 Ob) from which initial reaction rates were determined. These studies have revealed that the dimer and trimer are not cleaved by PLA. Furthermore, the reaction rates from (Gal/?A>4 onward increased with increasing DP up to DP = 8, which demonstrates that at least eight subsites are present on the enzyme. The latter is again in accordance with the results obtained by van Houdenhoven (1975) for PLII (=PLA). Digestion of the methyl-esterified oligoGal/?A (DP 4-10) consistently resulted in A4,5-unsaturated (GalpAb as the major product, indicating that the highest affinity of the enzyme for the substrate resides in subsites +1 to +3. Subsequently, the influence of the carboxyl substituent (C-6) was investigated. In Table 7.8 the results are shown for (6-0-CH3-GalpA)6, which is representative for all compounds tested of different DP. No PLA activity was detected using nonmethylated (GalpA)^. Upon replacing the methyl ester by an ethyl ester, the PLA activity dropped severely, indicating that the length of the ester is restricted to a methyl group. Changing the Olinked methyl ester into an AMinked hydroxyl or methyl group resulted in a complete loss of PLA activity. From these data it can be concluded that PLA is highly specific for methyl esters. Since PLA is active on partially methyl-esterified pectin it is conceivable that several subsites can accommodate a nonesterified GalpA. To shed more light on the subsites involved, the mode of action of PLA on partially methyl-esterified oligoGalpA was analyzed. Fully methyl-esterified (6-O-CH3-Gal/?A)5 and (60-CH3-Gal/?A)6 were quantitatively randomly de-esterified under alkaline conditions to DM of 80% and 86%, respectively, corresponding to on average one free carboxyl group per molecule for both the pentamer and the hexamer. The random de-esterification in theory yields all possible isomers. However, during esterification it turned out that the reducing and nonreducing ends are preferentially esterified (van Alebeek et al, 200Ib). It is therefore assumed that only the isomers with a carboxyl group of the inner Gal/?A residues are most likely to be present. Using these substrates resulted in a severe drop of activity and a change in bond cleavage frequency (BCF). For the hexamer substrate (Table 7.9) the BCFs changed from covering subsites +1 to +3 at the reducing

202

PECTINS AND THEIR MANIPULATION

Table 7.9 Effect of DM on BCF and reaction rate on (6-O-CH3-Gal/7A)6 and \-O-CH^(6-O-CU:, GalpA)6. The reducing end is indicated by bold type (G) Rate (mU/mg)

G

DM (<*} 97

G

86

43

DP

6

G

6

G

6

G

6

G

— 5 —

G G G

—

G

— 21 — 29 — 28 — 35

G G G G

— 65 — 59 — 72 — 47

G G G G

— 9 — 12 — — 18

G

—

G

538

G

—

G-0-CH3

97

107

G

—

G-O-CHj

73

5

end subsites to covering subsites +1 and +2 only, notably towards the end of the incubation period when about 80% of the initial substrate present was converted. The most straightforward conclusion that can be drawn from these experiments is that PLA is highly specific for fully methyl-esterified substrates and that the observed 'high' activities on polymer substrates are a result of stretches of fully methyl-esterified GalpA. or, in other words, heterogeneity of the substrate. Following the influence of the C-6 substituent on PLA activity, methyl glycosidation of the reducing end (C-l) was investigated. Methyl-glycosidated (6-O-CH3-Gal/?A)6 was subjected to PLA digestion. The activity was much lower compared to the nonglycosidated oligoGal/?A (Table 7.8). Furthermore, the only products formed were unsaturated l-0-CH3-(Gal/?A)3 and 1-O-CH?(Gal/?A)4. After partial de-esterification the BCFs changed and A4.5-unsaturated l-0-CH3-(Gal/?A)2 was detected as well. Furthermore, a large drop in the activity was again observed. HPAEC (pH 12) analysis revealed the presence of two forms of each of the methyl-glycosidated substrates and products. These have tentatively been identified as the a and P anomers. Only one of the anomers was degraded to either the a or P anomer of the unsaturated 1 -0-CH3-(GalpA)3. Currently, a detailed analysis of this phenomenon is in progress. The detailed analysis of PLA, which has been partially described here, has provided insight into affinities of individual subsites. PLA mutants have been generated at these subsites and the effect of these mutations is at hand. It is expected that the detailed characterization of the mutated enzymes will improve insight into pectin lyase(s) to the level of that of pectate lyases and polygalacturonases. 7.4

Heterogalacturonases

Rhamnogalacturonan (RG, also referred to as RG-I) is a heterogalacturonan that can have a backbone consisting of as many as 100 repeats of the disaccharide [-+2)-a-L-Rha/>(l-»4)-a-D-Gal/7A-(l^] (Albersheim et al.. 1996). The

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rhamnosyl residues can be substituted at O-4 with a single unit [|3-D-Gal/?~ (1 ->4)] or long neutral side chains. These side chains are often referred to as the 'hairs', and are mainly composed of galactosyl and/or arabinosyl residues. The GalpA residues can be acetylated at the O-2 and/or O-3 position. This section will focus on the enzymes that are required to convert RG to monosaccharides. Compared to homogalacturonan-degrading enzymes, RG-degrading enzymes are fairly new players in the field of carbohydrases. The recent attention has been attracted by industrial interest in these enzymes as a result of new fruit juice manufacturing techniques (the so-called liquefaction process; Voragen and Pilnik, 1989). Since these enzymes are usually minor components of commercial enzyme preparations and the enzymes produce complex mixtures of degradation products, their presence went unnoticed for a long time. Furthermore, only recently developed sophisticated techniques for identification and analysis have allowed their characterization. It is expected that the interest in these enzymes will continue to grow, as they may be important in tailoring bioactive oligosaccharides. Japanese studies suggest that RG fragments in particular have a wide spectrum of health-promoting effects (Yamada, 1994). As a consequence of their fairly recent discovery, the characterization of many RG-degrading enzymes is rather incomplete when compared to the PGs and pectic lyases. Fewer genes encoding such enzymes are known and systematic studies unraveling structure-function relationships still have to be undertaken. From the limited studies carried out, it has already become clear that the various RG-degrading enzymes differ considerably in specificity. 7.4,1 Endo-acting rhamnogalacturonan-degrading enzymes Schols et at. (1990) were the first to report a rhamnogalacturonan-degrading enzyme. This enzyme was discovered during fractionation of a commercial enzyme preparation derived from Aspergillus aculeatus. The enzyme acted exclusively on RG. By expression cloning, Kofod et al. (1994) obtained the gene encoding this enzyme (RGaseA), as well as a gene encoding another rhamnogalacturon-degrading activity (RGaseB) that later turned out to be a lyase instead of a hydrolase (Azadi et al., 1995; Mutter et al., 1996). Two genes encoding RG hydrolases A and B were cloned from A. niger (Suykerbuyk et al., 1997). To avoid confusion, the hydrolase and lyase have been renamed as endorhamnogalacturonan hydrolase (endoRGH) and endo-rhamnogalacturonan lyase (endoRGL), respectively. The GH28 family now contains four entries of endoRGHs: the one from A. aculeatus (Kofod etal, 1994; Suykerbuyk et al., 1995), rhgAmdrhgB from A. niger (Suykerbuyk et al., 1997), and one from Botryotiniafuckeliana (Fu et al, 2001). Comparison of the primary structure of the enzymes reveals a number of regions, some of which are pectinase-specific and some of which are endoRGHspecific (Fu et al, 2001). Typically, RHGB from A. niger and endoRGH from

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B. fuckeliana have C-terminal extensions that are not present in the two other RGHs. It is not known whether this extension affects the properties of these enzymes. The endoRGLs have so far been assigned to two polysaccharide lyase families. The A. aculeatus endoRGL belongs to family L4, whereas the recently discovered Pseudomonas cellulosa endoRGL forms a new family (LI 1). Family LI 1 also contains the uncharacterized proteins YesW and YesX from Bacillus subtilis, and PSP from Streptomyces coelicolor (McKie et al, 2001). The P. cellulosa endoRGL is equipped with a cellulose-binding module, which is very uncommon for pectinases. The significance of this binding domain is still obscure. The P. cellulosa endoRGL differs from the A. aculeatus endoRGL in having an absolute requirement for Ca2+ and having a higher pH optimum (9.5 versus 6). The 3D structure of the A. aculeatus endoRGH has been solved (Petersen et al., 1997), and appears to be very similar to that of other pectinases. EndoRGH folds into a large right-handed parallel p-helix (four parallel ^-sheets), with a core composed of 13 turns of ^-strands (Petersen et al., 1997). The activesite residues as identified in PGII (Armand et al, 2000) are also present in endoRGH. However, one of the aspartates, Asp202 (PGII numbering) is replaced by a glutamate. This glutamate also shares a water molecule with the D180 equivalent and hence the mechanism can be the same. The fact that a glutamate has replaced aspartate may be related to the fact that at subsite +1 a rhamnose is bound that may be oriented differently. RGH is among the most decorated pectinases, containing almost 6 kDa of carbohydrates in a total molecular mass of 52 kDa. Both N- and O-linked glycosylation is present. /V-glycans [Manal-6(Manal-3)Manpl-4GlcNAc^l4GlcNAc] are linked to Asn32 and Asn299. The glycosylation sites are not uniformly distributed over the enzyme. The O-linked glycosylation (single mannose residues) all occur at the C-terminal part of the enzyme (amino acid residues 367^422). Detailed structural characterization of reaction products has shown that endoRGL and endoRGH cleave the RG backbone at different positions (Colquhoun et al., 1990; Schols et al, 1994; Mutter et al, 1996, 1998a). The oligomeric products resulting from A. aculeatus endoRGH action consist of two or three Rhap-GalpA disaccharide units, with a rhamnosyl at the nonreducing and a galacturonosyl residue at the reducing end (Colquhoun et al., 1990; Schols et al, 1994). The rhamnosyl residues can be substituted with single units of galactose. Similar oligosaccharides were observed by Suykerbuyk et al (1997) for the two A. niger endoRGHs hydrolyzing saponified hairy regions (this is a RG preparation still containing arabinan and galactan side chains but devoid of methyl and acetyl groups). However, the ratio between the various oligosaccharides formed is different for the three enzymes. A. niger rhgB resulted in the accumulation of the largest products of the three endoRGHs, including a number of larger oligosaccharides that have not yet been characterized.

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The oligosaccharides produced by A. aculeatus endoRGL differ from those formed by endoRGH, in that they contain a A4,5-unsaturated GalpA residue at the nonreducing end and a Rhap at the reducing terminus (Mutter et al, 1996, L998a). Similar products were released from saponified hairy regions by the P. cellulosa rglllA (McKie et al., 2001). In addition, this enzyme seems to produce smaller fragments tentatively identified as a A4,5Gal/?A-Rhap disaccharide. The different actions of endoRGH and endoRGL are illustrated in Figure 7.11. From the formation of oligomeric products discussed above, one might conclude that endoRGH and endoRGL are active towards a similar part of the RG backbone. However, there is evidence that the various RGdegrading enzymes recognize different parts of the backbone. When the same saponified hairy regions were degraded using different RG-degrading enzymes, different shifts in the molecular weight distribution of the polymeric products were observed (Kofodetal., 1994; McKie et al., 2001). Removal of the arabinan hairs from saponified hairy regions resulted in an increased catalytic efficiency of A. aculeatus endoRGL, whereas removal of galactan hairs decreased the efficiency (Mutter et al, 1998a). The P. cellulosa rgll 1A was able to degrade a galactosylated oligosaccharide with a [Rhap-GalpA]3 backbone, whereas this oligosaccharide was not degraded when treated with a ^-galactosidase (McKie et al., 2001). These examples show that side chains play an important role in molecular recognition. In analogy to PGI, A, C and D, it turned out that endoRGH and endoRGL from A. aculeatus are processive enzymes. However, the enzymes differ in their degree of multiple attack: 4.0 and 2.5, respectively (Mutter et al., 1998a). Linear RG oligosaccharides have been used to further analyze the mode of action of RGdegrading enzymes. The substrates were prepared by treating pectin with dilute acid, and subsequent fractionation of the resulting fragments by chromatography (Renard et al., 1995). The linkage between Rhap and GalpA is more susceptible to hydrolysis by acid than the linkage between GalpA and Rhap, which results in linear RG oligosaccharides carrying a rhamnosyl residue at their reducing terminus. Figure 7.12 summarizes the mode of action of three different enzymes towards linear RG oligosaccharides. Both endoRGHs require a backbone of at least 9 or 10 glycosyl residues for activity, whereas endoRGL requires as many as 12 glycosyl residues. The A. aculeatus endoRGH hydrolyzes the fourth or fifth glycosidic linkage from the nonreducing terminus of the oligosaccharide, which is followed by processive degradation (Mutter et al., 1998b). The B. fuckeliana enzyme prefers the fifth or seventh glycosidic linkage from the nonreducing end and is not processive (Fu et al., 2001). The point of attack of this enzyme is not consistently at the same linkage of a particular substrate (see Figure 7.12). The A. aculeatus endoRGL prefers to cleave the smaller oligosaccharides four residues from the reducing rhamnose and the longest oligosaccharide used (DP 16) is cleaved at six units from the reducing terminus. These examples demonstrate that RG-degrading enzymes differ considerably in their mode of action.

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Fig. 7.12. Mode of action of Aspergillus aculeatus and Botryotinia fuckeliana RGH and Aspergillus aculeatus RGL on degalactosylated rhamnogalacturonan oligosaccharides of various lengths. Heavy arrows indicate preferred cleavage sites; less preferred sites are indicated with lighter arrows. Closed arrowheads marked with '2' indicate the second cleavage site of the enzyme. Open circles represent a-D~ GalpA residues; closed boxes represent a-L-Rha/? residues. V, A. aculeatus endoRGH; ti, B. fuckeliana endoRGH; A, A. aculeatus endoRGL.

7.4.2 Rhamnogalacturonan rhamnohydrolase Mutter et al. (1994) purified RG-rhamnohydrolase from a commercial pectinase preparation. This enzyme is an exo-acting pectinase with an absolute specificity to release terminal rhamnosyl residues (l->4)-linked to a-galacturonosyl residues (see Figure 7.11). To date no other report on this activity has been published. The enzyme was not able to split/?-nitrophenyl-ot-rhamnoside. Products resulting from endoRGH action are perfect substrates for RG-rhamnohydrolase. However, removal of the galactosyl residues from the RG oligosaccharides is required. The enzyme has not yet been assigned to a glycosyl hydrolase family as no sequence information is available. Pitson et al. (1998) have shown that the enzyme is an inverting hydrolase.

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7.4.3 Rhamnogalacturonan galacturonohydrolase The RG-galacturonohydrolase is also an exo-acting enzyme and can act perfectly in concert with RG-rhamnohydrolase in the degradation of products released from hairy regions by endoRGH (see Figure 7.11). Also, this enzyme is highly specific and releases galacturonosyl residues from the nonreducing end of RG oligosaccharides (Mutter et al., 1998c). OligoGalpA, as well as products released from hairy regions by endoRGL action, were not degraded by this enzyme. Highest reaction rates were observed for smaller RG oligosaccharides. As for RG-rhamnohydrolase, no sequence information for RG-galacturonohydrolase is available. This enzyme also acted by inversion of the anomeric configuration (Pitson et a/., 1998). 7.4.4 Synergy in rhamnogalacturonan degradation In the previous sections it has been shown that many enzymes are involved in the degradation of hairy regions. These RG-degrading enzymes can profit considerably from the concerted action of endogalactanase, endoarabinanase, arabinofuranosidase and fi-galactosidase. RG-acetylesterase (RGAE) is another important enzyme for the degradation of RG. This enzyme specifically removes 70% of the acetyl groups present in hairy regions, whereas acetyl esters present in homogalacturonans are not hydrolyzed (Searle-van Leeuwen et a/., 1992). Both A. aculeatus endoRGH and endoRGL are hampered by acetyl-esterification of the substrate (Kauppinen et al., 1995). RGAE is discussed in more detail in the next section. 7.5 Pectic esterases The pectin backbone, consisting of alternating rhamnogalacturonan and homogalacturonan, is decorated with small substituents such as methyl and acetyl groups. Both the presence of these groups on the pectin backbone and their distribution over the pectin backbone, can significantly influence the properties of pectins (Chapter 5) and the susceptibility of pectins for depolymerizing enzymes (see above). Pectic esterases are capable to hydrolyze these methyl and acetyl esters (EC 3.1.1-). So far three classes of esterases have been identified: (1) the pectin methylesterases (EC 3.1.1.11), (2) the pectin acetylesterases (EC 3.1.1.6) and (3) the rhamnogalacturonan acetylesterases (3.1.1.-) (Table 7.1). The pectin methylesterases (PMEs) hydrolyze the methyl esters, which are present at the carboxylic function (C-6) in the homogalacturonan backbone (Figure 7.13). The acetylesterases remove acetyl groups from O-2 and/or O3 of GalpA residues. The pectin acetylesterases (PAEs) are restricted to the homogalacturonan, whereas the rhamnogalacturonan acetylesterases (RGAEs) are restricted to the rhamnogalacturonan. The PMEs belong to family CE8

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Figure 7.13 Schematic representation of the esterases involved in the de-esterification of pectin, (a) homogalacturonan and (b) rhamnogalacturonan. Open circles represent a-D-GalpA residues; closed square represent a-L-Rhap residues; shaded triangles represent a-D-Gal/?; small shaded circles represent acetyl groups on the the O-2 and O-3 positions; and small open circles represent methyl groups at the C-6 position. 'NR' indicates the nonreducing end; 'R' indicates the reducing end.

in the carbohydrate esterase classification (Coutinho and Henrissat, 1999). The microbial RGAE and PAE have been grouped into family CE12. Other esterases, such as feruloylesterase, that are not active on the backbone but rather on side chains are not covered here. The properties of the pectin molecule are changed upon removal of the esters. Since these enzymes have different modes of action, pectins with different properties are generated. The properties and the mode of action of these enzymes will be addressed in next sections. 7.5.7 Pectin methylesterases Pectin methylesterases (PMEs) hydrolyze the ester bond between methanol and the carboxylic function of GalpA, resulting in the formation of a free carboxylic group and methanol. PMEs are common in fungi and bacteria. A few PMEs were also found in yeast (Gainvors et al., 1994; Nakagawa et al., 2000). In many plants several isoforms of PME are found both at the expression and genetic level. This variety of PME isoforms is not present in microorganisms. At the genetic level two PME isoforms have been identified in E. chrysanthemi (Laurent et al, 1993; Shevchik et al., 1996), whereas all other microorganisms studied so far have only one pme gene. Eukaryotic enzymes and proteins are often island/or O-linked glycosylated, which frequently leads to multiple isoforms of the same enzyme. However, there are no reports describing the analysis of

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(heterogeneous) glycosylation patterns of pectic esterase isoforms. Although PME activity is found in several microorganisms, only a limited number of enzymes have been purified and characterized in detail. The microbial PMEs are medium-sized enzymes (35-45 kDa) that are active as monomers. Many PMEs of fungal origin are glycoproteins and there is even a report of a bacterial PME from E. chrysanthemi, which in fact is a lipoprotein (Shevchik et al, 1996). The isoelectric points of PMEs vary from as low as 3.8 for fungal PME (Christgau et al, 1996) to 9.9 for Envinia PME (Plastow, 1988). In general, the bacterial PMEs have pH optima between pH 6 and 9. whereas most fungal PMEs have pH optima between pH 4 and 6. Thus far, only the 3D structure for the Envinia chrysanthemi PME has been elucidated (Jenkins etal, 2001; Pickersgill and Jenkins, 1999). The topology of the PME is similar to that found for the PEL, PL, PG, and RGHA (Yoder et al.. 1993; Mayans et al, 1997; Petersen et al., 1997; Pickersgill et al., 1998). The conservation of this unique topology among these enzymes strongly suggests that a complete set of different pectinases have evolved from the same ancestral gene. Based on the 3D structure of Erwinia PME and the primary sequence alignments, Pickersgill and Jenkins hypothesized a possible mechanism of action (Pickersgill and Jenkins, 1999). Two aspartates and one arginine are strictly conserved among PMEs. One of the Asp residues makes a hydrogen bond to the Arg and is therefore most likely unprotonated. The other Asp is likely to be protonated. A water molecule adjacent to the unprotonated Asp may be activated by transferring its proton to the Asp. Subsequently, the hydroxyl generated can attack the carbonyl-carbon of the methyl-esterified Gal/?A residue. Simultaneous protonation of one of the oxygen atoms results in the formation of a tetrahedral intermediate, which collapses with the release of methanol and results in de-esterification of the Gal/?A residue (Pickersgill and Jenkins. 1999). Microbial PMEs can differ in their mode of action. Fungal PMEs attack the methyl groups on the pectin randomly, resulting in a random distribution of the nonmethylated galacturonic acid residue, whereas the bacterial Envinia PME removes blocks of methyl groups on a single chain, similarly to the plant PMEs (T. Christensen, personal communication). However, PME action on (highly) methylated pectins does not result in complete de-esterification (Versteeg, 1979). Generally, about 20-30% of the methyl esters are not removed. In part, this is due to the presence of acetyl groups in certain pectins (e.g. sugar beet pectin). Pretreatment or simultaneous treatment with PAE results in a higher degree of de-esterification, although still not all methyl groups will be hydrolyzed (Oosterveld et al, 2000). Since pectin is too heterogeneously esterified for study of the mode of action of PMEs, A. niger PME has been analyzed on purified smaller compounds with a defined methyl-esterification pattern (Kester et al. 2000). Fully methylesterified oligoGal/?As (DP 3-6) were individually subjected to PME attack. No activity was detected on the dimer. However, the rate of de-esterification

MICROBIAL PECTINASES

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increased by a factor 100 when the DP increased from 3 to 6. The increase in reaction rate from DP 5 to 6 was still a factor 2. These results indicate that at least five or six subsites are present on the A. niger PME and that the presence of nonesterified residues somewhere along the chain is not a prerequisite for PME activity. However, when de-esterification was monitored in time using MALDI-TOF MS, the preference for one free carboxyl group in the substrate could be demonstrated (Figure 7.14). As soon as one ester was removed from a fully methyl-esterified (Gal/>A)4, this compound was almost immediately further de-esterified to (Gal/?A)4 containing two methyl esters, which was in turn de-esterified to the final product, (GalpA)4 containing one methyl ester (van Alebeek, unpublished). Similar observations were made for fully methylesterified (Gal/?A)5 and (GalpA)^, but not for the trimer. Apparently, at least four subsites have to be occupied before this phenomenon occurs, which suggests that a carboxyl function is preferred in the active-site cleft at either subsite +2, +3 or subsite —2, —3 (van Alebeek, unpublished). The order in which the methyl esters were removed during the course of reaction was determined using ESI-MS/MS (Kester et al., 2000). It appeared that for the methyl-esterified oligoGalpA analyzed, there was a preference for the internal residues followed by the methyl esters near the reducing end. However, the enzyme was not able to remove the methyl ester from the nonreducing Gal/?A residue. The distribution of the products observed after partial de-esterification demonstrates that this enzyme acts according to a multiple-chain mechanism: each encounter results in the removal of only one methyl group (Kester et al., 2000). Monomethylesterified trigalacturonides carrying the monomethyl group at a defined position (Kester et al., 1999) were used to study the mode of action of A. niger PME, These studies also showed that this fungal enzyme could not remove a methyl group from a GalpA at the nonreducing end. The methyl esters were removed from the other two positions, the middle and reducing end residue, although the activity was reduced drastically. This is in agreement with the MALDI-TOF MS patterns showing A. niger PME de-esterification of fully methyl-esterified (Gal/?A)3 (van Alebeek, unpublished). 7.5.2 Pectin homogalacturonan acetylesterases Pectin acetylesterase (PAE) is capable of hydrolyzing the acetyl esters from the O-2 and/or O-3 of GalpA residues located on the homogalacturonan part of pectin, generating acetate and a free hydroxyl group on the GalpA residue. So far, only few PAEs have been detected in microorganisms: one in the fungus A. niger (Searle-Van Leeuwen et al., 1996), and one in the bacterium E. chrysanthemi (Shevchik and Hugouvieux-Cotte-Pattat, 1997b). Both A. niger and E. chrysanthemi PAE are 60kDa proteins. The fungal enzyme has an optimal activity at a pH of 5.5 (50°C), whereas the bacterial PAE has a pH optimum of 8. A. niger PAE enzyme displayed the highest activity

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21 3

on beet pectin and reduced activity on modified hairy regions (MHR) and acetylated xylan polymers (Searle-Van Leeuwen et al., 1996), whereas E. chrysanthemi PAE was active on beet pectin but not on apple pectin (MHR is the main acetylated constituent) (Shevchik and Hugouvieux-Cotte-Pattat, 1997b). Furthermore, both enzymes are capable of deacetylation of the artificial substrates triacetin, p-nitrophenol acetate and 4-methylumbelliferyl acetate (Searle-Van Leeuwen et al., 1996; Shevchik and Hugouvieux-Cotte-Pattat, 1997b; van Alebeek, unpublished). Although beet pectin appeared to be the best substrate, A. niger PAE and E. chrysanthemi PAE could only remove 30% and 15% of the total amount of acetyl esters present, respectively. Pretreatment of the beet pectin with pectinolytic enzymes increased the initial activity of PAE. While the total release of acetate did not increase for the A. niger PAE, an increase to 67% was seen for the E. chrysanthemi PAE. This suggests that both PAEs have different specificities and that additional PAEs are needed for the complete deacetylation of beet pectin. Indeed, the presence of additional PAEs with a lower pi has been demonstrated in E. chrysanthemi using isoelectrofocusing (Shevchik and Hugouvieux-Cotte-Pattat, 1997b). There is only limited knowledge on the mode of action of PAE. Until now only PAE of A. niger has been investigated using NMR. 'H NMR spectra of beet pectin (degree of acetylation (DA) 34%; note that since acetylation can occur at two positions on each GalpA, DA can be as high as 200%) showed resonance signals at 1.95ppm and 2.10ppm specific for acetyl groups. Upon PAE treatment the resonance at 1.95 ppm disappeared completely, whereas the signal at 2.10 ppm was only slightly reduced (Searle-Van Leeuwen et al., 1996). However, this set of resonance signals is in a slightly lower ppm range compared to that reported by Ishii (1995) and Renard and Jarvis (1999), which is probably caused by differences in calibration. Renard et al. monitored the acetylation of homogalacturonan to DA 180 with *H NMR. They found resonance signals at 2.10 and 2.18 ppm corresponding to the singly-acetylated GalpA residues, whereas signals at 2.04 and 2.13 ppm were found for doubly-acetylated GalpA residues. Furthermore, it was concluded that the signal at 2.18 ppm corresponded to the O-2 position, whereas the signal at 2.10 ppm corresponded to the O-3 position. Since beet pectin showed only two peaks of resonance (1.95 and 2.10 pprn), it is most likely that only singly-acetylated GalpA residues are present in this beet pectin. Since the 1.95 ppm peak disappeared from the !H NMR spectrum upon PAE treatment it is most likely that this corresponds to the O-3 position of the GalpA residue. This would imply that this PAE is capable of removing the acetyl ester from the O-3 position of a singly-substituted GalpA residue. 7.5.3 Rhamnogalacturonan acetylesterases Rhamnogalacturonan acetylesterase (RGAE) specifically hydrolyzes the acetyl esters from the GalpA residue in rhamnogalacturonan, releasing acetate and

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generating a free hydroxyl group. Thus far, RGAEs have only been isolated from the fungi A. niger (Searle-van Leeuwen etal., 1996) and A. aculeatus (Searle-van Leeuwen etal., 1992; Kauppinen etal., 1995). The properties of both enzymes are quite similar. The cloned RGAE from A. aculeatus revealed a molecular weight of 32-35 kDa on SDS-PAGE, which was approximately 8-11 kDa above the calculated molecular weight due to glycosylation of the enzyme (Kauppinen et al., 1995). However, using gel-fitration, molecular weights of 40 and 42 kDa were determined for the A. niger and A. aculeatus RGAEs, respectively. Both enzymes are optimally active at pH 5.5-6 and 40-50=C (Searle-van Leeuwen et al., 1992, 1996; Kauppinen et al., 1995). The enzyme appeared to be very specific for a certain RG preparation (modified hairy regions: MHR) and had no activity towards beet pectin, acetylated xylan, triacetin or acetylated salicylic acid (Searle-van Leeuwen etal., 1992, 1996; Kauppinen et al., 1995; Beldman et al., 1996). Furthermore, RGAE was an essential enzyme in the breakdown of RG (Kauppinen et al., 1995; Beldman et al., 1996; De Vries et al.. 2000). After de-acetylation, endoRGH (Schols et al., 1990) and endoRGL (Mutter et al., 1998a) were able to degrade the hairy regions, which is of technological importance for the extraction of fruit and for fruit juice ultrafiltration (Beldman etal., 1996). So far, only the 3D structure of A. aculeatus RGAE (M01gaard et al., 2000) is available. RGAE folds into an a/p/a structure, which is completely different from the normally observed a/p structure common to esterases and lipases. As for the o/p enzymes, in the RGAE the active-site residues are Ser, Asp and His, in the same sequential order, but the Asp and His residues are only three residues apart instead of being located on different loops (M01gaard et al., 2000). Based on the strict sequence conservation in the esterase family 12 of Ser and His and two additional residues, Gly and Asn, which both act as hydrogen bond donors to the oxyanion hole, it has been proposed to name this group of esterases the SGNH-hydrolase family (M01gaard et al., 2000). For RGAE it was postulated that catalysis will proceed via the general mechanism observed for lipases. where the catalytic triad Asp-His-Ser, which forms a charge-relay system, will attack the ester bond via the activated serine (M01gaard et al., 2000). Little is known of the mode of action of RGAEs on RG or RG oligomers. RG oligomers from bamboo shoots have been characterized and appeared to be specifically acetylated at the O-2 or O-3 of Gah?A residues. Both singlyand doubly-acetylated GalpA residues were identified (Ishii, 1995). 'H NMR has been performed on apple MHR and demonstrated at least three resonance peaks in the acetyl group region, indicating the presence of both singly- and doubly-acetylated Gal/?A residues. Upon incubation with RGAE of A. niger RGAE all signals decreased, leaving the overall pattern the same (Searle-van Leeuwen et al., 1996). This indicates that all acetyl groups are subjected to RGAE hydrolysis to the same extent. Thus, apparently, RGAE has no preference for a specific position of the acetyl group as was observed for PAE. However.

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30-40% of the acetyl groups were still present in the MHR and could not be removed enzymatically. Perhaps side chains cause steric hindrance and prevent RGAEs from acting on highly branched parts of the rhamnogalacturonan backbone.

References Akita, M., Suzuki, A., Kobayashi, T., Ito, S. and Yamane, T. (2000) Crystallization and preliminary X-ray analysis of high-alkaline pectate lyase. Acta Crystallogr. Sect. D Biol. Crystallogr., 56, 749-750, Albersheim, P., Darvill, A.G., O'Neill, M.A., Schols, H.A. and Voragen, A.G.J. (1996) An hypothesis: the same six polysaccharides are components of the primary cell walls of all higher plants, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 47-53. Allen, J.D. and Thoma J.A. (1976) Subsite mapping of enzymes. Biochem. J., 159, 121-132. Armand, S., Wagemaker, M.J.M., Kester, H.C.M., et al. (2000) The active-site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis. /. Biol. Chern., 275, 691-696. Altaian, M.T. and Nagel, C.W. (1979) The role of calcium ions in the activity of an endo-pectic acid lyase on oligogalacturonides. / Food Biochem., 1, 185-206. Azadi, P., O'Neill, M.A., Bergmann, C, Darvill, A.G. and Albersheim, P. (1995) The backbone of the pectic polysaccharide rhamnogalacturonan I is cleaved by an endohydrolase and an endolyase. Glycobiology, 5, 783-789. Barras, E, van Gijsegem, F. and Chatterjee, A.K. (1994) Extracellular enzymes and pathogenesis of soft rot Erwinia. Annu. Rev. Phytopathol., 32, 221-234. Beldman, G., Mutter, M., Searle-van Leeuwen, M.J.F., Van de Broek, L.A.M., Schols, H.A. and Voragen, A.G.J. (1996) New enzymes active towards pectic structures, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 231-245. Benen, J.A.E., Kester, H.C.M. and Visser, J. (1999) Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. Eur. J. Biochem., 259, 577-585. Benen, J.A.E., Pafenicova, L., Kester, H.C.M. and Visser, J. (2000) Characterization of pectate lyase A from Aspergillus niger. Biochemistry, 39, 15563-15569. Biely, P., Benen, J.A.E., Heinrichova', K., Kester, H.C.M. and Visser, J. (1996) Inversion of configuration during hydrolysis of alpha-1,4-galacturonidic linkage by three Aspergillus polygalacturonases. FEBS Lett., 382, 249-255. Blanco, P., Sieiro, C., Reboredo, N.M. and Villa, T.G. (1997) Genetic determination of polygalacturonase production in wild type and laboratory strains of Saccharomyces cerevisiae. Arch. Microbioi, 167, 284-288. Bonnin, E., Le Goff, A., Korner, R., et al. (2001) Study of the mode of action of endopolygalacturonase from Fusarium moniliforme. Biochim. Biophys. Acta, 1526, 301—309. Brooks, A.D., He, S.Y., Gold, S., Keen, N.T., Collmer, A. and Hutcheson, S.W. (1990) Molecular cloning of the structural gene for exopolygalacturonate lyase from Erwinia chrysanthemi EC 16 and characterization of the enzyme product. J. Bacterial., 172, 6950-6958. Brown, I.E., Mallen, M.H., Charnock, S.J., Davies, G.J. and Black, G.W. (2001) Pectate lyase 10A from Pseudomonas cellulosa is a modular enzyme containing a family 2a carbohydrate-binding module. Biochem. J., 355, 155-165. Bussink, H.J.D., Buxton, P.P. and Visser, J. (199la) Expression and sequence comparison of the Aspergillus niger and Aspergillus tubingensis genes encoding polygalacturonase II. Curr. Genet., 19,467-474.

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Kauppinen, S., Christgau, S., Kofod, L.V., Halkier, T., Dorreich, K. and Dalb0ge, H. (1995) Molecular cloning and characterization of a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. Synergism between rhamnogalacturonan degrading enzymes. /. Biol. Chem., 270, 27172-27178. Kester, H.C.M. and Visser, J. (1990) Purification and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger. Biotechnol. Appl. Biochem., 12, 150-160. Kester, H.C.M. and Visser, J. (1994) Purification and characterization of pectin lyase B, a novel pectinolytic enzyme from Aspergillus niger. FEMS Lett., 120, 63-67. Kester, H.C.M., Kusters-van Someren, M.A., Miiller, Y. and Visser, J. (1996) Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis. Eur. J. Biochem., 240, 738-746. Kester, H.C.M., Magaud, D., Roy, C., et al. (1999) Performance of selected microbial pectinases on synthetic monomethyl-esterified di- and trigalacturonates. J. Biol. Chem., 274, 37053-37059. Kester, H.C.M., Benen, J.A.E., Visser, J., et al. (2000) Tandem mass spectrometric analysis of Aspergillus niger pectin methylesterase; mode of action on fully methylesterified oligogalacturonates. Biochem. J., 346, 469-474. Kita, N., Boyd, C.M., Garrett, M.R., Jurnak, F. and Keen, N.T. (1996) Differential effect of site-directed mutations in pelC on pectate lyase activity, plant tissue maceration, and elicitor activity. J. Biol. Chem. 271, 26529-26535. Kobayashi, T., Higaki, N., Suzumatsu, A., et al. (2001) Purification and properties of a high-molecularweight, alkaline exopolygalacturonase from a strain of Bacillus. Enzyme Microb. Technol., 29, 70-75. Kofod, L.V., Kauppinen, S., Christgau, S., et al. (1994) Cloning and characterization of two structurally and functionally divergent rhamnogalacturonases from Aspergillus aculeatus. J. Biol. Chem., 269, 29182-29189. Kohn, -R. (1971) The activity of calcium ions in aqueous solutions of the lower calcium oligogalacturonates. Carbohydr. Res., 20, 351-356. Korner, R., Limberg, G., Christensen, T.M.I.E., Mikkelsen, J.D. and Roepstorff, P. (1999) Sequencing of partially methyl-esterifled oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specificities. Anal. Chem., 71, 1421-1427. Laurent, F., Kotoujansky, A., Labesse, G. and Bertheau, Y. (1993) Characterization and overexpression of the pern gene encoding pectin methylesterase of Erwinia chrysanthemi strain 3937. Gene, 131, 17-25. Liao, C.H., Revear, L., Hotchkiss, A. and Savary, B. (1999) Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase from Yersinia enterocolitica. Can. J. Microbiol., 45, 396-403. Lietzke, S.E., Yoder, M.D., Keen, N.T. and Jurnak, F. (1994) The three-dimensional structure of pectate lyase E, a plant virulence factor from Erwinia chrysanthemi. Plant Physio!., 106, 849-862. Lojkowska, E., Masclaux, C., Boccara, M., Robert-Baudouy, J. and Hugouvieux-Cotte-Pattat, N. (1995) Characterization of the pelL gene encoding a novel pectate lyase of Erwinia chrysanthemi 3937. Mol. Microbiol, 16, 1183-1195. Mayans, O., Scott, M., Connerton, I., et al. (1997) Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substratebinding clefts of pectin and pectate lyases. Structure, 5, 677-689. McKie, V.A., Vincken, J.-P., Voragen, A.G.J., van den Broek, L.A.M., Stimson, E. and Gilbert, H.J. (2001) A new family of rhamnogalacturonan lyases contains an enzyme that binds to cellulose. Biochem. J., 355, 167-177. Meeuwsen, P.J.A., Vincken, J.-P., Beldman, G., Voragen, A.G.J. (2000) A universal assay for screening expression libraries for carbohydrases. /. Biosci. Bioeng., 89, 107-109. M01gaard, A., Kauppinen, S. and Larsen, S. (2000) Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Structure, 8, 373-383.

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Morris, E.R., Powell, D.A., Gidley, M.J. and Rees, D.A. (1982) Conformations and interactions of pectins. I. Polymorphism between gel and solid states of calcium polygalacturonate. /. Mol. Biol.. 155,507-516. Mutter, M., Beldman, G., Schols, H.A. and Voragen, A.G.J. (1994) Rhamnogalacturonan a-Lrhamnopyranosylhydrolase. A novel enzyme specific for the terminal nonreducing rhamnosyl unit in rhamnogalacturonan regions of pectin. Plant Physiol., 106, 241-250. Mutter, M., Colquhoun, I.J., Schols, H.A., Beldman, G. and Voragen, A.G.J. (19%) Rhamnogalacturonase B from Aspergillus aculeatus is a rhamnogalacturonan a-L-rhamnopyranosyl-( 1.4)-a-Dgalactopyranosyluronide lyase. Plant Physiol., 110, 73-77. Mutter. M., Colquhoun, I.J., Beldman, G., Schols, H.A., Bakx, E.J. and Voragen, A.G.J. (1998a) Characterization of recombinant rhamnogalacturonan a-L-rhamnopyranosyl-(l,4)-aD-galactopyranosyl-uronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturonan I regions of pectin. Plant Physiol.. 117, 141-152. Mutter, M., Renard, C.M.G.C., Beldman, G.. Schols, H.A. and Voragen. A.G.J. (1998b) Mode of action of RG-hydrolase and RG-lyase towards rhamnogalacturonan oligomers. Characterization of degradation products using RG-rhamnohydrolase and RG-galacturonohydrolase. Carbohydr. Res.. 311, 155-164. Mutter, M., Beldman, G., Pitson, S.M., Schols, H.A. and Voragen. A.G.J. (1998c) Rhamnogalacturonan a-D-galactopyranosyluronohydrolase. An enzyme that specifically removes the terminal nonreducing galacturonosyl residue in rhamnogalacturonan regions of pectin. Plant Physiol.. 117. 153-163. Nakagawa, T., Miyaji, T, Yurimoto, H., Sakai, Y.,Kato, N. and Tomizuka, N. (2000) A methylotrophic pathway participates in pectin utilization of Candida boidinii. Appl. Environ. Microbiol.. 66. 4253-4257. Oosterveld, A. Beldman, G. Searle-Van-Leeuwen, M.J.F. and Voragen, A.G.J. (2000) Effect of enzymatic deacetylation on gelation of sugar beet pectin in the presence of calcium. Carbohydr. Polym.. 43. 249-256. Pages, S., Heijne. W.H.M., Kester H.C.M., Visser, J. and Benen, J.A.E. (2000) Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis. J. Biol. Chem.. 275. 29348-29353. Pages, S., Kester, H.C.M., Visser, J. and Benen J.A.E. Changing a single amino acid residue switches processive and non-processive behavior of Aspergillus niger endopolygalacturonase I and II. / Biol. Chem.. 276, 33652-33656. Pafenicova, L., Benen, J.A.E., Kester, H.C.M. and Visser. J. (1998) pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. Eur. J. Biochem.. 251. 72-80. Pafenicova, L., Benen, J.A.E., Kester. H.C.M. and Visser, J. (2000a). pgaA and pgaB encode constitutively expressed members of the Aspergillus niger endopolygalacturonase gene family. Biochem. J., 345, 637-644. Pafenicova, L., Kester, H.C.M., Benen, J.A.E. and Visser, J. (2000b) pgaD encodes a new type of endopolygalacturonase from Aspergillus niger. FEES Lett.. 467. 333-336. Pasculli, R., Geraeds. C. Voragen, F. and Pilnik, W. (1991) Characterization of polygalacturonases from yeast and fungi. Lebensm.-Wissens. Technol., 24, 63-70. Pashova, S., Slokoska, L., Krumova, E. and Angelova, M. (1999) Induction of polymethylgalacturonase biosynthesis by immobilized cells of Aspergillus niger 26. Enzyme Microb. Technol.. 24. 535-540. Petersen, T.N., Kauppinen. S. and Larsen, S. (1997) The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix. Structure. 5. 533-544. Pickersgill, R.W. and Jenkins, J.A. (1999) Crystal structures of polygalacturonase and pectin methylesterase, in Recent Advances in Carbohydrate Bioengineering (eds H.J. Gilbert. G.J. Davies. B. Henrissat and B. Svensson), The Royal Society of Chemistry. Cambridge, pp. 144—149. Pickersgill, R., Jenkins, J., Harris, G., Nasser, W. and Robert-Baudouy. J. (1994) The structure of Bacillus subtilis pectate lyase in complex with calcium. Nature Structural Biology. 1.717-723.

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Pickersgill, R., Smith, D., Worboys, K. and Jenkins, J. (1998) Crystal structure of polygalacturonase from Erwinia camtovora ssp. carotovora. J. Biol. Chem., 273, 24660-24664. Pissavin, C., Robert-Baudouy, J. and Hugouvieux-Cotte-Pattat, N. (1996) Regulation ofpelZ, a gene of the pelB-pelC cluster encoding a new pectate lyase of Erwinia chrysanthemi 3937. J. Bacteriol., 178,7187-7196. Pitson, S.M., Mutter, M., van den Broek, L.A.M., Voragen, A.G.J. and Beldman, G. (1998) Stereochemical course of hydrolysis catalysed by a-L-rhamnosyl and a-D-galacturonosyl hydrolases from Aspergillus aculeatus. Biochem. Biophys. Res. Commun,, 242, 552-559. Plastow, G.S. (1988) Molecular cloning and nucleotide sequence of the pectin methylesterase gene of Erwinia chrysanthemi B374. Mol. Microbiol., 2, 247-254. Preston, J.F. Rice, J.D., Ingram, L.O. and Keen, N.T. (1992) Differential depolymerization mechanisms of pectate lyases secreted by Erwinia chrysanthemi EC16. J. Bacteriol.. 174, 2039-2042. Renard, C.M.G.C. and Jarvis M.C. (1999) Acetylation and methylation of homogalacturonans 1: Optimisation of the reaction and characterization of the products. Carbohydr. Po/ym., 39. 201-207. Renard, C.M.G.C., Thibault,J.-R, Mutter, M.,Schols,H.A. and Voragen, A.G.J. (1995) Some preliminary results on the action of rhamnogalacturonase on rhamnogalacturonan oligosaccharides from beet pulp. Int. J. Biolo. Macromol., 17, 333-336. Rexova-Benkova. L. (1973) The size of the substrate-binding site of an Aspergillus niger extracellular endopolygalacturonase. Eur. J. Biochem., 39, 109-115. Roy, C., Kester, H.C.M., Visser, J., et al. (1999) Mode of action of five different endo-pectate lyases from Envinia chrysanthemi. J. Bacteriol., 181, 3705-3709. Sakai, T., Sakamoto, T., Hallaert, J. and Vandamme, E.J. (1993) Pectin, pectinase, and protopectinase: production, properties, and applications. Adv. Appl. Microbiol., 39, 213-294. Scavetta, R.D., Herron, S.R., Hotchkiss, A.T., et al. (1999) Structure of plant cell wall fragment complexed to PelC. Plant Cell, 11, 1081-1092. Schols, H.A. and Voragen, A.G.J. (1996) Complex pectins: structure elucidation using enzymes, in Pectins and Pectinases (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp. 3-19. Schols, H.A., Geraeds, C.C.J.M., Searle-van Leeuwen, M.J.F., Kormelink, F.J.M. and Voragen A.G.J. (1990) Rhamnogalacturonase: a novel enzyme that degrades the hairy regions of pectins. Carbohydr. Res., 206, 105-115. Schols, H.A., Voragen, A.G.J. and Colquhoun, I.J. (1994) Isolation and characterization of rhamnogalacturonan oligomers, librated during degradation of pectic hairy regions by rhamnogalacturonase. Carbohydr. Res., 256, 97-111. Schwan, R.F. and Rose, A.H. (1994) Polygalacturonase production by Kluyveromyces marxianus: effect of medium composition. /. Appl. Microbiol., 76, 62-67. Searle-van Leeuwen, M.J.F., Van de Broek, L.A.M., Schols, H.A., Beldman, G. and Voragen, A.G.J. (I992) Rhamnogalacturonan acetylesterase: a novel enzyme from Aspergillus aculeatus, specific for the deacetylation of hairy (ramified) regions of pectin. Appl. Microbiol. Biotechnol, 38, 347-349. Searle-van Leeuwen, M.J.F., Vincken, J-P., Schipper, D., Voragen, A.G.J. and Beldman, G. (1996) Acetyl esterases of Aspergillus niger. purification and mode of action on pectins, in Pectins and Pectinase s (eds J. Visser and A.G.J. Voragen), Progress in Biotechnology 14, Elsevier Science, Amsterdam, pp 793-798. Shevchik, V.E., Condemine, G., Hugouvieux-Cotte-Pattat, N. and Robert-Baudouy, J. (1996) Characterization of pectin methylesterase B, an outer membrane lipoprotein of Erwinia chrysanthemi 3937. Mol. Microbiol., 19, 455-466. Shevchik, V.E., Robert-Baudouy, J. and Hugouvieux-Cotte-Pattat, N. (1997a) Pectate lyase Pell of Envinia chrysanthemi 3937 belongs to a new family. J. Bacteriol., 179, 7321-7330.

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Shevchik, V.E. and Hugouvieux-Cotte-Pattat, N. (1997b) Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937. Mol Microbiol., 24, 1285-1301. Shevchik, V.E., Kester, H.C.M., Benen, J.A.E., Visser, J., Robert-Baudouy, J. and Hugouvieux-CottePattat, N. (1999a) Characterization of the exo-pectate lyase PelX of En\'inia chrysanthemi. J. Bacterial., 181, 1652-1663. Shevchik, V.E., Condemine, G. Robert-Baudouy, J. and Hugouvieux-Cotte-Pattat, N. (1999b) Exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937. 7. Bacterial., 181, 3912-3919. Soriano, M., Blanco, A., Diaz, P. and Pastor, F.I.J. (2000) An unusual pectate lyase from a Bacillus sp with high activity on pectin: cloning and characterization. Microbiology UK. 146, 89-95. Suganuma, T, Matsuno, R., Ohnishi. M. and Hiromi, K. (1978) A study of the mechanism of action of Taka-amylase Al on linear oligosaccharides by product analysis and computer simulation. J. Biochem., 84, 293-316. Suykerbuyk, M.E.G., Schaap, P.J., Stam, H., Musters, W. and Visser J. (1995) Cloning, sequence and expression of the gene coding for rhamnogalacturonase of Aspergillus aculeatus: a novel pectinolytic enzyme. Appl. Microbiol. Biotechnol., 43, 861-870. Suykerbuyk, M.E.G., Kester, H.C.M., Schaap, P.J., Stam, H., Musters, W. and Visser, J. (1997) Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger. Appl. Environ. Microbiol., 63, 2507-2515. Tamaru,Y. and Doi, P.H. (2001) Pectate lyase A, an enzymatic subunit of the Clostridium cellulovorans cellulosome. Proc. Nail. Acad. Sci. USA, 98, 4125^129. Tardy, F, Nasser, W., Robert-Baudouy, J. and Hugouvieux-Cotte-Pattat, N. (1997) Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors. J. Bacterial., 179, 2503-2511. Thoma, J.A., Rao, G.V.K., Brothers, C. and Spradlin, J. (1971) Subsite mapping of enzymes. 7. Biol. Chem., 246, 5621-5635. Van Alebeek, G.J.W.M., Zabotina, O., Schols, H.A., Beldman. G. and Voragen, A.G.J. (2000) Esterification and glycosidation of oligogalacturonides: examination of the reaction products using MALDI-TOF MS and HPAEC. Carbohydr. Polym., 43. 39^6. Van Alebeek, G.J.W.M., Schols, H.A. and Voragen, A.G.J. (200la) Amidation of methyl-esterified oligogalacturonides: examination of the reaction products using MALDI-TOF MS. Carbohydr. Polym., 46, 311-321. Van Alebeek, G.J.W.M., Zabotina, O., Schols, H.A., Beldman, G. and Voragen, A.G.J. (2001b) Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. 7. Mass Spectrom.. 35. 831-840. Van der Vlugt-Bergmans, C.J.B., Meeuwsen, P.J.A., Voragen, A.G.J. and van Ooyen. A.J.J. (2000) Endo-xylogalacturonan hydrolase. a novel pectinolytic enzyme. Appl. Environ. Microbiol., 66, 36-41. Van Houdenhoven, F.E.A. (1975) Studies on pectin lyase, PhD dissertation. Wageningen University. Wageningen, The Netherlands. Van Rijssel, M., Gerwig, G.J. and Hansen, T.A. (1993) Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity. Appl. Environ. Microbiol., 59, 828-836. Van Santen, Y., Benen, J.A.E., Schroter, K.-H., et al. (1999) 1.86 A crystal structure of endopolygalacturonase II from Aspergillus niger and identification of active site residues by site-directed mutagenesis. 7. Biol. Chem., 274, 30474-30480. Versteeg, C. (1979) Pectinesterases from the orange fruit: their purification, general characteristics and juice cloud destabilizing properties, PhD dissertation. Wageningen University. Wageningen. The Netherlands. Vitali, J., Schick, B., Kester, H.C.M., Visser, J. and Jurnak, F. (1998) The three-dimensional structure of Aspergillus niger pectin lyase B at 1.7-A resolution. Plant Physiol.. 116, 69-80.

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Voragen, A.GJ. (1972) Characterization of pectin lyases on pectins and methyl oligogalacturonates, PhD dissertation, Wageningen University, Wageningen, The Netherlands. Voragen, A.GJ. and Pilnik, W. (1989) Pectin-degrading enzymes in fruit and vegetable processing, in Biocatalysis in Agricultural Biotechnology (eds J.R. Whithaker and P.E. Sonnet), ACS Symposium Series No. 389, Washington, DC, pp. 93-115. Yamada, H. (1994) Pectic polysaccharides from Chinese herbs: structure and biological activity. Carbohydr. Polym., 25, 269-276. Yoder, M.D., Keen, N.T. and Jurnak, F. (1993) New domain motif: the structure of pectate lyase C. a secreted plant virulence factor. Science, 260, 1503-1507.

8

Commercial pectin preparations Claus Rolin

8.1 Production 8.1.1 Raw materials Commercial pectin is produced almost exclusively from citrus peel or apple pomace, which are available as solid by-products from industrial juice manufacture. In addition, a comparatively small amount of pectin is produced from sugar beet solids, which come as a by-product of sugar manufacture. Apple pomace is the traditional source of pectin and, historically, the major part of the industry developed from utilization of apple. Today, dried peel of lemon and lime is the largest source. Sugar beet was used as a replacement for other pectin raw materials during the 1940s and early 1950s, but this use lapsed, because the pectin has inherently different properties. Nowadays, advantage is being taken of this difference in specific applications (see Section 8.5.3) where sugar beet pectin is preferred over other pectins. Citrus fruit becomes available to the pectin manufacturer after removal of juice and, in most cases, etheric oil by squeezing. The fruit solids, mostly peel, but also parts of the fruit interior, segments, etc., are washed in water to remove water-soluble material. The washed fruit solids can then be utilized immediately for pectin manufacture or dried for later use. Until it is dried, the traumatized fruit has poor storage stability, so facilities for drying peel, as well as facilities for producing pectin from fresh peel, are usually located in the immediate vicinity of juice producers. The various kinds of citrus fruit differ with respect to the properties of the extractable pectin. Lemon and, in particular, lime yield pectins of high specific viscosity in aqueous solution and low calcium sensitivity (i.e., low dependence of the viscosity upon the presence of Ca 2+ ), and when used for gelation they provide high breaking strength. Some varieties of orange and grapefruit yield pectins of somewhat lower specific viscosity and higher calcium sensitivity; these are less suited for classical gel applications with calcium-rich fruit. The raw material may vary from day to day owing to factors such as the weather prior to the harvest, differences between plantations with respect to the soil and hereditary traits of the trees, and so on. These variations affect the properties of pectin, and it is therefore necessary to standardize pectin (see Section 8.1.4) in order to achieve consistency from batch to batch.

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8.1.2 Production process for high-ester pectin In summary, the raw material is suspended in hot water with an appropriate amount of strong acid. After some time, the resulting solution is separated from the undissolved solids by filtration. The solution is optionally ion-exchanged, optionally concentrated, and then mixed with an alcohol, which precipitates the pectin. The precipitate is squeezed free of the alcohol, purified by washing in more alcohol, and finally dried and milled. The conditions of the extraction (the process in which peel is suspended in hot, acidified water) are typically within a window of pH 1-3, temperature 50-90°C, duration 3-12 hours. Nitric acid is commonly used for acidifying, because it is less corrosive than hydrochloric acid, for example. The peel to water ratio is chosen so that the resulting solution contains about 5-10 g/1 of pectin. The process turns protopectin (i.e. pectin, or precursor of pectin, as it exists in the living plant in which, from a practical point of view, it is not water-soluble) into water-soluble pectin; it further dissolves the pectin and leaches it from the undissolved plant material. The modification of protopectin to water-soluble pectin is not understood in detail, but, at least, it involves limited hydrolysis of molecular parts. In particular, the degree of esterification (DE) can be controlled within an interval of about 55 to about 80 by choosing extraction conditions that cause the desired number of methyl ester groups to hydrolyze. Further, since various molecular parts in the protopectin differ with respect to their rate of hydrolysis as a function of pH and temperature, subtle tailoring of pectin functionality is possible by selecting specific combinations of raw material and extraction conditions. Some plant materials are very different from citrus with respect to the extraction conditions that lead to the optimal result (in terms of high yield of material with high molecular weight). As an example, Aloe vera may advantageously be extracted with EDTA at pH 7-8.5, because the protopectin inherently has a relatively low degree of esterification (Ni et at., 1999). This pH would degrade pectin of higher DE owing to ^-elimination (Section 8.3.4). The undissolved solids are removed in a series of filtrations, which are difficult owing to a high solution viscosity, which, in turn, is the reason why the pectin concentration cannot exceed about 10 g/1. Insoluble filter aids such as wood cellulose and perlite may be used to facilitate this process. The undissolved plant material is typically used as cattle feed. The extract is then, optionally, ion-exchanged to remove Ca2+ ions, so that the resulting pectin becomes more soluble. Next, the solution can be concentrated by evaporation or membrane filtration before the pectin is precipitated by pouring the extract into an alcohol, such as 2-propanol. The stringy precipitate is squeezed by pressing and then washed in a fresh batch of alcohol. After thorough squeezing, the precipitate is dried. The dried precipitate is finally milled and sieved to a powder of controlled particle size.

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The alcohol that has been used for precipitation is recovered by distillation. Pectin production is generally rather energy-consuming owing to the processes of evaporation, distillation and drying for removing water. Removal of about 100-200 kg water per kilogram of product is necessary because of the limited concentration of pectin that is possible during filtration. Additional energy consumption is incurred for peel drying if dried peel is used as the raw material. 8.1.3 Production process for low-ester and amidated pectin If a degree of esterification less than about 55 is desired, a separate treatment for de-esterification usually takes place at some point after filtration. Otherwise, the process for low-ester pectin is the same as for high-ester pectin. Since the glycosidic bonds of the main chain of pectin are reasonably resistant to hydrolysis, it is possible to de-esterify dissolved pectin with acid while keeping the loss of molecular weight at an acceptable level. It is also possible to de-esterify pectin by use of strong alkali (e.g. NaOH), but the main chain of highester pectin is vulnerable to alkaline conditions, so it is necessary to perform this reaction at low temperature and/or while the pectin is not in aqueous solution. In industrial production such treatment may be done on the pectin precipitate obtained after the precipitation with alcohol by reaction with alkali while the precipitate is suspended in the alcohol. If ammonia is used in the place of other alkali, some ester groups are converted into amide groups, and the resulting product becomes what is known as 'amidated pectin'. 8.1.4

Standardization

Pectin as produced directly by the above procedures is referred to within the industry as 'unstandardized pectin'. It varies from batch to batch owing to variations in the raw material. Because it is a natural and polydisperse material, many of its properties can vary independently. This presents the pectin producer with a challenge, because pectin customers need batch-to-batch consistency. However, it also makes pectin versatile, and major pectin producers have taken advantage of the possibility of differentiation by creating products tailored for use in specific situations (Section 8.5). Standardized commercial pectin types are characterized by a set of specifications. Some specifications are general for all pectin (like those required by authorities, Section 8.2.1), while others, alone or in combination, characterize specific pectin types. The latter are put in place for ensuring batch-to-batch consistency with respect to properties relevant for the intended use. Specifications can be simple, such as a range for the DE or a maximum concentration of a possible contaminant. Other specifications are results of complex test protocols that simulate the industrial use and may even be developed for individual customers. In some cases, the result can be expressed as a grading strength. By definition, a

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grading strength is inversely proportional to the amount of pectin that is needed for imparting a specified effect to a specified application-simulating system, for example a specified firmness to a specified test gel. An example is the grade USA-SAG (Section 8.2.2). 'Standardization' involves the pectin manufacturer mixing batches of unstandardized pectin in appropriate amounts so that the mixture meets the specifications for the intended commercial product. If these specifications include a grading strength, the preparation is further 'diluted' to the specified strength with an appropriate amount of sucrose (or other food-acceptable material that does not contribute to the functionality). Commercial pectin for food use may contain up to almost 50% sucrose. Researchers studying pectin or developing new uses for pectin should be aware of the diversity of pectin and the concept of standardization. Given the broad diversity of pectin types and their substantially different properties, it is hardly satisfactory to undertake a comprehensive study on 'pectin' without paying attention to the details of the specific sample being used. If the sample is sourced from a fine chemicals supplier, one should ask about its history and relevant details before using it for investigations! If the sample is sourced from the pectin industry, be aware that the sample may contain sucrose or other material. Most manufacturers are willing to supply samples without sucrose (e.g. pharmaceutical grades) on request. When commercializing new concepts involving the use of pectin, one should verify that the product specifications for the pectin are adequate for the intended new use. The pectin manufacturer can only warrant batch-to-batch consistency with respect to the specified properties; other properties are still free to vary. 8.2 8.2.1

Commercial definitions and standards 'Pectin' as defined by national and international authorities

Commercial pectin for food use complies with national regulations in the country in which it is sold, and typically complies with the specifications stipulated by FAO (FAO, 1992) as well as those of the Food Chemicals Codex (FCC, 1996) and the European Union (EU, 1998). Pectin for use as a pharmaceutical excipient likewise complies with current national regulations where it is sold and typically with the United States Pharmacopoeia and National Foundry (USP, 2000). These publications all define pectin, devise tests for verifying its identity, and stipulate the minimum content of the main component and maximum contents of possible contaminants. The definition by FAO is as follows: Pectin consists mainly of the partial methyl esters of polygalacturonic acid and their sodium, potassium, calcium and ammonium salts. It is obtained by aqueous extraction of appropriate edible plant material, usually citrus

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fruits or apples. No organic precipitants shall be used other than methanol, ethanol and isopropanol. In some types a portion of the methyl esters may have been converted to primary amides by treatment with ammonia under alkaline conditions. The commercial product is normally diluted with sugars for standardization purposes, and mixed with suitable food-grade buffer salts required for pH control and desirable setting characteristics. The article of commerce may be further specified as to pH value, jelly strength, viscosity, degree of esterification, and setting characteristics.

The content of galacturonide, expressed as galacturonic acid, CftHioO? (Mr 194.1) must, according to FAO, FCC and EU be at least 65% on a sugar-, ashand moisture-free basis. In the analysis, the sample is first flushed with acidified aqueous ethanol, then washed free of the acid with aqueous ethanol and pure ethanol, and finally dried under prescribed conditions. This treatment removes sugar, replaces metal ions with protons, and brings the moisture to a uniform level for all samples. The '65%' means 65% of the sample that is left after the treatment. It should be noted that previous editions of some of the above documents stipulated slightly different criteria and used other formula weights for galacturonide, such as that of anhydrogalacturonic acid (MT 176.1), or that of methyl-esterified and amidated anhydrogalacturonic acid of the appropriate degree of esterification (DE) and degree of amidation (DA). The latter might, for example, be expressed as 176.1 + 0.140 • (DE) - 0.010 • (DA). The definition of the US Pharmacopoeia is different. It deals with pure pectin in the sense that there has been no intentional addition of sugar and buffer salts. It recognizes only citrus fruits and apple pomace as sources for pectin. It further stipulates 'Pectin yields not less than 6.7 percent of methoxy groups (—OCH3) and not less than 74.0 percent of galacturonic acid (CftHioO?), calculated on the dried basis'. The 6.7% correspond to a DE of about 50 (exactly 50 if the percentage of galacturonic acid is 83.8, which is within the range typical of citrus pectin). 8.2.2 Vocabulary of terminology and concepts The concept of 'degree of esterification' has already been used extensively in preceding chapters and the present chapter. It is commonly abbreviated DE, as in the present text, or alternatively as DM. When nothing else is specified, it signifies the percentage of the C-6 carboxylate groups that are esterified with methanol. Pectins are classified as high-methyl-ester pectins (HM pectins) if their DE is 50 or above, and low-methyl-ester pectins (LM pectins) if their DE is less than 50. The methyl ester groups were referred to as methoxyl groups in older literature, and terminologies like 'degree of methoxylation' and 'highmethoxyl pectin' are still commonly used despite their inaccuracy. For amidated pectins, the degree of amidation (DA) is the percentage of C-6 positions of the galacturonide that are amide groups.

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HM pectins are subdivided by some manufacturers according to their relative quickness of setting into 'rapid set', 'medium rapid set' and 'slow-set' types (see Sections 8.3.5 and 8.4.3 for details). HM pectin for gelation is usually standardized to a specific grading strength according to the method known as 'grade USA-SAG' (IFT, 1959). In this method the grading strength of the pectin is measured by using it for making a test gel, which is cast in a glass of specified dimensions and which contains 65% soluble solids at a pH of 2.30 ± 0.10. If the appropriate amount of pectin has been used, the gel must have such relative firmness that, once it is removed from the glass, it sags by 23.5% of its original height within 2 minutes. The typical specification '150 grade USA-SAG' means that one part of pectin is enough for making this gel from 150 parts of sucrose. The result of the test correlates nicely with the intrinsic viscosity of the pectin (Christensen, 1954), and with the elastic modulus determined by oscillatory rheology (Nielsen et al, 2001). HM pectin for stabilization of acidified milk beverages (Section 8.5.2) can be graded by preparing a series of beverages with various dosages of pectin and then measuring the sediment that forms upon centrifugation. A standard batch, which by definition is 100 grade, is run in parallel with the unknown sample, and the curve of sediment versus pectin dose is recorded for both samples. The grade is then 100 times the number by which all the pectin doses of the 'unknown' curve must be multiplied in order to make the curve superimpose as well as possible upon the 'standard' curve. One of the major manufacturers markets pectins standardized by grade SAM (testing with milk acidified by glucono 5lactone) and grade YOG (testing in yoghurt), but there is no universal agreement between manufacturers and customers about a grading test. LM pectin for gelation can be graded by procedures analogous to the USASAG test, and various types can be classified according to their calcium reactivity, like the HM pectin classification into rapid-set and slow-set types. Owing to the diversity of conditions under which LM pectins can be used, however, there are no conventions that share the same broad acceptance as those for HM pectins. 8.3 General properties 8.3.1

Composition

Commercial pectin, produced as described previously, is highly polydisperse. Samples differ with respect to the averages of molecular weight, DE, content of other substituents (such as amide groups and O-acetyl groups), and the proportion between galacturonide material and other polymeric material. Within a sample, these properties vary intermolecularly and can be described with fairly broad statistical distributions. Further, the intramolecular distribution of substituents can vary. The free (unesterified) C-6 carboxylate group, in

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particular, can be present in a blockwise arrangement, with local areas of consecutive unesterified anhydrogalacturonic moieties in the galacturonan chain (Speiser and Eddy, 1946; Daas et al, 1999,2000,2001). In other samples it may have a more uniform presence. Blocks of free carboxylate groups are created by pectinesterases, which are native to the raw materials and serve a physiological role in the living plants (Chapters 6 and 7). The pectin manufacturer can control the (average) DE. Depending on the pectin type it can have any value less than about 80. The weight-average molecular weight of commercial pectin samples is approximately 200 kDa as determined by liquid chromatography with lightscattering detection (see, e.g., Hourdet and Muller, 1991; Gamier etal., 1993). A natural tendency for aggregation of molecules, even in dilute solution, makes interpretation of light-scattering data uncertain (Hourdet and Muller, 1991; Kravtchenko et al, 1992a; Berth et al, 1994). As previously mentioned, commercial pectin preparations may contain sugar or other material that has been added for the purpose of standardization (Section 8.1.4). It may further contain salts, and contains cations (e.g. Na^) for partial neutralization of the carboxylate groups. The part of the pectin that does not dissolve upon washing in acidified alcohol followed by washing in pure alcohol—the 'pure gum'—must according to legislative standards account for at least 50%. The pure gum, in turn, must contain at least 65% galacturonide (see Section 8.2.1 for details and analytical method). The actual value differs according to the raw material: it is typically about 85% for lemon pectin, while 67% has been reported for apple pectin (Kravtchenko et al, 1992b) (the values reported in the original article as percent anhydrogalacturonic acid have been converted to galacturonic acid by multiplication by 1.102). Sugar beet pectin contains close to (but with commercial samples above) 65% galacturonide. The part of the pure gum that is not galacturonide yields neutral sugars, predominantly galactose, arabinose and rhamnose, upon hydrolysis. It occurs as relatively short side chains to rhamnosyl units in the otherwise linear polymer, and these side chains occur in a blockwise fashion, that is the pectin molecule contains so-called 'hairy regions' and 'smooth regions' (Chapter 1). 8.3.2 Acidic properties Pectin is a polyprotic, weak acid, and as such has no single, well-defined pK. As with all polyelectrolytes, the pK depends upon the current charge density of the polymer (and thus, in turn, on the pH). The apparent pK (the pK at 50% dissociation) depends somewhat upon the conditions, and various investigators report values ranging from 3.5 to 4.5 (Plaschina et al, 1978; Michel et al, 1984; Racape et al, 1989). Estimates for the intrinsic pK, that is the pK extrapolated to zero degree of dissociation, range from 2.9 to 3.3 (Michel et al, 1984; Racape etal, 1989).

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8.3.3 Interactions Pectin is soluble in pure water. It is insoluble in alcohol and most other solvents. It forms insoluble salts with di- and trivalent metal ions. Pectin further forms insoluble complexes with positively charged polymers (this is sometimes referred to as associative separation). With negatively charged polymers, such as proteins that are above their isoelectric point, pectin may undergo segregative separation (Tolstoguzov, 1988): the mixture develops into two coexisting aqueous phases, one phase enriched in pectin and the other enriched in protein. 8.3.4 Chemical stability Pectin generally has good stability in aqueous solution at pH around 3-4, which is the pH typical of the major food uses for pectin. Above this pH, the galacturonan chain of pectin depolymerizes by a mechanism known as J3elimination. The ^-elimination occurs at glycosidic bonds to the C-4 position of anhydrogalacturonic moieties that are methyl-esterified at their C-6 carboxylate group. HM pectin is particularly vulnerable (Albersheim et al, 1960; Krall and McFeeters, 1998), and decay is noticeable at pH above 5 unless the temperature is below room temperature. Owing to this intolerance to high pH, it is difficult to adjust the pH of pectin solutions in the upward direction: decay takes place at the interfaces that exist temporarily when the titrant meets the pectin solution. LM pectin is more robust, but still should not be exposed to combinations of high temperature and pH above 5. At low pH, some depolymerization of the galacturonan chain is caused by hydrolysis. The effect is noticeable if a pectin solution is left for several hours at high temperature and pH less than about 3, but it is not a problem with ordinary handling for food use. Pectin of low DE decays faster than pectin of high DE (Krall and McFeeters, 1998). Pectin can be degraded by various enzymes, some of which are produced by plants (Chapter 6). Microorganisms produce other enzymes for the purpose of catabolizing vegetable material (Chapter 7); such enzymes are produced commercially by fermenting with these microorganisms for use in the fruit processing industry. Failures in pectin use can sometimes be related to the presence of enzymes, for example if vegetable material that has previously been treated with pectolytic enzymes is used together with pectin without having been subjected to an adequate enzyme-denaturing heat treatment. 8.3.5 Parameters influencing thickening and gelling Pectin gels by a combined action of fundamentally different mechanisms. One mechanism is characteristic of HM pectin. It is preponderant when HM pectin is gelled under the conditions of classical jams: pH about 3 and in the presence of about 65% of dissolved solids (mainly sugar). Another mechanism is characteristic of LM pectin: it is preponderant when LM pectin is gelled at pH above 3

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in the presence of a sufficient amount of appropriate di- or trivalent metal ions, in food use typically Ca2+. Further, pectin of low DE may gel at low pH (less than about 2.5 depending upon other conditions). The rheology of a preparation, which contains dissolved pectin, depends upon properties of the pectin as well as properties of the system. Influential properties of the pectin are the molecular weight, the amount and distribution pattern of substituents such as C-6 methyl ester groups, C-6 amide groups, and O-acetyl groups. Relevant parameters of the system include the pH, presence of dissolved solids or solvents, ionic strength, presence of specific metal ions, the concentration of pectin and the temperature. The relationship between these parameters and the resulting rheology is complex, because the rheology is influenced by diverse mechanisms of molecular association that under appropriate circumstances lead to the kinds of gelation described above. For HM pectin in conditions close to those of jam (pH less than 3.5 and typically about 3, soluble solids at least 60% and typically about 65%). high degree of esterification (DE) means high gelation temperature when preparing gels by the usual procedure (Section 8.4.3) of mixing the hot ingredients and then solidifying by cooling. When the gel batch is cooled below the gelling temperature, gelation occurs after a delay, which is short with pectin of high DE and longer with pectin of lower DE. HM pectin is accordingly classified into 'rapid set', 'medium rapid set' and 'slow set' types. Rapid set typically has a DE around 72 and will solidify jam at about 75 C under normal conditions of use (Section 8.5.1), while slow-set has a DE around 62 and causes gelation at about 60°C under similar conditions. It should be cautioned, however, that the gelation temperature is a function of all of the aforementioned parameters, and the designations 'rapid set' and 'slow set' only signify relative differences between the pectins under their normal conditions of use. The gelation of HM pectin exhibits hysteresis in the sense that the melting temperature is higher than the setting temperature. With typical uses, the melting temperature is so high that from a practical point of view it is impossible to re-melt the gel without causing destruction, and gels with HM pectin are accordingly often characterized as 'thermo-irreversible'. The distribution of free carboxylate groups and methyl-esterified carboxylate groups along the molecular chain is of importance to the functionality. HM pectin with blocky DE distribution (Section 8.3.1) gels at higher temperature than pectin with the same (average) DE in a more uniform distribution. When used with calcium-rich fruit material, blocky pectin may create heterogeneous gels, in which the surface that forms upon cutting with a sharp knife contains visible grains. In some cases, the breaking strength of gels may increase with the amount of Ca2+, but in other cases the heterogeneity becomes so pronounced that gels have poor breaking strength and increased tendency to syneresis. Blocky HM pectin is for the same reason sometimes referred to as 'calcium sensitive' pectin. Blocky HM pectin, on the other hand, is superior to other pectin for

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stabilization of milk-containing beverages of pH about 4, such as drinking yoghurt (Section 8.5.2). LM pectin finds much use for jam with lower sugar content and slightly higher pH than regular jams, such as 55% soluble solids and pH 3.4. The relation between setting temperature and DE is the opposite of the corresponding relation for HM pectin: in the case of LM pectin the setting temperature is inversely correlated to the DE. The gelation with LM pectin exhibits little or no delay and little or no difference between gelling temperature and melting temperature. Amidated LM pectin is widely used for jam with reduced sugar content because, compared to conventional LM pectin, it yields gels with lower tendency to syneresis. Further, at typical conditions of use, the texture is not affected by moderate variations in the presence of Ca2+ (such variations might, for example, originate from variations in the fruit that is used for the jam). Amidated HM pectin gels under the conditions typical of uses for HM pectin at lower temperature than nonamidated pectin of similar DE (Ehrlich and Cox, 1974). Pectin from other sources than citrus may contain a considerable presence of ester-bound acetate of which at least part in the case of sugar beet pectin is (9-acetyl groups at the C-2 and C-3 positions in the homogalacturonan part (Rombouts and Thibault, 1986). Sugar beet pectin contains one ester-bound acetate group per four to five galacturonide moieties. This presence of esterbound acetate is sometimes used as an explanation for the inferior gelling properties of sugar beet pectin compared to citrus pectin, and it has accordingly been shown that citrus pectin loses its ability to form gels if 0-acetyl groups are introduced by reaction with acetic anhydride (Pippen et al., 1950). Commercial production of LM pectin by careful acid-hydrolysis of ester-bound acetate (and some C-6 methyl ester) took place in Europe during and after World War II, and this treatment increased the gelling strength of the pectin. The functionality of such acid-treated beet pectin is, however, still very different from that of citrus pectin. Apple pectin contains some ester-bound acetate (Voragen et a/., 1986), but it is an excellent gelling agent nevertheless. Thus the differences between various botanical sources of pectin with respect to their suitability for gelation is not merely a question of the presence of acetate. Sugar beet pectin has a unique ability to stabilize emulsions (Section 8.5.3), arguably due to the content of ester-bound acetate (Dea and Madden, 1986). The grading strength of pectin for gelation is positively correlated with the apparent molecular weight according to methods that infer the molecular weight from the intrinsic viscosity (Christensen, 1954; Kim et al., 1978; Van DeventerSchriemer and Pilnik, 1987). The gelling temperature and the firmness of gels are inversely correlated with pH (Smit and Bryant, 1968). This is true for HM pectin in the vicinity of its typical conditions of use (Section 8.5.1), as well as for LM pectin at its typical use conditions. The viscosity of pure aqueous solutions is likewise inversely correlated with pH, and pectin of low DE can gel at pH less than about 2.5

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(Speiser et al., 1947; Gilsenan et al., 2000). Moderate concentrations of alkali metal salts found in typical food systems will usually decrease the viscosity as compared to the pure solution, while very high concentrations suppress the solubility and, for example, pectate may be gelled with K + (Buhl, 1990). The presence of salts ofdi- or trivalent metals suppresses the solubility of pectin, and this can, depending upon the conditions, either reduce or increase the viscosity, or gel the solution. In food applications, Ca2+ is the ion, other than H + , that has the largest influence on pectin performance. An appropriate level of Ca2+ is required for gelling LM pectin, and the gelling temperature as well as the firmness of gels are both positively correlated with the concentration of Ca2^. The viscosity of an aqueous solution of pectin together with Ca2+ is usually positively correlated with pH within the interval of 2.5 < pH < 4.5, which is in contrast to the behavior of a pure pectin solution. Blocky HM pectin may form pseudoplastic, semigelled solutions (Rolin, 1994). In spite of its similarity to Ca2+, Mg2+ has much less impact on pectin rheology, and it is unable to gel LM pectin systems (Malovikova et al., 1994). A certain level of dissolved solids is required for HM pectin gelation and, in the vicinity of typical use conditions, gelling temperature and firmness of gels are both positively correlated to the content of dissolved solids. About 60% sucrose is needed, but other sugars differ slightly with respect to the minimum concentration that can create gelation. About 20% of alcohol (or other solvents) (Oakenful and Scott, 1984) can also gel an aqueous HM pectin solution. Syrup made from enzymatically depolymerized starch is often used in the place of sucrose. In this case, it is even more important to control the pH precisely in order to achieve the desired setting temperature, as compared to when sucrose is used, because the dependence of setting temperature upon pH is steeper with syrup (May, 1990; May and Stainsby 1986). LM pectin may gel in the absence of dissolved solids, apart from the pectin itself, but gels with less than about 20% soluble solids are not very resilient and have a high tendency to syneresis. 8.4 Handling advice 8.4.1 Making an aqueous solution Adding pectin directly to water with no or only moderate agitation is not recommended, because it will form lumps. The lumps consist of a sticky shell surrounding a core of dry powder. Lumps of partially hydrated pectin tend to stick to surfaces, spindles, etc.; and tearing the lumps apart takes a very long time. Pectin solutions can be made in three ways: •

Use high-shear mixing equipment (e.g. in the laboratory, a blender) and add the pectin gradually to the vortex. If the equipment has variable speed, start with slightly reduced speed and increase the speed as the viscosity

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•

•

23 3

increases. The temperature is not very critical, but about 60-80°C is optimal. Moisten the pectin with, or disperse it in, a liquid in which it is insoluble, then gradually add water while stirring efficiently (but not necessarily with high-shear mixing equipment). For laboratory purposes, ethanol and 2-propanol are convenient. For food use, the pectin can be dispersed in a concentrated sugar solution. Dry-mix the pectin with at least five parts of sugar, then add this powder mixture gradually to water while stirring efficiently (but not necessarily with high-shear mixing equipment).

Solids and solvents lower the solubility, and the refractometer solids of the final solution should be less than 25% in the case of HM pectin. In turn, this limits the possible pectin concentration if one of the latter two methods, involving dispersing agents, have been used. Heating solutions made with dispersing agents to about the boiling point of water in order to ensure full hydration is recommended. When dispersing at 60-80°C with high shear mixing equipment, it is possible to make a 12% solution of standardized pectin, but such a thick solution is almost impossible to handle or to mix with other ingredients. 8.4.2 Mixing pectin with other ingredients In some cases, it is difficult to blend pectin, or a pectin solution, uniformly with other ingredients. Taking classical jam as an example, it is on one hand desirable to minimize the addition of water, because water must be evaporated by boiling in order to reach a solids content of about 65% in the final jam; accordingly the pectin solution should be as concentrated as possible. On the other hand, agitation should be gentle in order to avoid rupturing fruit pieces (e.g. strawberries). When a concentrated, highly viscous pectin solution is added, it is difficult to ensure uniform distribution with such gentle agitation. This is particularly the case if the pectin is calcium sensitive and the fruit is rich in calcium: with moderate agitation, the concentrated pectin solution has enough time to gel at the (otherwise temporary) interfaces where the pectin solution meets the other ingredients. The compromise between these conflicting considerations is that classical jam usually is made with a 5-8% pectin solution (depending upon the pectin type and the fruit). In general, the following are recommended: • •

Avoid blending pectin, or a pectin solution, into a mixture of ingredients that cause pectin gelation. If there is no reason to limit the addition of water, then make a relatively dilute pectin solution (e.g. 1-2%) before blending that solution with the other ingredients.

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Use ion-exchanged or distilled water for the solution when possible. If use of a concentrated pectin solution is necessary, and the concentrated solution is going to be blended with Ca-rich ingredients, then the mixing process can be considerably improved if a Ca-sequestrant is included in the pectin solution. Since local gelation at a temporary interface is a kinetic phenomenon, it is not necessary to stoichiometrically compensate for all of the Ca2+ in the ingredients. About 50 mg sodium hexametaphosphate per gram of pectin is usually suitable (if it is acceptable in the final application).

8.4.3 Making gels Gels can generally be made either by mixing the ingredients at a temperature that is above the gelling temperature, followed by cooling under quiescent conditions, or by quickly mixing the ingredients at a temperature below the gelling temperature and then leaving the preparation quiescent until it sets. The first procedure, gelling by cooling, is more frequently used than the second. The following steps are recommended in making a gel with HM pectin: • •

•

•

• • •

•

Prepare a pectin solution, preferably with ion-exchanged water. Mix the other ingredients in such proportion so that the content of dissolved solids, once the pectin solution is added at a later time, becomes slightly below the value that supports gelling, and the pH becomes about 3.6. Allow time for osmotic equilibration between ingredients (e.g. between the continuous phase and fruit particles). Add the pectin solution to the warm or hot mixture of other ingredients under as good agitation as can be provided (while respecting other considerations such as avoiding rupture of fruit pieces). Evaporate water by boiling until the content of dissolved solids reaches the desired level, typically 65% (in commercial jam manufacture this is often done under vacuum in order to minimize decay of fruit color and aroma). Heat to pasteurization temperature (if the batch was boiled under vacuum it might not be hot enough at this stage). Adjust the pH to 3.0-3.1 with, for example, a solution of citric acid. Cool to the filling temperature (cooling may be desirable if gelation shortly after filling is required in order to prevent fruit flotation, or if the gel is cast in a large container that cools so slowly that heat-deterioration of the product would become unacceptable). Cast the gels and continue cooling under quiescent conditions.

The rationale behind the sequence in which these operations are done is that the conditions must not be too close to gelling conditions when the pectin is mixed

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with other ingredients. With HM pectin this is, in particular, ensured by keeping the pH above the value that can support gelation until shortly before rilling. A gel with LM pectin is essentially made by the same operations, namely, mixing at solids below and pH above those of the final product, then bringing the solids up, and finally bringing the pH down shortly before casting the gel. However, the applications with LM pectin are more diverse, so it is not possible to provide quantitative, generally valid, limits for solids content and pH. Cold gelation of HM pectin can be done, for example, by preparing two syrups of about the same volume and both containing the amount of solids that is desired in the final gel. The one preparation contains all of the pectin, and the pH of that syrup made too high for gelling. The other syrup contains sufficient acid for bringing the pH in the mixture of the two syrups to between 3.0 and 3.1. Gelation is induced by mixing the two syrups, and then leaving them quiescent to gel. Here, one takes advantage of the fact that the HM gelation is delayed, which leaves time for manipulation before gelation commences. Cold gelation of LM pectin is also possible, for example by making a pectincontaining preparation without Ca2+, and then inducing gelation by mixing the preparation with a Ca2+-containing liquid such as milk. The problem with this concept, however, is that LM pectin gelation with Ca2+ is almost instantaneous and leaves little time for manipulation. 8.4.4 Avoiding degradation—storing pectin Pectin loses less than 5% of its grading per year when stored under cool and dry conditions. 'Cool' means ambient temperature in places that only rarely exceed 25°C. More is lost at higher temperature or in moist conditions (Padival et al., 1981). Although it is not usually prescribed that pectin must be kept cold, use of a freezer (about — 18°C) is recommended with samples for scientific purposes and samples used for referencing, when they are to be kept for several years. Depending upon the conditions, pectin solutions can be kept for about a day at a temperature not exceeding 60°C. Good hygiene is needed because mold or other microorganisms can infect the solution. The stability declines with increasing temperature, and one must carefully observe the pH requirements of pectin (Section 8.3.4). Adjustment of pH with alkali may cause decay if it is not done carefully. The risk is worst with HM pectin and if the temperature is high and/or the mixing is inefficient and/or if hydroxides are used. 8.5 Uses 8.5.1 Sweet, fruit-flavored gels Jam is the oldest, and still the best-known, application for pectin. Traditional jam manufacture, before industrial pectin was available, utilized the natural pectin

236

PECTINS AND THEIR MANIPULATION

content of the fruit. The pectin was extracted by prolonged boiling, and it was necessary to include fruit of high pectin content in the recipe. Modern industrial jam manufacture is, in comparison, gentler to the color, aroma, and vitamins of the fruit, and the kind of fruit can be chosen more freely. The original concept of jam has today been expanded to a range of gels and thickened foods, which have in common a certain content of dissolved solids (typically sugar) and a pH of less than about 4 (compatible with fruit flavor). LM pectin may be used at pH of about 4.3. This is still acidic, but not more acidic, if the buffer capacity of the food system is not too high, than is compatible with neutral flavors such as vanilla, mint and toffee. Industrial pectin is designed to impart specific textures and specific gelling and re-melting properties. Products that represent characteristic examples of textures that are possible with pectin, include • • •

•

so-called 'fruit jellies' (rather firm, resilient, fruit-flavored candies) ordinary jam (gelled, but able to be spread on a slice of bread with a knife) fruit preparations for industrial yoghurt manufacture (similar to jams, but with a soft texture that allows for pumping the preparation through pipes, etc., while it is still strong enough to prevent fruit flotation) chutneys, ketchup and the like (thickened, pseudoplastic texture)

Various representatives for specific gelling and re-melting properties include • • • •

glazings for pies and cheesecakes (gels that can be melted and applied to hot tarts or cakes, where they float and create a uniform glazing before solidifying as the cake cools) heat-resistant jam for pastry (must withstand baking without melting) strawberry jam (should gel shortly after casting so that the fruit does not drift to the top of the jar) clear gels (should solidify after a delay that allows air bubbles to escape)

Some examples are summarized in Table 8.1. 8.5.2 Use with fermented or acidified milk Stabilization of beverages of pH about 4 that contain milk as an ingredient is a large application for HM pectin. One example is drinkable yoghurt with live culture and content of milk solids of about 8.5% on a fat-free basis (i.e. the natural content in cow's milk, abbreviated 8.5% MSNF, milk solids non fat). Another example is pasteurized soft drinks with about 2-3% MSNF, which are popular products in Japan and other Asia-Pacific countries. The purpose of adding pectin is to prevent whey-separation and formation of lumps, which generally tends to occur in the absence of added stabilizers. The ideal product should have a smooth mouthfeel, total absence of separated whey, minimal

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237

Table 8.1 Jams and other sweet, fruit-flavored gels Food product

Pectin type and use level

Dissolved solids (%)

Classical jam

0.3% HM pectin

>60; typically 65

3.0-3.1

Ca 2 + not necessary, but gel will not form at higher pH or lower solids than specified

Jam with reduced sugar

0.6% amidated LM pectin

35-55

3.1-3.4

Ca 2 + must be present, but the natural content in fruit is usually adequate

Heat-resistant jam for baked goods (I)

0.6-1.0% very Ca-reactive LM pectin

65

3.5-3.6

Pumpable preparation that becomes coherent on baking but does not flow. The combination of ingredients yields high melting point

0.6% HM pectin

65

3.2-3.3

Firm gel. Withstands baking while not flowing. Thermo-irreversibility of HM gels is utilized

Heat-reversible glazing

1.2% Ca-reactive amidated LM pectin

65

3.5

The gelled system is diluted with water, then melted and poured over hot tarts, etc.

Fruit preparation for yoghurt

0.3-0.45% amidated LM pectin

40-65 3.8-4.0 flotation,

Texture prevents fruit but allows for pumping through pipes, etc.

'Fruit jelly'with fruit flavor

0.8-1.5% slow-setting HM pectin

78

3.5

Firm, resilient gel

Jelly with toffee flavor

1.5-2.5% Amidated LM pectin of medium calcium reactivity

78

4.3

Firm, resilient gel

pH

Remarks

sediment, and a batch-to-batch consistent viscosity with little or no yield. Pectin, in particular LM pectin, is also used for increasing the firmness of spoonable yoghurt. Both uses can be explained as two regimes of the same mechanism. At pH 4.0 ± 0.4, typical of yoghurt, the milk protein has a weakly positive net charge. Most of the protein, at least the casein part, exists as water-insoluble particles. The positive charge is not enough to prevent these particles from sticking together. In spoonable yoghurt they form a network, which under quiescent conditions is kinetically (mechanically) prevented from rearranging further. In beverages the protein forms an unstable suspension in which the particles (if no stabilizer is added) are not prevented from growing bigger by continued flocculation. Pectin, being negatively charged, binds to the protein particles by electrosorption (possibly assisted by contributions from other molecular forces)

238

PECTINS AND THEIR MANIPULATION

(Glahn, 1982; Parker et al, 1994). At partial surface coverage the pectin causes flocculation, and this flocculation can reinforce the natural network of spoonable yoghurt and thus increase the firmness of the yoghurt. Owing to the blockiness of the substitution pattern of pectin (Section 8.3.1), however, various parts of the pectin have different affinity for the surface. At full surface coverage, those areas in the pectin molecules that bind strongly to the protein force other molecular areas with less strong affinity away from the surface. The latter low-affinity areas may be part of the same long molecules, which bind strongly at other points, and it may be envisioned that they protrude from the surface as loops and dangling tails as in the description of the general mechanism termed 'steric stabilization' (see, e.g., Everett, 1988). At sufficient pectin concentration, polymer-enriched zones will entirely surround the protein particles. Since pectin is generally watersoluble, these zones are more hydrated and not as sticky towards each other as are the bare protein particles. This gives stability in the sense of smooth mouthfeel, no whey exudation, and so on. 8.5.3 Stabilization of oil-in-water emulsions Sugar beet pectin may be used for stabilizing oil-in-water emulsions (Dea and Madden, 1986). Emulsions with sugar beet pectin can withstand substantial dilution with water without breaking immediately, as would happen, depending upon the water-solubility of the particular oil, with most other stabilizers owing to Ostwald ripening. Sugar beet pectin may thus be used as an alternative to gum arabic for flavor oil emulsions, which are typically 10-20% oil/water but are diluted with about 500 parts of other ingredients in the final application. 8.5.4 Fat replacement Water-insoluble beads may be prepared from Ca-reactive pectin and Ca-salt. A commercial product range, Slendid®, utilizes such beads to mimic the mouthfeel of fat in low-calorie foods (Hoefler et al., 1994; Glahn, 2001). The user can prepare the beads in controlled size, from about 30 jim to about 250 |im. 8.5.5 Pharmaceutical uses A rather large quantity of pectin is used in wound bandages and gaskets for receptacles for intestinal fluids from stomatized patients. Pectin is typically used together with other water-soluble or water-swellable biopolymers such as carboxymethylcellulose and gelatin. The biopolymers are mixed with a waterinsoluble polymer such as polyisobutylene and made into a thick film, which constitutes the skin-contacting part of the bandage (Chen, 1967). The most important function of the water-swellable polymers is to suck up wound exudate or other bodily fluids and to impart a suitable tackiness when contacting moist skin or a wound surface. Pectin may, in addition to fulfilling these mechanical

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239

objectives, contribute a more active role in wound healing, since the use in traditional medicine of certain plants for this purpose seems to be related to the pectin content of the plants. Pectin from Plantago major has been shown to enhance wound healing in mice; at least part of this beneficial effect is because the pectin stimulates the immune system of the wounded animal (Hetland et al, 2000). Looking beyond skin lesions and also including gastric ulcers, pectin of certain plants such as Panax ginseng (Sun etal, 1993) and Bupleurum falcatum (Sun et al., 1991) have documented healing effects. Pectin is used in medicine for treating so-called 'heartburn', irritation of the gullet (esophagus) when acidic gastric juice leaks ('refluxes') from the stomach. The medicine consists of pectin of low DE (less than about 10) in mixture with acid-neutralizing and CO2-evolving agents (Foldager et al., 1991; Waterhouse et al., 2000). When swallowed, the preparation, once it enters the stomach, produces a stable foam of pH above 3, which floats atop the stomach contents and prevents them from moving into the gullet without being partly neutralized. The advantages over other neutralizing agents are that it is not necessary to neutralize all of the stomach contents, and the retention time of the medicine in the stomach is longer. A proteinaceous substance, such as casein, may be incorporated to strengthen the foam and increase its buffer capacity. Acknowledgements The author is indebted to Jens Trudsoe, Heidi Nielsen, Dorthe Pedersen, Jeanette Kristensen and Todd Talashek for identifying relevant references, providing practical information about pectin handling, and critically reviewing the manuscript. References Albersheim, P., Neukom, H. and Deuel, H. (1960) Splitting of pectin chain molecules in neutral solutions. Arch. Biochem. Biophys., 90, 46. Berth, G., Dautzenberg, H. and Hartmann, J. (1994) Static light scattering technique applied to pectin in dilute solution. Part III: The tendency for association. Carbohydr. Polytn., 25, 197. Buhl, S. (1990) Gelation of very low DE pectin. Gums Stabiliz. Food Ind., 5, 233. Chen, J.L. (1967) Bandage for adhering to moist surfaces. US patent 3,339,546. Christensen, P.E. (1954) Methods of grading pectin in relation to the molecular weight (intrinsic viscosity) of pectin. Food Res., 19, 163. Daas, P.J.H., Meyer-Hansen, K., Schols, H. A., DeRuiter, G.A. and Voragen, A.G.J. (1999) Investigation of the non-esterified galacturonic acid distribution in pectin with endopolygalacturonase. Carbohydr. Res., 318, 135. Daas, P.J.H., Voragen, A.G.J. and Schols, H.A. (2000) Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase. Carbohydr. Res., 326, 120. Daas, P.J.H., Boxma, B., Hopman, A.M.C.P., Voragen, A.G.J. and Schols, H.A. (2001). Nonesterified galacturonic acid sequence homology of pectins. Biopolymers, 58, 1.

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Dea, I.C.M. and Madden, J.K. (1986) Acetylated pectic polysaccharides of sugar beet. Food Hydrocolloids, 1, 71. Ehrlich, R.M. and Cox, R.E. (1974) Slow set pectin and process for preparing same. US patent 3,835, HI. EU (1998) Directive of 11 November, 1998 (98/86/EEC). Everett, D.H. (1988) Basic Principles of Colloid Science, Royal Society of Chemistry Paperbacks, London. FAO (1992) FAO Food and Nutrition Paper, 52. FCC (1996) Food Chemicals Codex, 4th edn, Washington. DC. Foldager, J., Toftkjaer, H. and Kjornos, K. (1991) Antacid composition. US patent 5,068,109. Gamier, C., Axelos, M.A.V. and Thibault, J.-F. (1993) Phase diagrams of pectin-calcium systems: influence of pH, ionic strength, and temperature on the gelation of pectins with different degree of methylation, Carbohydr. Res., 240, 219. Gilsenan, P.M., Richardson, R.K. and Morris. E.R. (2000) Thermally reversible acid-induced gelation of low-methoxy pectin. Carbohydr. Polym., 41, 339. Glahn, P.E. (1982) Hydrocolloid stabilization of protein suspensions at low pH. Prog. Food Nutr. Sci.. 6, 171. Glahn, P.E. (2001) Pectin process and composition. US patent 6,207,194. Hetland, G., Samuelsen, A.B., Lovik, M.. et al. (2000) Protective effect of Plantago major L. pectin polysaccharide against systemic Streptococcus pneumoniae infection in mice. Scand. J. Immunol.. 52, 348. Hoefler, A.C., Sleap, J.A. and Trudsoe, I.E. (1994) Fat substitute. US patent 5,324,531. Hourdet. D. and Muller, G. (1991) Solution properties of pectin polysaccharides II. Conformation and molecular size of high galacturonic acid content isolated pectin chains. Carbohydr. Polym.. 16. 113. IFT (1959) Final report of the committee, pectin standardization. Food Technol., 13, 496. Kim. W.J., Rao, V.N.M. and Smit, C.J.B. (1978) Effect of chemical composition on compressive mechanical properties of low ester pectin gels. J. Food Sci., 43. 572. Krall, S.M. and McFeeters, R.F. (1998) Pectin hydrolysis: effect of temperature, degree of methylation. pH, and calcium on hydrolysis rates. J. Agric. Food Chem.. 46, 1311. Kravtchenko, T.P., Voragen, A.G.J. and Pilnik, W. (1992a) Analytical comparison of three industrial pectin preparations. Carbohydr. Polym., 18. 17. Kravtchenko, T.P., Berth, G., Voragen, A.G.J. and Pilnik, W. (1992b) Studies on the intermolecular distribution of industrial pectins by means of preparative size exclusion chromatography. Carbohydr. Polym., 18, 253. Malovikova, A., Rinaudo, M. and Milas, M. (1994) Comparative interactions of magnesium and calcium counterions with polygalacturonic acid. Biopolymers, 34. 1059. May, C.D. (1990) Industrial pectins: sources, production and applications. Carbohydr. Polym.. 12. 79. May, C.D. and Stainsby, G. (1986) Factors affecting pectin gelation. Gums Stabiliz. Food Ind.. 3, 515. Michel, F., Thibault, J.-F. and Doublier, J.-L. (1984) Viscosimetric and potentiometric study of highmethoxyl pectins in the presence of sucrose. Carbohydr. Polym., 4. 283. Ni, Y., Yates, K.M. and Zarzycki, R. (1999) Aloe pectins. US patent 5,929,051. Nielsen, H., Marr, B.U. and Hvidt, S. (2001) Correlation between sagging and shear elasticity in pectin. gelatin and polyacrylamide gels. Carbohydr. Polym., 45. 395. Oakenful, D. and Scott, A. (1984) Hydrophobic interaction in the gelation of high methoxyl pectins. J. Food Sci., 49, 1093. Padival, R.A., Ranganna, S. and Manjrekar, S.P. (1981) Stability of pectins during storage. J. Food Technol., 16, 367. Parker, A., Boulenguer, P. and Kravtchenko, T.P. (1984) Effect of the addition of high methoxyl pectin on the rheology and colloidal stability of acid milk drinks, in Food Hydrocolloids: Structures, Properties, and Functions, (eds K. Nishihari and E. Doi), Plenum Press, New York, p. 307. Pippen, E.L., McCready, R.M. and Owens, H.S. (1950) Gelation properties of partially acetylated pectins. J. Am. Chem. Soc., 72. 813.

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Plaschina, I.G., Braudo, E.E. and Tolstoguzov, V.B. (1978) Circular dicroism studies of pectin solutions. Carbohydr. Res., 60, 1. Racape, E., Thibault, J.-E, Reitsma, J.C.E. and Pilnik, W. (1989) Properties of amidated pectins II. Polyelectrolyte behavior and calcium binding of amidated pectins and amidated pectic acids. Biopolymers, 28, 1435. Rolin, C. (1994) Calcium sensitivity of high ester citrus pectins. Gums Stabiliz. Food Ind., 7, 413. Rombouts, EM. and Thibault, J.-F. (1986) Enzymic and chemical degradation and the fine structure of pectins from sugar beet pulp. Carbohydr. Res., 154, 189. Smit, C.J.B. and Bryant, E.F. (1968) Ester content and jelly pH influences on the grade of pectins. J. Food Set., 33, 262. Speiser, R. and Eddy, C.R. (1946) Effect of molecular weight and method of deesterification on the gelling behavior of pectin. J. Am. Chem. Soc., 68, 287. Speiser, R., Copley, MJ. and Nutting, G.C. (1947) Effect of molecular association and charge distribution on the gelation of pectin. J. Phys. Colloid Chem., 51, 117. Sun, X.-B., Matsumoto, T. and Yamada, H. (1991) Effects of a polysaccharide fraction from the roots of Bupleurum falcatum L. on experimental gastric ulcer models in rats and mice. /. Pharm. Pharmacol, 43, 699. Sun, X.-B., Matsumoto, T. and Yamada, H. (1993) Anti-ulcer activity and mode of action of the polysaccharide fraction from the leaves of Panax ginseng. Planta Med., 58, 432. Tolstoguzov, V.B. (1988) Concentration and purification of proteins by means of two-phase systems: membraneless osmosis process. Food Hydrocolloids, 2, 195. USP (2000) US Pharmacopeia & National Foundry USP24/NF19. Van Deventer-Schriemer, W.H. and Pilnik, W. (1987) Studies on pectin degradation. Acta Aliment., 16, 143. Voragen, A.G.J., Schols, H.A. and Pilnik, W. (1986) Determination of the degree of methylation and acetylation of pectins by HPLC. Food Hydrocolloids, 1, 65. Waterhouse, E.T., Washington, C. and Washington, N. (2000) An investigation into the efficacy of the pectin based anti-reflux formulation—Aflurax. Int. J. Pharmaceut., 209, 79.

Index

abscission 144, 150, 154, 162 Acacia Senegal 15 acericacid 10,61,71.72.82 acetate 78 [uC]acetate 87 acetyl esterases 208,213.214 groups 8, 33 transferase 52. 80 acetyl-CoA 76.87 ['4C]acetyl-CoA 87 acetyl-esterification 153 acetylate 73,78,86 acidic properties 228 acid-soluble pectins 23 actin 55 active sites 177 ['H]S-adenosylmethionine 76 5-adenosyl methionine 76, 77. 78 AGK1 61 AGPs 84 alcohol dehydrogenase 67 alkali-extractable pectins 119 alkali-soluble pectins 23 allosteric regulation 68 inhibition 68 amidated pectin 224 amide crosslinks 119 linkages 33 ammonium oxalate 37, 42 Anabaena flos-aquae 62 anion-exchange chromatography 22 antibodies 55, 87 CCRC-M1 133 CCRC-M2 133. 142 CCRC-M7 133 CCRC-R1 antiserum 133 2F4 55,133 JIM5 55, 133. 137. 144 JIM7 55. 133. 136

LM5 133, 136, 137. 139. 140. 141. 142. 143 LM6 133, 136, 137, 139. 140, 141. 142. 143 LM7 133, 136, 144 PAM1 133, 144 anti-pectin antibodies 133 antiporters 58, 59 antisense PE plants 164 apiobiose (Api) 70, 78 PU-Api/T 79,81 pl,3-Api/T 81 D-Api/T 79.81 [uC]apiobiose 81 apiogalacturonan 2. 5. 10. 53. 70. 78. 80. 81, 82 apiose 10.61.78,81 [MC]apiose 81 D-apiose 70 apiosyl residues 120 transferase 78,80,81 Pl,2-apiosyl transferase 80 D-apiosyl transferase 81 apoplast 121 apparent pK 228 apple pectin 8,288 apple pomace 1. 222 Arabidopsis 61-69, 71, 74. 82. 85. 133, 134. 141. 142, 154, 162, 163. 167 arabinans 2. 5. 12, 17. 37. 64. 82, 115 a(1.5)-linked arabinans 13, 30. 102, 114, 118.132. 145 arabinofuranose 64 arabinogalactan 2. 12. 37. 82. 84. 86 3.4-linked 30 3.6-linked 30. 35 P-( 1,3,6) 16 p-(1.4)-linked 14 proteins 12.46.64 type I 5. 12.13.14. 17 type II 5. 15. 17 arabinose (Ara) 55. 59. 62. 61. 64. 86

243

INDEX

ARA 1 61 kinase 61 a-(l,3)-linked 15 5-phosphate 72 L-arabinose 86 D-arabinose-5-phosphate 71 a-arabinosidase 152, 157 arabinosyl 203 transferase (AraT) 64, 86 1,4-Ara/T 83 1,5-L-Ara/T 83 al,2-L-Ara/T 83 al,3-L-Ara/T 83 a 1,5-L-Ara/T 83 pl,2-L-Ara/T 79 P 1,5-L-Ara/T 79 ex 1,4-L-ArapT 79 L-AraT 79,83,84 artificial composites 99 Asclepias speciosa 143 Aspergillus aculeatus 158 niger 154, 156, 157 niger pectin lyase A 199 atomicforce microscope 106,113 lever 114 Azolla 144 Azuki bean 74, 75 Bacillus subtilis 41 bacterial cellulose 99 polysaccharides 71 bean 86 bioactive 203 blockwise 197 esterified pectins 20 blocky DE distribution 230 bond cleavage frequencies 178 borate 120 esters 32, 121 borate-binding apiosyl residues 120 borodeuteride 42 bovine UDP-GLcDH 67 Brassica campestris 167 calcium binding 116 bridges 21 ions 116

ions in catalysis 195

sensitivity 222

calcium-galacturonan gels 115 calcium-pectate gel 21 Candida pseudotropicalis 65 Cap\J 63 carbodiimide 42 carbonate-soluble pectins 23 carboxyl substituent 201 carboxylic esters 121 carrot 8 calli 163 root apex 140 catalytic residues 194 cell adhesion 131, 144 differentiation 142 expansion 139 proliferation 139 suspensions 61 wall architecture 136 cellulose-hemicellulose complexes 163 cellulose microfibrils 134 chain conformation 111 Chara 131 chelator-soluble pectins 23 chemical stability 229 Chenopodium 120 Chlorella pyrenoidasa 64, 71 chlorite oxidation 42 cholesterol-lowering effects 21 chromatography 63, 76 chutneys 236 circular dichroism 198 citrus peel 1, 222 classical jams 229 CMP-Dha 72 CMP-Kdo 71, 72 CMP-Kdo synthetase 71 cold alkaline conditions 36 cold gelation 235 Colletotrichum linemuthianum 34 commercial jam manufacture 234 pectinase preparations 177 definitions and standards 225 competitive inhibitor 185 coniferyl alcohol 45, 46 cotton 8, 40 coumaroyl arabinoxylan 32 covalent crosslinks 107, 118 interactions 31

244 linkages 30 crystal structure 111 Cryptococcus neoformans 68 Cyamopsis tetragonoloba 66 cyclohexane-diaminetetraaceticacid (CDTA) 37, 38 cysteine-rich adhesin 46, 145 cytidine 5'-monophosphate-3-deoxy-D-mannooctulosonate (CMP-Kdo) 71 cytidine-5'-triphosphate (CTP) 71 DEAE-Sephadex 34,35 decarboxylation 72 de-esterification 55, 153, 162 deformation 100 degree of amidation 226 esterification 6, 223, 226 polymerization (DP) 73,75,199 dehiscence 144 4,6-dehydratase 64, 65, 69 2-dehydro-3-deoxy-D-octonate-8phosphate 71 3-deoxy-D-arabino-heptulosonate-7-phosphate synthetase 72 3-deoxy-D-lyxo-2-heptulosaric acid 72 deoxylyxo-heptulopyranosylaric acid (Dha) 61,71,72,82 D-DhaT 80 p2,3-DhaT 80 detergent 58,72-77,86 detergent-solubilized 75, 77, 78 developmental biology 131, 145 dideutero-galactose 42 diester linkages 120 diferulic acid 32 digalacturonic acid (diGalA) 74, 75 divalent cations 120 Driselase 32,42,45 dTDP-glucose 64 dTDP-D-glucose 65 dTDP-L-rhamnose 65 dTDP-rhamnose 64,65 dwarfism 69 Escherichia coli 65, 67, 69, 72, 156 'egg box' junction zones 115 structures 164 elastic behaviour 106 electrostatic binding 116

INDEX forces 109 p-elimination 36, 174, 190, 194 depolymerisations 18,21 endo-acting rhamnogalacturonan-degrading enzymes 203 endoarabinanase 39, 40 Pl,4-endogalactanase 85 endoglucanase 39 endo-pectate lyase 156,193 endoplasmic reticulum 58, 59. 86 endopolygalacturonase (endoPG) 34. 53,62. 74,75,78,85, 175, 181, 186 endorhamnogalacturonan hydrolase (endoRGH) 203 endorhamnogalacturonan lyase (endoRGL) 203 endo-xylogalacturonan hydrolase 181 enthalpic elasticity 114 mechanisms 107 springs 106, 113 stretching 122 entropic mechanisms 107 springs 106. 115 stretching 123 enzyme-substrate complex 192 3,5-epimerization 71 4-epimerization 62, 63 epitope 55.58,82 equilibrium constant 62. 63. 71 Erwinia 155. 156 carolovora 154 chrysanthemi 154, 156 ESI-MS/MS 211 exo-pectate lyases 193 exopolygalacturonase (exoPG) 175.178 exoxylogalacturonase 180 expression library 181 extensin 35. 36. 37.46 extraction procedure 22 fat replacement 238 feedback inhibition 67,69 Fenton reaction 158 ferulicacid 10.38 feruloyl acetylesterase 157 esterase 210 esters 119 feruloylated XG 32 flax 76.77,85.86

INDEX

flexibility 112 flocculation 238 fluorescence 198 food thickeners 18 fractionating procedures 22 French bean 67 fruit 154 jellies 236,237 ripening 150, 158 softening 168 fruit-flavoured gels 235 FTIR 117 fucose(Fuc) 59,61,62,82 acetyl transferase 80 L-fucose (L-Fuc) 69, 71, 79, 84 al,2-L-FucT 79 al,4-L-FucT 79 fucosyl transferase 82 a 1,2-fucosyl transferase 82 fungi 178 galactan 2, 12, 37, 82, 85, 115, 205 p-D-(l,3/l,6)-galactan 102 P-D-(l,4)-galactan 114,132 p-L-(l,4)-galactan 102 (l,3)-galactan 15 P(l,4)-linked galactan 85,118 galactan-degrading activity 161 [I4C] galactobiose 85 galactokinase 61 galactomannan 85 P-( 1,6)-linked galactopyranosyl residues 15 galactose (Gal) 55, 61, 62, 85 D-galactose 86 L-galactose 69, 86 GAL1 61 galactosidase 85 P-galactosidase 152, 157, 161, 165, 166, 205 p( 1,4)-galactosidic linkage 112 galactosyl 203 galactosyl transferase (GalT) 59, 85 a-D-1 -6-galactosyl transferase 85 Pl,4-galactosyl transferase 85 D-GalT 79,83 L-GalT 79 1,5-GalT 83 (31,2-GalT 79 Pl,3-Gal7 83,84 Pl,6-Gall 83 galacturonic acid 2, 4, 34, 55, 59, 61, 62, 7275,78,81,85,86, 102

245

a-D-galacturonic acid (UDP-GalA) 62 galacturonosyl transferase (GalAT) 58. 74, 75,76 al,4-galacturonosyl transferase (HGAGalAT) 59, 63, 73, 74, 75 a 1,2 GalAT 79,83 a 1,4 GalAT 74,79,81,82,83 P 1,3 GalAT 79 P 1,4 GalT 83,84,85 D-GalAT 79,81,82,83 gas-liquid chromatography 33 gastric ulcers 239 gel rigidity 113 state 100 structure 103 gel-permeation chromatography 43 gelling agents 18 conditions 234 temperature 231 gene silencing techniques 164 genes encoding PG 154 GER1 69 GER2 69 GH family 28 181 GH28 174 glucose (Glc) 55,59 GlcA 59,61,66 GlcA-l-P 61 pl,4-GlcAT 80 D-GlcAT 80,84 metabolism 1 glucuronic acid 62, 86 decarboxylase 59 p-glucuronidase 67 glucuronoarabinoxylan 46 glycan synthase 72, 73 glycosidation 199,204,210 glycosyl transferase 52, 56, 58, 59, 72, 73, 74, 78,79,80,81,82,83,84, 134 glycosylation 53 GMD1 69 Golgi 44, 53, 55, 56, 58, 59, 61, 68, 72, 74, 75, 76, 86 green alga 62 growth 150 GTPase(raW7) 166 6M guanidine thiocyanate 36 guanosine diphosphate (GDP) GDP-fucose 68,69 GDP-4-keto-6-deoxy-D-mannose 69

246 GDP-4-keto-6-deoxy-D-mannose 3,5epimerase 69 GDP-4-keto-6-deoxy-L-galactose 69 GDP-4-keto-L-fucose 69 GDP-D-mannase 69,71 3,5-epimerase 71 4,6-dehydratase 69, 71 pyrophosphorylase 61 GDP-keto-6-deoxy-mannose 3,5epimerase-4-reductase 69 GDP-L-galactose 71 GDP-mannose 69,71 guar 66 gum arable 15 gum tragacanth 181 hairy region 4, 228 health-promoting 203 heartburn 239 p-helix 174 hemicellulose 53, 82, 85, 86, 114 hemicellulose xyloglucan 82 heterogalacturonase 202 hexose phosphates 61 HGA-AT 78 high-ester pectin 223 highly branched galacturonan (HBG) 10 highly esterified pectins 20 high-methoxyl (HM) pectins 6, 266 high-methyl ester pectins 226, 230, 234, 235, 236 high-performance anion-exchange chromatography (HPAEC) 24 holoenzyme 73 homogalacturonan (HGA) 4, 5, 6-8, 30, 54, 55, 70, 73, 78, 79, 84, 131, 132. 137. 141, 145, 174 homogalacturonan 3-0-acetyl transferase (HGA-AT) 73, 78 homogalacturonan galacturonosyl transferase (HGA-GalAT) 58,74,76 homogalacturonan-methyl transferase (HGAMT) 58,73,74,76,77 horseradish peroxidase 158 human health 21 hydrofluoric acid 8 hydrogen bonding 118 hydrolysis 174 hydrophobic forces 109 hydroxyproline 31 hysteresis 113. 230

INDEX

immune system stimulants 1 immunocytochemistry/immunofluorescent labelling 55.58.82, 136 inactivating endogenous enzymes 21 industrial pectins 19 intercellular spaces 136 interconversion pathways 59. 61 interstrand contacts 195-197 ionization state 186 isoditryosine-like crosslinks 42 isodityrosine 37 jam 235-237 junction zone 103. 108, 109. 111. 122 ketchup 236 4-keto intermediate 62 ketodeoxymanno-octulopyranosylonic acid (Kdo) 61,71.82 Kdo-8-phosphate 71 phosphatase 71 synthetase 71,72 2,3-KdoT 80 D-KdoT 80 kinetic analysis 195 Km 62. 63. 66, 67. 68. 69. 71. 72. 75, 77. 81. 85.87 leaf 160 Lemna minor 70. 78. 81 lemon albedo 8 pectin 228 lignin precursor 46 Lilium longiflorum 67 Linum usitatissimum 85 lipids 55 lipopolysaccharides 71 liquefaction 203 low-methoxyl (LM) pectins 6 low-methyl ester pectins 226. 231. 235. 237 maize cell suspension-culture walls 45 MALDI-TOFMS 24.213 mannose (Man) 59. 62 mannose-1 -phosphate guanylyltrasnferase 61 mass spectrometric analysis 184 mechanical role of pectins 99 stress 99. 104. 121. 122 membrane 54. 62. 66. 68. 75. 76 meristem 143

147

INDEX

methyl 78 esterases 208 esterification 19, 41, 53-55, 58, 73, 74, 76, 78, 114, 153 transferase 52, 58, 78 methylated polysaccharides 33 methylation 76, 77, 78, 86 2-0-methylfucose 78 2-0-methyl-L-fucose 69 2-O-methylxylose 78 methylumbelliferyl acetate 213 microsomal membrane 72, 74-76, 84-87 milk beverages 227 mitochondria 58 molecular lever 109 molecular modelling arabinogalactan I 17 arabinogalactan II 17 multiple attack 182 multiple-chain mechanism 213 mung bean 63, 64, 74, 75, 76, 77, 84, 86 hypocotyls 36 mur mutants 64, 69, 71, 141 mutagenesis 186 mutants 69, 71, 166 mutorotation 64 myoinositol 61, 66 myosin 55 NajCO, 37,38,41 NAD+ 62, 63, 65, 66, 67, 68, 70 NADH 64,66,68,69 NADPH 69 Nicotiana 145 Nitella 131 NMR 23, 111, 115, 116, 118,214 nonprocessively 75 nucleophile 186 nucleophilic attack 186 nucleoside diphosphate 59 diphosphatase 56 monophosphate (NMP) 59, 61 triphospate (NTP) 61 nucleotide-5'-diphosphatase 59 nucleotide-sugars 52, 56, 58, 59, 60, 61, 72 interconversion pathways 59 oil-in-water emulsions 238 oligogalacturonan lyase 193 oligomeric substrates 178 oligosaccharides 15,42,138

oligosaccharins 138 onion 37 oxidative coupling 23 parsley 70 pea 58, 66, 67, 68, 72, 74, 75 cotyledon 143 hypocotyl 37 pectate lyase 111, 152, 155-156, 190, 193. 197 pectin acetyl esterase 156-157 esterase (PE) 6, 21, 55,78, 134, 152-154, 159,210 homogalacturonan acetyl esterases 213 manufacture 222 methyl esterases (PME) 6, 21, 55, 78, 134, 138, 140,141, 152-154,210 methyl transferase (PMT) 73, 77, 78, 86 rheology 232 side chains 35 pectinase 81, 150 pectin-cellulose composites 101 pectin-degrading enzymes 150 pectin-depolymerising activity 161 pectin-lignin crosslinks 46 pectin-polymer complex 31 pectin-protein interactions 117 pectolytic enzymes 229 peptide-galacturonan binding 117 peptidoglycan 30 peroxidases 44, 158 'persil-milF structure 15 Petunia 141 pH 62, 63, 66, 68, 69, 70, 71, 72, 75-77, 81, 85 pharmaceutical grades 225 uses 238 pharmacological activities 22 Phaseolus aureus 86 vulgaris 67, 69, 86, 155 phenolic acid ester-sugar linkages 32 phosphatases 44 phosphoenolpyruvate 71,72 photoaffinity 76 Pichia pastoris 40 Pisum sativum 58, 66 plasma membrane 55, 58 plastic deformation 107 pollen 67, 154

248 tube 46, 142, 145 polyamines 117 polydispersity 227 polyelectrolytes 101 gel 104 polygalacturonan endotransglycosylase 44 polygalacturonase (PG) 21,55,75, 138, 144. 152, 154, 155, 160, 161, 175 polygalacturonate (PGA) 73. 75 polyphenolics 43 polysaccharide lyase 174, 192 pore size 1,121 porosity 135 potato 72, 85, 87 suspension 85 tubers 143, 163 processing of fruits and vegetables 21 processive behavior 72. 73, 75, 76, 182. 190, 205 processivity 72, 73, 75 product progression 182 progression profiles 201 pronase 35 proteases 44, 45 protein-phenolic-protein crosslinks 43 protein-protein crosslinks 43 proteoglycan 64 pummelo 64 pyrophosphate 61,71 pyrophosphorylases 61 quartet mutants (qrt) of Arabidopsis 166 radish 74 random-coil conformation 105 randomly esterified pectins 20 rapid set 227, 230 reverse genetics 182 rhamnogalacturonan (RG) 2, 3, 4, 8-10, 41. 43, 102, 112, 174.203.207 'smooth' regions 3 acetylesterase (RGAE) 154,208,214 galacturonohydrolase 208 'hairy' regions 3 hydrolase 3 hydrolase (RGase A) 85 lyase (RGase B) 85. 87 0-acetyl esterase 87 rhamnogalacturonan I (RG-I) 3, 5, 8, 30, 5355, 62, 63, 64, 65, 66, 68, 72, 73, 76. 78, 82, 84, 85, 86, 87, 114. 119, 123, 132,138.142. 143

INDEX

acetyl transferase (RG-I-AT) 86. 87 arabinosyl transferase 86 GalA 2-0-acetyl transferase 84 GalA 3-0-acetyl transferase 84 GlcA 4-0-methyl transferase 84 methyl transferase (RG-I-MT) 86 rhamnogalacturonan II (RG-II) 4. 5. 10. 30, 32. 53. 54. 62. 63. 64, 65. 66, 68. 69. 71. 73. 76. 78. 79. 80. 82. 86, 102. 120. 123,132.137. 143 dimers 78 fucose 2-O-methyl transferase 80 methyl transferase (RG-II-MT) 78 rhamnogalacturonase 157 rhamnohydrolase 207 rhamnose (Rha) 59. 61. 62. 64. 79. 83. 85. 203 rhamnosylation 64 D-ribulose-5-phosphate 71 root 142. 160 rubber elasticity 104. 106 Saccharomyces cerevisiae 69 salicylic acid 214 saponified hairy regions 204 SDS-PAGE 43 secondary attack 184 wall formation 45 walls 67 SGHN-hydrolase 215 shoot primordia 155 Silene dioica 64 single-chain mechanics 105 site-directed mutagenesis 181 size-exclusion chromatography 3. 22 slow set 227.230 smooth region 4. 228 sodium hexametaphosphate 234 Solatium tuberosum 72 solubilization 75 soybean 67. 68.76.77 spinach 71 Spirodela polyrrhiza 70. 81 stabilizers 236 stoichiometry 184 storing pectin 235 Streptococcus pneumoniae 63 pyogenes 67 structural topology 174 structure-function relationships 132

INDEX

3D structures 192 subcellular localisation 56 subsites 177, 178, 180,201 substrate specificity 181 substrate-Ca"+ complex 197 sucrose gradients 58 sugar beet pectin 228 pulp 38 solids 222 sugar-1-phosphates 59,61 suspension-cultured carrot cells 140 rose cells 38 sycamore 62, 74, 75 syneresis 208, 232 synthetic wall composites 139 TBG-1 165 TBG-4 162, 165 texture 103 thickening and gelling 229 threose 72 tobacco 8, 62, 64, 74, 75, 76, 77 cell suspensions 61 microsomes 74 tomato 8, 74 colourless non ripening mutant (Cnr) 145, 167 fruit pericarp 137, 143 non ripening mutant (nor) 167 PG gene family 162 pollen 155 ripening 159 ripening inhibitor mutant (riri) 165 suspension-culture 42 transgenic plants 99, 164, 165 transporter 56, 58 triacetin 213,214 trifluoroacetic acid 33 trigalacturonic acid 74 turgor-imposed stresses 122 turnip 74 two-dimensional infrared-mechanical spectroscopy 99 tyrosine residues 45

uridine diphosphate (UDP) 62, 68 UDP glucose 4,6-dehydratase 64 UDP xylase 4-epimerase 63 UDP-['4C]apiose 81

249 UDP-['4C]Gal 63, 85 UDP-['4C]GalA 63, 74. 75 UDP-4-keto rhamnose reductase 64, 65 UDP-4-keto-6-deoxy-D-Glc 64 UDP-4-keto-6-deoxy-Glc 64 UDP-4-keto-6-dexyl-L-mannose 64. 65 UDP-4-keto-hexose 68 UDP-4-keto-L-rhamnose 3,5 epimerase 64, 65 UDP-apiose synthase 70 UDP-arabinose 64 UDP-arabinose 4-epimerase 63 UDPase 58 UDP-p-L-arabinopyranose 63 UDP-D-apiose 70 UDP-D-GalA 62 UDP-D-Glc 64,65,70 UDP-D-Glc 4-epimerase 65 UDP-D-GlcA 62,66,68,70 UDP-D-glucuronate decarboxylases 68 UDP-D-glucuronic acid 66 UDP-D-Xyl 63,68 UDP-GalA 62,63,76,81 UDP-Glc 65,66,67,68 4-epimerase 65, 66 6-dehydrogenase 66, 67 dehydrogenase 67 epimerase 66 UDP-Glc A 61, 62, 63, 66, 68, 70 4-epimerase 62, 63 carboxylase 68 decarboxylase 68 epimerase 63 UDP-GlcDH 67 UDP-methyl-D-GlcA 70 UDP-Rha 64, 65 UDP-Xyl 61, 63, 67, 68, 70, 82 UDP-Xyl synthase 70 LIDP-xylose 4-epimerase 63, 64 vascular bundles 155 Viciafaba 66 viscosity 222, 231 wall-associated kinases 45 water-soluble pectins 23 wheat germ 63, 66, 68 wounding 1 xylan 38 xylem 150

250

xylogalacturonan (XGA) 2, 3, 4, 5. 10-11. 30, 41,43,53,54.68,73,81.82, 102, 174. 180,181 xyloglucan (XG) 30. 33, 34, 35. 37. 38, 41. 44,71, 101, 134 chains 31 endotransglycosylases 139 XG-pectin complexes 39 XG-pectin link 35 xylose (Xyl) 59,61,62,70,82 2-0-methyl transferase 80 cd,3-XylT 80-82 xylosyl transferase 81 D-XylT 80,82 xylosylated oligogalacturonides 181 yoghurt 236, 237 Zinnia elegans 156

INDEX