Journal of Experimental Botany, Vol. 53, No. 377, pp. 2115-2119,
October 1, 2002
© 2002 Oxford University Press
Pectate lyases, cell wall degradation and fruit softening
Received 26 March 2002; Accepted 16 July 2002
1 Horticulture Research International, Plant Genetics and Biotechnology Department, Wellesbourne, Warwickshire CV35 9EF, UK
2 Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Kent ME4 4TB, UK
4 Present address: Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RR, UK.
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Abstract |
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 Top Abstract IntroductionMicrobial PLs: mode of...Plant PLsPL and fruit softeningConclusionReferences |
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This is a brief review of what is known about the role of pectate lyases in plants. The mode of action and three-dimensional structure of microbial pectate lyases is discussed first and then the limited information on the plant proteins is presented. Pectate lyase-like genes have been isolated from a wide range of plant tissues including germinating seeds, pollen, cell cultures, and ripening fruits. The abundance of ESTs for these genes in tomato and the presence of pectate lyase-like transcripts in many other fruits may indicate that these enzymes have a more important role in ripening than previously suspected.
Key words: Keywords: Cell wall, fruit softening, pectate lyase, pectin.
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Introduction |
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TopAbstractIntroductionMicrobial PLs: mode of...Plant PLsPL and fruit softeningConclusionReferences |
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Pectate lyases (PL, EC 4.2.2.2), otherwise known as pectate transeliminases, catalyse the eliminative cleavage of de-esterified pectin, which is a major component of the primary cell walls of many higher plants (Carpita and Gibeaut, 1993). The backbone of pectic polysaccharides is built of blocks of
-1,4 linked polygalactosyluronic acid residues interspersed with regions of alternating galactosyluronic acid and rhamnosyl residues (Willats et al., 2001). Cleavage by PL requires the presence of calcium ions and generates oligosaccharides with unsaturated galacturonosyl residues at their non-reducing ends. Until recently, it was thought that PLs were secreted mainly by plant pathogens, their action resulting in the maceration of plant tissues. However, the abundance of PL-like sequences in plant genomes (currently 27 genes in the Arabidopsis genome are thought to encode PL-like proteins, www.tigr.org, Arabidopsis database, July 2002) strongly suggests an important role for these enzymes in various plant developmental processes.
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Microbial PLs: mode of action and three-dimensional structures |
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TopAbstractIntroductionMicrobial PLs: mode of...Plant PLsPL and fruit softeningConclusionReferences |
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PL activity was first discovered in 1962 in cultures of Erwinia carotovora and Bacillus sp. (Starr and Morán, 1962) and their secretion by plant pathogenic bacteria is today well-documented (Collmer and Keen, 1986; Kotoujansky, 1987; Nakajima et al., 1999). PL action results not only in plant cell-wall degradation, but also in the activation of defence systems, presumably through the release of oligogalacturonides from the plant cell wall, which then function as defence elicitors (De Lorenzo et al., 1991).
The best-studied microbial PLs to date are those from Erwinia chrysanthemi, which causes devastating diseases involving maceration of parenchymatous tissues of various dicot plants (Pérombelon and Kelman, 1980; Keen et al., 1984; Kelemu and Collmer, 1993). These enzymes act by depolymerizing cell-wall polygalacturonides in the presence of calcium ions, thus destroying the integrity of the plant tissues (Collmer and Keen, 1986; Barras et al., 1994). This bacterium has been shown to express up to five independently regulated PL genes (pelA, B, C, D, and E) coding for five isozymes of PL (first reported by Lietzke et al., 1994, 1996). Erwinia isoforms, obtained by expression in E. coli, have been shown to act synergistically to extend the range of pectin substrates that the bacterium can degrade (Bartling et al., 1995).
The three-dimensional structures of various extracellular PLs (family 1 lyases as defined by http://afmb.cnrsmrs.fr/
pedro/CAZY/lya.html) have been determined, including PelC (Yoder et al., 1993a, b; Yoder and Jurnak, 1995), and PelE (Lietzke et al., 1994) from Erwinia chrysanthemi and BsPel from Bacillus subtilis (Pickersgill et al., 1994). All these enzymes share an unusual structure termed ‘the parallel ß helix’ in which ß-strands are folded into a large, right-handed superhelix (Fig. 1). Two PLs, which cleave methylated pectin, also belong to this family (Mayans et al., 1997; Vitali et al., 1998). There is also a brief preliminary description of the E. chrysanthemi PL, which also has parallel ß-helix architecture and PL activity, but no sequence similarity with the family 1 enzymes (Jenkins and Pickersgill, 2001). The PL structures differ in the size and conformation of the loops that protrude from the parallel ß-helix core, nevertheless they all share the same basic structure. It can be deduced from sequence similarity, the position of the calcium-binding site in BsPel and from site-directed mutagenesis (Kita et al., 1996), that the protruding loops on one side of the parallel ß helix form the pectolytic active site. The structural differences in the loops are probably related to subtle differences in the enzymatic and maceration properties of the proteins (Scavetta et al., 1999). Recent work on E. chrysanthemi PelC, the first protein in which the parallel ß-helix structure was recognized, shows that it appears to consist of two domains that strongly interact and unfold at pH 7, the co-operativity decreasing at higher and at lower pH (Kamen et al., 2000). However, the crystal structure of PelC only reveals a single domain.
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It is likely that the PLs secreted by plant pathogens share a common enzymatic mechanism but, unfortunately, the catalytic roles of the amino acid residues in the active site have not yet been identified. The crystal structure of a PelC mutant complexed to a plant cell-wall fragment has recently been published (Scavetta et al., 1999). The substrate binds in a cleft, interacting primarily with positively charged groups; either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex (Scavetta et al., 1999). The suggestion made by the authors is that an arginine, which is invariant in the PL superfamily, is the amino acid that initiates proton abstraction during ß elimination cleavage of polygalacturonic acid.
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Plant PLs |
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TopAbstractIntroductionMicrobial PLs: mode of...Plant PLsPL and fruit softeningConclusionReferences |
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PL-like sequences from higher plants were first reported from pollen (Wing et al., 1989). Two genes with sequence similarity to Erwinia PLs were expressed at maximal levels in mature tomato flowers, anthers and pollen. Since then, many other similar sequences have been shown to be expressed in pollen, anthers and pistils (Kulikauskas and McCormick, 1997) and a Japanese cedar pollen allergen has been positively identified as having PL activity (Taniguchi et al., 1995). Functions suggested for PL in pollen include the initial loosening of the pollen cell wall to enable pollen tube emergence and growth and breakdown of the cell wall of transmitting tissue in the style to facilitate penetration of pollen (Taniguchi et al., 1995; Wu et al., 1996). Genes encoding a variety of cell-wall-degrading enzymes, including polygalacturonase (Brown and Crouch, 1990; Niogret et al., 1991; Allen and Lonsdale, 1993), pectinesterase (Albani et al., 1991) and ß-galactosidase (Rogers et al., 2001) have also been reported in pollen.
In a search for genes involved in cell wall modifications during trans-differentiation of Zinnia elegans cells, two PL-like genes were isolated recently (Domingo et al., 1998; Milioni et al., 2001). The detailed study of ZePel1 (Domingo et al., 1998) showed that it was active at a very early stage in tracheary element induction and its expression was modulated by auxin. Furthermore, the recombinant protein made in E. coli exhibited calcium-dependent PL activity. The authors speculated that these enzymes may assist in the removal and modification of the existing pectin matrix to allow the deposition of newly synthesized wall polymers for a specialized function.
In the capsules of the opium poppy, latex-containing laticifers are abundant and the laticifer system develops through the gradual disappearance of adjacent cell walls between differentiating laticifer elements throughout the plant. Sequences with homology to PL and other cell-wall-degrading enzymes have recently been isolated from an opium poppy latex cDNA library and PL activity has also been observed in the latex (Pilatzke-Wunderlich and Nessler, 2001).
A search of the tomato EST database (http://www.tigr.org/tdb/lgi/index.html) shows that PL-like sequences were isolated from a host of cDNA libraries including those made from germinating seeds, developing flowers, ovaries, pollen, trichomes, and ripening fruits suggesting that PL gene expression is widespread, if not ubiquitous in plant tissues.
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PL and fruit softening |
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TopAbstractIntroductionMicrobial PLs: mode of...Plant PLsPL and fruit softeningConclusionReferences |
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Perishable horticultural commodities such as fleshy fruits have a relatively short post-harvest shelf life during which the fruit tissues undergo profound changes in texture, colour and flavour, as well as becoming more susceptible to pathogenic attack (Seymour et al., 1993). Fruit softening is associated with cell wall disassembly (Seymour and Gross, 1996) and modifications to the pectin fraction are some of the most apparent changes that take place in the cell wall during ripening.
The majority of work on the disassembly of fruit cell walls has focused on ripening in tomatoes. The ripe pericarp of these fruit is rich in polygalacturonase activity and it was long assumed that this was the principal enzyme responsible for fruit softening. However, transgenic experiments in which the accumulation of polygalacturonase mRNA was suppressed still softened normally (Smith et al., 1989a). Also, in other fruits such as strawberry and banana, polygalacturonase activity is very low or absent despite evidence for pectin solubilization and degradation (Huber, 1984; Smith et al., 1989b). Early experiments to measure the presence of PL activity in tomato fruit proved unsuccessful (Besford and Hobson, 1972). However, the tomato EST programme (http://www.tigr.org/tdb/lgi/index.html) suggests a high level of PL-like gene expression in ripe tomato fruits.
PL sequences have also been reported from banana (Domínguez-Puigjaner et al., 1997; Medina-Suárez et al., 1997; Pilatzke-Wunderlich and Nessler, 2001; Marín-Rodríguez, 2001) and strawberry fruits (Medina-Escobar et al., 1997) and from ripening grape berries (Nunan et al., 2001). In bananas, the expression of two distinct PL-like genes (Pel I and Pel II) has been detected during ripening. Both show different levels of expression in ripening pulp and peel, with Pel I predominating. An active PL protein was produced by expression of banana Pel I in yeast. More importantly, for the first time from fruit tissue, PL activity has been obtained directly from banana pulp with a substantial increase in activity during ripening (Marín-Rodríguez, 2001). Additionally, a PL sequence from strawberry has also been expressed in yeast giving an active protein, although the authors were unable to observe any endogenous enzyme activity in the fruits themselves (Medina-Escobar et al., 1997). More recently PL gene expression has been manipulated in transgenic strawberry fruits and suppression of the PL mRNA during ripening resulted in significantly firmer fruits (Jiménez-Bermúdez et al., 2002). The highest reduction in softening occurring during the transition from the white to the red stage.
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Conclusion |
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TopAbstractIntroductionMicrobial PLs: mode of...Plant PLsPL and fruit softeningConclusionReferences |
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The likely importance of PLs in plant development has been appreciated only recently as a result of genome sequencing and EST programmes and from biochemical studies where PL activity has been measured in various plant tissue extracts. While pectin degrading enzymes such as polygalacturonase have been the focus of significant research, PLs have been less well studied. However, the extent of PL-like gene expression in ripening fruits suggests that these enzymes could play a more important role in fruit softening than previously thought.
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Acknowledgements |
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MCM-R was funded by a postgraduate award from NRI, at University of Greenwich, UK. GBS is funded by the Biotechnology and Biological Sciences Research Council. We would like to that Professor Richard Pickersgill for providing Fig. 1 and for his helpful comments during the preparation of the manuscript.
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