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• Modulators of cAMP, calcium and G proteins were used to treat bean (Phaseolus vulgaris) cells before addition of an elicitor from Colletotrichum lindemuthianum in order to elucidate the early steps of signal transduction leading to the production of the apoplastic oxidative burst. • Hydrogen peroxide production by elicited bean cells was monitored with luminol-or xylenol-orange-based assays. • Pretreatment with forskolin, dibutyryl cAMP or the Ca

Background and Aims: \ud Cultured cell suspensions have been the preferred model to study the apoplast as well as to monitor metabolic and cell cycle-related changes. Previous work showed that methyl jasmonate (MeJA) inhibits leaf growth in a CORONATINE INSENSITIVE 1 (COI1)-dependent manner, with COI1 being the jasmonate (JA) receptor. Here, the effect of COI1 overexpression on the growth of stably transformed arabidopsis cell cultures is described.\ud \ud Methods: \ud Time-course experiments were carried out to analyse gene expression, and protein and metabolite levels.\ud \ud Key Results: \ud Both MeJA treatment and the overexpression of COI1 modify growth, by altering cell proliferation and expansion. DNA content as well as transcript patterns of cell cycle and cell wall remodelling markers were altered. COI1 overexpression also increases the protein levels of OLIGOGALACTURONIDE OXIDASE 1, BETA-GLUCOSIDASE/ENDOGLUCANASES and POLYGALACTURONASE INHIBITING PROTEIN2, reinforcing the role of COI1 in mediating defence responses and highlighting a link between cell wall loosening and growth regulation. Moreover, changes in the levels of the primary metabolites alanine, serine and succinic acid of MeJA-treated Arabidopsis cell cultures were observed. In addition, COI1 overexpression positively affects the availability of metabolites such as β-alanine, threonic acid, putrescine, glucose and myo-inositol, thereby providing a connection between JA-inhibited growth and stress responses.\ud \ud Conclusions: \ud This study contributes to the understanding of the regulation of growth and the production of metabolic resources by JAs and COI1. This will have important implications in dissecting the complex relationships between hormonal and cell wall signalling in plants. The work also provides tools to uncover novel mechanisms co-ordinating cell division and post-mitotic cell expansion in the absence of organ developmental control.

Reactive oxygen species (ROS) generated by NADPH oxidases or apoplastic peroxidases play an important role in the plant defense response. Diminished expression of at least two Arabidopsis thaliana peroxidase encoding genes, PRX33 (At3g49110) and PRX34 (At3g49120), as a consequence of anti-sense expression of a heterologous French bean peroxidase gene (asFBP1.1), were previously shown to result in reduced levels of ROS following pathogen attack, enhanced susceptibility to a variety of bacterial and fungal pathogens, and reduced levels of callose production and defense-related gene expression in response to the microbe associated molecular pattern (MAMP) molecules flg22 and elf26. These data demonstrated that the peroxidase-dependent oxidative burst plays an important role in the elicitation of pattern-triggered immunity (PTI). Further work reported in this paper, however, shows that asFBP1.1 antisense plants are not impaired in all PTI-associated responses. For example, some but not all flg22-elicited genes are induced to lower levels by flg22 in asFPB1.1, and callose deposition in asFPB1.1 is similar to wild-type following infiltration with a Pseudomonas syringae hrcC mutant or with non-host P. syringae pathovars. Moreover, asFPB1.1 plants did not exhibit any apparent defect in their ability to mount a hypersensitive response (HR). On the other hand, salicylic acid (SA)-mediated activation of PR1 was dramatically impaired in asFPB1.1 plants. In addition, P. syringae-elicited expression of many genes known to be SA-dependent was significantly reduced in asFBP1.1 plants. Consistent with this latter result, in asFBP1.1 plants the key regulator of SA-mediated responses, NPR1, showed both dramatically decreased total protein abundance and a failure to monomerize, which is required for its translocation into the nucleus.

Perception by plants of so-called microbe-associated molecular patterns (MAMPs) such as bacterial flagellin, referred to as pattern-triggered immunity, triggers a rapid transient accumulation of reactive oxygen species (ROS). We previously identified two cell wall peroxidases, PRX33 and PRX34, involved in apoplastic hydrogen peroxide (H2O2) production in Arabidopsis (Arabidopsis thaliana). Here, we describe the generation of Arabidopsis tissue culture lines in which the expression of PRX33 and PRX34 is knocked down by antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase cDNA construct. Using these tissue culture lines and two inhibitors of ROS generation, azide and diphenylene iodonium, we found that perxoxidases generate about half of the H2O2 that accumulated in response to MAMP treatment and that NADPH oxidases and other sources such as mitochondria account for the remainder of the ROS. Knockdown of PRX33/PRX34 resulted in decreased expression of several MAMP-elicited genes, including MYB51, CYP79B2, and CYP81F2. Similarly, proteomic analysis showed that knockdown of PRX33/PRX34 led to the depletion of various MAMP-elicited defense-related proteins, including the two cysteine-rich peptides PDF2.2 and PDF2.3. Knockdown of PRX33/PRX34 also led to changes in the cell wall proteome, including increases in enzymes involved in cell wall remodeling, which may reflect enhanced cell wall expansion as a consequence of reduced H2O2-mediated cell wall cross-linking. Comparative metabolite profiling of a CaCl2 extract of the PRX33/PRX34 knockdown lines showed significant changes in amino acids, aldehydes, and keto acids but not fatty acids and sugars. Overall, these data suggest that PRX33/PRX34-generated ROS production is involved in the orchestration of pattern-triggered immunity in tissue culture cells.

Tobacco plants expressing an antisense construct for a cationic peroxidase, which down-regulated lignin content at the presumed level of polymerisation, have been further analysed. T(1) plants were derived from a large-scale screen of T(0) mutant lines, previously published, which identified lines demonstrating consistent lignin down-regulation. Of these, line 1074 which had the most robust changes in lignin distribution through several generations was shown to have accompanying down-regulation of transcription of most lignin biosynthesis genes, except cinnamoyl-CoA reductase. The consistent 20% reduction in lignin was not accompanied by significant gross changes in vascular polysaccharide content and composition, despite a modest up-regulation of transcripts of genes involved in cellulose and hemicellulose synthesis. Morphologically, 1074 plants have under-developed xylem with both fibers and vessels having thin cell walls and limited secondary wall thickening with an abnormal S2 layer. However, they were not compromised in overall growth. Nevertheless, these and other lines showed improved potential industrial utility through a threefold increase in enzymic saccharification efficiency compared with wild-type (wt). Therefore, they were profiled for further un-intended effects of transgenesis that might compromise their value for industrial or biofuel processes. Other phenotypic changes included increased leaf thickness and bifurcation at the tip of the leaf. wt-Plants had smaller chloroplasts and higher stomatal numbers than mutants. Transgenic lines also showed a variable leaf pigment distribution with light-green areas that contained measurably less chlorophyll a, b, and carotenoids. Changes in epidermal pavement cells of mutant lines were also observed after exposure to various chemicals, while wt leaves retained their structural integrity. Despite these changes, the mutant plants grew and were viable indicating that lignification patterns can be manipulated considerably through targeting polymerisation without serious deleterious effects.

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490 bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.

The oxidative burst is an early response to pathogen attack leading to the production of reactive oxygen species (ROS) including hydrogen peroxide. Two major mechanisms involving either NADPH oxidases or peroxidases that may exist singly or in combination in different plant species have been proposed for the generation of ROS. We identified an Arabidopsis thaliana azide-sensitive but diphenylene iodonium-insensitive apoplastic oxidative burst that generates H(2)O(2) in response to a Fusarium oxysporum cell-wall preparation. Transgenic Arabidopsis plants expressing an anti-sense cDNA encoding a type III peroxidase, French bean peroxidase type 1 (FBP1) exhibited an impaired oxidative burst and were more susceptible than wild-type plants to both fungal and bacterial pathogens. Transcriptional profiling and RT-PCR analysis showed that the anti-sense (FBP1) transgenic plants had reduced levels of specific peroxidase-encoding mRNAs, including mRNAs corresponding to Arabidopsis genes At3g49120 (AtPCb) and At3g49110 (AtPCa) that encode two class III peroxidases with a high degree of homology to FBP1. These data indicate that peroxidases play a significant role in generating H(2)O(2) during the Arabidopsis defense response and in conferring resistance to a wide range of pathogens.

Plant secondary metabolism is highly regulated within the major pathways to terpenoids, phenolics and alkaloids. Such regulation can occur at multiple levels from transcription through to the compartmentation of the product. However, the possibility exists for cross-talk between these pathways, the regulation of which is largely unknown at present. Such phenomena are important to understand in the application of plant breeding, where unintended effects of transgenesis or mutation can have an impact on the environment or human health. In an effort to improve dietary antioxidant content of crop plants, the tomato has been a major focus of effort for engineering both lipophilic antioxidants such as carotenoids and hydrophilic antioxidants such as flavonoid glycosides. In this study, a panel of transgenic and mutant tomato lines has been subjected to metabolite profiling in comparison with wild type Ailsa Craig for both carotenoids and phenolics. A range of mutants and transgenic lines were selected showing a range of phenotypes varying from down-regulation through to increased levels of lycopene and beta-carotene. All mutants altered in structural genes for carotenoid biosynthesis showed that perturbations in carotenoid biosynthesis do not generally alter phenolic or flavonoids content significantly even when devoid of carotenoids. Reciprocally, the down-regulation of ferulate 5-hydroxylase had no effect on carotenoid content. In contrast mutants defective in light perception such as the high pigment (hp-1) and LA3771 possess elevated chlorogenic acid and rutin as well as increased carotenoid content. These lines can act as the hosts for further genetic manipulation for increased antioxidant content.

A 42kDa chitin-binding proline-rich protein (PRP) from French bean has been previously characterised through its involvement in plant–pathogen interactions. It is located at the plasmalemma-wall interface, intercellular spaces and binds to the pathogen Colletotrichum lindemuthianum in vitro and in planta. It is also present in cell wall appositions formed in response to an hrp mutant of Xanthomonas campestris. We now show that the 42kDa protein is composed of two components, a 25kDa polypeptide member of the PRP family of legumes and a 6.8kDa cysteine-rich peptide with high similarity to snakin-2 from potato. Snakins bind to pathogens and are antimicrobial. Molecular cloning of the longest PRP corresponding to the N-terminal sequence of the purified protein and the 6.8kDa component is reported. The cognate mRNAs show coordinate expression. The two-component protein complex has already been shown to be involved in binding and immobilising pathogens through oxidative cross-linking of the PRP components but could also function as a two-component chitin-receptor involved in plant–pathogen interactions through antimicrobial activity and/or signalling.

A tobacco peroxidase isoenzyme (TP60) was down-regulated in tobacco using an antisense strategy, this affording transformants with lignin reductions of up to 40-50% of wild type (control) plants. Significantly, both guaiacyl and syringyl levels decreased in essentially a linear manner with the reductions in lignin amounts, as determined by both thioacidolysis and nitrobenzene oxidative analyses. These data provisionally suggest that a feedback mechanism is operative in lignifying cells, which prevents build-up of monolignols should oxidative capacity for their subsequent metabolism be reduced. Prior to this study, the only known rate-limiting processes in the monolignol/lignin pathways involved that of Phe supply and the relative activities of cinnamate-4-hydroxylase/p-coumarate-3-hydroxylase, respectively. These transformants thus provide an additional experimental means in which to further dissect and delineate the factors involved in monolignol targeting to precise regions in the cell wall, and of subsequent lignin assembly. Interestingly, the lignin down-regulated tobacco phenotypes displayed no readily observable differences in overall growth and development profiles, although the vascular apparatus was modified.

UDP-glucuronic acid decarboxylase catalyses the reaction responsible for the formation of UDP-xylose and commits assimilate for the biosynthesis of cell wall polysaccharides and glycosylation of proteins. Xylose-rich polymers such as xylans are a feature of dicot secondary walls. Thus a cell culture system of tobacco transformed with the ipt gene from Agrobacterium tumefaciens for cytokinin production and which when manipulated with auxin and sucrose leads to induction of xylogenesis, has been used as a source for purification of the enzyme. UDP-glucuronic acid decarboxylase was purified by ion-exchange, gel filtration and affinity chromatography on Reactive Brown–Agarose. The native enzyme had an apparent Mr of 220,000 which yielded a single subunit of 87,000 when analysed on SDS–PAGE using silver staining. This appears to be a novel form of the enzyme since a gene family encoding polypeptides around Mr 40,000 with homology to the fungal enzyme also exists in plants. Using an antibody raised to the native 87 kDa form of the enzyme, this decarboxylase was localised mainly to to cambium and differentiating vascular tissue in tobacco stem, consistent with a role in the provision of UDP-xylose for the synthesis of secondary wall xylan. Further analysis using immunogold electron microscopy localised the 87 kDa UDP-glucuronic acid decarboxylase to the cytosol of developing vascular tissue.

Changes in protein kinase activity have been investigated during the early response of suspension cultured cells of French bean to fungal elicitor. One of the kinases activated has a known target, phenylalanine ammonia-lyase (PAL), which has an important role in plant defence responses, and was purified. Kinase acivity during purification was monitored for both the PAL-derived peptide and syntide-2, which it also phosphorylated. The kinase had an M r of 55 000 on the basis of gel migration, 45Ca2+ binding, autophosphorylation and phosphorylation of various substrates using in-gel assays. The kinase has been characterised with respect to kinetics and other properties in vitro and appears to be a CDPK. In-gel assays were also used to show that this kinase and a number of other CDPKs of similar M r showed complex changes in elicitor-treated suspension-cultured cells of French bean. An activation was observed within 10 min and was maintained for up to 4 h. The time course of activation was different from MAP kinase and casein kinase assayed in the same extracts. However, at 5 min after addition of elicitor there is a transient inactivation of the CDPKs before activation. This inactivation can be mimicked by adding forskolin to the cells 30 min before elicitation, which brings about changes in the cellular pH. Forskolin potentiates the oxidative burst when elicitor is subsequently added while the CDPK cannot be activated by elicitor upon forskolin treatment. In contrast, intracellular acidification brought about by forskolin brings about slight activation of MAPkinase.

The behaviour of four membrane-bound glycosyl transferases involved in cell wall polysaccharide synthesis has been studied in relation to the effects of a graded series of organic solvents on their activity and type of product formed. Relative enzyme inhibition observed for some solvents was in direct relationship to the hydrophilicity of the product. This was in the order of arabinan synthase > callose synthase> xylan synthase > beta-1,4-glucan synthase. The former two were always inhibited, the xylan synthase rather less so. However, the beta-1,4-glucan synthase showed significant increases in substrate incorporation in the presence of solvents. A graded series of primary alcohols were much more effective in enhancing activity than acetone, ethyl acetate and dimethyl formamide. In the presence of the most effective solvent, methanol, there was considerable activation of beta-1,4-glucan production. This reciprocal nature of the behaviour of the beta-1,4- and beta-1,3-glucan synthases in organic solvent is supportive of recent molecular data that the two types of glucans are catalysed by separate enzyme systems. However, the results reported here do not totally negate the proposition that either enzyme is capable of synthesising the other linkage in minor amounts in vitro.

A cell suspension culture of a tobacco (Nicotiana tabacum L. cv. Petit Havana) cell line derived from a cultivar transformed with the Tcyt gene from Agrobacterium, which leads to high endogenous levels of cytokinin, has been established. This cell line shows increased cell aggregation, elongated cells and a 5-fold increase in wall thickness. If allowed to carry on growing it can form a single mass without shedding cells into the medium. When analysed at an earlier growth stage, these cultures were found to produce improved levels of vascular nodule formation than in other systems that employ exogenous cytokinin. This differentiation was optimised with respect to sucrose and auxin signals in order to induce maximum production of cells with thickened walls and a morphology characteristic of fibre cells and tracheids, in addition to cells that remain meristematic. In order to establish the validity of this system for studying secondary wall formation, the walls and associated biosynthetic changes were analysed in these cells by chemical analysis of the walls, changes in activities of enzymes of xylan and monolignol synthesis, and expression of mRNAs coding for enzymes of lignin biosynthesis. The wall composition of the transformed cells was compared with that determined for primary walls from a typical untransformed tobacco cell line. Recovery of wall material was 50% greater in the transformed culture. In this material a major difference was found in the pectin fraction where there was a distinct difference in size distribution together with a lower level of methylation for the transformed line, which may be related to increased adhesiveness. There were increased amounts of xylan, although the ratio of xyloglucan to xylan content was not substantially different due to the mixture of cell types. There was also an increase in cellulose and phenolic components. Increased activity of enzymes involved in the synthesis of xylan as a marker for the secondary wall occurred around the time of tracheid differentiation and coincided with a broad peak of cinnamyl alcohol dehydrogenase activity. The expression of mRNAs coding for enzymes of the general phenylpropanoid pathway, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, catechol O-methyl transferase was relatively constitutive in the cultures while transcripts of ferulate 5-hydroxylase, cinnamoyl CoA-reductase, cinnamyl alcohol dehydrogenase and lignin peroxidase were induced. The walls of the transformed cells also showed considerable differences in the subset of extractable proteins from that found in primary walls of tobacco when these were subjected to proteomic analysis. Many of these proteins appear to be novel and not present in primary walls. However an M r-32,000 chitinase, an M r-34,000 peroxidase, an M r-65,000 polyphenoloxidase/laccase and possibly an M r-68,000 xylanase could be identified as well as structural proteins.

Understanding of the biosynthesis of plant cell wall polysaccharides is lacking at present. More attention has been paid to the synthesis of other carbohydrate sink-materials such as starch and sucrose and progress in the study of wall polysaccharides has been more directed towards understanding turnover up to the present time. The main reasons for this deficiency are the potential number and complexity of enzymes required for wall polysaccharide synthesis, difficulty in their purification and the consequent lack of identified genes. This review illustrates the progress in the light of work in the author’s laboratory which has been directed towards improving identification of the enzymes involved. Wider progress in the field is reviewed that has led to identification of the first set of polysaccharide synthase genes. Some aspects of regulation of cell wall polysaccharide synthesis during growth, differentiation and biotic stress are also highlighted. Progress in engineering the polysaccharide content of walls is also reviewed.

The site of phosphorylation of phenylalanine ammonia-lyase (PAL) has been identified as a threonine residue. A Ca2+-stimulated protein kinase of approximately 55 kDa has been partially purified from elicited cells. The kinase can phosphorylate a synthetic peptide derived from PAL and a recombinant poplar PAL. PAL phosphorylation was associated with a decrease in Vmax in agreement with the suggestion that protein phosphorylation is involved in marking PAL subunits for turnover. The phosphorylation site in French bean PAL is most likely Thr545 in the sequence VAKRTLTT (539–546). Conservation of the phosphorylation site in PAL from diverse species suggests that phosphorylation of PAL may be a ubiquitous regulatory mechanism in higher plants.

Qualitative sugar flux into cell wall polysaccharides has been determined for two model systems. The first, treatment of suspension-cultured French bean ( Phaseolus vulgaris L.) cells with an increase in the cytokinin/auxin ratio and in the concentration of sucrose, models some aspects of differentiation. Wall changes are characterised by up to a five-fold increase in thickness due to the laying down of extra wall material. Sugar flux following labelling of cells with [ 14 C]-sucrose was examined during the period of maximum extractable catalytic activities of the enzymes of sugar nucleotide conversion determined previously. Increased secretion was observed in all major groups of polysaccharides, particularly the cellulosic fraction. Analysis of the sugars in the hemicellulosic fraction indicated that the newly synthesised polysaccharide was most probably xylan. It was confirmed by immunolocalisation of xylan in these walls. This treatment thus increases incorporation into the wall of components characteristic of secondary wall. In the second system, which models the defence response, suspension cultures were treated with an elicitor from the walls of Colletotrichum lindemuthianum . Again, sugar flux was determined by labelling cells with [ 14 C]-sucrose and examined during the period determined previously of maximum extractable catalytic activities of the enzymes of sugar nucleotide conversion. Increased secretion into unextractable polymers was the major change and was consistent with the occurrence of oxidative processes leading to immobilisation of some wall components. Callose, a polysaccharide characteristic of the defence response was immunolocalised in these walls but not in those of control cells.

Prolyl 4-hydroxylase (EC 1.14.11.2), the enzyme responsible for the post-translational hydroxylation of peptide proline, has been well described in animals but has been little studied in plants. The best characterised example is the enzyme from French bean (Phaseolus vulgaris). In this study, the biochemical properties of this plant enzyme were examined in more detail and, using specific polyclonal antibodies, the localisation of the enzyme was determined. Both α- and β-subunits did not show multiple forms, suggesting a relatively broad specificity of the enzyme complex with respect to the target hydroxylated amino acid sequences. Antibodies to the mammalian and Chlamydomonas enzymes cross-react with the higher plant subunits, indicating that some epitopes are highly conserved. The P. vulgaris enzyme was inhibited by analogues of oxoglutarate, but was not susceptible to doxorubicin. Inhibition of the bean enzyme by an oxaloglycine derivative resulted in the retention of the target (hydroxy)proline-rich protein in the endomembrane system. Immunolocalisation of the enzyme showed close association with the endoplasmic reticulum and Golgi apparatus in root tip cells of P. vulgaris or Tropaeolum majus. This localisation was particularly pronounced in Golgi-associated vesicles of young root tip cells of T. majus, cell types where rapid synthesis and deposition of wall material was observed. These data are consistent with the hypothesis, proposed by Bolwell [G.P. Bolwell, Dynamic aspects of the plant extracellular matrix, Int. Rev. Cytol. 146 (1993) 261–324], that protein hydroxylation must be completed before the glycosylation of the target (hydroxy)proline-rich glycoproteins in the Golgi stack.

The parasite’s devastating eVect is accomplished prior to Striga’s emergence from the soil. Striga seeds germinate Developmentally-specific markers have been identi- and develop its parasitic proboscis, the haustorium, in fied in the germinating and haustorial stages of Striga response to host root signals. The germination, haustorial hermonthica seedlings. Four water-soluble proteins, induction and attachment stages are critical interdiction preferentially expressed in germinated seedlings, were points in Striga’s life cycle. EVorts should be addressed microsequenced. An haustorial-specific cDNA clone to understanding these mechanisms in order to develop was isolated by differential screening. Tissue specifi- more eVective methods of intervention. In this work an city for this clone was assessed by Northern blot attempt is made to analyse the Striga genome with hybridization analysis. particular reference to gene expression in the very early stages of the parasite’s development (free living state=

Summary Immunogold labelling was used to probe the responses of mesophyll cells in French bean (Phaseolus vulgaris L.) to anhrpAmutant ofXanthomonas campestrispv.vesicatoriaand a saprophytic strain ofX.c.The non-pathogenic strains both caused localized alterations to the plant cell wall and formation of large papillae in adjacent cells. Immunocytochemistry showed the co-localization, in the cell wall and paramural deposits, of anMr 42 000 proline-rich glycoprotein with chitin-binding activity (CBPRP) and the enzyme responsible for its immobilization, anMr 46 000 peroxidase. The CBPRP appeared to lose antigenicity after cross-linking, and, unlike the peroxidase, was not detected consistently in the extracellular matrix that encapsulated bacteria onto the plant cell wall. The peroxidase may have a dual function in both the generation and utilization of H2O2 for cross-linking of proteins and phenolics during the construction of papillae. A burst of H2O2 was detected 1–5 h after inoculation at reaction sites by histochemical staining with cerium chloride. Progressive expansion of papillae and cell-wall alterations was, however, not associated with the maintenance of high levels of H2O2. Co-localization of callose and anMr 65 000 polypeptide component of callose synthase was also demonstrated. Synthesis of callose appeared so rapid that the enzyme became embedded in the polysaccharide so that both were detected as integral to the developing papilla. Localized alterations to the cell wall and deposition of papillae were found to involve co-ordinated synthetic and oxidative activities at microsites within responding cells, without activation of the hypersensitive reaction.

Transfer of ferulic acid from feruloyl-CoA onto polymeric material can be readily demonstrated by microsomal membranes from suspension-cultured cells of French bean. Transfer onto endogenous components present in the microsomal preparations can be supplemented by addition of polysaccharide or lipid components. However, addition of catalase to reaction mixtures suppresses transfer onto polysaccharide components, indicating that this is peroxidative. In contrast, suppression of microsomal peroxidase activities leads to increased transfer onto hydroxy-hexadecanoic acid showing that any transferase is possibly specific for lipid and therefore involved in suberisation. This transferase was transiently induced by elicitation of the cells. The time course of induction was similar to that for the cytochrome P450, laurate ω-hydroxylase required for the hydroxylation of fatty acids. Solubilisation and partial purification of the transferase was accomplished and indicated an enzyme with a tendency to aggregate but with a probable minimum subunit M r of 40 000. © 1997 Elsevier Science Ltd. All rights reserved

The proteins of the primary cell walls of suspension cultured cells of five plant species, Arabidopsis, carrot, French bean, tomato, and tobacco, have been compared. The approach that has been adopted is differential extraction followed by SDS-polyacrylamide gel electrophoresis (PAGE), rather than two-dimensional gel analysis, to facilitate protein sequencing. Whole cells were washed sequentially with the following aqueous solutions, CaCl2, CDTA (cyclohexane diaminotetraacetic acid, DTT (dithiothreitol), NaCl, and borate. SDS-PAGE analysis showed consistent differences between species. From the 233 proteins that were selected for sequencing, 63% gave N-terminal data. This analysis shows that (i) patterns of proteins revealed by SDS-PAGE are strikingly different for all five species, (ii) a large number of these proteins cannot be identified by data base searches indicating that a significant proportion of wall proteins have not been previously described, (iii) the major proteins that can be identified belong to very different classes of proteins, (iv) the majority of proteins found in the extracellular growth media are absent from their respective cell wall extracts, and (v) the results of the extraction process are indicative of higher order structure. It appears that aspects of speciation reside in the complement of extracellular wall proteins. The data represent a protein resource for cell wall studies complementary to EST (expressed sequence tag) and DNA sequencing strategies.

Among many aspects of plant defence responses to pathogenic infection are changes in the composition of the exocellular matrix. To study potential defences in white lupin (Lupinus albus L.), suspension-cultured cells were treated for 24 h with one of three different elicitors : CuCl 2 , and two fungal elicitor preparations from purified cell walls of yeast and Colletotrichum lindemuthianum. Two subsets of exocellular proteins: ionicallybound wall proteins and proteins secreted into culture medium, were isolated, and their patterns compared following electrophoretic separation. Only a few proteins were observed in culture filtrates with dominating bands at 27, 33, and 42 kDa. About 30 proteins were observed in cell wall extracts. Changes in protein intensities evoked by elicitor treatments depended on the type of elicitor used, the age and composition of lupin cell culture, and concentration of applied fungal elicitor. Based on these observations, ten proteins were chosen for N-terminal sequencing, and sequences 5-30 amino acids long for nine proteins were obtained. Three of the major proteins sequenced were identified as acidic exocellular chitinase, polygalacturonase-inhibiting protein, and germin/oxalate oxidase.

A study has been made of changes in the enzymes of UDP-sugar metabolism in suspension cultured cells of French bean during their normal growth following subculture. These are compared to cells of the same cell suspension subcultured into a cytokinin enriched media which induces changes in their cell walls that resemble xylogenesis. The same enzymes have also been measured in seedling hypocotyls undergoing elongation growth and differentiation. Evidence was obtained for sucrose synthase and possibly amylase for having roles in the mobilization of sugar reserves during growth changes in suspension cultures. Furthermore, UDP-glucose dehydrogenase showed characteristic increased activity that indicated a key role in the provision of sugar nucleotides for cell wall biosynthesis. Less significant changes were observed for other enzymes involved in sugar nucleotide interconversion.

Stressed plant cells often show increased oxygen uptake which can manifest itself in the transient production of active oxygen species, the oxidative burst. There is a lack of information on the redox status of cells during the early stages of biotic stress. In this paper we measure oxygen uptake and the levels of redox intermediates NAD/NADH and ATP and show the transient induction of the marker enzyme for redox stress, alcohol dehydrogenase. Rapid changes in the redox potential of elicitor-treated suspension cultures of French bean cells indicate that, paradoxically, during the period of maximum oxygen uptake the levels of ATP and the NADH/NAD ratio fall in a way that indicates the occurrence of stress in oxidative metabolism. This period coincides with the maximum production of active oxygen species particularly H2O2. The cells recover and start producing ATP immediately of H2O2 production. This indicates that the increased O2 uptake is primarily incorporated into active O2 species. A second consequence of these changes is probably a transient compromising of the respiratory status of the cells as indicated in expression of alcohol dehydrogenase. Elicitor-induced bean ADH was purified to homogeneity and the M(r) 40,000 polypeptide was subjected to amino acid sequencing. 15% of the whole protein was sequenced from three peptides and was found to have nearly 100% sequence similarity to the amino acid sequence for pea ADH1 (PSADH1). The cDNA coding for the pea enzyme was used to demonstrate the transient induction of ADH mRNA in elicitor-treated bean cells. Enzyme activity levels also increased transiently subsequently. Increased oxygen uptake has previously been thought to be associated with provision of energy for the changes in biosynthesis that occur rapidly after perception of the stress signal. However the present work shows that this rapid increase in oxygen uptake as a consequence of elicitor action is not wholly associated with respiration.

This study focuses upon the developmental transition of the parasitic plant Striga hermonthica from its freeliving state (germinated seedling) to its parasitic state after development of an infection organ: the haustorium. A new method has been developed that allows the production of gram quantities of germinated and haustorially-induced Striga seedlings, thereby facilitating biochemical and molecular analysis of haustorial induction. Water-soluble proteins have been extracted from germinated seeds (stage A) and seedlings treated with 2,6-dimethoxy-p-benzoquinone (2,6-DMBQ) to induce haustorium (stage B). Samples were analysed by two-dimension al polyacrylamid e gel electrophoresis and quantitative as well as qualitative differences could be observed. In particular a group of four highly abundant acidic proteins (molecular weight 39 kDa, pi 5.1, 5.3, 5.3, 5.6) and three other proteins (molecular weight 12 kDa, pi 6.9; 17 kDa, pi 4.4; 17 kDa, pi 4.45) were seen in stage A while at least four proteins (molecular weight 21.5 kDa, pi 6.4; 21.5 kDa, pi 6.3; 31 kDa, pi 5.1; 34 kDa, pi 6.2) were present in greater abundance in stage B. In order to compare watersoluble protein with newly synthesized protein patterns, mRNAs from the two stages of development were isolated and cell-free translation products analysed by 2-D PAGE. Two-D gels of cell-free translation products showed the appearance of six proteins in stage B (molecular weight ranging from 10 to 35 kDa) and the presence of three acidic proteins in stage A with one protein (molecular weight 40 kDa) very similar in size to the triplet of proteins in the water-soluble protein 2-D gels.

Harnessing the toxic properties of reactive oxygen species (ROS) to fight off invading pathogens can be considered a major evolutionary success story. All aerobic organisms have evolved the ability to regulate the levels of these toxic intermediates, whereas some have evolved elaborate signalling pathways to dramatically increase the levels of ROS and use them as weapons in mounting a defence response, a process commonly referred to as the oxidative burst. The balance between steady state levels of ROS and the exponential increase in these levels during the oxidative burst has begun to shed light on complex signalling networks mediated by these molecules. Here, we discuss the different sources of ROS that are present in plant cells and review their role in the oxidative burst. We further describe two well-studied ROS generating systems, the NADPH oxidase and apoplastic peroxidase proteins, and their role as the primary producers of ROS during pathogen invasion. We then discuss what is known about the metabolic and proteomic fluxes that occur in plant cells during the oxidative burst and after pathogen recognition, and try to highlight underlying biochemical processes that may provide more insight on the complex regulation of ROS in plants.

In plants, reactive oxygen species (ROS) associated with the response to pathogen attack are generated by NADPH oxidases or apoplastic peroxidases. Antisense expression of a heterologous French bean (Phaseolus vulgaris) peroxidase (FBP1) cDNA in Arabidopsis thaliana was previously shown to diminish the expression of two Arabidopsis peroxidases (peroxidase 33 [PRX33] and PRX34), block the oxidative burst in response to a fungal elicitor, and cause enhanced susceptibility to a broad range of fungal and bacterial pathogens. Here we show that mature leaves of T-DNA insertion lines with diminished expression of PRX33 and PRX34 exhibit reduced ROS and callose deposition in response to microbe-associated molecular patterns (MAMPs), including the synthetic peptides Flg22 and Elf26 corresponding to bacterial flagellin and elongation factor Tu, respectively. PRX33 and PRX34 knockdown lines also exhibited diminished activation of Flg22-activated genes after Flg22 treatment. These MAMP-activated genes were also downregulated in unchallenged leaves of the peroxidase knockdown lines, suggesting that a low level of apoplastic ROS production may be required to preprime basal resistance. Finally, the PRX33 knockdown line is more susceptible to Pseudomonas syringae than wild-type plants. In aggregate, these data demonstrate that the peroxidase-dependent oxidative burst plays an important role in Arabidopsis basal resistance mediated by the recognition of MAMPs.

Antisera raised against l-phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and a cationic cell-wall peroxidase, which had all been purified from suspension-cultured cells of French bean, have been used to carry out immunogold localisations in the growing plant. Immunoglobulin-G fractions were prepared from each antiserum and used to study the distribution of the enzymes in differentiating and wounded hypocotyls by immunogold techniques and visualisation by both light and electron microscopy. Following silver enhancement to amplify the signal, proteins were detected by confocal microscopy in both developing (pre-xylem/ phloem) and later metaxylem stelar tissue. l-Phenylalanine ammonia-lyase and C4H also accumulated in cells adjacent to metaxylem, presumably involved in maintaining a supply of phenylpropanoid precursors to the enucleated xylem for further lignin synthesis. In these cells, PAL subunits were cytosolic although some were associated with endomembrane. Cinnamate-4-hydroxylase was wholly associated with membrane and particularly high concentrations were found in the Golgi bodies. The cationic peroxidase accumulated in xylem at sites of secondary thickening and in the middle lamella. The three proteins are also involved in defensive lignification. Thus when visualised by light microscopy, PAL and C4H were seen to accumulate to high levels throughout the cell types in wound sites and especially in the epidermal cells. An even more intense general distribution was found upon hyperinduction of wounded cells with α-aminooxy-β-phenylpropionic acid. At the subcellular level, PAL was found to be localised in the cytosol in the wounded cells; however, because of the loss of membrane through mechanical damage, association with membrane structures, particularly endoplasmic reticulum, in unwounded cells is not entirely ruled out. Cinnamate-4-hydroxylase was associated with membranes when these were preserved. In wounded tissue, the peroxidase was found at the growing edges of tylose-like structures in the vascular xylem.

A role for the phosphorylation of phenylalanine ammonia-lyase (PAL) which catalyses a key metabolic step in the biosynthesis of plant phenolics has been identified. Using immunoprecipitation of proteins radiolabelled with [ 32 P]orthophosphate in cells in vivo , the M r 70 000 subunit degradation product was found to be phosphorylated. This phosphorylation was amplified by treatment of the cells with high concentrations of forskolin, but not elicitor alone, relative to the level of [ 35 S]methionine labelled M r 77 000 and 70 000 subunit forms newly synthesised and turned-over as a consequence of elicitor action. Forskolin treatments bring about a correspondingly decreased specific activity of the extracted enzyme. Although a high level of phosphorylation was observed for a large number of species of polypeptides in cell-free extracts in vitro , including one of M r 69 000, this was not immunoprecipitated. The results are consistent with phosphorylation of the M r 70 000 PAL degradation product being specific and completed in vivo . Since forskolin brings about the down-regulation of elicitor-induced PAL, it is probable that the transient appearance of the enzyme activity and its turnover is in part regulated by phosphorylation, which is possibly cAMP dependent, in addition to other inactivating processes.

The utility of plant secondary cell wall biomass for industrial and biofuel purposes depends upon improving cellulose amount, availability and extractability. The possibility of engineering such biomass requires much more knowledge of the genes and proteins involved in the synthesis, modification and assembly of cellulose, lignin and xylans. Proteomic data are essential to aid gene annotation and understanding of polymer biosynthesis. Comparative proteomes were determined for secondary walls of stem xylem and transgenic xylogenic cells of tobacco and detected peroxidase, cellulase, chitinase, pectinesterase and a number of defence/cell death related proteins, but not marker proteins of primary walls such as xyloglucan endotransglycosidase and expansins. Only the corresponding detergent soluble proteome of secretory microsomes from the xylogenic cultured cells, subjected to ion-exchange chromatography, could be determined accurately since, xylem-specific membrane yields were of poor quality from stem tissue. Among the 109 proteins analysed, many of the protein markers of the ER such as BiP, HSP70, calreticulin and calnexin were identified, together with some of the biosynthetic enzymes and associated polypeptides involved in polymer synthesis. However 53% of these endomembrane proteins failed identification despite the use of two different MS methods, leaving considerable possibilities for future identification of novel proteins involved in secondary wall polymer synthesis once full genomic data are available.

Reactive oxygen species (ROS), superoxide, hydrogen peroxide and nitric oxide are produced at all levels of resistance reactions in plants. In basal resistance, they are linked to papilla formation and the assembly of barriers. In the hypersensitive response, they may be linked to programmed cell death, and in systemic acquired resistance, they interact with salicylate in signalling. Despite this importance, there is still a need to dissect the identities, activation and relative contributions of the ROS generating systems. Progress, however, is being made in identifying the molecular targets at the transcriptional, protein and cellular structure levels that are regulated by ROS in coordinating resistance responses.

A range of modulators have been used to investigate signal transduction involved in the response of suspension cultured cells of French bean to an elicitor preparation from the fungal pathogen Colletotrichum lindemuthianum. Elicitor action was effectively determined by the induction of phenylalanine ammonia-lyase(PAL) protein and activity. Three types of modulators were used. One group that potentially directly affected calcium mobilization and action had with the exception of caffeine and procaine no consistent effect on elicitor action. A number of ionophores and compounds that affect membrane ion transport weretested and the most effective was found to be monensin. Further studies showed that the inhibition of elicitation by monensin was more likely to be mediated by inhibition of uptake of elicitor than perturbation of ion transport. A third group of modulators were characterized by their ability in animal systems to interact with G proteins. A variety of effects were observed which were consistent with putative G protein involvement as a component of elicitor-induced signal transduction. Modulation of the induction of the synthesis of PAL protein and of PAL activity were both observed. Overall, the results were consistent with elicitor action being potentiated through multicomponent transduction pathways.

The present understanding of ROS generation in the defence response of Arabidopsis thaliana is reviewed. Evidence suggests that the apoplastic oxidative burst generated during basal resistance is peroxidase-dependent. The ROS generated during this basal resistance may serve to activate NADPH oxidase during the R-gene-mediated hypersensitive response. The processes involved in the production of reactive oxygen species in A. thaliana cell suspension cultures in response to an elicitor from Fusarium oxysporum are investigated in the present work. This system appears analogous to the production of ROS during the basal resistance response in French bean, which is peroxidase-dependent. A panel of modulators effective in other pathogen elicitor and plant cell systems has been used to investigate the Arabidopsis signalling pathways and the plant cell responses involved. Thus as in other systems, an early calcium influx into the cytosolic compartment, a rapid efflux of K(+) and Cl(-), and extracellular alkalinization of elicited cell cultures has been found. However the alkalinization is not sufficient to stimulate the apoplastic oxidative burst by itself, unlike in French bean, although vectorial ion fluxes are needed. A secretory component which is sensitive to monensin and N-ethylmaleimide and insensitive to brefeldin A may also be necessary for the release and provision of substrates for peroxidase-dependent generation of H(2)O(2).

The pathway from UDP-glucose to UDP-xylose has been characterised in differentiating tobacco tissue. A xylogenic suspension cell culture of tobacco has been used as a source for the purification of the enzymes responsible for the oxidation of UDP-glucose to UDP-glucuronic acid and its subsequent decarboxylation to UDP-xylose. Protein purification and transcriptional studies show that two possible candidates can contribute to the first reaction. Most of the enzyme activity in the cultured cells could be accounted for by a protein with an Mr of 43 kDa which had dual specificity for UDP-glucose and ethanol. The cognate cDNA, with similarity to alcohol dehydrogenases (NtADH2) was expressed in E. coli to confirm the dual specificity. A second UDP-glucose dehydrogenase, corresponding to the monospecific form, ubiquitous amongst plants and animals, could not be purified from the tobacco cell cultures. However, two cDNAs were cloned with high similarity to the family of UDP-glucose dehydrogenases. Transcripts of both types of dehydrogenase showed highest expression in tissues undergoing secondary wall synthesis. The UDP-glucuronate decarboxylase was purified as polypeptides of Mr 87 and 40 kDa. Peptide fingerprinting of the latter polypeptide identified it as a form of UDP-glucuronate decarboxylase and functionality was established by expressing the cognate cDNA in E. coli. Expression of 40 kDa polypeptide and its corresponding mRNA was also found to be highest in tissues associated with secondary wall formation.

Changes in protein kinase activity have been investigated during the early response of suspension cultured cells of French bean to fungal elicitor. One of the kinases activated has a known target, phenylalanine ammonia-lyase (PAL), which has an important role in plant defence responses, and was purified. Kinase acivity during purification was monitored for both the PAL-derived peptide and syntide-2, which it also phosphorylated. The kinase had an Mr of 55,000 on the basis of gel migration, 45Ca2+ binding, autophosphorylation and phosphorylation of various substrates using in-gel assays. The kinase has been characterised with respect to kinetics and other properties in vitro and appears to be a CDPK. In-gel assays were also used to show that this kinase and a number of other CDPKs of similar Mr showed complex changes in elicitor-treated suspension-cultured cells of French bean. An activation was observed within 10 min and was maintained for up to 4 h. The time course of activation was different from MAP kinase and casein kinase assayed in the same extracts. However, at 5 min after addition of elicitor there is a transient inactivation of the CDPKs before activation. This inactivation can be mimicked by adding forskolin to the cells 30 min before elicitation, which brings about changes in the cellular pH. Forskolin potentiates the oxidative burst when elicitor is subsequently added while the CDPK cannot be activated by elicitor upon forskolin treatment. In contrast, intracellular acidification brought about by forskolin brings about slight activation of MAPkinase.

The Golgi apparatus of plant cells is thought to be the main site of synthesis of cell wall matrix polysaccharides and the terminal glycosylation of glycoproteins. Much of this evidence still depends on earlier biochemical studies employing subcellular fractionation. However acquiring pure Golgi membranes is still difficult and the question of spatial organisation of glycosyl transferases can be addressed by immunolocation of the enzymes. An antibody to a xylan synthase-associated polypeptide from French bean, the enzyme which synthesises the core polysaccharide for secondary wall xylan, has been raised and shown to inhibit its activity. Xylan is deposited in secondary thickenings and the xylan synthase was only detected in appreciable amounts in developing xylem cells. The location within the Golgi stack was observed throughout the dictyosomes. Some enzyme subunits were also detected in post-Golgi vesicles. A second antibody to a non-catalytic M r 65 000 subunit of β 1,3- glucan (callose) synthase was used for a comparative study. Although the bulk of this enzyme has been detected in previous studies at plasmamembrane-wall interfaces in sieve plates and stressed tissue, a Golgi-location can be observed in root tip meristematic cells during cell plate formation. The enzyme was present throughout the stacks. Callose was also immunolocated in a similar manner to xylan in secondary walls and thickenings and in pits in developing xylem. In these cells, the callose synthase was detected at the surface of the growing thickenings and the plasmamembrane within the pits.

Arabinogalactan-proteins (AGPs) were initially defined as Hyp-containing, highly glycosylated glycoproteins or proteoglycans. They contain arabinogalactan chains, and the linkage between the carbohydrate and protein is normally through Gal-Hyp. The protein is generally neutral to acidic and frequently contains Hyp/Pro-Ala motifs. However, the discovery of a number of developmentally important proteins showing hybrid features has broadened the definition (Sommer-Knudsen et al 1998). Some examples are those that contain Cys-rich domains at the C-terminus (Du et al 1996, Schultz et al 1997). Similarly, hybrid solanaceous lectins such as that from potato have an extensin-like domain at the N-terminus and a Cys-rich C-terminus, which contains chitin-binding domains analogous to other chitin-binding lectins (Allen et al 1996). A major chitin-binding extracellular protein of French bean has been identified and shows similar hybrid features with a Cys-rich C-terminal domain, but the N-terminus is that of a Pro-rich protein in its repeat sequence (Bolwell 1986 1987, Millar et al 1992). This French bean protein has been classified as a proline/hydroxyproline-rich glycoprotein (P/HRGP) (Sommer-Knudsen et al 1998), but it also shows some features common to AGPs. Further properties of this protein suggest an important role in defense against pathogen attack. In terms of this work, the protein has been defined as a chitin-binding Pro-rich protein (CBPRP; Brown et al 1998).

It remains a major goal of modern biology to understand the molecular events involved in the differentiation of specialized cells. This chapter considers to what extent the experimental induction of vascular differentiation in cell and tissue cultures can be achieved and what insight such studies can generate into similar processes in the intact plant. The lack of complexity, relative homogeneity and isolation of events within individual or small groups of cells has great potential for study of aspects of signal transduction leading to the modulation of genes responsible for structural and other changes during differentiation. Although such systems cannot reconstitute cell lineages and position effects they could, in principle, help define the role of extracellular signals and other factors and their modulation by a largely empirical approach involving optimization of their effects under various nutrient and growth regulator requirements. Furthermore, biochemical studies may be able to identify aspects of metabolic regulation that are not accessible in the intact plant. Finally, tissue culture systems can be used to understand intracellular signal transduction pathways leading to changes in gene expression.

The possible role of cyclic AMP (cAMP) in plants has been an area of controversy for nearly 20 years: although cAMP and putative components of its synthesis, degradation and site of action can be demonstrated, there has been no clear identification of a signal transduction pathway dependent upon cAMP in plants. However, stronger evidence is accumulating for its possible role in stress signalling.

In this study the antioxidant activities of the hydroxycinnamic acids, chlorogenic, caffeic, ferulic and p-coumaric have been investigated in peroxidising lipid systems mediated by metmyoglobin. The results show that the order of effectiveness in increasing the resistance of LDL to peroxidation, in protecting LDL cholesterol from oxidation and preventing the oxidative modification of the LDL apoprotein B100 is caffeic = chlorogenic > ferulic > p-coumaric acid. Assessment of the rates of reaction of the hydroxycinnamates with ferrylmyoglobin, a product of the reductive decomposition of lipid hydroperoxides, reveals that the compounds are more effective as peroxyl radical scavengers than reductants of ferryl myoglobin in peroxidising LDL systems mediated by haem proteins.

Publisher Summary This chapter discusses dynamic aspects of the plant extracellular matrix. It describes the current status of knowledge of biochemical regulation that leads to differential patterns of synthesis of wall components, their site of synthesis and deposition, and the changes that occur in response to developmental and environmental cues. The plant-cell wall, with its connotations of rigidity and maintenance of shape, is possibly considered as an extracellular matrix with dynamic properties. Various classes of polysaccharides of the extracellular matrix are well-conserved. Cellulose microfibrils are spun into a matrix consisting of both pectinaceous and hemicellulosic polysaccharides. In addition to cellulose microfibrils, plant walls also contain hemicelluloses and pectins. Phenolics are deposited mainly in the secondary wall but are also important constituents within the primary wall. Wall phenolics are based upon the phenylpropanoid unit and are found as both conjugated acids and, more familiarly, lignin alcohols. The cell wall is intimately involved as part of the sensing of the environment and in responding through the ensuing changes.