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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.

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.

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 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.

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

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.

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.

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 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.

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.

In contrast to microsomal membranes from suspension cultured cells undergoing primary wall synthesis which incorporated arabinose directly from UDP- β-L-arabinose into arabinan, membranes from cells treated with fungal elicitor catalysed the formation of a lipid oligosaccharide intermediate in the arabinosylation of an inducible M r 42 500 glycoprotein. These variations in the patterns and mechanism of arabinosylation observed between the cells in response to the differing stimuli were detected in both the kinetics of incorporation and the demonstration that the lipid oligosaccharide after purification on ion-exchange chromatography could act as primary donor for the glycoprotein and not polysaccharide. These results distinguish mechanisms for the transfers of arabinose onto glycoprotein and polysaccharide by enzyme systems known to be immunologically distinct. The fungal elicitor-induced M r 42 500 glycoprotein binds to thyrogolubulin- and fetuin-Sepharoses in a specific manner and this binding is prevented by chitin oligomers. The glycoprotein thus appears to be a carbohydrate-binding protein and the sugar specificity together with the demonstration of hydroxyproline residues in acid hydrolysates of the glycoprotein purified by affinity chromatography indicates a close similarity to the arabinosylated hydroxyproline-rich lectins of the Solanaceae which can function as bacterial agglutinins. The M r 42 500 glycoprotein which undergoes rapid transient induction also clearly differs from other extensin-like hydroxyproline-rich glycoproteins, arabinogalactan proteins and the characteristic bean seed-lectin, phytohaemagglutinin, in a number of properties.

Addition of trans -cinnamic acid to bean cell suspension cultures after their treatment with fungal elicitor resulted in the loss of induced phenylalanine ammonia-lyase (PAL) enzyme activity. In contrast elicitor-induced chalcone synthase (CHS) activity was arrested but did not decline, whereas chalcone isomerase (CHI) activity was relatively unaffected. However, translational activities of extracted polysomal mRNAs encoding these three enzymes were depressed by cinnamate treatments as were levels of PAL and CHS polysomal mRNAs and rates of transcription of these genes measured in isolated nuclei. Treatment of elicitor-induced cultures with l -α-aminooxy-phenylpropionic acid (AOPP), a potent inhibitor of PAL activity (and therefore cinnamate production) in vivo , resulted in increased production of PAL and CHS mRNAs. Addition of cinnamate to elicitor-treated cultures inhibited the appearance of a number of polypeptides translated in vitro from polysomal mRNA, although at least nine polypeptides were specifically induced by cinnamate treatment. We conclude that cinnamic acid potentially could act as an in vivo modulator of the synthesis of phenylpropanoid pathway enzymes although it is not yet fully possible to rule out less specific inhibitory effects.

The dynamic qualities of the plant cell wall are explored. Recent developments in the understanding of how membrane systems operate in the synthesis of wall components is discussed. Particular attention is paid to the synthesis of polysaccharides and wall-bound phenolics, while the identification of some wall bound proteins and the subsequent study of their synthesis and processing is also described. Probable molecular controls, both qualitative and quantitative, governing the appearance of newly synthesized components within the wall are discussed. The conditions under which sets of components are modified specifically, by the accumulation of newly synthesized material or extracellular enzyme action, are outlined. Some deficiencies in understanding how these aspects may be interrelated, especially with regard to mechanisms of signal transduction are highlighted. However the potentially rapid capacity for implementation of changes in the extracellular matrix of the cell, probably commencing with processes of activation of specific subsets of genes in response to particular signals, is striking.