Acibenzolar acid, the first metabolite formed in planta from the defence inducer acibenzolar-S-methyl (ASM), has been shown to be an inhibitor of the enzyme shikimate hydroxycinnamoyltransferase (HST), extracted from grapevine or tobacco cell suspension cultures. Using a purified recombinant Arabidopsis thaliana HST, the inhibition was found to be competitive, acibenzolar acid binding reversibly to the shikimate binding site of the HST:p-coumaroyl-CoA complex, with a Ki value of 250 μM. The other hydroxycinnamoyltransferases tested in the course of this study, using either hydroxypalmitic acid, putrescine, tyramine, or quinic acid as acyl acceptors were not, or only slightly, inhibited by acibenzolar acid. To understand the specificity of the interaction of acibenzolar acid with HST, we analyzed the structure-activity relationship of a series of benzoic or acibenzolar acid analogues, tested either as AtHST substrates or as inhibitors. This analysis confirmed previously published data on the substrate flexibility of HST and demonstrated that both the carboxyl group and the thiadiazole moiety of acibenzolar acid are playing an important role in the interaction with the shikimate binding site. Acibenzolar acid, which cannot form an ester bond with p-coumaric acid, was however a less potent inhibitor than protocatechuic or 3-hydroxybenzoic acids, which are used as acyl acceptors by HST. Our results show that the interaction of acibenzolar acid with HST, which is probably directly linked to the substrate promiscuity of HST, is unlikely to play a direct role in the defence-inducing properties of ASM in plants.

In a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader's hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall-degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards.

Crude extracts of Vitis vinifera canes represent a natural source of stilbene compounds with well characterized antifungals properties. In our trials, exogenous application of a stilbene extract (SE) obtained from grape canes on grapevine leaves reduces the necrotic lesions caused by Botrytis cinerea. The SE showed to possess a direct antifungal activity by inhibiting the mycelium growth. The activation of some grapevine defense mechanism was also investigated. H2O2 production and activation of mitogen-activated protein kinase (MAPK) phosphorylation cascades as well as accumulation of stilbenoid phytoalexins were explored on grapevine cell suspension. Moreover, the transcription of genes encoding for proteins affecting defense responses was analyzed on grapevine plants. The SE induced some grapevine defense mechanisms including MAPK activation, and the expression of pathogenesis-related (PR) genes and of a gene encoding the glutathione-S-transferase 1 ( GST1) . By contrast, treatment of grapevine leaves with SE negatively regulates de novo stilbene production.

Grapevine trutk diseases, especially Esca, are of major concern since they gradually alter vineyards worldwide and cause heavy economic losses. The expression of Esca disease symptoms depends on several factors, including the grapevine cultivar. In this context, a possible clone-dependent expression of the Esca disease was studied. Two clones of 'Chardonnay' grown in the same plot were compared according to their developmental and physiological traits, metabolome, and foliar symptom expression. Analysis of their leaf metabolome highlighted differences related to symptom expression. Interestingly, the content of a few specific metabolites exhibited opposite variations in leaves of symptomatic shoots of clones 76 and 95. Altogether this study showed a clone-dependent expression of Esca disease in 'Chardonnay' and the relevance of GC-MS and 3D fluorescence methods to analyze the impact of the disease on the leaf metabolome.

Laminaran, a β-(1→3)-glucan extracted from Laminaria digitata, is a known elicitor of plant defenses, but provides only low level of disease control in vineyard trials. In this context, laminaran was partly hydrophobized by grafting from 1.6 to 7.6 lauryl chains to the native saccharidic chain and the impact of sulfation of the hydrophobized glucans was studied. The activity of the different synthetized laminaran derivatives as antimicrobial agents against Plasmopara viticola, the causal agent of grape downy mildew, and as elicitors of defense reactions in planta, was evaluated. Our results showed that acylation imparts an antimicrobial activity to laminaran which is related to the degree of acylation, AL3, with 7.6 lauryl chains, being the most effective derivative. Sulfation of the acylated laminarans did not further increase the antimicrobial activity. Our results also demonstrated that the efficacy of AL3 against Plasmopara viticola was most likely due to the direct antimicrobial activity of the lauryl chains rather than to an elicitation of plant defenses.

Elicitors trigger plant defense responses, including phytoalexin production and cell-wall reinforcement. Primary metabolism plays an important role in these responses as it fuels the associated energetic costs and provides precursors for the synthesis of the numerous secondary metabolites involved in defenses against pathogens. In this context, we aimed to determine whether oligosaccharidic elicitors differing in their capacity to activate defense-associated secondary metabolism in grapevine would differently impact primary metabolism. To answer this question, cell suspensions were treated with two elicitors: an oligogalacturonide, and the β-glucan laminarin. Enzymatic activity assays together with targeted (HPLC) and global (GC-MS) analyses of metabolites were next performed to compare their impact on plant primary or secondary metabolism. The results showed that the oligogalacturonide, which induced the highest level of the phytoalexin resveratrol and the highest activity of stilbene synthase, also induced the highest activity of shikimate hydroxycinnamoyltransferase, a key enzyme involved in the synthesis of lignin. The oligogalacturonide-induced defenses had a significant impact on primary metabolism 24 h following elicitor treatment, with a reduced abundance of pyruvate and 2-oxoglutarate, together with an increase of a set of metabolites including carbohydrates and amino acids. Interestingly, an accumulation of galacturonate and gentiobiose was observed in the oligogalacturonide- and laminarin-treated cells, respectively, suggesting that both elicitors are rapidly hydrolyzed in grapevine cell suspension cultures.

Protecting vineyards from cryptogamic diseases such as downy mildew, caused by Plasmopara viticola, generally requires a massive use of phytochemicals. However, the issues on unintentional secondary effects on environment and human health, and the occurrence of P. viticola resistant strains, are leading to the development of alternative strategies, such as the use of biocontrol products. In this paper, we evidenced the ability of a plant extract to protect grapevine from P. viticola. Further experiments carried out both on cell suspensions and on plants revealed that plant extract activates typical defense-related responses such as the production of H2O2, the up-regulation of genes encoding pathogenesis-related proteins and stilbene synthase, as well as the accumulation of resveratrol or its derivative piceid. We also brought to light a strong direct effect of PE on the release and motility of P. viticola zoospores. Furthermore, we found out that PE application left dried residues on leaf surface, impairing zoospores to reach stomata. Altogether, our results highlight the different modes of action of a new biocontrol product able to protect grapevine against downy mildew.

A Gram-negative bacterium able to grow using chlorogenic acid (5-caffeoylquinic acid) as sole carbon source has been isolated from the roots of tomato plants inoculated with the arbuscular mycorrhizal fungus Rhizophagus irregularis. An intracellular esterase exhibiting very high affinity (K-m = 2 mu M) for chlorogenic acid has been extracted and purified by FPLC from the chlorogenate-grown cultures of this bacterium. The molecular mass of the purified esterase determined by SDS-PAGE was 61 kDa and its isoelectric point determined by chromatofocusing was 7.75. The esterase hydrolysed chlorogenic acid analogues (caffeoylshikimate, and the 4- and 3-caffeoylquinic acid isomers), feruloyl esterases substrates (methyl caffeate and methyl ferulate), and even caffeoyl-CoA in vitro but all of them were less active than chlorogenic acid, demonstrating that the esterase is a genuine chlorogenic acid esterase. It was also induced when the bacterial strain was cultured in the presence of hydroxycinnamic acids (caffeic, p-coumaric or ferulic acid) as sole carbon source, but not in the presence of simple phenolics such as catechol or protocatechuic acid, nor in the presence of organic acids such as succinic or quinic acids. The purified esterase was remarkably stable in the presence of methanol, rapid formation of methyl caffeate occurring when its activity was measured in aqueous solutions containing 10-60% methanol. Our results therefore show that this bacterial chlorogenase can catalyse the transesterification reaction previously detected during the methanolic extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots. Data are presented suggesting that colonisation by Rhizophagus irregularis could increase chlorogenic acid exudation from tomato roots, especially in nutrient-deprived plants, and thus favour the growth of chlorogenate-metabolizing bacteria on the root surface or in the mycorhizosphere. (C) 2016 Elsevier Masson SAS. All rights reserved.

Acetosyringone (3',5'-dimethoxy-4'-hydroxyacetophenone) is a well-known and very effective inducer of the virulence genes of Agrobacterium tumefaciens but the precise pathway of its biosynthesis in plants is still unknown. We have used two tobacco cell lines, cultured in suspension and exhibiting different patterns of accumulation of acetosyringone in their culture medium upon treatment with methyl jasmonate, to study different steps of acetosyringone biosynthesis. In the two cell lines studied, treatment with 100 μM methyl jasmonate triggered a rapid and transient increase in acetovanillone synthase activity followed by a progressive increase in S-adenosyl-L-methionine: 5-hydroxyacetovanillone 5-O-methyltransferase activity which paralleled the rise in acetosyringone concentration in the culture medium. This O-methyltransferase displayed Michaelis-Menten kinetics with an apparent Km value of 18 μM for 5-hydroxyacetovanillone and its activity was magnesium-independent. Its molecular mass was estimated by gel permeation on an FPLC column and was found to be of ca. 81 kDa. 5-Hydroxyacetovanillone was the best substrate among the different o-diphenolic compounds tested as methyl acceptors in the O-methyltransferase assay. No formation of 5-hydroxyacetovanillone could be detected in vitro from 5-hydroxyferuloyl-CoA and NAD in the extracts used to measure acetovanillone synthase activity, indicating that 5-hydroxyacetovanillone is probably formed by direct hydroxylation of acetovanillone rather than by β-oxidation of 5-hydroxyferulic acid. Taken together our results strongly support the hypothesis that acetosyringone biosynthesis in tobacco proceeds from feruloyl-CoA via acetovanillone and 5-hydroxyacetovanillone.

When Glomus intraradices-colonised tomato roots were extracted in methanol at 6 degrees C, chlorogenic acid (5-caffeoylquinic acid), naturally present in the extract, was slowly converted by transesterification into methyl caffeate. The progress of the reaction could be monitored by HPLC. The reaction only occurred when the ground roots were left in contact with the hydro-alcoholic extract and required the presence of 15-35% water in the mixture. When the roots were extracted in ethanol, chlorogenic acid was transformed to ethyl caffeate in the same conditions. The reaction was also detected in Glomus mosseae-colonised tomato root extracts. It was also detectable in non-mycorrhizal root extracts but was 10-25 times slower. By contrast it was undetectable in extracts of the aerial parts of tomato plants, which also contain high amounts of chlorogenic acid, whether or not these plants were inoculated by the arbuscular mycorrhizal fungus. We found that this transesterification reaction is catalysed by a tomato enzyme, which remains active in hydro-alcoholic mixtures and exhibits chlorogenate-dependant caffeoyltransferase activity in the presence of methanol or ethanol. This transferase activity is inhibited by phenylmethanesulfonyl fluoride. The 4- and 3-caffeoylquinic acid isomers were also used as substrates but were less active than chlorogenic acid. Highest activity was detected in mycorrhizal roots of nutrient-deprived tomato plants. Surprisingly this caffeoyltransferase activity could also be detected in hydro-alcoholic extracts of G. intraradices-colonised roots of leek, sorghum or barrel medic. (C) 2013 Elsevier Masson SAS. All rights reserved.

With the aim of obtaining new strategies to control plant diseases, we investigated the ability of antagonistic lipopolypeptides (paenimyxin) from Paenibacillus sp. strain B2 to elicit hydrogen peroxide (H₂O₂) production and several defense-related genes in the model legume Medicago truncatula. For this purpose, M. truncatula cell suspensions were used and a pathosystem between M. truncatula and Fusarium acuminatum was established. In M. truncatula cell cultures, the induction of H₂O₂ reached a maximum 20 min after elicitation with paenimyxin, whereas concentrations higher than 20 μM inhibited H₂O₂ induction and this was correlated with a lethal effect. In plant roots incubated with different concentrations of paenimyxin for 24 h before inoculation with F. acuminatum, paenimyxin at a low concentration (ca. 1 μM) had a protective effect and suppressed 95% of the necrotic symptoms, whereas a concentration higher than 10 μM had an inhibitory effect on plant growth. Gene responses were quantified in M. truncatula by semiquantitative reverse transcription-PCR (RT-PCR). Genes involved in the biosynthesis of phytoalexins (phenylalanine ammonia-lyase, chalcone synthase, chalcone reductase), antifungal activity (pathogenesis-related proteins, chitinase), or cell wall (invertase) were highly upregulated in roots or cells after paenimyxin treatment. The mechanisms potentially involved in plant protection are discussed.

A soluble enzyme, extracted from tobacco cell-suspension cultures 24 h after treatment with 100 mu M methyl jasmonate. has been shown to synthesize acetovanillone (apocynin) from feruloyl-CoA in the presence of NAD. The enzyme displayed Michaelis-Menten kinetics with apparent K-m values of 5.6 mu M for feruloyl-CoA and 260 mu M for NAD and exhibited very high specificity for its substrates. The increase in acetovanillone synthase activity was followed by an increase in the concentration of both acetovanillone and acetosyringone in the culture medium. No intermediate could be detected when analysing the reaction medium by HPLC during the formation of acetovanillone in cell-free extracts. The apparent molecular mass estimated by gel permeation on an FPLC column was ca. 79 kDa. To our knowledge, this is the first report of an enzymic system catalysing the synthesis of an acetophenone. This work demonstrates that the biosynthesis of acetophenones in tobacco proceeds from hydroxycinnamic acids through a CoA-dependent beta-oxidation pathway. Interestingly in methyl jasmonate-treated cells, which synthesize very large amounts of hydroxycinnamoylputrescines, inhibition of the synthesis of these conjugates increased the concentration of acetovanillone and acetosyringone in the culture medium, suggesting that the two metabolic pathways can compete for their common precursors, i.e. hydroxycinnamoyl-CoA thioesters. (C) 2010 Elsevier Ltd. All rights reserved.

A burst of evolutionary duplication upon land colonization seems to have led to the large superfamily of cytochromes P450 in higher plants. Within this superfamily some clans and families are heavily duplicated. Others, such as genes involved in the phenylpropanoid pathway have led to fewer duplication events. Eight coding sequences belonging to the CYP98 family reported to catalyze the 3-hydroxylation step in this pathway were isolated from Triticum aestivum (wheat) and expressed in yeast. Comparison of the catalytic properties of the recombinant enzymes with those of CYP98s from other plant taxa was coupled to phylogenetic analyses. Our results indicate that the unusually high frequency of gene duplication in the wheat CYP98 family is a direct or indirect result from ploidization. While ancient duplication led to evolution of enzymes with different substrate preferences, most of recent duplicates underwent silencing via degenerative mutations. Three of the eight tested CYP98s from wheat have phenol meta-hydroxylase activity, with p-coumaroylshikimate being the primary substrate for all of these, as it is the case for CYP98s from sweet basil and Arabidopsis thaliana. However, CYP98s from divergent taxa have acquired different additional subsidiary activities. Some of them might be significant in the metabolism of various free or conjugated phenolics in different plant species. One of the most significant is meta-hydroxylation of p-coumaroyltyramine, predominantly by the wheat enzymes, for the synthesis of suberin phenolic monomers. Homology modeling, confirmed by directed mutagenesis, provides information on the protein regions and structural features important for some observed changes in substrate selectivity. They indicate that the metabolism of quinate ester and tyramine amide of p-coumaric acid rely on the same recognition site in the protein.

ABSTRACT Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae , produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B 1 , whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B 1 , with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.

Colonization of Pisum sativum L. cv. Frisson roots with the arbuscular mycorrhizal fungus Glomus mosseae leads to the induction of four acidic symbiosis-related chitinase (SR-chi) isoforms (EC 3.2.1.14). These isoforms were characterized as 30-kDa proteins with isoelectric points ranging between 5.2 and 5.85. One of these SR-chis was purified by affinity and anion-exchange chromatographies, and isoelectric focusing electrophoresis. The sequences of four internal peptides were obtained. They showed high homology to a class-I chitinase isoform from pea shoots. Parts of the conserved regions of class-I chitinases were found in this SR-chi. This result strongly supports the argument that this SR-chi isoform is of plant origin. The functional role of the SR-chis in arbuscular mycorrhizal symbiosis is discussed.

l-Tyrosine β-naphthylamide, a synthetic substrate designed to measure tyrosine aminopeptidase activity, is a potent inhibitor of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) purified from elicited tobacco cell-suspension cultures. The inhibition is competitive, with the inhibitor binding reversibly to the tyramine binding site of the enzyme. Similar results were obtained with THT extracted from elicited potato cell-suspension cultures. Ki values were found to be 0.66 μM for the enzyme from tobacco and 0.3 μM for the enzyme from potato. l-Tyrosine 7-amido-4-methylcoumarin, a fluorogenic substrate for tyrosine aminopeptidases, the structure of which is close to that of l-tyrosine β-naphthylamide, was also a powerful inhibitor, but slightly less effective with Ki values of 0.72 and 0.42 μM for tobacco and potato THT, respectively. l-Tyrosine β-naphthylamide was rapidly hydrolysed when fed in vivo to tobacco or potato cell cultures or when incubated in crude enzymic extracts prepared from these cultures. This hydrolysis, which is presumably catalysed by aminopeptidases, precludes the use of l-tyrosine amides as inhibitors of THT in vivo.

The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A)+RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5' leader and 3' UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl acetic acid 1,1-dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.

Arbuscular mycorrhizal (AM) symbioses are known to play a role in increased resistance of plants against soilborne pathogens. Mechanisms involved in this phenomenon are not yet well understood. This work investigates possible roles of endoproteolytic activities in bioprotection of Pisum sativum roots by Glomus mosseae against Aphanomyces euteiches. First, it is demonstrated that bioprotection occurs only in pre‐mycorrhizal plants. Second, endoproteolytic activities were analysed qualitatively and quantitatively during AM symbiosis, in plants infected with either zoospores or mycelium of A. euteiches, and in mycorrhizal plants infected with the pathogen. In mycorrhizal symbiosis a progressive increase in endoproteolytic activities was observed following root colonization by G. mosseae. By contrast, in roots inoculated with A. euteiches, a drastic increase in endoproteolytic activities was observed which was correlated with the amount of pathogen occurring in roots. Qualitative differences were seen among the endoproteolytic activities detected in roots inoculated with zoospores or mycelium. The constitutive as well as mycorrhizal and pathogen‐induced activities were further characterized as ‘trypsin‐like’ serine endoproteases. Interestingly, in a situation of bioprotection, only low levels of the activities normally associated with the infection by A. euteiches were detected, suggesting that the synthesis of these proteins is directly linked to the growth or virulence of the pathogen.

Caffeoyl-coenzyme A (CoA) O-methyltransferase (CCoAOMT) has been proposed to be involved in an alternative methylation pathway of lignin biosynthesis. However, no direct evidence has been available to confirm that CCoAOMT is essential for lignin biosynthesis. To understand further the methylation steps in lignin biosynthesis, we used an antisense approach to alter O-methyltransferase (OMT) gene expression and investigated the consequences of this alteration. We generated transgenic tobacco plants with a substantial reduction in CCoAOMT as well as plants with a simultaneous reduction in both CCoAOMT and caffeic acid O-methyltransferase (CAOMT). Lignin analysis showed that the reduction in CCoAOMT alone resulted in a dramatic decrease in lignin content. The reduction in CCoAOMT also led to a dramatic alteration in lignin composition. Both guaiacyl lignin and syringyl lignin were reduced in the transgenic plants. However, guaiacyl lignin was preferentially reduced, which resulted in an increase in the S/G (syringl/guaiacyl) ratio. We have also analyzed lignin content and composition in transgenic plants having a simultaneous reduction in both CCoAOMT and CAOMT. The reduction in both OMTs resulted in a further decrease in total lignin content. This is in sharp contrast to the effect that resulted from the reduction in CAOMT alone, which only decreased the syringl lignin unit without a reduction in overall lignin content. These results unequivocally demonstrate that methylation reactions in lignin biosynthesis are catalyzed by both CCoAOMT and CAOMT.