Jonathan Negrel

Ether-linked ferulic acid amides in natural and wound periderms of potato tuber

Abstract The occurrence of ether-linked ferulic acid amides (feruloyltyramine and/or feruloyloctopamine) in suberin-enriched samples of natural and wound periderms of potato tubers was established by thioacidolysis and desulphuration, followed by GC-mass spectrometry of diagnostic dihydroferuloyltyramine. Analysis of samples premethylated in the presence of diazomethane showed that the major part of the ether bonds involved the ferulic moiety of the amides; 20% of the amides released upon thioacidolysis were nevertheless attached to cell walls through the tyramine phenolic group. In addition, dicovalently linked ferulic acid amides were evidenced. These bridges were twice as abundant in the wound than in the natural periderm.

Impact of different levels of cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants

Abstract. The effects of cinnamyl alcohol dehydrogenase (CAD, EC.1.1.1.195) down-regulation on lignin profiles of plants were analysed in four selected transgenic lines of tobacco (Nicotiana tabacum L. cv. Samsun) exhibiting different levels of CAD activity (8–56% of the control). A significant decrease in thioacidolysis yields (i.e. yield of β-O-4 linked monomers) and in the ratio of syringyl to guaiacyl monomers (S/G) was observed for three transgenic lines and the most drastic reduction (up to 50%) was correlated with the lowest level of CAD activity. Higher lignin extractability by mild alkali treatment was confirmed, and, in addition to a tenfold increase in C6-C1 aldehydes, coniferyl aldehyde was detected by high-performance liquid chromatography in the alkali extracts from the xylem of transgenic plants. In-situ polymerisation of cinnamyl aldehydes in stem sections of untransformed tobacco gave a xylem cell wall coloration strikingly similar to the reddish-brown coloration of the xylem of antisense CAD-down-regulated plants. Overall, these data provide new arguments for the involvement of polymerised cinnamyl aldehydes in the formation of the red-coloured xylem of CAD-down-regulated plants.

The phenolic domain of potato suberin : Structural comparison with lignins

Abstract Natural and wound-healing potato periderms were analysed using thioacidolysis, a specific lignin degradative method. The nature and relative frequencies of phenylpropane units and of inter-unit bonds comprising this lignin-like domain were determined. Results confirmed that this domain contains guaiacyl G and syringyl S units, similar to lignins of woody angiosperms. The recovery yield of the G and S thioacidolysis monomers was, however, about one-tenth of that obtained for wood or straw lignified cell walls. This result suggests that G and S phenylpropane units are relatively few in suberized samples. These units occur in different ratios in the natural and wound potato periderms and are assembled with the same inter-unit bonds as those found in lignins of woody angiosperms, although in different proportions. Both the scarcity of the β - O −4 and β−1 bonds, and the high terminal to internal units ratio, suggest that this lignin-like domain is highly cross-linked.

Formation of ω-feruloyloxypalmitic acid by an enzyme from wound-healing potato tuber discs

Abstract An enzyme, extracted from the outermost cell layers of wound-healing potato tuber discs, has been shown to transfer ferulic acid from feruloyl-CoA to ω-hydroxypalmitic acid. p -Coumaroyl-CoA and sinapoyl-CoA also acted as substrates but were less active than feruloyl-CoA. Good correlation was found between the development of the enzymic activity and the accumulation of alkali-soluble ferulic acid in the suberized cell walls. This transferase (hydroxycinnamoyl-CoA: ω-hydroxypalmitic acid O -hydroxycinnamoyltransferase, HHT, EC 2.3.1.—) could be involved in suberin biosynthesis. Several 1-alkanols were also conjugated to ferulic acid in crude enzymic extracts of wound-healing potato tuber discs. Maximal activity was obtained with 1-tetradecanol, suggesting that ω-hydroxypalmitic acid and 1-tetradecanol could be conjugated by distinct enzymes.

Modulation of the Activity of two Hydroxycinnamoyl Transferases in Wound-Healing Potato Tuber Discs in Response to Pectinase or Abscisic Acid

Summary The activity of two enzymes known to be involved in the biosynthesis of cell-wall phenolics in wound-healing potato tuber tissues was monitored in tuber discs treated with abscisic acid (ABA), pectinase, arachidonic acid, or jasmonic acid. Tyramine hydroxycinnamoyl transferase (THT) activity was strongly stimulated by pectinase and, to a lower extent, by ABA. By contrast, ω-hydroxypalmitic acid hydroxycinnamoyl transferase (HHT), which is a good marker of suberin biosynthesis, was induced by ABA, but not by pectinase treatments. Neither arachidonic acid, a known elicitor of phytoalexin biosynthesis in potato tubers, nor jasmonic acid, a putative intermediate in the signal transduction to wounding, significantly modified the time course or the intensity of induction of THT or HHT activities.

Distribution of hydroxycinnamoyl-CoA: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase in higher plants

Abstract The occurrence of hydroxycinnamoyl-CoA: ω-hydroxypalmitic acid O -hydroxycinnamoyltransferase in roots of plants from various families is reported. Among the 21 species investigated, 17 plants representing 11 different families of angiosperms and gymnosperms showed extractable activity. In intact tobacco plants, the enzyme which was undetectable in leaves and stems, was apparently restricted to roots. Relatively high enzymatic activity was found however in cell suspension cultures grown from tobacco pith.

Purification and characterization of hydroxycinnamoyl-coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase from tobacco (Nicotiana tabacum L.) cell-suspension cultures

An acyltransferase hydroxycinnamoyl-Coenzyme A: ω-hydroxypalmitic acid O-hydroxycinnamoyltransferase (HHT; EC 2.3.1.-), which transfers hydroxycinnamic acids from hydroxycinnamoyl-CoA thioesters to several hydroxylated fatty acid derivatives, was characterized from tobacco (Nicotiana tabacum L. cv. Xanthi nc) cell-suspension cultures. It exhibited the same properties as the enzyme previously detected in wound-healing potato tuber discs (Lotfy et al., 1994, Phytochemistry 35: 1419–1424), and especially a marked specificity for ω-hydroxypalmitic acid and feruloyl-CoA. It was purified 300-fold to near homogeneity from late logarithmic-phase cell suspensions. The apparent molecular mass of the native protein was 55 kDa and its isoelectric point, estimated by electrofocusing, was 4.6. The purified enzyme conjugated ferulic acid to ω-hydroxypalmitic acid and to 1-tetradecanol, its main lipidic substrates, suggesting that the same enzyme probably synthesizes the different esters of 1-alkanols and of ω-hydroxy fatty acids which are formed in vitro.

vir-Gene-inducing activities of hydroxycinnamic acid amides in Agrobacterium tumefaciens

Expression of Agrobacterium tumefaciens virulence genes and transformation of dicots by this organism are dependent upon host plant phenolic compounds. Several alkylsyringamides have recently been shown to be powerful inducers of these vir-genes. These synthetic amides, and especially ethylsyringamide, are much stronger inducers than syringic acid. In this work, four alkylamides derived from ferulic or sinapic acids were synthesized by a dicyclohexylcarbodiimide method and tested for their potential to induce vir-gene expression on A. tumefaciens strains harbouring virB::lacZ or virE::lacZ fusion plasmids. Their effectiveness was compared to that of ethylsyringamide and tyraminylferulamide, a naturally occurring amide in plants. Whatever the amine moiety of the amide (ethylamine, propylamine, tyramine or beta-alanine ethyl ester) conjugation of the acid functional group clearly diminished the toxicity to the bacteria of the respective acid at high concentration and thereby increased the vir-inducing potential. However, none of the inducers tested exhibited higher activity than acetosyringone, the reference compound for vir-gene induction, with the exception of ethylsyringamide at concentrations above 1mM. When tested on Agrobacterium tumefaciens strain A348(pSM243cd), ethylferulamide and ethylsinapamide were more efficient than the corresponding phenolic acids but only above 100 microM.

Characterization and purification of a bacterial chlorogenic acid esterase detected during the extraction of chlorogenic acid from arbuscular mycorrhizal tomato roots

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 (Kmxa0=xa02xa0μM) 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 61xa0kDa 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 inxa0vitro 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.

Combined enzymatic and metabolic analysis of grapevine cell responses to elicitors

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 24xa0h 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.