Philippe Hugueney

Xanthophyll Biosynthesis CLONING, EXPRESSION, FUNCTIONAL RECONSTITUTION, AND REGULATION OF β-CYCLOHEXENYL CAROTENOID EPOXIDASE FROM PEPPER (CAPSICUM ANNUUM)

Pepper (Capsicum annuum) β-cyclohexenyl xanthophyll epoxidase cDNA was cloned and the corresponding enzyme overexpressed and purified from Escherichia coli, for investigation of its catalytic activity. The recombinant protein did not directly accept NADPH for epoxidation of cyclohexenyl carotenoids, nor did it operate according to a peroxygenase-based mechanism. Instead, the reducing power of NADPH was transferred to the epoxidase via reduced ferredoxin as shown by reconstitution of epoxidase activity in the presence of NADPH, ferredoxin oxidoreductase, and ferredoxin. Bacterial rubredoxin could be substituted for ferredoxin. The pepper epoxidase acted specifically on the β-ring of xanthophylls such as β-cryptoxanthin, zeaxanthin, and antheraxanthin. The proposed reaction mechanism for epoxidation involves the formation of a transient carbocation. This characteristic allows selective inhibition of the epoxidase activity by different nucleophilic diethylamine derivatives, p-dimethylaminobenzenediazonium fluoroborate and N,N-dimethyl-2-phenylaziridinium. It was also shown that the epoxidase gene was up-regulated during oxidative stress and when chloroplasts undergo differentiation into chromoplasts in pepper fruit.

Regulation and activation of phytoene synthase, a key enzyme in carotenoid biosynthesis, during photomorphogenesis

Abstract.u2002During photomorphogenesis in higher plants, a coordinated increase occurs in the chlorophyll and carotenoid contents. The carotenoid level is under phytochrome control, as reflected by the light regulation of the mRNA level of phytoene synthase (PSY), the first enzyme in the carotenoid biosynthetic pathway. We investigated PSY protein levels, enzymatic activity and topological localization during photomorphogenesis. The results revealed that PSY protein levels and enzymatic activity increase during de-etiolation and that the enzyme is localized at thylakoid membranes in mature chloroplasts. However, under certain light conditions (e.g., far-red light) the increases in PSY mRNA and protein levels are not accompanied by an increase in enzymatic activity. Under those conditions, PSY is localized in the prolamellar body fraction in a mostly enzymatically inactive form. Subsequent illumination of dark-grown and/or in far-red light grown seedlings with white light causes the decay of these structures and a topological relocalization of PSY to developing thylakoids which results in its enzymatic activation. This light-dependent mechanism of enzymatic activation of PSY in carotenoid biosynthesis shares common features with the regulation of the NADPH:protochlorophyllide oxidoreductase, the first light-regulated enzyme in chlorophyll biosynthesis. The mechanism of regulation described here may contribute to ensuring a spatially and temporally coordinated increase in both carotenoid and chlorophyll contents.

A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment.

Developmental and stress regulation of gene expression for plastid and cytosolic isoprenoid pathways in pepper fruits.

Plant cells synthesize a myriad of isoprenoid compounds in different subcellular compartments, which include the plastid, the mitochondria, and the endoplasmic reticulum cytosol. To start the study of the regulation of these parallel pathways, we used pepper (Capsicum annuum) fruit as a model. Using different isoprenoid biosynthetic gene probes from cloned cDNAs, we showed that only genes encoding the plastid enzymes (geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and capasanthin-capsorubin synthase) are specifically triggered during the normal period of development, at the ripening stage. This pattern of expression can be mimicked and precociously induced by a simple wounding stress. Concerning the cytosol-located enzymes, we observed that the expression of the gene encoding farnesyl pyrophosphate synthase is constitutive, whereas that of farnesyl pyrophosphate cyclase (5-epi-aristolochene synthase) is undetectable during the normal development of the fruit. The expression of these later genes are, however, only selectively triggered after elicitor treatment. The results provide evidence for developmental control of isoprenoid biosynthesis occurring in plastids and that cytoplasmic isoprenoid biosynthesis is regulated, in part, by environmental signals.

A novel cation-dependent O-methyltransferase involved in anthocyanin methylation in grapevine

Anthocyanins are major pigments in colored grape (Vitis vinifera) berries, and most of them are monomethoxylated or dimethoxylated. We report here the functional characterization of an anthocyanin O-methyltransferase (AOMT) from grapevine. The expression pattern in two cultivars with different anthocyanin methylation profiles (Syrah and Nebbiolo) showed a peak at start ripening (véraison), when the concentrations of all methylated anthocyanins begin to increase. The purified recombinant AOMT protein was active on both anthocyanins and flavonols in vitro, with Km in the micromolar range, and was dependent on divalent cations for activity. AOMT showed a preference for 3′,5′ methylation when a 3′,4′,5′ hydroxylated anthocyanin substrate was tested. In order to assess its in planta activity, we performed transient expression of AOMT in tobacco (Nicotiana benthamiana) leaves expressing the Production of Anthocyanin Pigment1 (PAP1) transcription factor from Arabidopsis (Arabidopsis thaliana). PAP1 expression in leaves induced the accumulation of the nonmethylated anthocyanin delphinidin 3-rutinoside. The coexpression of PAP1 and AOMT resulted in an accumulation of malvidin 3-rutinoside. We also showed that AOMT localized exclusively in the cytoplasm of tobacco leaf cells. These results demonstrate the ability of this enzyme to methylate anthocyanins both in vitro and in vivo, indicating that AOMT plays a major role in anthocyanin biosynthesis in grape berries.

Identification of a novel gene coding for neoxanthin synthase from Solanum tuberosum

The polymerase chain reaction analysis of potato plants, transformed with capsanthin capsorubin synthase ccs, revealed the presence of a highly related gene. The cloned cDNA showed at the protein level 89.6% identity to CCS. This suggested that the novel enzyme catalyzes a mechanistically similar reaction. Such a reaction is represented by neoxanthin synthase (NXS), forming the xanthophyll neoxanthin, a direct substrate for abscisic acid formation. The function of the novel enzyme could be proven by transient expression in plant protoplasts and high performance liquid chromatography analysis. The cloned NXS was imported in vitro into plastids, the compartment of carotenoid biosynthesis.

Role of Petal-Specific Orcinol O-Methyltransferases in the Evolution of Rose Scent

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominanty in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses.

Link between carrot leaf secondary metabolites and resistance to Alternaria dauci

Alternaria Leaf Blight (ALB), caused by the fungus Alternaria dauci, is the most damaging foliar disease affecting carrots (Daucus carota). In order to identify compounds potentially linked to the resistance to A. dauci, we have used a combination of targeted and non-targeted metabolomics to compare the leaf metabolome of four carrot genotypes with different resistance levels. Targeted analyses were focused on terpene volatiles, while total leaf methanolic extracts were subjected to non-targeted analyses using liquid chromatography couple to high-resolution mass spectrometry. Differences in the accumulation of major metabolites were highlighted among genotypes and some of these metabolites were identified as potentially involved in resistance or susceptibility. A bulk segregant analysis on F3 progenies obtained from a cross between one of the resistant genotypes and a susceptible one, confirmed or refuted the hypothesis that the metabolites differentially accumulated by these two parents could be linked to resistance.

The Aphid-Transmitted Turnip yellows virus Differentially Affects Volatiles Emission and Subsequent Vector Behavior in Two Brassicaceae Plants

Aphids are important pests which cause direct damage by feeding or indirect prejudice by transmitting plant viruses. Viruses are known to induce modifications of plant cues in ways that can alter vector behavior and virus transmission. In this work, we addressed whether the modifications induced by the aphid-transmitted Turnip yellows virus (TuYV) in the model plant Arabidopsis thaliana also apply to the cultivated plant Camelina sativa, both belonging to the Brassicaceae family. In most experiments, we observed a significant increase in the relative emission of volatiles from TuYV-infected plants. Moreover, due to plant size, the global amounts of volatiles emitted by C. sativa were higher than those released by A. thaliana. In addition, the volatiles released by TuYV-infected C. sativa attracted the TuYV vector Myzus persicae more efficiently than those emitted by non-infected plants. In contrast, no such preference was observed for A. thaliana. We propose that high amounts of volatiles rather than specific metabolites are responsible for aphid attraction to infected C. sativa. This study points out that the data obtained from the model pathosystem A. thaliana/TuYV cannot be straightforwardly extrapolated to a related plant species infected with the same virus.