Cellular and Subcellular Compartmentation of the 2C-Methyl-D-Erythritol 4-Phosphate Pathway in the Madagascar Periwinkle

The Madagascar periwinkle (Catharanthus roseus) synthesizes the highly valuable monoterpene indole alkaloids (MIAs) through a long metabolic route initiated by the 2C-methyl-D-erythritol 4-phosphate (MEP) pathway. In leaves, a complex compartmentation of the MIA biosynthetic pathway occurs at both the cellular and subcellular levels, notably for some gene products of the MEP pathway. To get a complete overview of the pathway organization, we cloned four genes encoding missing enzymes involved in the MEP pathway before conducting a systematic analysis of transcript distribution and protein subcellular localization. RNA in situ hybridization revealed that all MEP pathway genes were coordinately and mainly expressed in internal phloem-associated parenchyma of young leaves, reinforcing the role of this tissue in MIA biosynthesis. At the subcellular level, transient cell transformation and expression of fluorescent protein fusions showed that all MEP pathway enzymes were targeted to plastids. Surprisingly, two isoforms of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase initially exhibited an artifactual aggregated pattern of localization due to high protein accumulation. Immunogold combined with transmission electron microscopy, transient transformations performed with a low amount of transforming DNA and fusion/deletion experiments established that both enzymes were rather diffuse in stroma and stromules of plastids as also observed for the last six enzymes of the pathway. Taken together, these results provide new insights into a potential role of stromules in enhancing MIA precursor exchange with other cell compartments to favor metabolic fluxes towards the MIA biosynthesis

Field-Based Metabolomics of Vitis vinifera L. Stems Provides New Insights for Genotype Discrimination and Polyphenol Metabolism Structuring

Grape accumulates numerous polyphenols with abundant health benefit and organoleptic properties that in planta act as key components of the plant defense system against diseases. Considerable advances have been made in the chemical characterization of wine metabolites particularly volatile and polyphenolic compounds. However, the metabotyping (metabolite-phenotype characterization) of grape varieties, from polyphenolic-rich vineyard by-product is unprecedented. As this composition might result from the complex interaction between genotype, environment and viticultural practices, a field experiment was setting up with uniform pedo-climatic factors and viticultural practices of growing vines to favor the genetic determinism of polyphenol expression. As a result, UPLC-MS-based targeted metabolomic analyses of grape stems from 8 Vitis vinifera L. cultivars allowed the determination of 42 polyphenols related to phenolic acids, flavonoids, procyanidins, and stilbenoids as resveratrol oligomers (degree of oligomerization 1–4). Using a partial least-square discriminant analysis approach, grape stem chemical profiles were discriminated according to their genotypic origin showing that polyphenol profile express a varietal signature. Furthermore, hierarchical clustering highlights various degree of polyphenol similarity between grape varieties that were in agreement with the genetic distance using clustering analyses of 22 microsatellite DNA markers. Metabolite correlation network suggested that several polyphenol subclasses were differently controlled. The present polyphenol metabotyping approach coupled to multivariate statistical analyses might assist grape selection programs to improve metabolites with both health-benefit potential and plant defense traits

Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses

Transcription factors play a fundamental role in plants by orchestrating temporal and spatial gene expression in response to environmental stimuli. Several R2R3-MYB genes of the Arabidopsis subgroup 4 (Sg4) share a C-terminal EAR motif signature recently linked to stress response in angiosperm plants. It is reported here that nearly all Sg4 MYB genes in the conifer trees Picea glauca (white spruce) and Pinus taeda (loblolly pine) form a monophyletic clade (Sg4C) that expanded following the split of gymnosperm and angiosperm lineages. Deeper sequencing in P. glauca identified 10 distinct Sg4C sequences, indicating over-represention of Sg4 sequences compared with angiosperms such as Arabidopsis, Oryza, Vitis, and Populus. The Sg4C MYBs share the EAR motif core. Many of them had stress-responsive transcript profiles after wounding, jasmonic acid (JA) treatment, or exposure to cold in P. glauca and P. taeda, with MYB14 transcripts accumulating most strongly and rapidly. Functional characterization was initiated by expressing the P. taeda MYB14 (PtMYB14) gene in transgenic P. glauca plantlets with a tissue-preferential promoter (cinnamyl alcohol dehydrogenase) and a ubiquitous gene promoter (ubiquitin). Histological, metabolite, and transcript (microarray and targeted quantitiative real-time PCR) analyses of PtMYB14 transgenics, coupled with mechanical wounding and JA application experiments on wild-type plantlets, allowed identification of PtMYB14 as a putative regulator of an isoprenoid-oriented response that leads to the accumulation of sesquiterpene in conifers. Data further suggested that PtMYB14 may contribute to a broad defence response implicating flavonoids. This study also addresses the potential involvement of closely related Sg4C sequences in stress responses and plant evolution

WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana

The plant-specific WRKY transcription factor (TF) family with 74 members in Arabidopsis thaliana appears to be involved in the regulation of various physiological processes including plant defence and senescence. WRKY53 and WRKY70 were previously implicated as positive and negative regulators of senescence, respectively. Here the putative function of other WRKY group III proteins in Arabidopsis leaf senescence has been explored and the results suggest the involvement of two additional WRKY TFs, WRKY 54 and WRKY30, in this process. The structurally related WRKY54 and WRKY70 exhibit a similar expression pattern during leaf development and appear to have co-operative and partly redundant functions in senescence, as revealed by single and double mutant studies. These two negative senescence regulators and the positive regulator WRKY53 were shown by yeast two-hydrid analysis to interact independently with WRKY30. WRKY30 was expressed during developmental leaf senescence and consequently it is hypothesized that the corresponding protein could participate in a senescence regulatory network with the other WRKYs. Expression in wild-type and salicylic acid-deficient mutants suggests a common but not exclusive role for SA in induction of WRKY30, 53, 54, and 70 during senescence. WRKY30 and WRKY53 but not WRKY54 and WRKY70 are also responsive to additional signals such as reactive oxygen species. The results suggest that WRKY53, WRKY54, and WRKY70 may participate in a regulatory network that integrates internal and environmental cues to modulate the onset and the progression of leaf senescence, possibly through an interaction with WRKY30

Caractérisation fonctionnelle d'HCT, une acyltransférase impliquée dans le métabolisme des phénylpropanoïdes

Le métabolisme des phénylpropanoïdes est un métabolisme secondaire, spécifique du règne végétal. Il conduit, à partir de la phénylalanine, à la synthèse d'une grande variété de composés phénoliques tels que la lignine, les flavonoïdes, les isoflavonoïdes, les stilbènes, les coumarines ou encore des esters d'acides hydroxycinnamiques. Ces métabolites secondaires interviennent dans de nombreux processus physiologiques, à la fois au cours du développement de la plante et lors des réponses de défense aux stress biotiques et abiotiques. Mon travail de thèse a consisté en l'étude d'acyltransférases impliquées dans le métabolisme des phénylpropanoïdes. Il a fait suite à la caractérisation, au laboratoire, d'une enzyme de N. tabacum à activité Hydroxycinnamoyl-CoA : Shikimate/Quinate Hydroxycinnamoyltransférase (HCT), capable in vitro de catalyser la synthèse des esters shikimique et quinique de l'acide pcoumarique qui sont les substrats d'une 3-hydroxylase à P450 intervenant dans le métabolisme des phénylpropanoïdes. La caractérisation fonctionnelle d'HCT a été réalisée chez le tabac et Arabidopsis. HCT est exprimée spécifiquement dans les tissus lignifiés et la répression du gène par VIGS chez N. benthamiana et par RNAi chez Arabidopsis a démontré une profonde perturbation de la synthèse des phénylpropanoïdes et en particulier de la lignine. Dans les plants d'Arabidopsis où HCT est réprimée, le flux métabolique est détourné vers les flavonoïdes. L'accumulation des flavonoïdes conduit à l'inhibition du transport d'auxine et à une forte réduction de la croissance des plantes. Quand la synthèse des flavonoïdes est bloquée par la répression de la chalcone synthase, les plantes retrouvent une croissance normale. Des analyses bioinformatiques ont permis d'identifier d'autres acyltransférases putatives chez Arabidopsis . Sur la base d'homologies de séquences protéiques avec HCT, deux de ces acyltransférases putatives sont probablement impliquées dans le métabolisme des phénylpropanoïdes. Les profils d'expression de ces deux enzymes sont restreints aux anthères pour l'une et au phloème des hampes florales pour l'autre. Des plantes transgéniques sous exprimant ces acyltransférases ont été obtenues et devraient constituer de bons outils pour la recherche des fonctions biologiques.Phenylpropanoid metabolism is specific of plants. Phenylalanine is the general precursor of the pathway that leads to a large variety of phenolic compounds as lignin, flavonoids, isoflavonoids, stilbenes, coumarins or hydroxycinnamic acid esters. These secondary compounds provide a vast array of physiological functions, being involved in development and defence responses for instance. During my PhD, I studied acyltransferases implicated in the phenylpropanoid metabolism. This work was initated, after the identification of a N. tabacum enzyme with Hydroxycinnamoyl-CoA : Shikimate/Quinate Hydroxycinnamoyltransferase activity (HCT). This acyltransferase catalyzes in vitro , the formation of shikimate and quinate esters of p-coumarate, that are substrates of a P450 3- hydroxylase implicated in phenylpropanoid pathway. Functional characterization of HCT was performed in tobacco and Arabidopsis . It was shown that is expressed in all lignified tissues and HCT gene silencing by VIGS in N. benthamiana or by RNAi in Arabidopsis deeply affected phenylpropanoid biosynthesis, in particular that of lignin. In HCT-repressed Arabidopsis plants, the metabolic flux was redirected towards flavonoid synthesis through chalcone synthase activity. Consequently, flavonoid accumulation inhibited auxin transport and plant growth. Normal plant development and auxin transport were restored by chalcone synthase repression that inhibited flavonoid synthesis in HCT-deficient plants. Putative acyltransferases of Arabidopsis were identified by bioinformatic analysis. Two of these enzymes have high identity to HCT and are probably implicated in phenylpropanoid pathway. One of them is expressed in stem phloem and the second is specific of anthers. Transgenic plants repressed for these enzymes were obtained and will be important tools to identify biological functions

Caractérisation fonctionnelle d'HCT, une acyltransférase impliquée dans le métabolisme des phénylpropanoïdes

Le métabolisme des phénylpropanoïdes est un métabolisme secondaire, spécifique du règne végétal. Il conduit, à partir de la phénylalanine, à la synthèse d'une grande variété de composés phénoliques tels que la lignine, les flavonoïdes, les isoflavonoïdes,Phenylpropanoid metabolism is specific of plants. Phenylalanine is the general precursor of the pathway that leads to a large variety of phenolic compounds as lignin, flavonoids, isoflavonoids, stilbenes, coumarins or hydroxycinnamic acid esters. These s

Efficient Terpene Production by Marine Thraustochytrids: Shedding Light on the Thermodynamic Driving Force

International audienceTerpenoids, such as squalene, are valuable compounds for cosmetic and drug industries, the supply of which is often limited by natural sources. Alternative production strategies have been investigated for decades but remain challenging due to low yields

Boosting lignan-precursor synthesis in yeast cell factories through co-factor supply optimization

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Gaining access to acetyl-CoA by peroxisomal surface display

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Peroxisomes: A New Hub for Metabolic Engineering in Yeast

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