Summary1. Plant hormones play important roles in regulating plant growth and defence by mediating developmental processes and signalling networks involved in plant responses to a wide range of parasitic and mutualistic biotic interactions. 2. Plants are known to rapidly respond to pathogen and herbivore attack by reconfiguring their metabolism to reduce pathogen/herbivore food acquisition. This involves the production of defensive plant secondary compounds, but also an alteration of the plant primary metabolism to fuel the energetic requirements of the direct defence. 3. Cytokinins are plant hormones that play a key role in plant morphology, plant defence, leaf senescence and source-sink relationships. They are involved in numerous plant-biotic interactions. 4. These phytohormones may have been the target of arthropods and pathogens over the course of the evolutionary arms race between plants and their biotic partners to hijack the plant metabolism, control its physiology and/or morphology and successfully invade the plant. In the case of arthropods, cytokinin-induced phenotypes can be mediated by their bacterial symbionts, giving rise to intricate plant-microbe-insect interactions. 5. Cytokinin-mediated effects strongly impact not only plant growth and defence but also the whole community of insect and pathogen species sharing the same plant by facilitating or preventing plant invasion. This suggests that cytokinins (CKs) are key regulators of the plant growth-defence trade-off and highlights the complexity of the finely balanced responses that plants use while facing both invaders and mutualists.

Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high-and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.

Our objective was to determine the respective roles of the couple glutamine synthetase/glutamate synthase (GS/GOGAT) and glutamate dehydrogenase (GDH) in ammonium and amino acid metabolism during germination and post-germinative growth in the model legume Medicago truncatula Gaertn. For this aim, amino acids were analyzed by HPLC and changes in gene expression of several enzymes involved in N and C metabolism were studied by real-time quantitative reverse transcription-polymerase chain reaction. Among the enzymes studied, GDH showed the highest increase in gene expression (80-fold), specifically in the embryo axis and concomitant with the increase in ammonium content during post-germinative growth. In cotyledons, GDH gene expression was very low. Although in vitro GDH aminating activity was several times higher than its deaminating activity, in vivo 15NH4 incorporation into amino acids was completely inhibited by methionine sulfoximine, a GS inhibitor, indicating that GDH is not involved in ammonium assimilation/detoxification. Changes in the expressions of GS and GOGAT isoforms revealed that GS1b (EC 6.3.1.2) in concert with NADH-dependent GOGAT (EC 1.4.1.14) constitute the major route of assimilation of ammonium derived from reserve mobilization and glutamic acid/glutamine synthesis in germinating M. truncatula seeds. However, during post-germinative growth, although germination was held in darkness, expression of GS2 and Fd-GOGAT (EC 1.4.7.1) increased and expression of GS1b decreased in cotyledons but not in the embryo axis. 2-Oxoglutarate, the substrate of the transamination reaction, was provided by the cytosolic isoform of isocitrate dehydrogenase (EC 1.1.1.42). We suggest that GDH during post-germinative growth, specifically in the developing embryo axis, contributes to ammonium delivery to GS for glutamine synthesis in the absence of primary NO3- assimilation. Interestingly, this reaction also produces reducing power (NADH) in organs deprived of photosynthesis.

Grape canes are vineyard waste products containing valuable phytochemicals of medicine and agriculture interest. Grape canes storage is critical for the accumulation of these bioactive compounds. In the present study, we investigated the changes in stilbenoid phytochemical composition during grape cane storage and the influence of the temperature on final concentrations. A strong increase in the concentration of the monomer E-resveratrol (approximately 40-fold) was observed during the first 6 weeks of storage at 20 °C in eight different grape varieties without any change in oligomer concentrations. The E-resveratrol accumulation was temperature-dependent with an optimal range at 15-20 °C. A 2 h heat-shock treatment aiming at protein denaturation inhibited E-resveratrol accumulation. The constitutive expression of key genes involved in the stilbene precursor biosynthesis along with an induction of stilbene synthase (STS) expression during the first weeks of storage contribute to a de novo biosynthesis of E-resveratrol in pruned wood grapes.

The modulation of primary nitrogen metabolism by hypoxic stress was studied in young Medicago truncatula seedlings. Hypoxic seedlings were characterized by the up-regulation of glutamate dehydrogenase 1 (GDH1) and mitochondrial alanine aminotransferase (mAlaAT), and down-regulation of glutamine synthetase 1b (GS1b), NADH-glutamate synthase (NADH-GOGAT), glutamate dehydrogenase 3 (GDH3), and isocitrate dehydrogenase (ICDH) gene expression. Hypoxic stress severely inhibited GS activity and stimulated NADH-GOGAT activity. GDH activity was lower in hypoxic seedlings than in the control, however, under either normoxia or hypoxia, the in vivo activity was directed towards glutamate deamination. 15NH4 labelling showed for the first time that the adaptive reaction of the plant to hypoxia consisted of a concerted modulation of nitrogen flux through the pathways of both alanine and glutamate synthesis. In hypoxic seedlings, newly synthesized 15N-alanine increased and accumulated as the major amino acid, asparagine synthesis was inhibited, while 15N-glutamate was synthesized at a similar rate to that in the control. A discrepancy between the up-regulation of GDH1 expression and the down-regulation of GDH activity by hypoxic stress highlighted for the first time the complex regulation of this enzyme by hypoxia. Higher rates of glycolysis and ethanol fermentation are known to cause the fast depletion of sugar stores and carbon stress. It is proposed that the expression of GDH1 was stimulated by hypoxia-induced carbon stress, while the enzyme protein might be involved during post-hypoxic stress contributing to the regeneration of 2-oxoglutarate via the GDH shunt.

We have developed an approach combining physiology and quantitative genetics to enhance our understanding of nitrogen (N) metabolism during kernel germination. The physiological study highlighted the central role of glutamine (Gln) synthetase (GS) and Gln synthesis during this developmental process because a concomitant increase of both the enzyme activity and the amino acid content was observed. This result suggests that Gln is acting either as a sink for ammonium released during both storage protein degradation and amino acid deamination or as a source for amino acid de novo synthesis by transamination. In the two parental lines used for the quantitative genetics approach, we found that the increase in Gln occurred earlier in Io compared with F 2 , a result consistent with its faster germinating capacity. The genetic study was carried out on 140 F6 recombinant inbred lines derived from the cross between F 2 and Io. Quantitative trait locus mapping identified three quantitative trait loci (QTLs) related to germination trait (T50, time at which 50% of the kernels germinated) that explain 18.2% of the phenotypic variance; three QTLs related to a trait linked to germination performance, kernel size/weight (thousand kernels weight), that explain 17% of the phenotypic variance; two QTLs related to GS activity at early stages of germination that explain 17.7% of the phenotypic variance; and one QTL related to GS activity at late stages of germination that explains 7.3% of the phenotypic variance. Coincidences of QTL for germination efficiency and its components with genes encoding cytosolic GS (GS1) and the corresponding enzyme activity were detected, confirming the important role of the enzyme during the germination process. A triple colocalization on chromosome 4 between gln3 (a structural gene encoding GS1) and a QTL for GS activity and T50 was found; whereas on chromosome 5, a QTL for GS activity and thousand kernels weight colocalized with gln4, another structural gene encoding GS1. This observation suggests that for each gene, the corresponding enzyme activity is of major importance for germination efficiency either through the size of the grain or through its faster germinating capacity. Consistent with the possible nonoverlapping function of the two GS1 genes, we found that in the parental line Io, the expression of Gln3 was transiently enhanced during the first hours of germination, whereas that of gln4 was constitutive.Seeds are complex structures consisting anatomically and genetically of three parts: the embryo, the endosperm, and the seed coat. During their development, seeds are supplied by the mother plant with assimilates. From a physiological point of view, seeds behave as sink organs accumulating C and N metabolites stored as starch, proteins, amino acids, or lipids. C and N metabolism in developing seeds has been extensively studied in various monocot species, i.e. maize (Zea mays) and wheat (Triticum aestivum), as well as dicot species, i.e. pea (Pisum sativum), soybean (Glycine max), and alfalfa ...

Phenolamides, so called hydroxycinnamic acid amides, are specialized metabolites produced in higher plants, involved in development, reproduction and serve as defence compounds in biotic interactions. Among them, trihydroxycinnamoyl spermidine derivatives were initially found to be synthetized by a spermidine hydroxycinnamoyltransferase (AtSHT) in Arabidopsis thaliana and to accumulate in the pollen coat. This study reports the identification, in Malus domestica, of an acyltransferase able to complement the sht mutant of Arabidopsis. The quantitative RT-PCR expression profile of MdSHT reveals a specific expression in flowers coordinated with anther development and tapetum cell activities. Three phenolamides including N (1),N (5),N (10)-tricoumaroyl spermidine and N (1),N (5)-dicoumaroyl-N (10)-caffeoyl spermidine identified by LC/MS, were shown to accumulate specifically in pollen grain coat of apple tree. Moreover, in vitro biochemical characterization confirmed MdSHT capacity to synthesize tri-substituted spermidine derivatives with a substrate specificity restricted to p-coumaroyl-CoA and caffeoyl-CoA as an acyl donor. Further investigations of the presence of tri-substituted hydroxycinnamoyl spermidine conjugates in higher plants were performed by targeted metabolic analyses in pollens coupled with bioinformatic analyses of putative SHT orthologues in a wide range of available plant genomes. This work highlights a probable early evolutionary appearance in the common ancestral core Eudicotyledons of a novel enzyme from the BAHD acyltransferase superfamily, dedicated to the synthesis of trihydroxycinnamoyl spermidines in pollen coat. This pathway was maintained in most species; however, recent evolutionary divergences have appeared among Eudicotyledons, such as an organ reallocation of SHT gene expression in Fabales and a loss of SHT in Malvales and Cucurbitales.

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