Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation.

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Genetic mechanisms underlying the methylation level of anthocyanins in grape (Vitis vinifera L.).

BACKGROUND: Plant color variation is due not only to the global pigment concentration but also to the proportion of different types of pigment. Variation in the color spectrum may arise from secondary modifications, such as hydroxylation and methylation, affecting the chromatic properties of pigments. In grapes (Vitis vinifera L.), the level of methylation modifies the stability and reactivity of anthocyanin, which directly influence the color of the berry. Anthocyanin methylation, as a complex trait, is controlled by multiple molecular factors likely to involve multiple regulatory steps. RESULTS: In a Syrah × Grenache progeny, two QTLs were detected for variation in level of anthocyanin methylation. The first one, explaining up to 27% of variance, colocalized with a cluster of Myb-type transcription factor genes. The second one, explaining up to 20% of variance, colocalized with a cluster of O-methyltransferase coding genes (AOMT). In a collection of 32 unrelated cultivars, MybA and AOMT expression profiles correlated with the level of methylated anthocyanin. In addition, the newly characterized AOMT2 gene presented two SNPs associated with methylation level. These mutations, probably leading to a structural change of the AOMT2 protein significantly affected the enzyme specific catalytic efficiency for the 3'-O-methylation of delphinidin 3-glucoside. CONCLUSION: We demonstrated that variation in methylated anthocyanin accumulation is susceptible to involve both transcriptional regulation and structural variation. We report here the identification of novel AOMT variants likely to cause methylated anthocyanin variation. The integration of QTL mapping and molecular approaches enabled a better understanding of how variation in gene expression and catalytic efficiency of the resulting enzyme may influence the grape anthocyanin profile.

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 K(m) 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.

Grapevine MATE-type proteins act as vacuolar H+-dependent acylated anthocyanin transporters.

In grapevine (Vitis vinifera), anthocyanins are responsible for most of the red, blue, and purple pigmentation found in the skin of berries. In cells, anthocyanins are synthesized in the cytoplasm and accumulated into the vacuole. However, little is known about the transport of these compounds through the tonoplast. Recently, the sequencing of the grapevine genome allowed us to identify genes encoding proteins with high sequence similarity to the Multidrug And Toxic Extrusion (MATE) family. Among them, we selected two genes as anthocyanin transporter candidates and named them anthoMATE1 (AM1) and AM3. The expression of both genes was mainly fruit specific and concomitant with the accumulation of anthocyanin pigment. Subcellular localization assays in grapevine hairy roots stably transformed with AM1 or AM3green fluorescent protein fusion protein revealed that AM1 and AM3 are primarily localized to the tonoplast. Yeast vesicles expressing anthoMATEs transported acylated anthocyanins in the presence of MgATP. Inhibitor studies demonstrated that AM1 and AM3 proteins act in vitro as vacuolar H(+)-dependent acylated anthocyanin transporters. By contrast, under our experimental conditions, anthoMATEs could not transport malvidin 3-O-glucoside or cyanidin 3-O-glucoside, suggesting that the acyl conjugation was essential for the uptake. Taken together, these results provide evidence that in vitro the two grapevine AM1 and AM3 proteins mediate specifically acylated anthocyanin transport.

Ectopic expression of VvMybPA2 promotes proanthocyanidin biosynthesis in grapevine and suggests additional targets in the pathway.

Grapevine (Vitis vinifera) proanthocyanidins contribute to plant defense mechanisms against biotic stress and also play a critical role in organoleptic properties of wine. In grapevine berry, these compounds are mainly accumulated in exocarps and seeds in the very early stages of development. A previous study has already identified VvMybPA1 as the first transcription factor involved in the regulation of the proanthocyanidin pathway during seed development in grapevine. A novel Myb factor, VvMybPA2, which is described in this study, is in contrast mainly expressed in the exocarp of young berries and in the leaves. This transcription factor shows very high protein sequence homology with other plant Myb factors, which regulate flavonoid biosynthesis. Ectopic expression of either VvMybPA1 or VvMybPA2 in grapevine hairy roots induced qualitative and quantitative changes of the proanthocyanidin profiles. High-throughput transcriptomic analyses of transformed grapevine organs identified a large set of putative targets of the VvMybPA1 and VvMybPA2 transcription factors. Both genes significantly activated enzymes of the flavonoid pathway, including anthocyanidin reductase and leucoanthocyanidin reductase 1, the specific terminal steps in the biosynthesis of epicatechin and catechin, respectively, but not leucoanthocyanidin reductase 2. The functional annotation of the genes whose expression was modified revealed putative new actors of the proanthocyanidin pathway, such as glucosyltransferases and transporters.

Validation of an extraction method on whole pericarp of grape berry (Vitis vinifera L. cv. Shiraz) to study biochemical and molecular aspects of flavan-3-ol synthesis during berry development.

An extraction method on grape berry was optimized for the total flavan-3-ol content measurement with regard to the nature of the sample and the duration of its extraction. This extraction was performed for the first time on the whole pericarp. Flavan-3-ol extractions were achieved on Shiraz ripe samples of pericarp versus skin within different durations: the best results were obtained for the whole pericarp and 1 h duration. Therefore, this more convenient protocol was used to investigate the flavan-3-ol content at different stages through berry development, in parallel with the abundance of transcripts involved in their biosynthesis. Furthermore, flavan-3-ol extractions on pericarp analysis confirmed their presence in both pulp and skin. For the first time, the flavan-3-ol biosynthesis in pulp was demonstrated with both biochemical and transcriptomic analyses since the presence of leucoanthocyanidin reductase (LAR2) and anthocyanin reductase (ANR) transcripts was revealed by real-time PCR. In addition, the percentage of epigallocatechin was different in pulp and skin.