MYB24 orchestrates terpene and flavonol metabolism as light responses to anthocyanin depletion in variegated grape berries.

Variegation is a rare type of mosaicism not fully studied in plants, especially fruits. We examined red and white sections of grape (Vitis vinifera cv. 'Béquignol') variegated berries and found that accumulation of products from branches of the phenylpropanoid and isoprenoid pathways showed an opposite tendency. Light-responsive flavonol and monoterpene levels increased in anthocyanin-depleted areas in correlation with increasing MYB24 expression. Cistrome analysis suggested that MYB24 binds to the promoters of 22 terpene synthase (TPS) genes, as well as 32 photosynthesis/light-related genes, including carotenoid pathway members, the flavonol regulator HY5 HOMOLOGUE (HYH), and other radiation response genes. Indeed, TPS35, TPS09, the carotenoid isomerase gene CRTISO2, and HYH were activated in the presence of MYB24 and MYC2. We suggest that MYB24 modulates ultraviolet and high-intensity visible light stress responses that include terpene and flavonol synthesis and potentially affects carotenoids. The MYB24 regulatory network is developmentally triggered after the onset of berry ripening, while the absence of anthocyanin sunscreens accelerates its activation, likely in a dose-dependent manner due to increased radiation exposure. Anthocyanins and flavonols in variegated berry skins act as effective sunscreens but for different wavelength ranges. The expression patterns of stress marker genes in red and white sections of 'Béquignol' berries strongly suggest that MYB24 promotes light stress amelioration but only partly succeeds during late ripening.

ABA-mediated responses to water deficit separate grapevine genotypes by their genetic background.

BACKGROUND: ABA-mediated processes are involved in plant responses to water deficit, especially the control of stomatal opening. However in grapevine it is not known if these processes participate in the phenotypic variation in drought adaptation existing between genotypes. To elucidate this question, the response to short-term water-deficit was analysed in roots and shoots of nine Vitis genotypes differing in their drought adaptation in the field. The transcript abundance of 12 genes involved in ABA biosynthesis, catabolism, and signalling were monitored, together with physiological and metabolic parameters related to ABA and its role in controlling plant transpiration. RESULTS: Although transpiration and ABA responses were well-conserved among the genotypes, multifactorial analyses separated Vitis vinifera varieties and V. berlandieri x V. rupestris hybrids (all considered drought tolerant) from the other genotypes studied. Generally, V. vinifera varieties, followed by V. berlandieri x V. rupestris hybrids, displayed more pronounced responses to water-deficit in comparison to the other genotypes. However, changes in transcript abundance in roots were more pronounced for Vitis hybrids than V. vinifera genotypes. Changes in the expression of the cornerstone ABA biosynthetic gene VviNCED1, and the ABA transcriptional regulator VviABF1, were associated with the response of V. vinifera genotypes, while changes in VviNCED2 abundance were associated with the response of other Vitis genotypes. In contrast, the ABA RCAR receptors were not identified as key components of the genotypic variability of water-deficit responses. Interestingly, the expression of VviSnRK2.6 (an AtOST1 ortholog) was constitutively lower in roots and leaves of V. vinifera genotypes and higher in roots of V. berlandieri x V. rupestris hybrids. CONCLUSIONS: This study highlights that Vitis genotypes exhibiting different levels of drought adaptation differ in key steps involved in ABA metabolism and signalling; both under well-watered conditions and in response to water-deficit. In addition, it supports that adaptation may be related to various mechanisms related or not to ABA responses.

A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity.

BACKGROUND: Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR). In grapevine (Vitis vinifera L.), several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. RESULTS: In this work, we describe the effects of a single amino acid substitution (R69L) located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5b(L), the native protein being referred as VvMYB5b(R)) was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5b(L) exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5b(R) and VvMYB5b(L) genes in tobacco. Flowers from 35S::VvMYB5b(L) transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5b(R) and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. CONCLUSIONS: Taken together, our findings indicate that VvMYB5b(L) is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s). In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment of protein-protein interactions between MYB and bHLH transcription factors. Mechanisms underlying these interactions are discussed and a model is proposed to explain the transcriptional activity of VvMYB5(L) observed in the tobacco model.

Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway.

Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

[Identification and characterization of "rd22" dehydration responsive gene in grapevine (Vitis vinifera L.)]. / Identification et caractérisation d'un gène de réponse à la déshydratation "rd22" chez la vigne (Vitis vinifera L.).

To identify and isolate genes related to abiotic stresses (salinity and drought) tolerance in grapevine, a candidate gene approach was developed and allowed isolating a full-length cDNA of rd22 gene from the Cabernet Sauvignon variety. The latter, named Vvrd22, is a dehydration-responsive gene that is usually induced by the application of exogenous ABA. Details of the physicochemical parameters and structural properties (molecular mass, secondary structure, conserved domains and motives, putative post-translational modification sites...) of the encoded protein have also been elucidated. The expression study of Vvrd22 was carried out at the berry growth stages and at the level of plant organs and tissues as well as under both drought and salt stresses. The results showed that Vvrd22 is constitutively expressed at a low level in all analyzed tissues. Moreover, salt stress induced Vvrd22 expression, particularly for the tolerant variety (Razegui), contrary to the sensitive one (Syrah), which did not display any expression variation during the stress, which means that Vvrd22 is involved in salt stress response and that its expression level depends on regulatory mechanisms that are efficient only for the tolerant variety. On the other hand, under drought stress, Vvrd22 is induced in an identical manner for both tolerant and sensitive varieties. In addition, stress signal molecules such as ABA (lonely applied or in combination with sucrose) induced Vvrd22 expression, even at a low level. A minimal knowledge about the role and the functionality of this gene is necessary and constitutes a prerequisite condition before starting and including Vvrd22 in any program of improvement of grapevine's abiotic stress tolerance.

Effect of methyl jasmonate in combination with carbohydrates on gene expression of PR proteins, stilbene and anthocyanin accumulation in grapevine cell cultures.

Grapevine (Vitis vinifera L.) is subject to a number of diseases which affect yield and wine quality. After veraison, berries become strongly susceptible to pathogens due to different physiological changes including the accumulation of glucose and fructose, on the one hand, and to the decrease of anti-microbial compounds called stilbenes, on the other. To obtain berry protection, pesticides are excessively used leading to important cost to the grower and to undesirable environmental impact of the residues, especially in grape, soil and water. As a consequence, alternative strategies have to be developed. Exogenously applied biotic elicitors induce defense responses. We studied the effects of methyl jasmonate in combination with sucrose on defense-related gene expression, stilbene and anthocyanin production in grapevine cell suspensions. The methyl jasmonate/sucrose treatment was effective in stimulating phenylalanine ammonia lyase, chalcone synthase, stilbene synthase, UDP-glucose: flavonoid-O-glucosyltransferase, proteinase inhibitor and chitinase gene expression, and triggered accumulation of both piceids and anthocyanins in cells, and trans-resveratrol and piceids in the extracellular medium. Methyl jasmonate treatment might be an efficient natural strategy to protect grapevine berries in vineyard.

Overexpression of VvWRKY2 in tobacco enhances broad resistance to necrotrophic fungal pathogens.

WRKY genes encode proteins belonging to a large family of transcription factors that are involved in various developmental and physiological processes and in plant responses to pathogen infections. In the present work, a full-length cDNA from a Vitis vinifera L. cv. Cabernet Sauvignon grape berry library was isolated and characterized. The cDNA, designated VvWRKY2, encodes a polypeptide of 536 amino acids that shows the structural features of group I of WRKY protein family. VvWRKY2 is expressed in the different organs of healthy grapevine plants. In leaves, VvWRKY2 is induced by wounding and after infection with Plasmopara viticola. Constitutive expression of VvWRKY2 in tobacco reduced the susceptibility of transgenic tobacco to three types of fungal pathogens infecting different parts of the plant: Botrytis cinerea (leaves), Pythium spp. (roots) and Alternaria tenuis (seeds). The results indicate that VvWRKY2 may be involved in the resistance of grapevine against the pathogens.

Overexpression of a grapevine R2R3-MYB factor in tomato affects vegetative development, flower morphology and flavonoid and terpenoid metabolism.

Although the terpenoid pathway constitutes, with the phenylpropanoid metabolism, the major pathway of secondary metabolism in plants, little is known about its regulation. Overexpression of a Vitis vinifera R2R3-MYB transcription factor (VvMYB5b) in tomato induced pleiotropic changes including dwarfism, modified leaf structure, alterations of floral morphology, pigmented and glossy fruits at the "green-mature" stage and impaired seed germination. Two main branches of secondary metabolism, which profoundly influence the organoleptic properties of the fruit, were affected in the opposite way by VvMYB5b overexpression. Phenylpropanoid metabolism was down regulated whereas the amount of beta-carotene was up regulated. This is the first example of the independent regulation of phenylpropanoid and carotenoid metabolism. The strongest modification concerns a decrease in beta-amyrin, the precursor of the oleanolic acid, which is the major component of grape waxes. Scanning electron microscopy analysis of fruits and leaves confirms the alteration of wax metabolism and a modification of cell size and shape. This may potentially impact resistance/tolerance to biotic and abiotic stresses. The results are compared with a similar approach using heterologous expression of VvMYB5b in tobacco.

Identification of grapevine aquaporins and expression analysis in developing berries.

Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.

The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries.

Among the dramatic changes occurring during grape berry (Vitis vinifera) development, those affecting the flavonoid pathway have provoked a number of investigations in the last 10 years. In addition to producing several compounds involved in the protection of the berry and the dissemination of the seeds, final products of this pathway also play a critical role in berry and wine quality. In this article, we describe the cloning and functional characterization of VvMYB5b, a cDNA isolated from a grape berry (V. vinifera 'Cabernet Sauvignon') library. VvMYB5b encodes a protein belonging to the R2R3-MYB family of transcription factors and displays significant similarity with VvMYB5a, another MYB factor recently shown to regulate flavonoid synthesis in grapevine. The ability of VvMYB5a and VvMYB5b to activate the grapevine promoters of several structural genes of the flavonoid pathway was confirmed by transient expression of the corresponding cDNAs in grape cells. Overexpression of VvMYB5b in tobacco (Nicotiana tabacum) leads to an up-regulation of genes encoding enzymes of the flavonoid pathway and results in the accumulation of anthocyanin- and proanthocyanidin-derived compounds. The ability of VvMYB5b to regulate particularly the anthocyanin and the proanthocyanidin pathways is discussed in relation to other recently characterized MYB transcription factors in grapevine. Taken together, data presented in this article give insight into the transcriptional mechanisms associated with the regulation of the flavonoid pathway throughout grape berry development.

Isolation and characterization of a Vitis vinifera transcription factor, VvWRKY1, and its effect on responses to fungal pathogens in transgenic tobacco plants.

Pathogen attack represents a major problem for viticulture and for agriculture in general. At present, the use of phytochemicals is more and more restrictive, and therefore it is becoming essential to control disease by having a thorough knowledge of resistance mechanisms. The present work focused on the trans-regulatory proteins potentially involved in the control of the plant defence response, the WRKY proteins. A full-length cDNA, designated VvWRKY1, was isolated from a grape berry library (Vitis vinifera L. cv. Cabernet Sauvignon). It encodes a polypeptide of 151 amino acids whose structure is characteristic of group IIc WRKY proteins. VvWRKY1 gene expression in grape is regulated in a developmental manner in berries and leaves and by various signal molecules involved in defence such as salicylic acid, ethylene, and hydrogen peroxide. Biochemical analysis indicates that VvWRKY1 specifically interacts with the W-box in various nucleotidic contexts. Functional analysis of VvWRKY1 was performed by overexpression in tobacco, and transgenic plants exhibited reduced susceptibility to various fungi but not to viruses. These results are consistent with a possible role for VvWRKY1 in grapevine defence against fungal pathogens.

Characterization of a grapevine R2R3-MYB transcription factor that regulates the phenylpropanoid pathway.

The ripening of grape (Vitis vinifera) berry is characterized by dramatic changes in gene expression, enzymatic activities, and metabolism that lead to the production of compounds essential for berry quality. The phenylpropanoid metabolic pathway is one of the components involved in these changes. In this study, we describe the cloning and functional characterization of VvMYB5a, a cDNA isolated from a grape L. cv Cabernet Sauvignon berry library. VvMYB5a encodes a protein belonging to a small subfamily of R2R3-MYB transcription factors. Expression studies in grapevine indicate that the VvMYB5a gene is mainly expressed during the early steps of berry development in skin, flesh, and seeds. Overexpression of VvMYB5a in tobacco (Nicotiana tabacum) affects the expression of structural genes controlling the synthesis of phenylpropanoid and impacts on the metabolism of anthocyanins, flavonols, tannins, and lignins. Overexpressing VvMYB5a induces a strong accumulation of several phenolic compounds, including keracyanin (cyanidin-3-rhamnoglucoside) and quercetin-3-rhamnoglucoside, which are the main anthocyanin and flavonol compounds in tobacco. In addition, VvMYB5a overexpression increases the biosynthesis of condensed tannins and alters lignin metabolism. These findings suggest that VvMYB5a may be involved in the control of different branches of the phenylpropanoid pathway in grapevine.

Isogene specific oligo arrays reveal multifaceted changes in gene expression during grape berry (Vitis vinifera L.) development.

The transition from a green, hard, and acidic pericarp to a sweet, soft, coloured, and sugar-rich ripe fruit occurs in many unrelated fruit species. High throughput identification of differentially expressed genes in grape berry has been achieved by the use of 50-mers oligoarrays bearing a set of 3,200 Unigenes from Vitis vinifera to compare berry transcriptome at nine developmental stages. Analysis of transcript profiles revealed that most activations were triggered simultaneously with softening, occurring within only 24 h for an individual berry, just before any change in colouration or water, sugar, and acid content can be detected. Although most dramatically induced genes belong to unknown functional categories, numerous changes occur in the expression of isogenes involved in primary and secondary metabolism during ripening. Focusing on isogenes potentially significant in development regulation (hormonal control of transcription factor) revealed a possible role for several hormones (cytokinin, gibberellin, or jasmonic acid). Transcription factor analysis revealed the induction of RAP2 and WRKY genes at véraison, suggesting increasing biotic and abiotic stress conditions during ripening. This observation was strengthened by an increased expression of multiple transcripts involved in sugar metabolism and also described as induced in other plant organs during stress conditions. This approach permitted the identification of new isogenes as possible control points: a glutathione S-transferase exhibits the same expression profile as anthocyanin accumulation and a new putative sugar transporter is induced in parallel with sugar import.

Plasma membrane aquaporins play a significant role during recovery from water deficit.

The role of plasma membrane aquaporins (PIPs) in water relations of Arabidopsis was studied by examining plants with reduced expression of PIP1 and PIP2 aquaporins, produced by crossing two different antisense lines. Compared with controls, the double antisense (dAS) plants had reduced amounts of PIP1 and PIP2 aquaporins, and the osmotic hydraulic conductivity of isolated root and leaf protoplasts was reduced 5- to 30-fold. The dAS plants had a 3-fold decrease in the root hydraulic conductivity expressed on a root dry mass basis, but a compensating 2.5-fold increase in the root to leaf dry mass ratio. The leaf hydraulic conductance expressed on a leaf area basis was similar for the dAS compared with the control plants. As a result, the hydraulic conductance of the whole plant was unchanged. Under sufficient and under water-deficient conditions, stomatal conductance, transpiration rate, plant hydraulic conductance, leaf water potential, osmotic pressure, and turgor pressure were similar for the dAS compared with the control plants. However, after 4 d of rewatering following 8 d of drying, the control plants recovered their hydraulic conductance and their transpiration rates faster than the dAS plants. Moreover, after rewatering, the leaf water potential was significantly higher for the control than for the dAS plants. From these results, we conclude that the PIPs play an important role in the recovery of Arabidopsis from the water-deficient condition.