RESUMEN
Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate-cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C3-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed.
Asunto(s)
Flavonoles/metabolismo , Oxigenasas/metabolismo , Proteínas de Plantas/metabolismo , Vitis/metabolismo , Oxidación-Reducción , Polifenoles/metabolismo , Especificidad por SustratoRESUMEN
Methoxypyrazines (MPs) are strongly odorant volatile molecules with vegetable-like fragrances that are widespread in plants. Some grapevine (Vitis vinifera) varieties accumulate significant amounts of MPs, including 2-methoxy-3-isobutylpyrazine (IBMP), which is the major MP in grape berries. MPs are of particular importance in white Sauvignon Blanc wines. The typicality of these wines relies on a fine balance between the pea pod, capsicum character of MPs and the passion fruit/grapefruit character due to volatile thiols. Although MPs play a crucial role in Sauvignon varietal aromas, excessive concentrations of these powerful odorants alter wine quality and reduce consumer acceptance, particularly in red wines. The last step of IBMP biosynthesis has been proposed to involve the methoxylation of the nonvolatile precursor 2-hydroxy-3-isobutylpyrazine to give rise to the highly volatile IBMP. In this work, we have used a quantitative trait loci approach to investigate the genetic bases of IBMP biosynthesis. This has led to the identification of two previously uncharacterized S-adenosyl-methionine-dependent O-methyltransferase genes, termed VvOMT3 and VvOMT4. Functional characterization of these two O-methyltransferases showed that the VvOMT3 protein was highly specific and efficient for 2-hydroxy-3-isobutylpyrazine methylation. Based on its differential expression in high- and low-MP-producing grapevine varieties, we propose that VvOMT3 is a key gene for IBMP biosynthesis in grapevine.
Asunto(s)
Metiltransferasas/genética , Proteínas de Plantas/genética , Pirazinas/metabolismo , Vitis/genética , Vitis/metabolismo , Vino , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Calidad de los Alimentos , Regulación de la Expresión Génica de las Plantas , Metilación , Metiltransferasas/química , Metiltransferasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Odorantes , Proteínas de Plantas/metabolismo , Conformación Proteica , Sitios de Carácter Cuantitativo , Homología de Secuencia de AminoácidoRESUMEN
Grafting is particularly important to the cultivation of perennial crops such as grapevine (Vitis vinifera) because rootstocks can provide resistance to soil-borne pests and diseases as well as improve tolerance to some abiotic stresses. Successful grafting is a complex biochemical and structural process beginning with the adhesion of the two grafted partners, followed by callus formation and the establishment of a functional vascular system. At the molecular level, the sequence of events underlying graft union formation remains largely uncharacterized. The present study investigates the transcriptome of grapevine rootstock and graft interface tissues sampled 3 d and 28 d after grafting of over-wintering stems in the spring. Many genes were differentially expressed over time, from 3 d to 28 d after grafting, which could be related to the activation of stem growth and metabolic activity in the spring. This hypothesis is supported by the up-regulation of many genes associated with cell wall synthesis, and phloem and xylem development. Generally, there was an up-regulation of gene expression in the graft interface tissue compared with the rootstock, particularly genes involved in cell wall synthesis, secondary metabolism, and signalling. Although there was overlap between the genes differentially expressed over time (from 3 d to 28 d after grafting) with the gene differentially expressed between the rootstock and the graft interface, numerous graft interface-specific genes were identified.
Asunto(s)
Pared Celular/metabolismo , Perfilación de la Expresión Génica , Reguladores del Crecimiento de las Plantas/metabolismo , Proteínas de Plantas/genética , Metabolismo Secundario , Vitis/metabolismo , Botánica , Pared Celular/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/metabolismo , Transducción de Señal , Vitis/genética , Vitis/crecimiento & desarrolloRESUMEN
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.
Asunto(s)
Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Vitis/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Flavonoides/biosíntesis , Flavonoides/genética , Regulación de la Expresión Génica de las Plantas , Genes myb , Modelos Moleculares , Datos de Secuencia Molecular , Fenotipo , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína , ARN de Planta/genética , Nicotiana/genética , Nicotiana/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Vitis/metabolismoRESUMEN
Previous work has shown that transgenic tobacco plants constitutively over-expressing the Vitis vinifera L. transcription factor VvWRKY2 exhibit reduced susceptibility to necrotrophic fungal pathogens, suggesting that this transcription factor plays a role in grapevine response to phytopathogens. The work presented here characterizes the modifications in cell wall structure observed in the stems and petioles of these transgenic plants. Histochemical stainings of stem and petiole cross-sections using phloroglucinol or Maüle reagents revealed a delay in xylem formation, particularly in the petioles, and differences in lignin composition. Evaluation of lignin quantity and quality showed a decrease in the syringyl/guaiacyl ratio in both stem and petioles. Expression analysis using RT-PCR and potato microarrays showed that tobacco plants over-expressing VvWRKY2 exhibited altered expression of genes involved in lignin biosynthesis pathway and cell wall formation. The ability of VvWRKY2 to activate the promoter of the VvC4H gene, which is involved in the lignin biosynthetic pathway, was confirmed by transient transcriptional activation assays in tobacco protoplasts. Moreover, in situ hybridization revealed that VvWRKY2 is specifically expressed in cells undergoing lignification in young grapevine stems. Together, these results confirm that VvWRKY2 plays a role in regulating lignification in grapevine, possibly in response to biotic or abiotic stresses.
Asunto(s)
Lignina/biosíntesis , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo , Proteínas de Plantas/fisiología , Factores de Transcripción/fisiología , Vitis/genética , Xilema/crecimiento & desarrollo , Hibridación in Situ , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nicotiana/genética , Factores de Transcripción/genética , Xilema/genética , Xilema/metabolismoRESUMEN
Anthocyanidin synthase from Vitis vinifera ( VvANS) catalyzes the in vitro transformation of the natural isomer of leucocyanidin, 2 R,3 S,4 S- cis-leucocyanidin, into 2 R,4 S-flavan-3,3,4-triol ([M + H]+, m/ z 323) and quercetin. The C3-hydroxylation product 2 R,4 S-flavan-3,3,4-triol is first produced and its C3,C4-dehydration product is in tautomeric equilibrium with (+)-dihydroquercetin. The latter undergoes a second VvANS-catalyzed C3-hydroxylation leading to a 4-keto-2 R-flavan-3,3-gem-diol which upon dehydration gives quercetin. The unnatural isomer of leucocyanidin, 2 R,3 S,4 R- trans-leucocyanidin, is similarly transformed into quercetin upon C3,C4-dehydration, but unlike 3,4- cis-leucocyanidin, it also undergoes some C2,C3-dehydration followed by an acid-catalyzed hydroxyl group extrusion at C4 to give traces of cyanidin. Overall, the C3,C4- trans isomer of leucocyanidin is transformed into 2 R,4 R-flavan-3,3,4-triol (M + 1, m/ z 323), (+)-DHQ, (-)-epiDHQ, quercetin, and traces of cyanidin. Our data bring the first direct observation of 3,4- cis-leucocyanidin- and 3,4- trans-leucocyanidin-derived 3,3-gem-diols, supporting the idea that the generic function of ANS is to catalyze the C3-hydroxylation of its substrates. No cyanidin is produced with the natural cis isomer of leucocyanidin, and only traces with the unnatural trans isomer, which suggests that anthocyanidin synthase requires other substrate(s) for the in vivo formation of anthocyanidins.