Floral Development on Vitis vinifera Is Associated with MADS-Box Transcription Factors through the Transcriptional Regulation of VviZIP3.

Several grapevine (Vitis vinifera L.) cultivars show a tendency to develop parthenocarpic seedless grapes, affecting fruit yield and quality. This reproductive disorder originates in defective ovule fertilization due to a failure in pollen tube growth. Zinc (Zn) is a crucial trace element, playing a vital role in various physiological and metabolic processes. It is particularly essential for the healthy growth of flowers and fruits. Insufficient zinc has been suggested as a potential reason for issues in this development process. This microelement is taken up through a mechanism that involves transporters, including the ZRT-IRT-like protein (ZIP) gene family, associated with the influx of Zn into the cell. In grapevines, 20 genes for ZIP-type transporters have been described. In this study, we analyzed the expression pattern of VviZIP3 during flower development and employ transgenic methods to assess its transcriptional regulation. Furthermore, through computational examination of the promoter region, we identified two CArG boxes, recognized as responsive elements to MADS transcription factors. These factors play a key role in shaping various components of a flower, such as pollen. Our investigation of the VviZIP3 promoter confirms the functionality of these CArG boxes. Overall, our results suggest that the increased expression of VviZIP3 during flowering is likely under the influence of MADS transcription factors.

AtZAT4, a C2H2-Type Zinc Finger Transcription Factor from Arabidopsis thaliana, Is Involved in Pollen and Seed Development.

Pollen plays an essential role in plant fertility by delivering the male gametes to the embryo sac before double fertilization. In several plant species, including Arabidopsis, C2H2-type zinc-finger transcription factors (TFs) have been involved in different stages of pollen development and maturation. ZINC FINGER of Arabidopsis thaliana 4 (AtZAT4) is homologous to such TFs and subcellular localization analysis has revealed that AtZAT4 is located in the nucleus. Moreover, analysis of AtZAT4 expression revealed strong levels of it in flowers and siliques, suggesting a role of the encoded protein in the regulation of genes that are associated with reproductive development. We characterized a T-DNA insertional heterozygous mutant Atzat4 (+/−). The relative gene expression analysis of Atzat4 (+/−) showed significant transcript reductions in flowers and siliques. Furthermore, the Atzat4 (+/−) phenotypic characterization revealed defects in the male germline, showing a reduction in pollen tube germination and elongation. Atzat4 (+/−) presented reduced fertility, characterized by a smaller silique size compared to the wild type (WT), and a lower number of seeds per silique. Additionally, seeds displayed lower viability and germination. Altogether, our data suggest a role for AtZAT4 in fertilization and seed viability, through the regulation of gene expression associated with reproductive development.

Overexpression of a SDD1-Like Gene From Wild Tomato Decreases Stomatal Density and Enhances Dehydration Avoidance in Arabidopsis and Cultivated Tomato.

Stomata are microscopic valves formed by two guard cells flanking a pore, which are located on the epidermis of most aerial plant organs and are used for water and gas exchange between the plant and the atmosphere. The number, size and distribution of stomata are set during development in response to changing environmental conditions, allowing plants to minimize the impact of a stressful environment. In Arabidopsis, STOMATAL DENSITY AND DISTRIBUTION 1 (AtSDD1) negatively regulates stomatal density and optimizes transpiration and water use efficiency (WUE). Despite this, little is known about the function of AtSDD1 orthologs in crop species and their wild stress-tolerant relatives. In this study, SDD1-like from the stress-tolerant wild tomato Solanum chilense (SchSDD1-like) was identified through its close sequence relationship with SDD1-like from Solanum lycopersicum and AtSDD1. Both Solanum SDD1-like transcripts accumulated in high levels in young leaves, suggesting that they play a role in early leaf development. Arabidopsis sdd1-3 plants transformed with SchSDD1-like under a constitutive promoter showed a significant reduction in stomatal leaf density compared with untransformed sdd1-3 plants. Additionally, a leaf dehydration shock test demonstrated that the reduction in stomatal abundance of transgenic plants was sufficient to slow down dehydration. Overexpression of SchSDD1-like in cultivated tomato plants decreased the stomatal index and density of the cotyledons and leaves, and resulted in higher dehydration avoidance. Taken together, these results indicate that SchSDD1-like functions in a similar manner to AtSDD1 and suggest that Arabidopsis and tomatoes share this component of the stomatal development pathway that impinges on water status.

Involvement of SchRabGDI1 from Solanum chilense in endocytic trafficking and tolerance to salt stress.

Physiological responses of plants to salinity stress requires the coordinated activation of many genes. A salt-induced gene was isolated from roots of the wild tomato species Solanum chilense and named SchRabGDI1 because it encodes a protein with high identity to GDP dissociation inhibitors of plants. These proteins are regulators of the RabGTPase cycle that play key roles in intracellular vesicular trafficking. The expression pattern of SchRabGDI1 showed an early up-regulation in roots and leaves under salt stress. Functional activity of SchRabGDI1 was shown by restoring the defective phenotype of the yeast sec19-1 mutant and the capacity of SchRabGDI1 to interact with RabGTPase was demonstrated through BiFC assays. Expression of SchRabGDI1 in Arabidopsis thaliana plants resulted in increased salt tolerance. Also, the root cells of transgenic plants showed higher rate of endocytosis under normal growth conditions and higher accumulation of sodium in vacuoles and small vesicular structures under salt stress than wild type. Our results suggest that in salt tolerant species such as S. chilense, bulk endocytosis is one of the early mechanisms to avoid salt stress, which requires the concerted expression of regulatory genes involved in vesicular trafficking of the endocytic pathway.

Differential Roles for VviGST1, VviGST3, and VviGST4 in Proanthocyanidin and Anthocyanin Transport in Vitis vinífera.

In plant cells, flavonoids are synthesized in the cytosol and then are transported and accumulated in the vacuole. Glutathione S-transferase-mediated transport has been proposed as a mechanism involved in flavonoid transport, however, whether binding of flavonoids to glutathione S-transferase (GST) or their transport is glutathione-dependent is not well understood. Glutathione S-transferases from Vitis vinífera (VviGSTs) have been associated with the transport of anthocyanins, however, their ability to transport other flavonoids such as proanthocyanidins (PAs) has not been established. Following bioinformatics approaches, we analyzed the capability of VviGST1, VviGST3, VviGST4, and Arabidopsis TT19 to bind different flavonoids. Analyses of protein-ligand interactions indicate that these GSTs can bind glutathione and monomers of anthocyanin, PAs and flavonols. A total or partial overlap of the binding sites for glutathione and flavonoids was found in VviGST1, and a similar condition was observed in VviGST3 using anthocyanin and flavonols as ligands, whereas VviGST4 and TT19 have both sites for GSH and flavonoids separated. To validate the bioinformatics predictions, functional complementation assays using the Arabidopsis tt19 mutant were performed. Overexpression of VviGST3 in tt19-1 specifically rescued the dark seed coat phenotype associated to correct PA transport, which correlated with higher binding affinity for PA precursors. VviGST4, originally characterized as an anthocyanin-related GST, complemented both the anthocyanin and PA deposition, resembling the function of TT19. By contrast, VviGST1 only partially rescued the normal seed color. Furthermore the expression pattern of these VviGSTs showed that each of these genes could be associated with the accumulation of different flavonoids in specific tissues during grapevine fruit development. These results provide new insights into GST-mediated PA transport in grapevine and suggest that VviGSTs present different specificities for flavonoid ligands. In addition, our data provide evidence to suggest that GST-mediate flavonoid transport is glutathione-dependent.

New member of the R2R3-MYB transcription factors family in grapevine suppresses the anthocyanin accumulation in the flowers of transgenic tobacco.

In grapevine, anthocyanins and proanthocyanidins are the main flavonoids in berries, which are associated to organoleptic properties in red wine such as color and astringency. Flavonoid pathway is specifically regulated at transcriptional level and several R2R3-MYB proteins have shown to act as positive regulators. However, some members of this family have shown to repress the flavonoid biosynthesis. In this work, we present the characterization of VvMYB4-like gene, which encodes a putative transcriptional factor highly expressed in the skin of berries at the pre veraison stage in grapevine. Its over-expression in tobacco resulted in the loss of pigmentation in flowers due a decrease in anthocyanin accumulation. Severity in anthocyanin suppression observed in petals could be associated with the expression level of the VvMYB4-like transgene. Expression analysis of flavonoid structural genes revealed the strong down-regulation of the flavonoid-related genes anthocyanidin synthase (ANS) and dihydroflavonol reductase (DFR) genes and also the reduction of the anthocyanin-related gene UDP glucose:flavonoid 3-O-glucosyl transferase (UFGT), which was dependent of the transgene expression. In addition, expression of VvMYB4-like in the model plant Arabidopsis showed similar results, with the higher down-regulation observed in the AtDFR and AtLDOX genes. These results suggest that VvMYB4-like may play an important role in regulation of anthocyanin biosynthesis in grapevine acting as a transcriptional repressor of flavonoid structural genes.

VvMATE1 and VvMATE2 encode putative proanthocyanidin transporters expressed during berry development in Vitis vinifera L.

KEY MESSAGE: VvMATE1 and VvMATE2 encode putative PA transporters expressed during seed development in grapevine. The subcellular localization of these MATE proteins suggests different routes for the intracellular transport of PAs. Proanthocyanidins (PAs), also called condensed tannins, protect plants against herbivores and are important quality components of many fruits. PAs biosynthesis is part of the flavonoid pathway that also produces anthocyanins and flavonols. In grape fruits, PAs are present in seeds and skin tissues. PAs are synthesized in the cytoplasm and accumulated into the vacuole and apoplast; however, little is known about the mechanisms involved in the transport of these compounds to such cellular compartments. A gene encoding a Multidrug And Toxic compound Extrusion (MATE) family protein suggested to transport anthocyanins-named VvMATE1-was used to identify a second gene of the MATE family, VvMATE2. Analysis of their deduced amino acid sequences and the phylogenetic relationship with other MATE-like proteins indicated that VvMATE1 and VvMATE2 encode putative PA transporters. Subcellular localization assays in Arabidopsis protoplasts transformed with VvMATE-GFP fusion constructs along with organelle-specific markers revealed that VvMATE1 is localized in the tonoplast whereas VvMATE2 is localized in the Golgi complex. Major expression of both genes occurs during the early stages of seed development concomitant with the accumulation of PAs. Both genes are poorly expressed in the skin of berries while VvMATE2 is also expressed in leaves. The presence of putative cis-acting elements in the promoters of VvMATE1 and VvMATE2 may explain the differential transcriptional regulation of these genes in grapevine. Altogether, these results suggest that these MATE proteins could mediate the transport and accumulation of PAs in grapevine through different routes and cellular compartments.

Epigenetic repressor-like genes are differentially regulated during grapevine (Vitis vinifera L.) development.

Grapevine sexual reproduction involves a seasonal separation between inflorescence primordia (flowering induction) and flower development. We hypothesized that a repression mechanism implicating epigenetic changes could play a role in the seasonal separation of these two developmental processes in grapevine. Therefore, the expression of five grapevine genes with homology to the Arabidopsis epigenetic repressor genes FERTILIZATION INDEPENDENT ENDOSPERM (FIE), EMBRYONIC FLOWER 2 (EMF2), CURLY LEAF (CLF), MULTICOPY SUPPRESSOR OF IRA 1 (MSI1) and SWINGER (SWN) was analyzed during the development of buds and vegetative and reproductive organs. During bud development, the putative grapevine epigenetic repressor genes VvCLF, VvEMF2, VvMSI1, VvSWN and VvFIE are mainly expressed in latent buds at the flowering induction period, but also detected during bud burst and inflorescence/flower development. The overlapping expression patterns of grapevine PcG-like genes in buds suggest that chromatin remodeling mechanisms could be operating during grapevine bud development for controlling processes such as seasonal flowering, dormancy and bud burst. Furthermore, the expression of grapevine PcG-like genes was also detected in fruits and vegetative organs, suggesting that epigenetic changes could be at the basis of the regulation of various proliferation-differentiation cell transitions that occur during grapevine development.

VvCO and VvCOL1, two CONSTANS homologous genes, are regulated during flower induction and dormancy in grapevine buds.

Two previously uncharacterized Vitis vinifera CONSTANS-like genes (VvCO, VvCOL1), which are predicted to encode proteins with homology to members of the Arabidopis CONSTANS family, were identified. Under controlled conditions, both genes show a diurnal expression pattern with peak at dawn. During grapevine bud development, VvCOL1 is mainly expressed in dormancy, suggesting a participation in the transcriptional photoperiod control of bud dormancy induction and maintenance in this species. On the other hand, VvCO expression in latent buds is in agreement with a function during flowering induction. A spatial and temporal relationship in the expression of VvCO, VFY and VvMADS8 (the Arabidopsis LEAFY and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 orthologues) in latent buds is observed, suggesting that these genes are involved in the seasonal periodicity of flowering in grapevines. Furthermore, our results provide a new molecular insight into tendril development showing that grapevine CO homologues are also expressed in this distinctive organ.