Rodrigo Loyola

Post-veraison sunlight exposure induces MYB-mediated transcriptional regulation of anthocyanin and flavonol synthesis in berry skins of Vitis vinifera

Anthocyanins, flavan-3-ols, and flavonols are the three major classes of flavonoid compounds found in grape berry tissues. Several viticultural practices increase flavonoid content in the fruit, but the underlying genetic mechanisms responsible for these changes have not been completely deciphered. The impact of post-veraison sunlight exposure on anthocyanin and flavonol accumulation in grape berry skin and its relation to the expression of different transcriptional regulators known to be involved in flavonoid synthesis was studied. Treatments consisting of removing or moving aside the basal leaves which shade berry clusters were applied. Shading did not affect sugar accumulation or gene expression of HEXOSE TRANSPORTER 1, although in the leaf removal treatment, these events were retarded during the first weeks of ripening. Flavonols were the most drastically reduced flavonoids following shading and leaf removal treatments, related to the reduced expression of FLAVONOL SYNTHASE 4 and its putative transcriptional regulator MYB12. Anthocyanin accumulation and the expression of CHS2, LDOX, OMT, UFGT, MYBA1, and MYB5a genes were also affected. Other regulatory genes were less affected or not affected at all by these treatments. Non-transcriptional control mechanisms for flavonoid synthesis are also suggested, especially during the initial stages of ripening. Although berries from the leaf removal treatment received more light than shaded fruits, malvidin-3-glucoside and total flavonol content was reduced compared with the treatment without leaf removal. This work reveals that flavonol-related gene expression responds rapidly to field changes in light levels, as shown by the treatment in which shaded fruits were exposed to light in the late stages of ripening. Taken together, this study establishes MYB-specific responsiveness for the effect of sun exposure and sugar transport on flavonoid synthesis.

The Phenylpropanoid Pathway Is Controlled at Different Branches by a Set of R2R3-MYB C2 Repressors in Grapevine

A set of transcriptional repressors negatively regulates the expression of genes involved in different branches of the phenylpropanoid pathway. Because of the vast range of functions that phenylpropanoids possess, their synthesis requires precise spatiotemporal coordination throughout plant development and in response to the environment. The accumulation of these secondary metabolites is transcriptionally controlled by positive and negative regulators from the MYB and basic helix-loop-helix protein families. We characterized four grapevine (Vitis vinifera) R2R3-MYB proteins from the C2 repressor motif clade, all of which harbor the ethylene response factor-associated amphiphilic repression domain but differ in the presence of an additional TLLLFR repression motif found in the strong flavonoid repressor Arabidopsis (Arabidopsis thaliana) AtMYBL2. Constitutive expression of VvMYB4a and VvMYB4b in petunia (Petunia hybrida) repressed general phenylpropanoid biosynthetic genes and selectively reduced the amount of small-weight phenolic compounds. Conversely, transgenic petunia lines expressing VvMYBC2-L1 and VvMYBC2-L3 showed a severe reduction in petal anthocyanins and seed proanthocyanidins together with a higher pH of crude petal extracts. The distinct function of these regulators was further confirmed by transient expression in tobacco (Nicotiana benthamiana) leaves and grapevine plantlets. Finally, VvMYBC2-L3 was ectopically expressed in grapevine hairy roots, showing a reduction in proanthocyanidin content together with the down-regulation of structural and regulatory genes of the flavonoid pathway as revealed by a transcriptomic analysis. The physiological role of these repressors was inferred by combining the results of the functional analyses and their expression patterns in grapevine during development and in response to ultraviolet B radiation. Our results indicate that VvMYB4a and VvMYB4b may play a key role in negatively regulating the synthesis of small-weight phenolic compounds, whereas VvMYBC2-L1 and VvMYBC2-L3 may additionally fine tune flavonoid levels, balancing the inductive effects of transcriptional activators.

The photomorphogenic factors UV-B RECEPTOR 1, ELONGATED HYPOCOTYL 5, and HY5 HOMOLOGUE are part of the UV-B signalling pathway in grapevine and mediate flavonol accumulation in response to the environment

By performing molecular studies coupled to radiation experiments and in silico systems analyses, we have ascertained the role of the grapevine UV-B receptor and two HY5 homologues in regulating flavonol synthesis.

Phytoplasma and virus detection in commercial plantings of Vitis vinifera cv. Merlot exhibiting premature berry dehydration

A new and devastating physiological disorder of Vitis vinifera cv. Merlot was recently reported, known as premature berry dehydration (PBD), which is characterized by plant growth reduction, induction of general senescence and pedicel necrosis in the fruit, causing significant reductions in vineyard production. The causes of this disease remain unclear and previous reports suggest that it may be associated with phloem disruption and water provision. For this reason, any factor causing phloem disturbances could cause an important change in the berry water status. As some micro-organisms have been reported to disrupt phloem flow, we analyzed the occurrence of phytoplasma and viruses in commercial vineyards presenting PBD. In this study, a phytoplasma was detected by electron microscopy and nested PCR while virus infections were diagnosed by RT-PCR in samples collected during two growing seasons. The presence of phytoplasma only in samples from grape plants with PBD suggests that this pathogen may be one of the causal agents of this disorder. We suggest that the influence of other factors, such as virus infections, agronomic handling and environmental conditions also modulate berry dehydration. This is the first study at the microscopic and molecular levels that correlates phytoplasma presence with PBD.

Transcriptome-Wide Identification of Novel UV-B- and Light Modulated Flavonol Pathway Genes Controlled by VviMYBF1

Flavonols constitute a group of flavonoids with important photoprotective roles in plants. In addition, flavonol content and composition greatly influences fruit quality. We previously demonstrated that the grapevine R2R3-MYB transcription factor (TF) VviMYBF1 promotes flavonol accumulation by inducing the expression of flavonol synthase (VviFLS1/VviFLS4), a key step of the initial flavonol pathway. Despite this, gene networks underlying flavonol modification in grapevine including both structural and regulatory genes remain poorly understood. In order to identify flavonol modifying genes and TFs acting downstream of VviMYBF1 a microarray-based transcriptome analysis was performed on grapevine hairy roots ectopically expressing VviMYBF1 or a Green Fluorescent Protein as control. VviFLS1 was induced in VviMYBF1 transgenic roots and glycosylated flavonols accumulated significantly compared with control lines. Among the differentially expressed genes, potential flavonol-modifying enzymes with predicted rhamnosyltransferase (e.g., RhaT1) or glycosyltransferase (e.g., GT3) activities were identified. In addition, important TFs of the MYB and bZIP families such as the proanthocyanidin regulator VviMYBPA1 and the UV-B light responsive HY5 homolog VviHYH were significantly altered in their expression pattern by overexpression of VviMYBF1. Co-temporal expression analysis demonstrated positive correlation of VviMYBF1 with VviFLS1, VviGT3, and VviRhaT1 during berry development and in fruits ripened with different light and UV-B radiation conditions at field. These results show that VviMYBF1 overexpression led to the identification of novel genes of the flavonol pathway and that the flavonol modifying machinery can be influenced by agricultural practices to optimize flavonol composition in grapes.

Grapevine Biotechnology: Molecular Approaches Underlying Abiotic and Biotic Stress Responses

Grapevine is one of the most abundant crops worldwide, with varieties destined for fresh and dry consumption, as well as wine production. Unfortunately, grapevine plants are affected by both biotic and abiotic stresses, generating significant economic losses. These conditions can negatively impact grape cultivation at different stages: plant and berry development during preand post-harvest, production, fresh fruit processing and export, along with wine quality. Most of the grapevine varieties are susceptible to several pathogens and within this chapter, particular attention is given to fungi (Botrytis cinerea and Erysiphe necator) and viruses, since they are a worldwide concern. Within the latter, special focus is given to the grapevine leafroll disease, a complex and destructive infection. On the other hand, abiotic stress is also relevant in grapevine, and in this chapter it will be exemplified by UV-B radiation and its impact on growth and fruit development, plant adaptive responses and its relationship with the quality of grape berries for winemaking. The main biotic and abiotic grapevine stress factors are reviewed in this chapter, considering a special focus on biotechnological approaches carried out in order to address them and minimize their detrimental consequences.

The role of UV-B light on small RNA activity during grapevine berry development

UV-B regulation of anthocyanin biosynthesis in vegetative and grapevine berry tissues has been extensively described. However, its relation with UV-B-regulated microRNAs (miRNAs) has not been addressed before in this species. We explored by deep sequencing of small RNA libraries the developmental dynamics and UV-B effects on miRNAs and associated phased small interfering RNA (phasi-RNAs)-producing loci abundances in in vitro-grown plantlets, in field-grown berry skins of cv. Cabernet Sauvignon, and low- and high UV-B fluence treatments of greenhouse-grown berries at several time points around veraison. We observed by RNA blotting a differential effect of low-versus high-fluence UV-B on miR828 abundances (an effector of anthocyanins and UV-absorbing polyphenolics) across berry development, and identified other miRNAs that correlated with miR828 dynamics. The functional significance of the observed UV-coordinated miRNA responses to UV was supported by degradome evidences of AGO-programmed slicing of mRNAs. Inverse co-expression of the up-regulated miRNAs miR156, miR482, miR530, and miR828 with cognate target gene expressions in response to high fluence UV-B measured by quantitative real-time PCR. These UV-response relationships were also corroborated by analyzing three published transcriptome datasets (berries subjected to UV-C for 1 hr [at pre-veraison], UV-B for five weeks post-veraison, and five red-skinned varieties across four berry development time points). Based on observed significant changes by UV-B on miRNA and derivative phasi-RNA abundances, we propose a regulatory network model of UV responses impacting anti-oxidant and stress-associated polyphenolic compound biosynthesis. In this model high-fluence UV-B increases miR168 (validated in a UV-B small RNA-derived degradome library to target ARGONAUTE1, which spawns phasi-RNAs) and miR530 (targets a novel Plus-3 domain mRNA), while decreasing miR403 abundances (validated to target ARGONAUTE2), thereby coordinating post-transcriptional gene silencing activities by different AGOs. Up-regulation of miR3627/4376 (validated to target Ca2+-transporting ATPase10 that spawns phasi-RNAs) could facilitate anthocyanin accumulation. miR395 and miR399, induced by sulfur and phosphorus starvation in other species (conditions known to trigger anthocyanin accumulation) respond positively to UV-B radiation and are shown to slice cognate targets in grapevine. miR156/miR535 is shown to target SQUAMOSA PROMOTER-BINDING transcription factor genes that potentially regulate the activities of MYB-bHLH-WD40 complexes and thereby anthocyanin biosynthesis. Increases in MYB-bHLH-WD40 TFs could also contribute to the observed up-regulation of miR828 via the conserved and degradome-validated auto-regulatory loop involving miR828/TAS4abc to regulate MYBA6/A7/A5-MYB113-like levels and thereby anthocyanin levels. These results and meta-analysis provide a basis for systems approaches to better understand non-coding RNA functions in response to UV.