Patricio Arce-Johnson

Analysis of the grape MYB R2R3 subfamily reveals expanded wine quality-related clades and conserved gene structure organization across Vitis and Arabidopsis genomes

BackgroundThe MYB superfamily constitutes the most abundant group of transcription factors described in plants. Members control processes such as epidermal cell differentiation, stomatal aperture, flavonoid synthesis, cold and drought tolerance and pathogen resistance. No genome-wide characterization of this family has been conducted in a woody species such as grapevine. In addition, previous analysis of the recently released grape genome sequence suggested expansion events of several gene families involved in wine quality.ResultsWe describe and classify 108 members of the grape R2R3 MYB gene subfamily in terms of their genomic gene structures and similarity to their putative Arabidopsis thaliana orthologues. Seven gene models were derived and analyzed in terms of gene expression and their DNA binding domain structures. Despite low overall sequence homology in the C-terminus of all proteins, even in those with similar functions across Arabidopsis and Vitis, highly conserved motif sequences and exon lengths were found. The grape epidermal cell fate clade is expanded when compared with the Arabidopsis and rice MYB subfamilies. Two anthocyanin MYBA related clusters were identified in chromosomes 2 and 14, one of which includes the previously described grape colour locus. Tannin related loci were also detected with eight candidate homologues in chromosomes 4, 9 and 11.ConclusionThis genome wide transcription factor analysis in Vitis suggests that clade-specific grape R2R3 MYB genes are expanded while other MYB genes could be well conserved compared to Arabidopsis. MYB gene abundance, homology and orientation within particular loci also suggests that expanded MYB clades conferring quality attributes of grapes and wines, such as colour and astringency, could possess redundant, overlapping and cooperative functions.

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.

Isolation of WDR and bHLH genes related to flavonoid synthesis in grapevine (Vitis vinifera L.).

Anthocyanins and tannins are two of the most abundant flavonoids found in grapevine, and their synthesis is derived from the phenylpropanoid pathway. As described for model species such as Arabidopsis thaliana, maize and petunia, the end-point branches of this pathway are tightly regulated by the combinatorial interaction of three families of regulatory factors; MYB, bHLH (also known as MYC) and WDR proteins. Among these, only MYB genes have been previously identified in grapes. Here, we report the isolation of the first members from the WDR and bHLH families found in Vitis vinifera, named WDR1, WDR2 and MYCA1. WDR1 contributed positively to the accumulation of anthocyanins when it was overexpressed in A. thaliana, although it was not possible to determine the function of WDR2 by ectopic expression. The sub-cellular localizations of WDR1 and MYCA1 were observed by means of GFP-fusion proteins, indicating both cytoplasm and nuclear localization, in contrast to the localization of a MYB factor exclusively in the nucleus. The expression patterns of these genes were quantified in coloured reproductive organs throughout development, and correlated with anthocyanin accumulation and the expression profiles of the flavonoid-related MYBA1-2,UFGT, and ANR genes. In vitro grapevine plantlets grown under high salt concentrations showed a cultivar-dependent response for anthocyanin accumulation, which correlated with the expression of MYBA1-2, MYCA1 and WDR1 genes. These results suggest that MYCA1 may regulate ANR and UFGT and that this last control is easier to distinguish whenever MYBA genes are absent or in low abundance. Future studies should address the specific interactions of these proteins and their quantitative contribution to flavonoid synthesis in grape berries.

Gene expression associated with compatible viral diseases in grapevine cultivars

Viral diseases affect grapevine cultures without inducing any resistance response. Thus, these plants develop systemic diseases and are chronically infected. Molecular events associated with viral compatible infections responsible for disease establishment and symptoms development are poorly understood. In this study, we surveyed viral infection in grapevines at a transcriptional level. Gene expression in the Vitis vinifera red wine cultivars Carménère and Cabernet-Sauvignon naturally infected with GLRaV-3 were evaluated using a genome-wide expression profiling with the Vitis vinifera GeneChip® from Affymetrix. We describe numerous genes that are induced or repressed in viral infected grapevines leaves. Changes in gene expression involved a wide spectrum of biological functions, including processes of translation and protein targeting, metabolism, transport, and cell defense. Considering cellular localization, the membrane and endomembrane systems appeared with the highest number of induced genes, while chloroplastic genes were mostly repressed. As most induced genes associated with the membranous system are involved in transport, the possible effect of virus in this process is discussed. Responses of both cultivars are analyzed and the results are compared with published data from other species. This is the first study of global gene profiling in grapevine in response to viral infections using DNA microarray.

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.

Molecular and physiological strategies to increase aluminum resistance in plants

Aluminum (Al) toxicity is a primary limitation to plant growth on acid soils. Root meristems are the first site for toxic Al accumulation, and therefore inhibition of root elongation is the most evident physiological manifestation of Al toxicity. Plants may resist Al toxicity by avoidance (Al exclusion) and/or tolerance mechanisms (detoxification of Al inside the cells). The Al exclusion involves the exudation of organic acid anions from the root apices, whereas tolerance mechanisms comprise internal Al detoxification by organic acid anions and enhanced scavenging of free oxygen radicals. One of the most important advances in understanding the molecular events associated with the Al exclusion mechanism was the identification of the ALMT1 gene (Al-activated malate transporter) in Triticum aestivum root cells, which codes for a plasma membrane anion channel that allows efflux of organic acid anions, such as malate, citrate or oxalate. On the other hand, the scavenging of free radicals is dependent on the expression of genes involved in antioxidant defenses, such as peroxidases (e.g. in Arabidopsisthaliana and Nicotiana tabacum), catalases (e.g. in Capsicum annuum), and the gene WMnSOD1 from T. aestivum. However, other recent findings show that reactive oxygen species (ROS) induced stress may be due to acidic (low pH) conditions rather than to Al stress. In this review, we summarize recent findings regarding molecular and physiological mechanisms of Al toxicity and resistance in higher plants. Advances have been made in understanding some of the underlying strategies that plants use to cope with Al toxicity. Furthermore, we discuss the physiological and molecular responses to Al toxicity, including genes involved in Al resistance that have been identified and characterized in several plant species. The better understanding of these strategies and mechanisms is essential for improving plant performance in acidic, Al-toxic soils.

Compatible GLRaV-3 viral infections affect berry ripening decreasing sugar accumulation and anthocyanin biosynthesis in Vitis vinifera

Virus infections in grapevine cause important economic losses and affect fruit quality worldwide. Although the phenotypic symptoms associated to viral infections have been described, the molecular plant response triggered by virus infection is still poorly understood in Vitis vinifera. As a first step to understand the fruit changes and mechanisms involved in the compatible grapevine-virus interaction, we analyzed the berry transcriptome in two stages of development in the red wine cultivar Cabernet Sauvignon infected with Grapevine leaf-roll-associated virus-3 (GLRaV-3). Analysis of global gene expression patterns indicate incomplete berry maturation in infected berries as compared to uninfected fruit suggesting viral infection interrupts the normal berry maturation process. Genes with altered expression in berries harvested from GLRaV-3-infected vines as compared to uninfected tissue include anthocyanin biosynthesis and sugar metabolism genes. The reduction in transcript accumulation for sugar and anthocyanin metabolism during fruit development is consistent with a dramatic reduction in anthocyanin biosynthesis as well as reduced sugar levels in berries, a hallmark phenotypic change observed in virus infected grapevines. Analysis of key regulatory factors provides a mechanism for the observed gene expression changes. Our results provide insight into commonly observed phenotypic alterations in virus infected vines and the molecular mechanisms associated with the plant response to the virus during berry ripening.

An update on sugar transport and signalling in grapevine

In addition to their role as a source of reduced carbon, sugars may directly or indirectly control a wide range of activities in plant cells, through transcriptional and post-translational regulation. This control has been studied in detail using Arabidopsis thaliana, where genetic analysis offers many possibilities. Much less is known about perennial woody species. For several years, various aspects of sugar sensing and signalling have been investigated in the grape (Vitis vinifera L.) berry, an organ that accumulates high concentrations of hexoses in the vacuoles of flesh cells. Here we review various aspects of this topic: the molecular basis of sugar transport and its regulation by sugars in grapevine; the functional analysis of several sugar-induced genes; the effects of some biotic and abiotic stresses on the sugar content of the berry; and finally the effects of exogenous sugar supply on the ripening process in field conditions. A picture of complex feedback and multiprocess regulation emerges from these data.

A molecular framework for the inhibition of Arabidopsis root growth in response to boron toxicity

Boron is an essential micronutrient for plants and is taken up in the form of boric acid (BA). Despite this, a high BA concentration is toxic for the plants, inhibiting root growth and is thus a significant problem in semi-arid areas in the world. In this work, we report the molecular basis for the inhibition of root growth caused by boron. We show that application of BA reduces the size of root meristems, correlating with the inhibition of root growth. The decrease in meristem size is caused by a reduction of cell division. Mitotic cell number significantly decreases and the expression level of key core cell cycle regulators is modulated. The modulation of the cell cycle does not appear to act through cytokinin and auxin signalling. A global expression analysis reveals that boron toxicity induces the expression of genes related with abscisic acid (ABA) signalling, ABA response and cell wall modifications, and represses genes that code for water transporters. These results suggest that boron toxicity produces a reduction of water and BA uptake, triggering a hydric stress response that produces root growth inhibition.

The grapevine guard cell-related VvMYB60 transcription factor is involved in the regulation of stomatal activity and is differentially expressed in response to ABA and osmotic stress

BackgroundUnder drought, plants accumulate the signaling hormone abscisic acid (ABA), which induces the rapid closure of stomatal pores to prevent water loss. This event is trigged by a series of signals produced inside guard cells which finally reduce their turgor. Many of these events are tightly regulated at the transcriptional level, including the control exerted by MYB proteins. In a previous study, while identifying the grapevine R2R3 MYB family, two closely related genes, VvMYB30 and VvMYB60 were found with high similarity to AtMYB60, an Arabidopsis guard cell-related drought responsive gene.ResultsPromoter-GUS transcriptional fusion assays showed that expression of VvMYB60 was restricted to stomatal guard cells and was attenuated in response to ABA. Unlike VvMYB30, VvMYB60 was able to complement the loss-of-function atmyb60-1 mutant, indicating that VvMYB60 is the only true ortholog of AtMYB60 in the grape genome. In addition, VvMYB60 was differentially regulated during development of grape organs and in response to ABA and drought-related stress conditions.ConclusionsThese results show that VvMYB60 modulates physiological responses in guard cells, leading to the possibility of engineering stomatal conductance in grapevine, reducing water loss and helping this species to tolerate drought under extreme climatic conditions.