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Grapevine (Vitis vinifera L.) crops are continuously exposed to biotic and abiotic stresses, which can cause genetic and epigenetic alterations. To determine the possible effects of grapevine cryopreservation on the regulation of DNA demethylase genes, this work studied the expression of DNA demethylase genes in cryopreserved and post-cryopreserved grapevine tissues. V. vinifera DNA demethylases were characterized by in silico analysis, and gene expression quantification was conducted by RT‒qPCR. Three DNA demethylase sequences were found: VIT_13s0074g00450 (VvDMT), VIT_08s0007g03920 (VvROS1), and VIT_06s0061g01270 (VvDML3). Phylogenetic analysis revealed that the sequences from V. vinifera and A. thaliana had a common ancestry. In the promoters of responsive elements to transcription factors such as AP-2, Myb, bZIP, TBP, and GATA, the conserved domains RRM DME and Perm CXXC were detected. These responsive elements play roles in the response to abiotic stress and the regulation of cell growth. These data helped us characterize the V. vinifera DNA demethylase genes. Gene expression analysis indicated that plant vitrification solution 2 (PVS2) treatment does not alter the expression of DNA demethylase genes. The expression levels of VvDMT and VvROS1 increased in response to cryopreservation by vitrification. Furthermore, in post-cryopreservation, VvROS1 was highly induced, and VvDML3 was repressed in all the treatment groups. Gene expression differences between different treatments and tissues may play roles in controlling methylation patterns during gene regulation in tissues stressed by cryopreservation procedures and in the post-cryopreservation period during plant growth and development.

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Germin-like proteins (GLPs) play an important role against various stresses. Vitis vinifera L. genome contains 7 GLPs; many of them are functionally unexplored. However, the computational analysis may provide important new insight into their function. Currently, physicochemical properties, subcellular localization, domain architectures, 3D structures, N-glycosylation & phosphorylation sites, and phylogeney of the VvGLPs were investigated using the latest computational tools. Their functions were predicted using the Search tool for the retrieval of interacting genes/proteins (STRING) and Blast2Go servers. Most of the VvGLPs were extracellular (43%) in nature but also showed periplasmic (29%), plasma membrane (14%), and mitochondrial- or chloroplast-specific (14%) expression. The functional analysis predicted unique enzymatic activities for these proteins including terpene synthase, isoprenoid synthase, lipoxygenase, phosphate permease, receptor kinase, and hydrolases generally mediated by Mn+ cation. VvGLPs showed similarity in the overall structure, shape, and position of the cupin domain. Functionally, VvGLPs control and regulate the production of secondary metabolites to cope with various stresses. Phylogenetically VvGLP1, -3, -4, -5, and VvGLP7 showed greater similarity due to duplication while VvGLP2 and VvGLP6 revealed a distant relationship. Promoter analysis revealed the presence of diverse cis-regulatory elements among which CAAT box, MYB, MYC, unnamed-4 were common to all of them. The analysis will help to utilize VvGLPs and their promoters in future food programs by developing resistant cultivars against various biotic (Erysiphe necator and in Powdery Mildew etc.) and abiotic (Salt, drought, heat, dehydration, etc.) stresses.

As proteínas do tipo germin (GLPs) desempenham um papel importante contra vários estresses. O genoma de Vitis vinifera L. contém 7 GLPs; muitos deles são funcionalmente inexplorados. No entanto, a análise computacional pode fornecer informações importantes sobre sua função. Atualmente, as propriedades físico-químicas, localização subcelular, arquitetura de domínio, estruturas 3D, sítios de N-glicosilação e fosforilação e estudos filogenéticos dos VvGLPs foram conduzidos usando as ferramentas computacionais mais recentes. Suas funções foram previstas usando a ferramenta Search para recuperação de genes/proteínas em interação (STRING) e servidores Blast2Go. A maioria dos VvGLPs são extracelulares (43%) na natureza, mas também mostraram expressão periplasmática (29%), na membrana plasmática (14%) e específica para mitocôndrias ou cloroplastos (14%). A análise funcional previu atividades enzimáticas únicas para essas proteínas, incluindo terpeno sintase, isoprenoide sintase, lipoxigenase, fosfato permease, receptor quinase e hidrolases geralmente mediadas por cátion Mn +. VvGLPs mostraram similaridade na estrutura geral, forma e posição do domínio cupin. Funcionalmente, os VvGLPs controlam e regulam a produção de metabólitos secundários para lidar com vários estresses. Filogeneticamente, VvGLP1, -3, -4, -5 e VvGLP7 mostraram maior similaridade devido à duplicação, enquanto VvGLP2 e VvGLP6 revelaram uma relação distante. A análise do promotor revelou a presença de diversos elementos cis-reguladores, entre os quais CAAT box, MYB, MYC, sem nome-4, sendo comum a todos eles. A análise ajudará a utilizar VvGLPs e seus promotores em programas alimentares futuros, desenvolvendo cultivares resistentes contra vários estresses bióticos (Erysiphe necator e no oídio, etc.) e abióticos (sal, seca, calor, estresse hídrico, etc.).

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Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of 4 important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot, and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivar's reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

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RESUMO

Germin-like proteins (GLPs) play an important role against various stresses. Vitis vinifera L. genome contains 7 GLPs; many of them are functionally unexplored. However, the computational analysis may provide important new insight into their function. Currently, physicochemical properties, subcellular localization, domain architectures, 3D structures, N-glycosylation & phosphorylation sites, and phylogeney of the VvGLPs were investigated using the latest computational tools. Their functions were predicted using the Search tool for the retrieval of interacting genes/proteins (STRING) and Blast2Go servers. Most of the VvGLPs were extracellular (43%) in nature but also showed periplasmic (29%), plasma membrane (14%), and mitochondrial- or chloroplast-specific (14%) expression. The functional analysis predicted unique enzymatic activities for these proteins including terpene synthase, isoprenoid synthase, lipoxygenase, phosphate permease, receptor kinase, and hydrolases generally mediated by Mn+ cation. VvGLPs showed similarity in the overall structure, shape, and position of the cupin domain. Functionally, VvGLPs control and regulate the production of secondary metabolites to cope with various stresses. Phylogenetically VvGLP1, -3, -4, -5, and VvGLP7 showed greater similarity due to duplication while VvGLP2 and VvGLP6 revealed a distant relationship. Promoter analysis revealed the presence of diverse cis-regulatory elements among which CAAT box, MYB, MYC, unnamed-4 were common to all of them. The analysis will help to utilize VvGLPs and their promoters in future food programs by developing resistant cultivars against various biotic (Erysiphe necator and in Powdery Mildew etc.) and abiotic (Salt, drought, heat, dehydration, etc.) stresses.

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Using tolerant genotypes and the correct use of fertilizers can mitigate the negative effect of elevated Cu levels in the growing medium. In this context, the study aimed to evaluate the effects of excess Cu in the root system and the effectiveness of phosphorus (P) in minimizing the phytotoxicity of Cu in three genotypes: IAC 572 [(Vitis riparia x V. rupestris) x V. caribaea], Magnolia (V. rotundifolia) and Paulsen 1103 (V. berlandieri x V. rupestris). The plants were grown in nutrient solutions and were supplemented with the following treatments: 0.3 µM Cu (Control), 60 µM Cu (Cu) and 60 µM Cu and 62 mg L-1 P (Cu + P). Root samples were sectioned for microscopy analyses, and the shoot lengths, shoot and root dry matter, relative growth rates (RGR) and tissue nutrient contents were also evaluated. The roots of the genotypes that were cultivated with high Cu concentrations produced greater numbers of branches and larger diameters, except for Magnolia genotype that was cultivated in a Cu + P solution, which had an organization similar to the control. Excess Cu caused accumulations of phenolic compounds and decreased shoot lengths, dry matter and RGR in the genotypes. In the treatments with excess Cu, there were increases in this element in the tissues, but P decreased the metal concentrations in Magnolia roots. Therefore, Cu accumulations alter the root system development patterns, growth parameters and tissue nutrient contents in the studied genotypes. Magnolia has a higher tolerance and is also the only genotype for which the use of P has been shown to be effective.

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Grapevine cultivars are clonally propagated to preserve their varietal attributes. However, genetic variations accumulate due to the occurrence of somatic mutations. This process is anthropically influenced through plant transportation, clonal propagation and selection. Malbec is a cultivar that is well-appreciated for the elaboration of red wine. It originated in Southwestern France and was introduced in Argentina during the 1850s. In order to study the clonal genetic diversity of Malbec grapevines, we generated whole-genome resequencing data for four accessions with different clonal propagation records. A stringent variant calling procedure was established to identify reliable polymorphisms among the analyzed accessions. The latter procedure retrieved 941 single nucleotide variants (SNVs). A reduced set of the detected SNVs was corroborated through Sanger sequencing, and employed to custom-design a genotyping experiment. We successfully genotyped 214 Malbec accessions using 41 SNVs, and identified 14 genotypes that clustered in two genetically divergent clonal lineages. These lineages were associated with the time span of clonal propagation of the analyzed accessions in Argentina and Europe. Our results show the usefulness of this approach for the study of the scarce intra-cultivar genetic diversity in grapevines. We also provide evidence on how human actions might have driven the accumulation of different somatic mutations, ultimately shaping the Malbec genetic diversity pattern.

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KEY MESSAGE: The transcriptomes of wild and cultivated grapes consists of similar expressed genes but distinct wiring of co-expressed modules associated with environmental conditions. Grapevine is an important fruit crop worldwide, with high economic value and widespread distribution. Commercial production is based on Vitis vinifera, and, to a lesser extent, on hybrids with American grapes, such as V. labrusca. Wild grape relatives are important sources of resistance against biotic and abiotic factors; however, their global gene expression patterns remain poorly characterized. We associated genome-wide transcript profiling to phenotypic analyses to investigate the responses of cultivated and wild vines to vineyard conditions. The expressed genes in the Vitis reference transcriptome are largely shared by wild grapes, V. labrusca hybrids and vinifera cultivars. In contrast, significant differential regulation between wild and vinifera genotypes represents 80% of gene expression variation, regardless of the environment. In wild grapes, genes associated to regulatory processes are downregulated, whereas those involved in metabolic pathways are upregulated, in comparison to vinifera. Photosynthesis-related ontologies are overrepresented in the induced genes, in agreement with higher contents of chlorophyll in wild grapes. Co-regulated gene network analyses provide evidence of more complex transcriptome organization in vinifera. In wild grapes, genes involved in signaling pathways of stress-related hormones are overrepresented in modules associated with the environment. Consensus network analyses revealed high preservation within co-regulated gene modules between cultivated and wild grapes, but divergent relationships among the expression clusters. In conclusion, the distinct phenotypes of wild and cultivated grapes are underlain by differences in gene expression, but also by distinct higher-order organization of the transcriptome and contrasting association of co-expressed gene clusters with the environment.

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MAIN CONCLUSION: In deciduous fruit trees in which dormancy is induced by low temperatures, the expression of DORMACY-ASSOCIATED MADS-BOX genes (DAM) is regulated by CBF/DREB1 transcription factors. In Vitis vinifera, in which dormancy is induced by the photoperiod, VvDAM-SVPs gene expression is regulated by FLOWERING LOCUS T (VvFT). Using the sequences of the six peach (Prunus persica) DORMACY-ASSOCIATED MADS-box genes (DAM) as query, eight putative DAM genes belonging to the family of MADS-box transcription factors and related to the Arabidopsis floral regulators SHORT VEGETATIVE PHASE (SVP) and AGAMOUS LIKE 24 (AGL24) were identified in the V. vinifera genome. Among these, five belong to the subfamily SVP-like genes which have been associated with the regulation of flowering and dormancy in annual and perennial plants, respectively. It has been proposed that they play a direct role in the induction and maintenance of endodormancy (ED) through the regulation of the FLOWERING LOCUS T (FT) gene. In the present study, it is demonstrated that in V. vinifera: (1) VvDAM-SVPs genes are not regulated by ABA/low temperature-induced VvCBFs transcription factors as described for other species of deciduous fruit trees. (2) A contrasting expression pattern between VvDAM3-SVP and VvFT was found under different experimental conditions related to the entry and exit of grapevine buds from ED. (3) Overexpression of VvFT in somatic grapevine embryos (SGE) repressed the expression of VvDAM3-SVP and VvDAM4-SVP. Taken together, the results suggest that VvDAM3-SVP could be associated with ED in grapevine buds, and that its expression could be regulated by VvFT.

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KEY MESSAGE: By studying three cv. Malbec clones cultivated in two vineyards with contrasting environmental conditions, we demonstrated that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent. Clonal selection and vegetative propagation determine low genetic variability in grapevine cultivars, although it is common to observe diverse phenotypes. Environmental signals may induce epigenetic changes altering gene expression and phenotype. The range of phenotypes that a genotype expresses in different environments is known as phenotypic plasticity. DNA methylation is the most studied epigenetic mechanism, but only few works evaluated this novel source of variability in grapevines. In the present study, we analyzed the effects on phenotypic traits and epigenome of three Vitis vinifera cv. Malbec clones cultivated in two contrasting vineyards of Mendoza, Argentina. Anonymous genome regions were analyzed using methylation-sensitive amplified polymorphism (MSAP) markers. Clone-dependent phenotypic and epigenetic variability between vineyards were found. The clone that presented the clearer MSAP differentiation between vineyards was selected and analyzed through reduced representation bisulfite sequencing. Twenty-nine differentially methylated regions between vineyards were identified and associated to genes and/or promoters. We discuss about a group of genes related to hormones homeostasis and sensing that could provide a hint of the epigenetic role in the determination of the different phenotypes observed between vineyards and conclude that DNA methylation has an important role in the phenotypic plasticity and that epigenetic modulation is clone-dependent.

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