Grapes are one of the world’s oldest and most important fruit crops. They are of high economic value in many countries, but the susceptibility of the dominant winegrape species Vitis vinifera to fungal disease is a significant problem. The Chinese wild grape species are a rich source of disease-resistance genes and these can be used to discover how disease resistance in V. vinifera grapevines might be enhanced. Pathogenesis-related (PR) 10 proteins are involved in the disease-response. Here, we use the genomic DNA of the Chinese wild species Vitis pseudoreticulata accession “Baihe-35-1” as the template to design specific primers based on VvPR10s sequences. We used overlap extension PCR to obtain the sequences: VpPR10.4, VpPR10.6, VpPR10.7 and VpPR10.9. The coding sequences of the VpPR10s were then cloned into the pGEX-4T-1 vector. The purified proteins VpPR10.4, VpPR10.6, VpPR10.7 and VpPR10.9 were used to analyse nuclease activity. Meanwhile, functional analysis of VpPR10s under different biotic and abiotic stresses was carried out to further clarify the disease-resistance mechanisms of the Chinese wild grapevine VpPR10 genes. The analysis of protein structure indicates that VpPR10.4 and VpPR10.7 had the P-loop domain and the Bet v 1 motif, which are a consistent feature of plant PR10. However, there was no P-loop domain or Bet v 1 motif in VpPR10.9 and we could not find the Bet v 1 motif in VpPR10.6. The results of the nuclease activity assay and of the functional analyses of VpPR10s under different biotic and abiotic stresses also confirm that VpPR10.4 and VpPR10.7 proteins have marked RNase, DNase, anti-fungal activities and respond to abiotic stresses. The VpPR10.6 and VpPR10.9 proteins do not have these activities and functions.
Vitis pseudoreticulata; pathogenesis-related protein 10; nuclease activity; anti-fungal activity; abiotic stresses
Vitis vinifera (grape) is one of the most economically significant fruit crops in the world. The availability of the recently released grape genome sequence offers an opportunity to identify and analyze some important gene families in this species. Subtilases are a group of subtilisin-like serine proteases that are involved in many biological processes in plants. However, no comprehensive study incorporating phylogeny, chromosomal location and gene duplication, gene organization, functional divergence, selective pressure and expression profiling has been reported so far for the grape.
In the present study, a comprehensive analysis of the subtilase gene family in V. vinifera was performed. Eighty subtilase genes were identified. Phylogenetic analyses indicated that these subtilase genes comprised eight groups. The gene organization is considerably conserved among the groups. Distribution of the subtilase genes is non-random across the chromosomes. A high proportion of these genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the subtilase gene family. Analyses of divergence and adaptive evolution show that while purifying selection may have been the main force driving the evolution of grape subtilases, some of the critical sites responsible for the divergence may have been under positive selection. Further analyses of real-time PCR data suggested that many subtilase genes might be important in the stress response and functional development of plants.
Tandem duplications as well as purifying and positive selections have contributed to the functional divergence of subtilase genes in V. vinifera. The data may contribute to a better understanding of the grape subtilase gene family.
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Vitis vinifera; Subtilase; Gene family; Evolution; Positive selection; Differential expression
Sorbitol dehydrogenase (SDH, EC 220.127.116.11) is the key enzyme involved in sorbitol metabolism in higher plants. SDH genes in some Rosaceae species could be divided into two groups. L-idonate-5-dehydrogenase (LIDH, EC 18.104.22.1684) is involved in tartaric acid (TA) synthesis in Vitis vinifera and is highly homologous to plant SDHs. Despite efforts to understand the biological functions of plant SDH, the evolutionary history of plant SDH genes and their phylogenetic relationship with the V. vinifera LIDH gene have not been characterized.
A total of 92 SDH genes were identified from 42 angiosperm species. SDH genes have been highly duplicated within the Rosaceae family while monocot, Brassicaceae and most Asterid species exhibit singleton SDH genes. Core Eudicot SDHs have diverged into two phylogenetic lineages, now classified as SDH Class I and SDH Class II. V. vinifera LIDH was identified as a Class II SDH. Tandem duplication played a dominant role in the expansion of plant SDH family and Class II SDH genes were positioned in tandem with Class I SDH genes in several plant genomes. Protein modelling analyses of V. vinifera SDHs revealed 19 putative active site residues, three of which exhibited amino acid substitutions between Class I and Class II SDHs and were influenced by positive natural selection in the SDH Class II lineage. Gene expression analyses also demonstrated a clear transcriptional divergence between Class I and Class II SDH genes in V. vinifera and Citrus sinensis (orange).
Phylogenetic, natural selection and synteny analyses provided strong support for the emergence of SDH Class II by positive natural selection after tandem duplication in the common ancestor of core Eudicot plants. The substitutions of three putative active site residues might be responsible for the unique enzyme activity of V. vinifera LIDH, which belongs to SDH Class II and represents a novel function of SDH in V. vinifera that may be true also of other Class II SDHs. Gene expression analyses also supported the divergence of SDH Class II at the expression level. This study will facilitate future research into understanding the biological functions of plant SDHs.
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Sorbitol dehydrogenase; L-idonate-5-dehydrogenase; Gene duplication; Functional divergence; Tartaric acid; Ascorbic acid; Grapevine
GRAS transcription factors are involved in many processes of plant growth and development (e.g., axillary shoot meristem formation, root radial patterning, nodule morphogenesis, arbuscular development) as well as in plant disease resistance and abiotic stress responses. However, little information is available concerning this gene family in grapevine (Vitis vinifera L.), an economically important woody crop. We performed a model curation of GRAS genes identified in the latest genome annotation leading to the identification of 52 genes. Gene models were improved and three new genes were identified that could be grapevine- or woody-plant specific. Phylogenetic analysis showed that GRAS genes could be classified into 13 groups that mapped on the 19 V. vinifera chromosomes. Five new subfamilies, previously not characterized in other species, were identified. Multiple sequence alignment showed typical GRAS domain in the proteins and new motifs were also described. As observed in other species, both segmental and tandem duplications contributed significantly to the expansion and evolution of the GRAS gene family in grapevine. Expression patterns across a variety of tissues and upon abiotic and biotic conditions revealed possible divergent functions of GRAS genes in grapevine development and stress responses. By comparing the information available for tomato and grapevine GRAS genes, we identified candidate genes that might constitute conserved transcriptional regulators of both climacteric and non-climacteric fruit ripening. Altogether this study provides valuable information and robust candidate genes for future functional analysis aiming at improving the quality of fleshy fruits.
abiotic stress; biotic stress; fruit ripening; grapevine; GRAS gene family; transcription factor
Dehydrins (DHNs) protect plant cells from desiccation damage during environmental stress, and also participate in host resistance to various pathogens. In this study, we aimed to identify and characterize the DHN gene families from Vitis vinifera and wild V. yeshanensis, which is tolerant to both drought and cold, and moderately resistant to powdery mildew.
Four DHN genes were identified in both V. vinifera and V. yeshanensis, which shared a high sequence identity between the two species but little homology between the genes themselves. These genes were designated DHN1, DHN2, DHN3 and DHN4. All four of the DHN proteins were highly hydrophilic and were predicted to be intrinsically disordered, but they differed in their isoelectric points, kinase selectivities and number of functional motifs. Also, the expression profiles of each gene differed appreciably from one another. Grapevine DHN1 was not expressed in vegetative tissues under normal growth conditions, but was induced by drought, cold, heat, embryogenesis, as well as the application of abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA). It was expressed earlier in V. yeshanensis under drought conditions than in V. vinifera, and also exhibited a second round of up-regulation in V. yeshanensis following inoculation with Erysiphe necator, which was not apparent in V. vinifera. Like DHN1, DHN2 was induced by cold, heat, embryogenesis and ABA; however, it exhibited no responsiveness to drought, E. necator infection, SA or MeJA, and was also expressed constitutively in vegetative tissues under normal growth conditions. Conversely, DHN3 was only expressed during seed development at extremely low levels, and DHN4 was expressed specifically during late embryogenesis. Neither DHN3 nor DHN4 exhibited responsiveness to any of the treatments carried out in this study. Interestingly, the presence of particular cis-elements within the promoter regions of each gene was positively correlated with their expression profiles.
The grapevine DHN family comprises four divergent members. While it is likely that their functions overlap to some extent, it seems that DHN1 provides the main stress-responsive function. In addition, our results suggest a close relationship between expression patterns, physicochemical properties, and cis-regulatory elements in the promoter regions of the DHN genes.
Grapevine; Dehydrin; Stress-induced expression; Powdery mildew; Promoter
Grape powdery mildew is caused by the North American native pathogen Erysiphe necator. Eurasian Vitis vinifera varieties were all believed to be susceptible. REN1 is the first resistance gene naturally found in cultivated plants of Vitis vinifera.
REN1 is present in 'Kishmish vatkana' and 'Dzhandzhal kara', two grapevines documented in Central Asia since the 1920's. These cultivars have a second-degree relationship (half sibs, grandparent-grandchild, or avuncular), and share by descent the chromosome on which the resistance allele REN1 is located. The REN1 interval was restricted to 1.4 cM using 38 SSR markers distributed across the locus and the segregation of the resistance phenotype in two progenies of collectively 461 offspring, derived from either resistant parent. The boundary markers delimit a 1.4-Mbp sequence in the PN40024 reference genome, which contains 27 genes with known functions, 2 full-length coiled-coil NBS-LRR genes, and 9 NBS-LRR pseudogenes. In the REN1 locus of PN40024, NBS genes have proliferated through a mixture of segmental duplications, tandem gene duplications, and intragenic recombination between paralogues, indicating that the REN1 locus has been inherently prone to producing genetic variation. Three SSR markers co-segregate with REN1, the outer ones confining the 908-kb array of NBS-LRR genes. Kinship and clustering analyses based on genetic distances with susceptible cultivars representative of Central Asian Vitis vinifera indicated that 'Kishmish vatkana' and 'Dzhandzhal kara' fit well into local germplasm. 'Kishmish vatkana' also has a parent-offspring relationship with the seedless table grape 'Sultanina'. In addition, the distant genetic relatedness to rootstocks, some of which are derived from North American species resistant to powdery mildew and have been used worldwide to guard against phylloxera since the late 1800's, argues against REN1 being infused into Vitis vinifera from a recent interspecific hybridisation.
The REN1 gene resides in an NBS-LRR gene cluster tightly delimited by two flanking SSR markers, which can assist in the selection of this DNA block in breeding between Vitis vinifera cultivars. The REN1 locus has multiple layers of structural complexity compared with its two closely related paralogous NBS clusters, which are located some 5 Mbp upstream and 4 Mbp downstream of the REN1 interval on the same chromosome.
The RESPONSIVE TO DEHYDRATION 22 (RD22) gene is a molecular link between abscisic acid (ABA) signalling and abiotic stress responses. Its expression has been used as a reliable ABA early response marker. In Arabidopsis, the single copy RD22 gene possesses a BURP domain also located at the C-terminus of USP embryonic proteins and the beta subunit of polygalacturonases. In grapevine, a RD22 gene has been identified but putative paralogs are also found in the grape genome, possibly forming a large RD22 family in this species. In this work, we searched for annotations containing BURP domains in the Vitis vinifera genome. Nineteen proteins were defined by a comparative analysis between the two genome predictions and RNA-Seq data. These sequences were compared to other plant BURPs identified in previous genome surveys allowing us to reconceive group classifications based on phylogenetic relationships and protein motif occurrence. We observed a lineage-specific evolution of the RD22 family, with the biggest expansion in grapevine and poplar. In contrast, rice, sorghum and maize presented highly expanded monocot-specific groups. The Vitis RD22 group may have expanded from segmental duplications as most of its members are confined to a region in chromosome 4. The inspection of transcriptomic data revealed variable expression of BURP genes in vegetative and reproductive organs. Many genes were induced in specific tissues or by abiotic and biotic stresses. Three RD22 genes were further studied showing that they responded oppositely to ABA and to stress conditions. Our results show that the inclusion of RNA-Seq data is essential while describing gene families and improving gene annotations. Robust phylogenetic analyses including all BURP members from other sequenced species helped us redefine previous relationships that were erroneously established. This work provides additional evidence for RD22 genes serving as marker genes for different organs or stresses in grapevine.
Oligopeptide transporters (OPTs) are a group of membrane-localized proteins that have a broad range of substrate transport capabilities and that are thought to contribute to many biological processes. The OPT proteins belong to a small gene family in plants, which includes about 25 members in Arabidopsis and rice. However, no comprehensive study incorporating phylogeny, chromosomal location, gene structure, expression profiling, functional divergence and selective pressure analysis has been reported thus far for Populus and Vitis.
In the present study, a comprehensive analysis of the OPT gene family in Populus (P. trichocarpa) and Vitis (V. vinifera) was performed. A total of 20 and 18 full-length OPT genes have been identified in Populus and Vitis, respectively. Phylogenetic analyses indicate that these OPT genes consist of two classes that can be further subdivided into 11 groups. Gene structures are considerably conserved among the groups. The distribution of OPT genes was found to be non-random across chromosomes. A high proportion of the genes are preferentially clustered, indicating that tandem duplications may have contributed significantly to the expansion of the OPT gene family. Expression patterns based on our analyses of microarray data suggest that many OPT genes may be important in stress response and functional development of plants. Further analyses of functional divergence and adaptive evolution show that, while purifying selection may have been the main force driving the evolution of the OPTs, some of critical sites responsible for the functional divergence may have been under positive selection.
Overall, the data obtained from our investigation contribute to a better understanding of the complexity of the Populus and Vitis OPT gene family and of the function and evolution of the OPT gene family in higher plants.
The AP2/ERF protein family contains transcription factors that play a crucial role in plant growth and development and in response to biotic and abiotic stress conditions in plants. Grapevine (Vitis vinifera) is the only woody crop whose genome has been fully sequenced. So far, no detailed expression profile of AP2/ERF-like genes is available for grapevine.
An exhaustive search for AP2/ERF genes was carried out on the Vitis vinifera genome and their expression profile was analyzed by Real-Time quantitative PCR (qRT-PCR) in different vegetative and reproductive tissues and under two different ripening stages.
One hundred and forty nine sequences, containing at least one ERF domain, were identified. Specific clusters within the AP2 and ERF families showed conserved expression patterns reminiscent of other species and grapevine specific trends related to berry ripening. Moreover, putative targets of group IX ERFs were identified by co-expression and protein similarity comparisons.
The grapevine genome contains an amount of AP2/ERF genes comparable to that of other dicot species analyzed so far. We observed an increase in the size of specific groups within the ERF family, probably due to recent duplication events. Expression analyses in different aerial tissues display common features previously described in other plant systems and introduce possible new roles for members of some ERF groups during fruit ripening. The presented analysis of AP2/ERF genes in grapevine provides the bases for studying the molecular regulation of berry development and the ripening process.
Fifty-nine VvWRKY genes were identified. Phylogenetic tree and synteny analysis revealed the specific evolutionary relationship of these genes. Meanwhile, differential expression patterns indicated their possible roles in specific tissues and under different stresses.
WRKY proteins comprise a large family of transcription factors that play important roles in plant defence regulatory networks, including responses to various biotic and abiotic stresses. To date, no large-scale study of WRKY genes has been undertaken in grape (Vitis vinifera L.). In this study, a total of 59 putative grape WRKY genes (VvWRKY) were identified and renamed on the basis of their respective chromosome distribution. A multiple sequence alignment analysis using all predicted grape WRKY genes coding sequences, together with those from Arabidopsis thaliana and tomato (Solanum lycopersicum), indicated that the 59 VvWRKY genes can be classified into three main groups (I–III). An evaluation of the duplication events suggested that several WRKY genes arose before the divergence of the grape and Arabidopsis lineages. Moreover, expression profiles derived from semiquantitative PCR and real-time quantitative PCR analyses showed distinct expression patterns in various tissues and in response to different treatments. Four VvWRKY genes showed a significantly higher expression in roots or leaves, 55 responded to varying degrees to at least one abiotic stress treatment, and the expression of 38 were altered following powdery mildew (Erysiphe necator) infection. Most VvWRKY genes were downregulated in response to abscisic acid or salicylic acid treatments, while the expression of a subset was upregulated by methyl jasmonate or ethylene treatments.
Evolution; expression profile analysis; grape (Vitis vinifera L.); phylogenetic analysis; synteny analysis; WRKY genes.
Latest research shows that small antimicrobial peptides play a role in the innate defense system of plants. These peptides typically contribute to preformed defense by developing protective barriers around germinating seeds or between different tissue layers within plant organs. The encoding genes could also be upregulated by abiotic and biotic stimuli during active defense processes. The peptides display a broad spectrum of antimicrobial activities. Their potent anti-pathogenic characteristics have ensured that they are promising targets in the medical and agricultural biotechnology sectors.
A berry specific cDNA sequence designated Vv-AMP1, Vitis vinifera antimicrobial peptide 1, was isolated from Vitis vinifera. Vv-AMP1 encodes for a 77 amino acid peptide that shows sequence homology to the family of plant defensins. Vv-AMP1 is expressed in a tissue specific, developmentally regulated manner, being only expressed in berry tissue at the onset of berry ripening and onwards. Treatment of leaf and berry tissue with biotic or abiotic factors did not lead to increased expression of Vv-AMP1 under the conditions tested. The predicted signal peptide of Vv-AMP1, fused to the green fluorescent protein (GFP), showed that the signal peptide allowed accumulation of its product in the apoplast. Vv-AMP1 peptide, produced in Escherichia coli, had a molecular mass of 5.495 kDa as determined by mass spectrometry. Recombinant Vv-AMP1 was extremely heat-stable and showed strong antifungal activity against a broad spectrum of plant pathogenic fungi, with very high levels of activity against the wilting disease causing pathogens Fusarium oxysporum and Verticillium dahliae. The Vv-AMP1 peptide did not induce morphological changes on the treated fungal hyphae, but instead strongly inhibited hyphal elongation. A propidium iodide uptake assay suggested that the inhibitory activity of Vv-AMP1 might be associated with altering the membrane permeability of the fungal membranes.
A berry specific cDNA clone, Vv-AMP1, was isolated and characterized and shown to encode a plant defensin. Recombinant Vv-AMP1 displayed non-morphogenic antifungal activity against a broad spectrum of fungi, probably altering the membrane permeability of the fungal pathogens. The expression of this peptide is highly regulated in Vitis vinifera, hinting at an important defense role during berry-ripening.
WRKY III genes have significant functions in regulating plant development and resistance. In plant, WRKY gene family has been studied in many species, however, there still lack a comprehensive analysis of WRKY III genes in the woody plant species poplar, three representative lineages of flowering plant species are incorporated in most analyses: Arabidopsis (a model plant for annual herbaceous dicots), grape (one model plant for perennial dicots) and Oryza sativa (a model plant for monocots).
In this study, we identified 10, 6, 13 and 28 WRKY III genes in the genomes of Populus trichocarpa, grape (Vitis vinifera), Arabidopsis thaliana and rice (Oryza sativa), respectively. Phylogenetic analysis revealed that the WRKY III proteins could be divided into four clades. By microsynteny analysis, we found that the duplicated regions were more conserved between poplar and grape than Arabidopsis or rice. We dated their duplications by Ks analysis of Populus WRKY III genes and demonstrated that all the blocks were formed after the divergence of monocots and dicots. Strong purifying selection has played a key role in the maintenance of WRKY III genes in Populus. Tissue expression analysis of the WRKY III genes in Populus revealed that five were most highly expressed in the xylem. We also performed quantitative real-time reverse transcription PCR analysis of WRKY III genes in Populus treated with salicylic acid, abscisic acid and polyethylene glycol to explore their stress-related expression patterns.
This study highlighted the duplication and diversification of the WRKY III gene family in Populus and provided a comprehensive analysis of this gene family in the Populus genome. Our results indicated that the majority of WRKY III genes of Populus was expanded by large-scale gene duplication. The expression pattern of PtrWRKYIII gene identified that these genes play important roles in the xylem during poplar growth and development, and may play crucial role in defense to drought stress. Our results presented here may aid in the selection of appropriate candidate genes for further characterization of their biological functions in poplar.
This article was reviewed by Prof Dandekar and Dr Andrade-Navarro.
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WRKY III; Microsynteny; Gene duplication; Expression; Populus
An important objective in genome research is to relate genome structure to gene function. Sequence comparisons among orthologous and paralogous genes and their allelic variants can reveal sequences of functional significance. Here, we describe a 379-kb region on chromosome 1 of maize that enables us to reconstruct chromosome breakage, transposition, non-homologous end-joining, and homologous recombination events. Such a high-density composition of various mechanisms in a small chromosomal interval exemplifies the evolution of gene regulation and allelic diversity in general. It also illustrates the evolutionary pace of changes in plants, where many of the above mechanisms are of somatic origin. In contrast to animals, somatic alterations can easily be transmitted through meiosis because the germline in plants is contiguous to somatic tissue, permitting the recovery of such chromosomal rearrangements. The analyzed region contains the P1-wr allele, a variant of the genetically well-defined p1 gene, which encodes a Myb-like transcriptional activator in maize. The P1-wr allele consists of eleven nearly perfect P1-wr 12-kb repeats that are arranged in a tandem head-to-tail array. Although a technical challenge to sequence such a structure by shotgun sequencing, we overcame this problem by subcloning each repeat and ordering them based on nucleotide variations. These polymorphisms were also critical for recombination and expression analysis in presence and absence of the trans-acting epigenetic factor Ufo1. Interestingly, chimeras of the p1 and p2 genes, p2/p1 and p1/p2, are framing the P1-wr cluster. Reconstruction of sequence amplification steps at the p locus showed the evolution from a single Myb-homolog to the multi-gene P1-wr cluster. It also demonstrates how non-homologous end-joining can create novel gene fusions. Comparisons to orthologous regions in sorghum and rice also indicate a greater instability of the maize genome, probably due to diploidization following allotetraploidization.
Plant genomes analyzed to date contain 15% or more genes that are arranged in tandem arrays. Tandem duplications are a source for allelic variability since their homologous sequences can serve in recombination events. For example, unequal crossing over between amplified genes can result in contraction and expansion of the array. Tandem gene multiplications are also subject to repeat induced gene silencing (RIGS). Most importantly, gene duplications create the evolutionary potential for genetic novelty (neo- or subfunctionalization). In addition to homologous recombination during meiosis, illegitimate recombination in somatic tissues of plants can create events that potentially can be transmitted through reproductive tissue to further enrich genetic diversity. Here we illustrate the evolution from a single Myb homolog to a multigene cluster that exemplifies the evolution of the maize genome. We used the p locus to demonstrate how plant genomes expand by polyploidization, gene duplication, and transposition. We characterized in detail the structural changes at the p cluster that resulted from genomic instability. Because structure determines function, we linked genomic rearrangements at the P1-wr cluster to functional consequences. At the P1-wr locus, structural changes caused regulatory/transcriptional modifications that in turn give rise to phenotypic alterations.
ABA-mediated processes are involved in plant responses to water deficit, especially the control of stomatal opening. However in grapevine it is not known if these processes participate in the phenotypic variation in drought adaptation existing between genotypes. To elucidate this question, the response to short-term water-deficit was analysed in roots and shoots of nine Vitis genotypes differing in their drought adaptation in the field. The transcript abundance of 12 genes involved in ABA biosynthesis, catabolism, and signalling were monitored, together with physiological and metabolic parameters related to ABA and its role in controlling plant transpiration.
Although transpiration and ABA responses were well-conserved among the genotypes, multifactorial analyses separated Vitis vinifera varieties and V. berlandieri x V. rupestris hybrids (all considered drought tolerant) from the other genotypes studied. Generally, V. vinifera varieties, followed by V. berlandieri x V. rupestris hybrids, displayed more pronounced responses to water-deficit in comparison to the other genotypes. However, changes in transcript abundance in roots were more pronounced for Vitis hybrids than V. vinifera genotypes. Changes in the expression of the cornerstone ABA biosynthetic gene VviNCED1, and the ABA transcriptional regulator VviABF1, were associated with the response of V. vinifera genotypes, while changes in VviNCED2 abundance were associated with the response of other Vitis genotypes. In contrast, the ABA RCAR receptors were not identified as key components of the genotypic variability of water-deficit responses. Interestingly, the expression of VviSnRK2.6 (an AtOST1 ortholog) was constitutively lower in roots and leaves of V. vinifera genotypes and higher in roots of V. berlandieri x V. rupestris hybrids.
This study highlights that Vitis genotypes exhibiting different levels of drought adaptation differ in key steps involved in ABA metabolism and signalling; both under well-watered conditions and in response to water-deficit. In addition, it supports that adaptation may be related to various mechanisms related or not to ABA responses.
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Abscisic acid; ABA signalling; Genotypic variability; Grapevine; Roots; Shoot; Transpiration; Water potential; Water-deficit
MicroRNA (miRNA) is a class of functional non-coding small RNA with 19-25 nucleotides in length while Amur grape (Vitis amurensis Rupr.) is an important wild fruit crop with the strongest cold resistance among the Vitis species, is used as an excellent breeding parent for grapevine, and has elicited growing interest in wine production. To date, there is a relatively large number of grapevine miRNAs (vv-miRNAs) from cultivated grapevine varieties such as Vitis vinifera L. and hybrids of V. vinifera and V. labrusca, but there is no report on miRNAs from Vitis amurensis Rupr, a wild grapevine species.
A small RNA library from Amur grape was constructed and Solexa technology used to perform deep sequencing of the library followed by subsequent bioinformatics analysis to identify new miRNAs. In total, 126 conserved miRNAs belonging to 27 miRNA families were identified, and 34 known but non-conserved miRNAs were also found. Significantly, 72 new potential Amur grape-specific miRNAs were discovered. The sequences of these new potential va-miRNAs were further validated through miR-RACE, and accumulation of 18 new va-miRNAs in seven tissues of grapevines confirmed by real time RT-PCR (qRT-PCR) analysis. The expression levels of va-miRNAs in flowers and berries were found to be basically consistent in identity to those from deep sequenced sRNAs libraries of combined corresponding tissues. We also describe the conservation and variation of va-miRNAs using miR-SNPs and miR-LDs during plant evolution based on comparison of orthologous sequences, and further reveal that the number and sites of miR-SNP in diverse miRNA families exhibit distinct divergence. Finally, 346 target genes for the new miRNAs were predicted and they include a number of Amur grape stress tolerance genes and many genes regulating anthocyanin synthesis and sugar metabolism.
Deep sequencing of short RNAs from Amur grape flowers and berries identified 72 new potential miRNAs and 34 known but non-conserved miRNAs, indicating that specific miRNAs exist in Amur grape. These results show that a number of regulatory miRNAs exist in Amur grape and play an important role in Amur grape growth, development, and response to abiotic or biotic stress.
Amur grape; microRNA; Sequences evolution; Solexa sequencing; miR-RACE; qRT-PCR
WRKY proteins are a class of transcription factors (TFs) involved in the regulation of various physiological processes, including the plant response to biotic and abiotic stresses. Recent studies in Arabidopsis have revealed that some WRKY TFs interact with a class of proteins designed as VQ proteins because of their typical conserved motif (FxxxVQxLTG). So far, no information is available about the genomic organization and the function of VQ motif-containing protein in grapevine (Vitis vinifera L). In the current study, we analyzed the 12X V1 prediction of the nearly homozygous PN40024 genotype identifying up to 18 predicted VQ genes (VvVQ). VvVQs phylogenetic and bioinformatic analyses indicated that the intron-exon structures and motif distribution are highly divergent between different members of the grapevine VQ family. Moreover, the analysis of the V. vinifera cv. Corvina expression atlas revealed a tissue- and stage-specific expression of several members of the family which also showed a significant correlation with WRKY TFs. Grapevine VQ genes also exhibited altered expression in response to drought, powdery mildew infection, salicylic acid (SA) and ethylene (ETH) treatments. The present study represents the first characterization of VQ genes in a grapevine genotype and it is a pivotal foundation for further studies aimed at functionally characterizing this mostly unknown grapevine multigenic family.
grapevine; VQ gene family; gene expression; drought treatment; biotic stress; WRKY protein; co-expression network
The homeodomain leucine zipper (HD-Zip) transcription factor family is one of the largest plant specific superfamilies, and includes genes with roles in modulation of plant growth and response to environmental stresses. Many HD-Zip genes are characterized in Arabidopsis (Arabidopsis thaliana), and members of the family are being investigated for abiotic stress responses in rice (Oryza sativa), maize (Zea mays), poplar (Populus trichocarpa) and cucumber (Cucmis sativus). Findings in these species suggest HD-Zip genes as high priority candidates for crop improvement.
In this study we have identified members of the HD-Zip gene family in soybean cv. ‘Williams 82’, and characterized their expression under dehydration and salt stress. Homology searches with BLASTP and Hidden Markov Model guided sequence alignments identified 101 HD-Zip genes in the soybean genome. Phylogeny reconstruction coupled with domain and gene structure analyses using soybean, Arabidopsis, rice, grape (Vitis vinifera), and Medicago truncatula homologues enabled placement of these sequences into four previously described subfamilies. Of the 101 HD-Zip genes identified in soybean, 88 exist as whole-genome duplication-derived gene pairs, indicating high retention of these genes following polyploidy in Glycine ~13 Mya. The HD-Zip genes exhibit ubiquitous expression patterns across 24 conditions that include 17 tissues of soybean. An RNA-Seq experiment performed to study differential gene expression at 0, 1, 6 and 12 hr soybean roots under dehydration and salt stress identified 20 differentially expressed (DE) genes. Several of these DE genes are orthologs of genes previously reported to play a role under abiotic stress, implying conservation of HD-Zip gene functions across species. Screening of HD-Zip promoters identified transcription factor binding sites that are overrepresented in the DE genes under both dehydration and salt stress, providing further support for the role of HD-Zip genes in abiotic stress responses.
We provide a thorough description of soybean HD-Zip genes, and identify potential candidates with probable roles in dehydration and salt stress. Expression profiles generated for all soybean genes, under dehydration and salt stress, at four time points, will serve as an important resource for the soybean research community, and will aid in understanding plant responses to abiotic stress.
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Soybean; HD-Zip; Transcription factor; Gene family; Whole-genome duplication; RNA-Seq; Dehydration stress; Salt stress; Abiotic stress
The SBP-box gene family is specific to plants and encodes a class of zinc finger-containing transcription factors with a broad range of functions. Although SBP-box genes have been identified in numerous plants including green algae, moss, silver birch, snapdragon, Arabidopsis, rice and maize, there is little information concerning SBP-box genes, or the corresponding miR156/157, function in grapevine.
Eighteen SBP-box gene family members were identified in Vitis vinifera, twelve of which bore sequences that were complementary to miRNA156/157. Phylogenetic reconstruction demonstrated that plant SBP-domain proteins could be classified into seven subgroups, with the V. vinifera SBP-domain proteins being more closely related to SBP-domain proteins from dicotyledonous angiosperms than those from monocotyledonous angiosperms. In addition, synteny analysis between grape and Arabidopsis demonstrated that homologs of several grape SBP genes were found in corresponding syntenic blocks of Arabidopsis. Expression analysis of the grape SBP-box genes in various organs and at different stages of fruit development in V. quinquangularis ‘Shang-24’ revealed distinct spatiotemporal patterns. While the majority of the grape SBP-box genes lacking a miR156/157 target site were expressed ubiquitously and constitutively, most genes bearing a miR156/157 target site exhibited distinct expression patterns, possibly due to the inhibitory role of the microRNA. Furthermore, microarray data mining and quantitative real-time RT-PCR analysis identified several grape SBP-box genes that are potentially involved in the defense against biotic and abiotic stresses.
The results presented here provide a further understanding of SBP-box gene function in plants, and yields additional insights into the mechanism of stress management in grape, which may have important implications for the future success of this crop.
Zinc (Zn) deficiency is one of the most widespread mineral nutritional problems that affect normal development in plants. Because Zn cannot passively diffuse across cell membranes, it must be transported into intracellular compartments for all biological processes where Zn is required. Several members of the Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) gene family have been characterized in plants, and have shown to be involved in metal uptake and transport. This study describes the first putative Zn transporter in grapevine. Unravelling its function may explain an important symptom of Zn deficiency in grapevines, which is the production of clusters with fewer and usually smaller berries than normal.
We identified and characterized a putative Zn transporter from berries of Vitis vinifera L., named VvZIP3. Compared to other members of the ZIP family identified in the Vitis vinifera L. genome, VvZIP3 is mainly expressed in reproductive tissue - specifically in developing flowers - which correlates with the high Zn accumulation in these organs. Contrary to this, the low expression of VvZIP3 in parthenocarpic berries shows a relationship with the lower Zn accumulation in this tissue than in normal seeded berries where its expression is induced by Zn. The predicted protein sequence indicates strong similarity with several members of the ZIP family from Arabidopsis thaliana and other species. Moreover, VvZIP3 complemented the growth defect of a yeast Zn-uptake mutant, ZHY3, and is localized in the plasma membrane of plant cells, suggesting that VvZIP3 has the function of a Zn uptake transporter.
Our results suggest that VvZIP3 encodes a putative plasma membrane Zn transporter protein member of the ZIP gene family that might play a role in Zn uptake and distribution during the early reproductive development in Vitis vinifera L., indicating that the availability of this micronutrient may be relevant for reproductive development.
Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera.
MAP kinase; Vitis vinifera; signal transduction; protein phosphorylation
In Vitis vinifera L., domestication induced a dramatic change in flower morphology: the wild sylvestris subspecies is dioecious while hermaphroditism is largely predominant in the domesticated subsp. V. v. vinifera. The characterisation of polymorphisms in genes underlying the sex-determining chromosomal region may help clarify the history of domestication in grapevine and the evolution of sex chromosomes in plants. In the genus Vitis, sex determination is putatively controlled by one major locus with three alleles, male M, hermaphrodite H and female F, with an allelic dominance M > H > F. Previous genetic studies located the sex locus on chromosome 2. We used DNA polymorphisms of geographically diverse V. vinifera genotypes to confirm the position of this locus, to characterise the genetic diversity and traces of selection in candidate genes, and to explore the origin of hermaphroditism.
In V. v. sylvestris, a sex-determining region of 154.8 kb, also present in other Vitis species, spans less than 1% of chromosome 2. It displays haplotype diversity, linkage disequilibrium and differentiation that typically correspond to a small XY sex-determining region with XY males and XX females. In male alleles, traces of purifying selection were found for a trehalose phosphatase, an exostosin and a WRKY transcription factor, with strikingly low polymorphism levels between distant geographic regions. Both diversity and network analysis revealed that H alleles are more closely related to M than to F alleles.
Hermaphrodite alleles appear to derive from male alleles of wild grapevines, with successive recombination events allowing import of diversity from the X into the Y chromosomal region and slowing down the expansion of the region into a full heteromorphic chromosome. Our data are consistent with multiple domestication events and show traces of introgression from other Asian Vitis species into the cultivated grapevine gene pool.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0229-z) contains supplementary material, which is available to authorized users.
Dioecy; Domestication; Hermaphroditism; Sex chromosome; Vitis vinifera L
Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5′ untranslated regions (5′UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.
Many immune genes are multigene families, presumably in response to pathogen variation. Some multigene families undergo expansion and contraction, leading to copy number variation (CNV), presumably due to more intense selection. Recently, the butyrophilin family in humans and other mammals has come under scrutiny, due to genetic associations with autoimmune diseases as well as roles in immune co-regulation and antigen presentation. Butyrophilin genes exhibit allelic polymorphism, but gene number appears stable within a species. We found that the BG homologues in chickens are very different, with great changes between haplotypes. We characterised one haplotype in detail, showing that there are two single BG genes, one on chromosome 2 and the other in the major histocompatibility complex (BF-BL region) on chromosome 16, and a family of BG genes in a tandem array in the BG region nearby. These genes have specific expression in cells and tissues, but overall are expressed in either haemopoietic cells or tissues. The two singletons have relatively stable evolutionary histories, but the BG region undergoes dynamic expansion and contraction, with the production of hybrid genes. Thus, chicken BG genes appear to evolve much more quickly than their closest homologs in mammals, presumably due to increased pressure from pathogens.
Vitis vinifera berry development is characterised by an initial phase where the fruit is small, hard and acidic, followed by a lag phase known as veraison. In the final phase, berries become larger, softer and sweeter and accumulate an array of organoleptic compounds. Since the physiological and biochemical makeup of grape berries at harvest has a profound impact on the characteristics of wine, there is great interest in characterising the molecular and biophysical changes that occur from flowering through veraison and ripening, including the coordination and temporal regulation of metabolic gene pathways. Advances in deep-sequencing technologies, combined with the availability of increasingly accurate V. vinifera genomic and transcriptomic data, have enabled us to carry out RNA-transcript expression analysis on a global scale at key points during berry development.
A total of 162 million 100-base pair reads were generated from pooled Vitis vinifera (cv. Shiraz) berries sampled at 3-weeks post-anthesis, 10- and 11-weeks post-anthesis (corresponding to early and late veraison) and at 17-weeks post-anthesis (harvest). Mapping reads from each developmental stage (36-45 million) onto the NCBI RefSeq transcriptome of 23,720 V. vinifera mRNAs revealed that at least 75% of these transcripts were detected in each sample. RNA-Seq analysis uncovered 4,185 transcripts that were significantly upregulated at a single developmental stage, including 161 transcription factors. Clustering transcripts according to distinct patterns of transcription revealed coordination in metabolic pathways such as organic acid, stilbene and terpenoid metabolism. From the phenylpropanoid/stilbene biosynthetic pathway at least 46 transcripts were upregulated in ripe berries when compared to veraison and immature berries, and 12 terpene synthases were predominantly detected only in a single sample. Quantitative real-time PCR was used to validate the expression pattern of 12 differentially expressed genes from primary and secondary metabolic pathways.
In this study we report the global transcriptional profile of Shiraz grapes at key stages of development. We have undertaken a comprehensive analysis of gene families contributing to commercially important berry characteristics and present examples of co-regulation and differential gene expression. The data reported here will provide an invaluable resource for the on-going molecular investigation of wine grapes.
Grapevine; Illumina; Shiraz; RNA-seq; Transcriptome
Worldwide, grapes and their derived products have a large market. The cultivated grape species Vitis vinifera has potential to become a model for fruit trees genetics. Like many plant species, it is highly heterozygous, which is an additional challenge to modern whole genome shotgun sequencing. In this paper a high quality draft genome sequence of a cultivated clone of V. vinifera Pinot Noir is presented.
We estimate the genome size of V. vinifera to be 504.6 Mb. Genomic sequences corresponding to 477.1 Mb were assembled in 2,093 metacontigs and 435.1 Mb were anchored to the 19 linkage groups (LGs). The number of predicted genes is 29,585, of which 96.1% were assigned to LGs. This assembly of the grape genome provides candidate genes implicated in traits relevant to grapevine cultivation, such as those influencing wine quality, via secondary metabolites, and those connected with the extreme susceptibility of grape to pathogens. Single nucleotide polymorphism (SNP) distribution was consistent with a diffuse haplotype structure across the genome. Of around 2,000,000 SNPs, 1,751,176 were mapped to chromosomes and one or more of them were identified in 86.7% of anchored genes. The relative age of grape duplicated genes was estimated and this made possible to reveal a relatively recent Vitis-specific large scale duplication event concerning at least 10 chromosomes (duplication not reported before).
Sanger shotgun sequencing and highly efficient sequencing by synthesis (SBS), together with dedicated assembly programs, resolved a complex heterozygous genome. A consensus sequence of the genome and a set of mapped marker loci were generated. Homologous chromosomes of Pinot Noir differ by 11.2% of their DNA (hemizygous DNA plus chromosomal gaps). SNP markers are offered as a tool with the potential of introducing a new era in the molecular breeding of grape.
Basic leucine zipper (bZIP) transcription factor gene family is one of the largest and most diverse families in plants. Current studies have shown that the bZIP proteins regulate numerous growth and developmental processes and biotic and abiotic stress responses. Nonetheless, knowledge concerning the specific expression patterns and evolutionary history of plant bZIP family members remains very limited.
We identified 55 bZIP transcription factor-encoding genes in the grapevine (Vitis vinifera) genome, and divided them into 10 groups according to the phylogenetic relationship with those in Arabidopsis. The chromosome distribution and the collinearity analyses suggest that expansion of the grapevine bZIP (VvbZIP) transcription factor family was greatly contributed by the segment/chromosomal duplications, which may be associated with the grapevine genome fusion events. Nine intron/exon structural patterns within the bZIP domain and the additional conserved motifs were identified among all VvbZIP proteins, and showed a high group-specificity. The predicted specificities on DNA-binding domains indicated that some highly conserved amino acid residues exist across each major group in the tree of land plant life. The expression patterns of VvbZIP genes across the grapevine gene expression atlas, based on microarray technology, suggest that VvbZIP genes are involved in grapevine organ development, especially seed development. Expression analysis based on qRT-PCR indicated that VvbZIP genes are extensively involved in drought- and heat-responses, with possibly different mechanisms.
The genome-wide identification, chromosome organization, gene structures, evolutionary and expression analyses of grapevine bZIP genes provide an overall insight of this gene family and their potential involvement in growth, development and stress responses. This will facilitate further research on the bZIP gene family regarding their evolutionary history and biological functions.
bZIP transcription factor family; Grapevine; Gene expression; Drought response; Heat stress response