Chaohong Zhang

Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress

BackgroundDehydrins (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.ResultsFour 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.ConclusionsThe 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.

Expression pattern, genomic structure, and promoter analysis of the gene encoding stilbene synthase from Chinese wild Vitis pseudoreticulata

The gene encoding stilbene synthase (STS) plays a central role in many biochemical and physiological actions, and its metabolite resveratrol possesses broad-spectrum resistance to pathogens, as well as diverse pharmacological properties, notably an anticancer effect. Here, we report the expression analysis of the gene encoding STS and its promoter function from a powdery mildew (PM)-resistant Chinese wild Vitis pseudoreticulata, and compare it with two PM-susceptible cultivated grapevines, Vitis vinifera cvs. Carignane and Thompson Seedless. We show an unusual expression pattern of STS in V. pseudoreticulata, which differs markedly from that of the cultivated species. Sequence comparisons reveal that the genomic DNA sequences encoding STS in the three grapevines are highly conserved, but a novel residue mutation within the key motif of STS is solely present in V. pseudoreticulata. Moreover, the STS promoter in V. pseudoreticulata displays a significantly different structure from that found in the two V. vinifera. The three promoter-driven GUS differential expression patterns in transformed tobacco plants induced with Alternaria alternata, methyl jasmonate, and wounding indicated that the structurally different STS promoter of V. pseudoreticulata is responsible for its specific regulatory function. We also demonstrate that the expression of STS genes from their native promoters are functional in transformed tobacco and retain pathogen inducibility. Importantly, the genomic DNA-2 of V. pseudoreticulata under its native promoter shows good induction and the maximum level of resveratrol content. These findings further our understanding of the regulation of STS expression in a resistant grapevine and provide a new pathogen-inducible promoter system for the genetic improvement of plant disease resistance.

The metacaspase gene family of Vitis vinifera L.: characterization and differential expression during ovule abortion in stenospermocarpic seedless grapes.

In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1-VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape.

The Novel Gene VpPR4-1 from Vitis pseudoreticulata Increases Powdery Mildew Resistance in Transgenic Vitis vinifera L.

Pathogenesis-related proteins (PRs) can lead to increased resistance of the whole plant to pathogen attack. Here, we isolate and characterize a PR-4 protein (VpPR4-1) from a wild Chinese grape Vitis pseudoreticulata which shows greatly elevated transcription following powdery mildew infection. Its expression profiles under a number of abiotic stresses were also investigated. Powdery mildew, salicylic acid, and jasmonic acid methyl ester significantly increased the VpPR4-1 induction while NaCl and heat treatments just slightly induced VpPR4-1 expression. Abscisic acid and cold treatment slightly affected the expression level of VpPR4-1. The VpPR4-1 gene was overexpressed in 30 regenerated V. vinifera cv. Red Globe via Agrobacterium tumefaciens-mediated transformation and verified by the Western blot. The 26 transgenic grapevines exhibited higher expression levels of PR-4 protein content than wild-type vines and six of them were inoculated with powdery mildew which showed that the growth of powdery mildew was repressed. The powdery mildew-resistance of Red Globe transformed with VpPR4-1 was enhanced inoculated with powdery mildew. Moreover, other powdery mildew resistant genes were associated with feedback regulation since VpPR4-1 is in abundance. This study demonstrates that PR-4 protein in grapes plays a vital role in defense against powdery mildew invasion.

Overexpression of VpEIFP1, a novel F-box/Kelch-repeat protein from wild Chinese Vitis pseudoreticulata, confers higher tolerance to powdery mildew by inducing thioredoxin z proteolysis

An F-box protein (VpEIFP1) induced by Erysiphe necator was isolated from Vitis pseudoreticulata, a wild Chinese grapevine species naturally resistant to powdery mildew (PM). It contains an F-box domain and two Kelch-repeat motifs. Expression profiles indicate the VpEIFP1 is strongly induced at both transcriptional and translational levels by PM infection. A subcellular localisation assay showed that VpEIFP1 is predominantly located in the nucleus and cytoplasm. Overexpression of VpEIFP1 accelerated the accumulation of hydrogen peroxide (H2O2) and up-regulated the expressions of ICS2, NPR1 and PR1 involved in defence responses, resulting in suppression of PM germination and growth. As an F-box protein, VpEIFP1 interacts with thioredoxin z (VpTrxz) in the yeast-two-hybrid (Y2H) assay and in the bimolecular fluorescence complementation (BiFC) assay. Decreased amounts of VpTrxz protein in transgenic grapevine leaves overexpressing VpEIFP1 were restored by proteasome inhibitor MG132, implying that VpEIFP1 mediated VpTrxz for degradation through the SCFVpEIFP1 (Skp1-Cullin-F-box) E3 ubiquitin ligase complex. The RNA interference line of VpTrxz showed increased H2O2 accumulation following PM inoculation. We propose VpEIFP1 positively modulates the grapevine defence response to PM by inducing the degradation of VpTrxz via the ubiquitin/26S proteasome system.

RING-H2-type E3 gene VpRH2 from Vitis pseudoreticulata improves resistance to powdery mildew by interacting with VpGRP2A

Grapevine is one of the worlds most important fruit crops. European cultivated grape species have the best fruit quality but show almost no resistance to powdery mildew (PM). PM caused by Uncinula necator is a harmful disease that has a significant impact on the economic value of the grape crop. In this study, we examined a RING-H2-type ubiquitin ligase gene VpRH2 that is associated with significant PM-resistance of Chinese wild-growing grape Vitis pseudoreticulata accession Baihe-35-1. The expression of VpRH2 was clearly induced by U. necator inoculation compared with its homologous gene VvRH2 in a PM-susceptible grapevine V. vinifera cv. Thompson Seedless. Using a yeast two-hybrid assay we confirmed that VpRH2 interacted with VpGRP2A, a glycine-rich RNA-binding protein. The degradation of VpGRP2A was inhibited by treatment with the proteasome inhibitor MG132 while VpRH2 did not promote the degradation of VpGRP2A. Instead, the transcripts of VpRH2 were increased by over-expressing VpGRP2A while VpRH2 suppressed the expression of VpGRP2A. Furthermore, VpGRP2A was down-regulated in both Baihe-35-1 and Thompson Seedless after U. necator inoculation. Specifically, we generated VpRH2 overexpression transgenic lines in Thompson Seedless and found that the transgenic plants showed enhanced resistance to powdery mildew compared with the wild-type. In summary, our results indicate that VpRH2 interacts with VpGRP2A and plays a positive role in resistance to powdery mildew.

VpPUB24, a novel gene from Chinese grapevine, Vitis pseudoreticulata, targets VpICE1 to enhance cold tolerance

The ubiquitination system plays important roles in the degradation and modification of substrate proteins. In this study, we characterize a putative U-box type E3 ubiquitin ligase gene, VpPUB24 (plant U-box protein 24), from Chinese wild grapevine, Vitis pseudoreticulata accession Baihe-35-1. We show that VpPUB24 is induced by a number of stresses, especially cold treatment. Real-time PCR analysis indicated that the PUB24 transcripts were increased after cold stress in different grapevine species, although the relative expression level was different. In grapevine protoplasts, we found that VpPUB24 was expressed at a low level at 22 °C but accumulated rapidly following cold treatment. A yeast two-hybrid assay revealed that VpPUB24 interacted physically with VpICE1. Further experiments indicated that VpICE1 is targeted for degradation via the 26S proteasome and that the degradation is accelerated by VpHOS1, and not by VpPUB24. Immunoblot analyses indicated that VpPUB24 promotes the accumulation of VpICE1 and suppresses the expression of VpHOS1 to regulate the abundance of VpICE1. Furthermore, VpICE1 promotes transcription of VpPUB24 at low temperatures. We also found that VpPUB24 interacts with VpHOS1 in a yeast two-hybrid assay. Additionally, over-expression of VpPUB24 in Arabidopsis thaliana enhanced cold tolerance. Collectively, our results suggest that VpPUB24 interacts with VpICE1 to play a role in cold stress.

Gene Cloning, Expression and Enzyme Activity of Vitis vinifera Vacuolar Processing Enzymes (VvVPEs).

Vacuolar processing enzymes (VPEs) have received considerable attention due to their caspase-1-like activity and ability to regulate programmed cell death (PCD), which plays an essential role in the development of stenospermocarpic seedless grapes ovules. To characterize VPEs and the relationship between stenospermocarpic grapes and the VPE gene family, we identified 3 Vitis vinifera VPE genes (VvβVPE, VvγVPE, and VvδVPE) from the PN40024 grape genome and cloned the full-length complementary DNAs (cDNAs) from the ‘Vitis vinifera cv. Pinot Noir’ and ‘Vitis vinifera cv. Thompson Seedless’ varietals. Each of the VPEs contained a typical catalytic dyad [His (177), Cys (219)] and substrate binding pocket [Arg (112), Arg (389), Ser (395)], except that Ser (395) in the VvγVPE protein sequence was replaced with alanine. Phylogenetic analysis of 4 Arabidopsis thaliana and 6 Vitis vinifera VPEs revealed that the 10 VPEs form 3 major branches. Furthermore, the 6 grapevine VPEs share a similar gene structure, with 9 exons and 8 introns. The 6 grapevine VPEs are located on 3 different chromosomes. We also tested the enzymatic activity of recombinant VPEs expressed in the Pichia Pastoris expression system and found that the VvVPEs exhibit cysteine peptidase activity. Tissue-specific expression analysis showed that VvδVPE is only expressed in flowers, buds and ovules, that VvγVPE is expressed in various tissues, and that VvβVPE was expressed in roots, flowers, buds and ovules. The results of quantitative real-time PCR (qRT-PCR) suggested that VvβVPE in seeded grapes increased significantly at 30 days after full-bloom (DAF), close to the timing of endosperm abortion at 32 DAF. These results suggested that VvβVPE is related to ovule abortion in seedless grapes. Our experiments provide a new perspective for understanding the mechanism of stenospermocarpic seedlessness and represent a useful reference for the further study of VPEs.

Vacuolar processing enzyme (VvβVPE) from Vitis vinifera, processes seed proteins during ovule development, and accelerates seed germination in VvβVPE heterologously over-expressed Arabidopsis

Vacuolar processing enzymes (VPEs), belonging to cysteine protease, are responsible for processing seed protein during maturation. Stenospermocarpic grapes occur self-abortion in fertilized embryos during the ovule development, which affects the formation of matured seed proteins. However, little is known about VPE functions in ovule self-defeating. Here, we investigated the role of one seed-type VPE gene, VvβVPE. Sequence analysis showed that all ORFs (Open reading frames) of VvβVPE from 19 seed/seedless genotypes are highly conserved. At the transcriptional level, VvβVPE was specifically expressed during ovule development, with distinct expression patterns: it increased gradually in seeded grapes; while weakly expressed in seedless grapes. Whereas, at the translational level, 3 forms of VvβVPE were expressed during ovule development in seeded grape: precursor βVPE (pβVPE), intermediate βVPE (iβVPE) and finally, active mature βVPE (mβVPE). By contrast, in seedless grape, VvβVPE only exists as pβVPE at whole developmental stage of ovule. for confirming these expression patterns, 12 seeded/seedless genotypes were sampled and analyzed. Furthermore, VPE enzyme activity was increased in Arabidopsis overexpressing VvβVPE, leading to faster germination. Our study indicated that VvβVPE is essential for grapevine ovule maturation through various forms and is involved in seed germination.

Genome-wide Identification, Phylogenetic Analysis, and Expression Profiling of CONSTANS-like (COL) Genes in Vitis vinifera

The CONSTANS (CO) gene plays an important role in the flowering of plants. However, the other precise roles of the CO gene are poorly understood. We carried out a genomic census and analysis of expression patterns for CONSTANS-like genes in Vitis vinifera (VviCOLs) to reveal the molecular characteristics of VviCOLs. Twelve VviCOLs were identified and 11 of their full-length complementary DNAs were cloned. Multiple sequence alignment suggested the VviCOLs contained B-box and CCT conserved domains. We further classified the VviCOLs into three groups according to the variability of the second B-box domain. Synteny analysis showed that eight orthologous gene pairs were identified between grapevine and Arabidopsis, suggesting that eight pairs may descend from a common evolutionary ancestor. Tissue expression analysis of COL genes in cv. Pinot Noir showed VviCOL11a and VviCOL11b were specifically expressed in flower bud, whereas VviCOL16b was only expressed in leaves. Ten VviCOLs were expressed in the developing ovule and six of them showed higher expression in the ovule of cv. Thompson Seedless than that of cv. Pinot Noir, indicating that VviCOLs were involved in the process of seed development or ovule abortion. Furthermore, nine of twelve VviCOLs were expressed in cv. Pinot Noir leaves and all of these nine genes had a response to exogenous hormone application. In summary, our findings provide a new insight into the further studies of VviCOLs, especially in terms of seed development and hormone response.