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BackgroundBud dormancy is a strategic mechanism plants developed as an adaptation to unfavorable environments. The grapevine (Vitis vinifera) is one of the most ancient fruit vine species and vines are planted all over the world due to their great economic benefits. To better understand the molecular mechanisms underlying bud dormancy between adjacent months, the transcriptomes of ‘Rosario Bianco’ grape buds of 6 months and three nodes were analyzed using RNA-sequencing technology and pair-wise comparison. From November to April of the following year, pairwise comparisons were conducted between adjacent months.ResultsA total of 11,647 differentially expressed genes (DEGs) were obtained from five comparisons. According to the results of cluster analysis of the DEG profiles and the climatic status of the sampling period, the 6 months were divided into three key processes (November to January, January to March, and March to April). Pair-wise comparisons of DEG profiles of adjacent months and three main dormancy processes showed that the whole grapevine bud dormancy period was mainly regulated by the antioxidant system, secondary metabolism, cell cycle and division, cell wall metabolism, and carbohydrates metabolism. Additionally, several DEGs, such asVvGA2OX6andVvSS3, showed temporally and spatially differential expression patterns, which normalized to a similar trend during or before April.ConclusionConsidering these results, the molecular mechanisms underlying bud dormancy in the grapevine can be hypothesized, which lays the foundation for further research.

Background Papain-like cysteine proteases (PLCPs), a large group of cysteine proteases, are structurally related to papain. The members belonging to PLCPs family contribute to plant immunity, senescence, and defense responses in plants. The PLCP gene family has been identified in Arabidopsis, rice, soybean, and cotton. However, no systematic analysis of PLCP genes has been undertaken in grapevine. Since Plasmopara viticola as a destructive pathogen could affect immunity of grapes in the field, we considered that the members belonged to PLCPs family could play a crucial role in defensive mechanisms or programmed cell death. We aimed to evaluate the role of PLCPs in 2 different varieties of grapevines and compared the changes of their expressions with the transcriptional data in response to P. viticola. Results In this study, 23 grapevine PLCP (VvPLCP) genes were identified by comprehensive bioinformatics analysis. Subsequently, the chromosomal localizations, gene structure, conserved domains, phylogenetic relationship, gene duplication, and cis-acting elements were analyzed. Numerous cis-acting elements related to plant development, hormone, and stress responses were identified in the promoter of the VvPLCP genes. Phylogenetic analysis grouped the VvPLCP genes into nine subgroups. The transcription of VvPLCP in different inoculation time points and varieties indicated that VvPLCP may have vital functions in grapevine defense against Plasmopara viticola. According to transcriptome data and qPCR analysis, we observed the increasing expression levels of VvRD21–1 at 72 h after inoculation in resistant variety, inferring that it was related to grape downy mildew resistance. Meanwhile, 3 genes including VvXBCP1, VvSAG12–1, and VvALP1 showed higher expression at 24 h after pathogen inoculation in the susceptible variety and might be related to the downy mildew phenotype. We nominated these four genes to function during hypersensitive response (HR) process, inferring that these genes could be associated with downy mildew resistance in grapes. Conclusions Our results provide the reference for functional studies of PLCP gene family, and highlight its functions in grapevine defense against P. viticola. The results help us to better understand the complexity of the PLCP gene family in plant immunity and provide valuable information for future functional characterization of specific genes in grapevine.

A significant body of evidence supports the important roles of miRNAs in grape berry developments. However, their specific molecular functions during stone-hardening stage development remain unclear. Here, a total of 161 conserved and 85 species-specific miRNAs/miRNAs*(precursor) were identified in grape stone hardening stage berries using Solexa sequencing. Out of them, 30 VvmiRNAs were tissue-specific and identified as stone-hardening related to VvmiRNAs, whereas 52 exhibited differential expression profiles during berry development, potentially participating in modulation of development, and qRT-PCR analysis verified their expression patterns. Interestingly, high SNP variations in VvmiRNA sequences might result into generation of new VvmiRNA family members like VvmiR168, VvmiR479, VvmiR3636 families and so on. Through GO and KEGG pathway analyses, we revealed that 13 VvmiRNAs involved in the regulation of embryo development, 11 in lignin and cellulose biosynthesis, and 28 in the modulation of hormone signaling, sugar and proline metabolism. Furthermore, the target genes for novel VvmiRNAs related to berry development were validated using RLM-RACE and PPM-RACE methods, and it was revealed their cleavage sites mainly happened at the 9th-11th sites from the 5’ ends of miRNAs at their binding regions. The potential roles of these VvmiRNAs in gibberellins repressing grape stone-hardening and embryo development by potentially inducing the expression of VvmiR31-3p and VvmiR8-5p to increase the cleavage product accumulation levels of corresponding target genes like lignin biosynthesis genes, CAFFEOYL COENZYME A-3-O-METHYLTRANSFERASE (VvCCoAOMT) and DDB1-CUL4 ASSOCIATED FACTOR1 (VvDCAF1), as a potential key molecular mechanism involved in GA-induced grape seedless berry development. Based on the characterization of stone-hardening stage related to VvmiRNAs, a schematic model of miRNA-mediated grape seed and stone-hardening development was proposed in this work. This is the first report about the regulatory role of VvmiRNAs in the regulation of stone-hardening stage of grape berries, which provides valuable genetic information for the breeding of seedless grape varieties.

Signal transductions require calcium (Ca2+) or reactive oxygen species (ROS) signatures, which act as chemical and electrical signals in response to various biotic and abiotic stresses. Calcium as an ion or second messenger affects the membrane potential and microtubules (MTs) dynamicity, while MTs can modulate auto-propagating waves of calcium and ROS signatures in collaboration with ion channels depending on the stimulus type. Thus, in the current review, we highlight advances in research focused on the relationship between dynamic MTs and calcium and ROS signatures in short-distance transmission. The challenges of Ca2+-MTs-ROS crosstalk in cold sensing are addressed, which could suggest the prioritization of ROS or Ca2+ in signalling.

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.

The grapevine VvβVPE promoter is specifically expressed in the seed. The − 1306~− 1045 bp core region restricts expression in other tissues and organs. Vacuolar processing enzyme (VPE) is a cysteine proteinase regulating vacuolar protein maturation and executing programmed cell death (PCD) in plants. Vitis vinifera (Vv)βVPE is a β-type VPE showing seed-specific expression that processes seed proteins during ovule development. However, the regulation of the seed-specific gene expression is far from understood. In this study, we characterize VvβVPE promoter (pVvβVPE) from 12 seeded and seedless grape genotypes. 94.56% of the pVvβVPE coding sequence is consistent. Two βVPE promoters were constructed and transformed into Arabidopsis thaliana via β-glucuronidase (GUS) fused expression vectors, using cv. Pinot Noir and cv. Thompson as seed and seedless candidates. GUS staining in different tissues and organs revealed that VvβVPE expresses specifically in the embryo, including the cotyledon, hypocotyl and suspensor, but not in the leaf, stem, root or flowers of the seedling. Using promoter deletion analysis, we created four incomplete VvβVPE promoters and found each pVvβVPE deletion could drive GUS gene to express in seeds. Interestingly, seed specificity disappeared when the promoter missed the core − 1306~− 1045 bp region. All deletion promoters presenting various quantified GUS activities indicate that the region − 1704~− 1306 bp inhibits, and the region − 705~− 861 bp promotes gene expression of VvβVPE. Our results demonstrate that pVvβVPE is a seed-specific promoter in both seeded and seedless grapes. Moreover, the core region of pVvβVPE (− 1306~− 1045 bp) is the key one responsible for seed-specific expression.

Metacaspase, as hypersensitive response (HR) executors, has been identified in many plant species. Previously, the entire gene family of metacaspase has been uncovered, but there are still questions that remain unclear regarding HR-regulating gene members. In this study, based on metacaspase expression during different grapevine genotypes interacting with Plasmopara viticola, we identified MC2 and MC5 as candidates involved in HR. We overexpressed both metacaspases as GFP fusions in tobacco BY-2 cells to address subcellular localization and cellular functions. We found MC2 located at the ER, while MC5 was nucleocytoplasmic. In these overexpressor lines, cell death elicited by the bacterial protein harpin, is significantly enhanced, indicating MC2 and MC5 mediated defence-related programmed cell death (PCD). This effect was mitigated, when the membrane-located NADPH oxidase was inhibited by the specific inhibitor diphenylene iodonium, or when cells were complemented with methyl jasmonate, a crucial signal of basal immunity. Both findings are consistent with a role of MC2 and MC5 in cell death-related immunity. Using a dual-luciferase reporter system in grapevine cells we demonstrated both MC2 and MC5 promoter alleles from V. rupestris were more responsive to harpin than those from V. vinifera cv ‘Muller-Thurgau’, while they were not induced by MeJA as signal linked with basal immunity. These findings support a model, where MC2 and MC5 act specifically as executors of the HR.

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.

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 abnormal seedlings, a common physiological anomalies, emerged during embryo rescue severely restricted grape breeding. To enhance the efficiency of the seedless grape breeding by reducing the production of abnormal seedlings in the course of embryo rescue, we investigated the effects of genotype, media type, embryo style, pre-chilling on the deformity rate of the abnormal seedlings during embryo rescue. The abnormal seedlings were firstly classified into seven categories based on their morphology. Our results indicated that the emergence of abnormal seedlings was highly dependent on the female parent genotype. Polyembryony was advantageous to diminish the number of abnormal plantlets and the germination rate of embryo was 100%. We also found that pre-chilling treatment could reduce the number of abnormal plantlets and promote the embryo germination. The abnormal plantlets were reduced significantly by the addition of ZnSO₄ 10 μmol?L -1 or mashed-banana 500 mg?L -1 to either embryo development or germination media. Transferring the abnormal seedlings onto the suitable fresh media in 4 weeks after embryo germination provided an effective way to transform them into normal seedlings.

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.