The evolution and formation of centromeric repeats analysis in Vitis vinifera.

MAIN CONCLUSION: Six grape centromere-specific markers for cytogenetics were mined by combining genetic and immunological assays, and the possible evolution mechanism of centromeric repeats was analyzed. Centromeric histone proteins are functionally conserved; however, centromeric repetitive DNA sequences may represent considerable diversity in related species. Therefore, studying the characteristics and structure of grape centromere repeat sequences contributes to a deeper understanding of the evolutionary process of grape plants, including their origin and mechanisms of polyploidization. Plant centromeric regions are mainly composed of repetitive sequences, including SatDNA and transposable elements (TE). In this research, the characterization of centromere sequences in the whole genome of grapevine (Vitis vinifera L.) has been conducted. Five centromeric tandem repeat sequences (Vv1, Vv2, Vv5, Vv6, and Vv8) and one long terminal repeat (LTR) sequence Vv24 were isolated. These sequences had different centromeric distributions, which indicates that grape centromeric sequences may undergo rapid evolution. The existence of extrachromosomal circular DNA (eccDNA) and gene expression in CenH3 subdomain region may provide various potential mechanisms for the generation of new centromeric regions.

Characterization and Potential Action Mode Divergences of Homologous ACO1 Genes during the Organ Development and Ripening Process between Non-Climacteric Grape and Climacteric Peach.

Ethylene is one crucial phytohormone modulating plants' organ development and ripening process, especially in fruits, but its action modes and discrepancies in non-climacteric grape and climacteric peach in these processes remain elusive. This work is focused on the action mode divergences of ethylene during the modulation of the organ development and ripening process in climacteric/non-climacteric plants. We characterized the key enzyme genes in the ethylene synthesis pathway, VvACO1 and PpACO1, and uncovered that their sequence structures are highly conserved, although their promoters exhibit important divergences in the numbers and types of the cis-elements responsive to hormones, implying various responses to hormone signals. Subsequently, we found the two have similar expression modes in vegetative organ development but inverse patterns in reproductive ones, especially in fruits. Then, VvACO1 and PpACO1 were further validated in promoting fruit ripening functions through their transient over-expression/RNAi-expression in tomatoes, of which the former possesses a weaker role than the latter in the fruit ripening process. Our findings illuminated the divergence in the action patterns and function traits of the key VvACO1/PpACO1 genes in the tissue development of climacteric/non-climacteric plants, and they have implications for further gaining insight into the interaction mechanism of ethylene signaling during the modulation of the organ development and ripening process in climacteric/non-climacteric plants.

VvATG6 contributes to copper stress tolerance by enhancing the antioxidant ability in transgenic grape calli.

Autophagy, a conserved degradation and reuse process, plays a crucial role in plant cellular homeostasis during abiotic stress. Although numerous autophagy-related genes (ATGs) that regulate abiotic stress have been identified, few functional studies have shown how they confer tolerance to copper (Cu) stress. Here, we cloned a novel Vitis vinifera ATG6 gene (VvATG6) which was induced by 0.5 and 10 mM Cu stress based on transcriptomic data, and transgenic Arabidopsis thaliana, tobacco (Nicotiana tabacum), and grape calli were successfully obtained through Agrobacterium-mediated genetic transformation. The overexpression of VvATG6 enhanced the tolerance of transgenic lines to Cu. After Cu treatment, the lines that overexpressed VvATG6 grew better and increased their production of biomass compared with the wild-type. These changes were accompanied by higher activities of antioxidant enzymes and a lower accumulation of deleterious malondialdehyde and hydrogen peroxide in the transgenic plants. The activities of superoxide dismutase, peroxidase, and catalase were enhanced owing to the elevation of corresponding antioxidant gene expression in the VvATG6 overexpression plants under Cu stress, thereby promoting the clearance of reactive oxygen species (ROS). Simultaneously, there was a decrease in the levels of expression of RbohB and RbohC that are involved in ROS synthesis in transgenic plants under Cu stress. Thus, the accelerated removal of ROS and the inhibition of its synthesis led to a balanced ROS homeostasis environment, which alleviated the damage from Cu. This could benefit from the upregulation of other ATGs that are necessary for the production of autophagosomes under Cu stress. To our knowledge, this study is the first to demonstrate the protective role of VvATG6 in the Cu tolerance of plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01415-y.

The physiology of drought stress in two grapevine cultivars: Photosynthesis, antioxidant system, and osmotic regulation responses.

Drought stress impedes viticultural plant growth and development by modifying various metabolic pathways. However, the regulatory network response underlying drought stress is not yet clear. In this study, the leaves and roots of "Shine Muscat" ("SM," Vitis labruscana × Vitis vinifera) and "Thompson Seedless" ("TS," V. vinifera L. cv.) were subjected to drought stress to study the regulatory network used by drought stress. Morphophysiological results showed that the malondialdehyde content after 28 days of drought stress increased more significantly in "TS" than "SM." Furthermore, the multiomics analysis studies showed that a total of 3036-6714 differentially expressed genes and 379-385 differentially abundant metabolites were identified in "SM" and "TS" grapevine cultivars under drought stress. Furthermore, the retained intron was the major form of differential alternative splicing event under drought stress. The photosynthesis pathway, antioxidant system, plant hormone signal transduction, and osmotic adjustment were the primary response systems in the two grapevine cultivars under drought stress. We have identified GRIK1, RFS2, and LKR/SDH as the hub genes in the coexpression network of drought stress. In addition, the difference in the accumulation of pheophorbide-a reveals different drought resistance mechanisms in the two grapevine cultivars. Our study explained the difference in drought response between cultivars and tissues and identified drought stress-responsive genes, which provides reference data for further understanding the regulatory network of drought tolerance in grapevine.

Characterization of VvmiR166s-Target Modules and Their Interaction Pathways in Modulation of Gibberellic-Acid-Induced Grape Seedless Berries.

Exogenous GA is widely used to efficiently induce grape seedless berry development for significantly improving berry quality. Recently, we found that VvmiR166s are important regulators of response to GA in grapes, but its roles in GA-induced seedless grape berry development remain elusive. Here, the precise sequences of VvmiR166s and their targets VvREV, VvHB15 and VvHOX32 were determined in grape cv. 'Rosario Bianco', and the cleavage interactions of VvmiR166s-VvHB15/VvHOX32/VvREV modules and the variations in their cleavage roles were confirmed in grape berries. Exogenous GA treatment significantly induced a change in their expression correlations from positive to negative between VvmiR166s and their target genes at the seeds during the stone-hardening stages (32 DAF-46 DAF) in grape berries, indicating exogenous GA change action modes of VvmiR166s on their targets in this process, in which exogenous GA mainly enhanced the negative regulatory roles of VvmiR166s on VvHB15 among all three VvmiR166s-target pairs. The transient OE-VvmiR166a-h/OE-VvHB15 in tobacco confirmed that out of the VvmiR166 family, VvmiR166h/a/b might be the main factors in modulating lignin synthesis through inhibiting VvHB15, of which VvmiR166h-VvHB15-NtPAL4/NtCCR1/NtCCR2/NtCCoAMT5/NtCOMT1 and VvmiR166a/b-VvHB15-NtCAD1 are the potential key regulatory modules in lignin synthesis. Together with the GA-induced expression modes of VvmiR166s-VvHB15 and genes related to lignin synthesis in grape berries, we revealed that GA might repress lignin synthesis mainly by repressing VvCAD1/VvCCR2/VvPAL2/VvPAL3/Vv4CL/VvLac7 levels via mediating VvmiR166s-VvHB15 modules in GA-induced grape seedless berries. Our findings present a novel insight into the roles of VvmiR66s that are responsive to GA in repressing the lignin synthesis of grape seedless berries, with different lignin-synthesis-enzyme-dependent action pathways in diverse plants, which have important implications for the molecular breeding of high-quality seedless grape berries.

Assessment of 'Cabernet Sauvignon' Grape Quality Half-Véraison to Maturity for Grapevines Grown in Different Regions.

Grapes are widely cultivated around the world and their quality has distinct regional characteristics. In this study, the qualitative characteristics of the 'Cabernet Sauvignon' grape variety in seven regions, from half-véraison to maturity, were analyzed comprehensively at physiological and transcriptional levels. The results indicated that the quality traits of 'Cabernet Sauvignon' grapes in different regions were significantly different with obvious regionality. Total phenols, anthocyanins, and titratable acids were the main factors of the regionality of berry quality, which were very sensitive to changes in the environment. It should be noted that the changes in titrating acids and total anthocyanin of berries vary greatly from half-véraison to maturity between regions. Moreover, the transcriptional analysis showed that the co-expressed genes between regions characterized the core transcriptome of berry development, while the unique genes of each region reflected the regionality of berries. The differentially expressed genes (DEGs) between half-véraison and maturity can be used to demonstrate that the environment of the regions could promote or inhibit gene expression. The functional enrichment suggested that these DEGs help to understand the interpretation of the plasticity of the quality composition of grapes according to the environment. Taken together, the information generated by this study could contribute to the development of viticultural practices aimed at making better use of native varieties for the development of wines with regional characteristics.

Genome-wide association study of the candidate genes for grape berry shape-related traits.

BACKGROUND: In the breeding of new horticultural crops, fruit shape is an important selection characteristic. A variety of fruit shapes appeared during the gradual process of selection and domestication. However, few studies have been conducted on grape berry shape, especially studies related to mining candidate genes. To discover candidate genes related to grape berry shape, the present study first took the berry shape parameters analyzed by Tomato Analyzer as the target traits and used a genome-wide association analysis to analyze candidate genes. RESULTS: In total, 122 single-nucleotide polymorphism (SNP) loci had significant correlations with multiple berry shape traits in both years, and some candidate genes were further mined. These genes were mainly related to LRR receptor-like serine/threonine-protein kinase (At1g05700 and At1g07650), transcription factors (GATA transcription factor 23-like, transcription factor VIP1, transcription initiation factor TFIID, and MADS-box transcription factor 6), ubiquitin ligases (F-box protein SKIP19 and RING finger protein 44), and plant hormones (indole-3-acetic acid-amido synthetase GH3.6 and ethylene-responsive transcription factor ERF061). In addition, some important SNP loci were associated with multiple berry-shape traits. The study further revealed some genes that control multiple traits simultaneously, indicating that these berry shape traits are subject to the coordinated regulation of some genes in controlling berry shape. CONCLUSIONS: In the present work, we identified interesting genetic determinants of grape berry shape-related traits. The identification of molecular markers that are closely related to these berry-shape traits is of great significance for breeding specific berry-shaped grape varieties.

Genome-wide identification and expression analysis of magnesium transporter gene family in grape (Vitis vinifera).

BACKGROUND: Magnesium ion is one of the essential mineral elements for plant growth and development, which participates in a variety of physiological and biochemical processes. Since there is no report on the research of magnesium ion transporter in grape, the study of the structure and function of magnesium ion transporters (MGT) is helpful to understand the dynamic balance mechanism of intracellular magnesium ions and their inter- or intra-cellular activities. RESULT: In this study, we identified the members of MGT protein family in grape and performed the phylogenetic and expression analysis. We have identified nine VvMGT genes in grape genome, which are distributed on eight different chromosomes. Phylogenetic analysis showed that MGT family members of grapes were divided into five subfamilies and had obvious homology with Arabidopsis, maize, and pear. Based on transcriptome data from the web databases, we analyzed the expression patterns of VvMGTs at different development stages and in response to abiotic stresses including waterlogging, drought, salinity, and copper. Using qRT-PCR method, we tested the expression of grape VvMGTs under magnesium and aluminum treatments and found significant changes in VvMGTs expression. In addition, four of the MGT proteins in grape were located in the nucleus. CONCLUSION: Overall, in this study we investigated the structural characteristics, evolution pattern, and expression analysis of VvMGTs in depth, which laid the foundation for further revealing the function of VvMGT genes in grape.

Jasmonate increases terpene synthase expression, leading to strawberry resistance to Botrytis cinerea infection.

KEY MESSAGE: Jasmonate induced FaTPS1 to produce terpene, and overexpression FaTPS1 led to fruit resistant against B. cinerea infection, FaMYC2 induced FaTPS1 by binding to its promoter that downstream of jasmonate. Jasmonic acid (JA) and its derivatives are associated with plant defence responses against pathogenic organisms. In the present study, a total of 10,631 differentially expressed genes, 239 differentially expressed proteins, and 229 differential metabolites were screened and found to be mainly involved in pathogen perception, hormone biosynthesis and signal transduction, photosynthesis, and secondary metabolism. In strawberry fruits, methyl jasmonate (MeJA) induced FaTPS1 expression and quickly increased the terpene content. Furthermore, FaTPS1 overexpression increased the emission of sesquiterpenes, especially germacrene D, and improved strawberry resistance against Botrytis cinerea infection, although the knockdown of FaTPS1 increased its susceptibility to the same pathogen. Using a yeast one-hybrid assay and transient expression analysis, we demonstrated that FaMYC2 can bind to the G-box element in the promoter region of FaTPS1, thus inducing FaTPS1 expression. MeJA also stimulated FaMYC2 expression and regulated downstream signalling cascades. Moreover, we presented a possible model of the new signalling pathway of MeJA-mediated strawberry resistance to B. cinerea.

Metabolomic profiling of brassinolide and abscisic acid in response to high-temperature stress.

KEY MESSAGE: Hormone treatment enhanced the content of osmotic substances under high-temperature conditions. The effect of ABA and BR treated separately is better than treated together. To determine the effect of abscisic acid (ABA), brassinolide (BR) and ABA + BR on grape quality under high-temperature stress, various metabolites were analyzed. Compared with the control (CK), DL-tryptophan, D-raffinose, geniposidic acid, dodecanedioic acid and polyphenols were found to be higher after ABA treatment. After BR treatment, amino acids and poricoic acid B were higher than in CK. And carbohydrates and amino acids were up-regulated after ABA + BR treatment. BR and ABA + BR treatment also induced higher endogenous ABA and epibrassinolide contents. In addition, treated grape had higher soluble solid concentrations and soluble sugar content, and delayed the degradation of middle lamella and microfibrils. Antioxidant and heat shock-related genes were examined, which significantly increased in treated grape. The finding of this study suggested that ABA, BR and ABA + BR are very useful for alleviating high-temperature damage by increasing the accumulation of osmotic adjustment substances, and endogenous hormones content.

Identification and Characterization of AUXIN Response Factor Gene Family Reveals Their Regulatory Network to Respond the Multi-Hormones Crosstalk during GA-Induced Grape Parthenocarpic Berry.

Exogenous gibberellin (GA) was widely used to improve berry quality through inducing parthenocarpic seedless berries in grapes. We revealed that auxin response factors (ARFs), the key transcription factors in response to auxin, might respond to GA involving modulation of grape parthenocarpy. However, the underlying molecular mechanism in this process remains yet unclear. Here, a total of 19 VvARF members were identified in the ovaries during GA-induced grapes' parthenocarpy. Interestingly, almost all members were GA-responsive factors, of which 9 could be classified in plant hormone signal transduction (KO04075) and involved in the tryptophan metabolic pathway (K14486). Moreover, VvARFs were predicted to have 310 interacted proteins involved in 19 KEGG pathways. Of them, 32 interacted proteins participated in the KO04075 pathway, including auxin (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signaling pathways by responding to GA-mediated multi-hormone crosstalk. Further analysis demonstrated that VvARF4-2 might be the major factor in the modulation of GA-induced parthenocarpy via the crosstalk of IAA, CTK, SA, and ethylene signaling, followed by VvARF6-1 and VvARF9 involved in SA and ABA signaling pathways, respectively. Finally, we developed a VvARFs-mediated regulatory network by responding to GA-mediated multi-hormone crosstalk during grape parthenocarpy. Collectively, our findings provided novel insights into the regulatory network of VvARFs in GA-guided multi-hormone signaling to modulate grape parthenocarpy, which has great implications for the molecular breeding of high quality seedless grape berries.

miR3633a-GA3ox2 Module Conducts Grape Seed-Embryo Abortion in Response to Gibberellin.

Seedlessness is one of the important quality and economic traits favored by grapevine consumers, which are mainly affected by phytohormones, especially gibberellin (GA). GA is widely utilized in seedless berry production and could effectively induce grape seed embryo abortion. However, the molecular mechanism underlying this process, like the role of RNA silencing in the biosynthesis pathway of GA remains elusive. Here, Gibberellin 3-ß dioxygenase2 (GA3ox2) as the last key enzyme in GA biosynthesis was predicated as a potential target gene for miR3633a, and two of them were identified as a GA response in grape berries. We also analyzed the promoter regions of genes encoding GA biosynthesis and found the hormone-responsive elements to regulate grape growth and development. The cleavage interaction between VvmiR3633a and VvGA3ox2 was validated by RLM-RACE and the transient co-transformation technique in tobacco in vivo. Interestingly, during GA-induced grape seed embryo abortion, exogenous GA promoted the expression of VvmiR3633a, thereby mainly repressing the level of VvGA3ox2 in seed embryos. We also observed a negative correlation between down-regulated VvGA20ox2/VvGA3ox2 and up-regulated VvGA2ox3/VvGA2ox1, of which GA inactivation was greater than GA synthesis, inhibited active GA content, accompanied by the reduction of VvSOD and VvCAT expression levels and enzymatic activities. These series of changes might be the main causes of grape seed embryo abortion. In conclusion, we have preliminarily drawn a schematic mode of GA-mediated VvmiR3633a and related genes regulatory network during grape seed abortion induced by exogenous GA. Our findings provide novel insights into the GA-responsive roles of the VvmiR3633a-VvGA3ox2 module in the modulation of grape seed-embryo abortion, which has implications for the molecular breeding of high-quality seedless grape berries.

Effects of ethylene on berry ripening and anthocyanin accumulation of 'Fujiminori' grape in protected cultivation.

BACKGROUND: Although the grape berries are deliberated as a non-climacteric fruit, ethylene seems to be involved in grape berry ripening. However, the precise role of ethylene in regulating the ripening of non-climacteric fruits is poorly understood. RESULTS: Exogenous ethephon (ETH) can stimulate the concentration of internal ethylene and accelerate the accumulation of anthocyanins in berries of 'Fujiminori', including malvidin-, delphinidin-, and petunidin-derivatives (3',4',5'-trihydroxylated anthocyanins) and cyanidin-derivatives (3',4'-dihydroxylated anthocyanins). The content of 3',4',5'-trihydroxylated anthocyanins was extremely higher than 3',4'-dihydroxylated anthocyanins, and ethylene did not affect the composition of anthocyanins in grape. Furthermore, we observed the expression of anthocyanin structural and regulatory genes as well as ethylene biosynthesis and response genes in response to ETH treatment. The anthocyanins accumulation is significantly associated with increased expression of anthocyanin structural (VvPAL, Vv4CH, VvCHS, VvCHI, VvF3H, and VvUFGT) and regulatory genes (VvMYBA1, VvMYBA2, and VvMYBA3), which persisted over the 12 days. In addition, exogenous ETH affected the endogenous ethylene biosynthesis (VvACO2 and VvACO4) and the downstream ethylene regulatory network (VvERS1, VvETR2, VvCTR1, and VvERF005). CONCLUSIONS: These findings bring new insights into the physiological and molecular function of ethylene during berry development and ripening in grapes. © 2021 Society of Chemical Industry.

"The PLCP gene family of grapevine (Vitis vinifera L.): characterization and differential expression in response to Plasmopara Viticola".

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.

Expressional diversity of grapevine 3-Hydroxy-3-methylglutaryl-CoA reductase (VvHMGR) in different grapes genotypes.

BACKGROUND: 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a key enzyme in the mevalonate (MVA) pathway, which regulates the metabolism of terpenoids in the cytoplasm and determines the type and content of downstream terpenoid metabolites. RESULTS: Results showed that grapevine HMGR family has three members, such as VvHMGR1, VvHMGR2, and VvHMGR3. The expression of VvHMGRs in 'Kyoho' has tissue specificity, for example, VvHMGR1 keeps a higher expression, VvHMGR2 is the lowest, and VvHMGR3 gradually decreases as the fruit development. VvHMGR3 is closely related to CsHMGR1 and GmHMGR9 and has collinearity with CsHMGR2 and GmHMGR4. By the prediction of interaction protein, it can interact with HMG-CoA synthase, MVA kinase, FPP/GGPP synthase, diphosphate mevalonate decarboxylase, and participates in the synthesis and metabolism of terpenoids. VvHMGR3 have similar trends in expression with some of the genes of carotenoid biosynthesis and MEP pathways. VvHMGR3 responds to various environmental and phytohormone stimuli, especially salt stress and ultraviolet (UV) treatment. The expression level of VvHMGRs is diverse in grapes of different colors and aroma. VvHMGRs are significantly higher in yellow varieties than that in red varieties, whereas rose-scented varieties showed significantly higher expression than that of strawberry aroma. The expression level is highest in yellow rose-scented varieties, and the lowest in red strawberry scent varieties, especially 'Summer Black' and 'Fujiminori'. CONCLUSION: This study confirms the important role of VvHMGR3 in the process of grape fruit coloring and aroma formation, and provided a new idea to explain the loss of grape aroma and poor coloring during production. There may be an additive effect between color and aroma in the HMGR expression aspect.

Identification of miRNAs-mediated seed and stone-hardening regulatory networks and their signal pathway of GA-induced seedless berries in grapevine (V. vinifera L.).

BACKGROUND: Stone-hardening stage is crucial to the development of grape seed and berry quality. A significant body of evidence supports the important roles of MicroRNAs in grape-berry development, but their specific molecular functions during grape stone-hardening stage remain unclear. RESULTS: Here, a total of 161 conserved and 85 species-specific miRNAs/miRNAs* (precursor) were identified in grape berries at stone-hardening stage using Solexa sequencing. Amongst them, 30 VvmiRNAs were stone-hardening stage-specific, whereas 52 exhibited differential expression profiles during berry development, potentially participating in the modulation of berry development as verified by their expression patterns. GO and KEGG pathway analysis showed that 13 VvmiRNAs might be involved in the regulation of embryo development, another 11 in lignin and cellulose biosynthesis, and also 28 in the modulation of hormone signaling, sugar, and proline metabolism. Furthermore, the target genes for 4 novel VvmiRNAs related to berry development were validated using RNA Ligase-Mediated (RLM)-RACE and Poly(A) Polymerase-Mediated (PPM)-RACE methods, and their cleavage mainly occurred at the 9th-11th sites from the 5' ends of miRNAs at their binding regions. In view of the regulatory roles of GA in seed embryo development and stone-hardening in grape, we investigated the expression modes of VvmiRNAs and their target genes during GA-induced grape seedless-berry development, and we validated that GA induced the expression of VvmiR31-3p and VvmiR8-5p to negatively regulate the expression levels of CAFFEOYL COENZYME A-3-O-METHYLTRANSFERASE (VvCCoAOMT), and DDB1-CUL4 ASSOCIATED FACTOR1 (VvDCAF1). The series of changes might repress grape stone hardening and embryo development, which might be a potential key molecular mechanism in GA-induced grape seedless-berry development. Finally, a schematic model of miRNA-mediated grape seed and stone-hardening development was proposed. CONCLUSION: This work identified 30 stone-hardening stage-specific VvmiRNAs and 52 significant differential expression ones, and preliminary interpreted the potential molecular mechanism of GA-induced grape parthenocarpy. GA negatively manipulate the expression of VvCCoAOMT and VvDCAF1 by up-regulation the expression of VvmiR31-3p and VvmiR8-5p, thereby repressing seed stone and embryo development to produce grape seedless berries.

The role of VvMYBA2r and VvMYBA2w alleles of the MYBA2 locus in the regulation of anthocyanin biosynthesis for molecular breeding of grape (Vitis spp.) skin coloration.

In grape, MYBA1 and MYBA2 at the colour locus are the major genetic determinants of grape skin colour, and the mutation of two functional genes (VvMYBA1 and VvMYBA2) from these loci leads to white skin colour. This study aimed to elucidate the regulation of grape berry coloration by isolating and characterizing VvMYBA2w and VvMYBA2r alleles. The overexpression of VvMYBA2r up-regulated the expression of anthocyanin biosynthetic genes and resulted in higher anthocyanin accumulation in transgenic tobacco than wild-type (WT) plants, especially in flowers. However, the ectopic expression of VvMYBA2w inactivated the expression of anthocyanin biosynthetic genes and could not cause obvious phenotypic modulation in transgenic tobacco. Unlike in VvMYBA2r, CA dinucleotide deletion shortened the C-terminal transactivation region and disrupted the transcriptional activation activity of VvMYBA2w. The results indicated that VvMYBA2r positively regulated anthocyanin biosynthesis by forming the VvMYBA2r-VvMYCA1-VvWDR1 complex, and VvWDR1 enhanced anthocyanin accumulation by interacting with the VvMYBA2r-VvMYCA1 complex; however, R44 L substitution abolished the interaction of VvMYBA2w with VvMYCA1. Meanwhile, both R44 L substitution and CA dinucleotide deletion seriously affected the efficacy of VvMYBA2w to regulate anthocyanin biosynthesis, and the two non-synonymous mutations were additive in their effects. Investigation of the colour density and MYB haplotypes of 213 grape germplasms revealed that dark-skinned varieties tended to contain HapC-N and HapE2, whereas red-skinned varieties contained high frequencies of HapB and HapC-Rs. Regarding ploidy, the higher the number of functional alleles present in a variety, the darker was the skin colour. In summary, this study provides insight into the roles of VvMYBA2r and VvMYBA2w alleles and lays the foundation for the molecular breeding of grape varieties with different skin colour.

Identification of GH17 gene family in Vitis vinifera and expression analysis of GH17 under various adversities.

Glycoside hydrolase (GH, EC 3.2.1) is a group of enzymes that hydrolyzes glycosidic bonds and play a role in the hydrolysis and synthesis of sugars in living organisms. Vitis vinifera is an important fruit crop and it harbors GH17 gene family however, their function in grapes has not been systematically investigated. In this study, a total of 870 GH17 genes were identified from 14 plant species and their structural domain, sequence alignment, phylogenetic tree, collinear analysis, with the expression profiles of VviGH17 gene family was performed. The promoter analysis of VviGH17 gene showed the presence of cis-acting elements, which are responsive to plant growth and development. In addition, elements for plant hormones were found that are triggered in response to abiotic/biological stress. Transcriptomic data led to the identification of several VviGH17 genes, which are associated with bud dormancy and in response to abiotic stress. Transcript analysis was carried out for some of the selected VviGH17 genes RT-qPCR. VviGH17-16 and VviGH17-30 genes were differentially expressed during bud dormancy, fruit development and different abiotic stresses. Moreover, VviGH17-37 and VviGH17-44 were differentially expressed at fruit development, in response to abiotic stress. In addition, subcellular localization predicts that the VviGH17-16, VviGH17-30, and VviGH17-37 genes were located in the cell membrane, while VviGH17-44 gene was located in the vacuole. In conclusion, our study led to the identification of several GH17s and their probable role in development and stress. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01014-1.

Transcriptional and metabolite analysis reveal a shift in fruit quality in response to calcium chloride treatment on "Kyoho" grapevine.

'Kyoho' grapevine (Vitis vinifera) treated by calcium ions solution has been proved as an effective treatment to extend grape quality during storage to reduce disease, but its molecular mechanism was not clear yet. In the current work, grape berries were treated with different concentration of Calcium chloride (CaCl2) solution, and their effects on antioxidant enzyme activity and transcriptome and metabolome in fruit were investigated. CaCl2 treatments reduced weight loss and inhibited the decrement of flesh firmness. 80 mM CaCl2 significantly increased the activity of antioxidant enzymes POD, SOD and CAT, which was the optimum experimental concentration. The study showed that the expression level of heat shock transcription factor and UBX which involved in endoplasmic reticulum stress and degradation pathway increased significantly. Moreover, the corresponding metabolites, such as heat shock protein and organic acid, also increased significantly. The misfolded proteins are transported to the cytosol for degradation, so that the preservation ability of grape is improved.

Characterization of the regulation mechanism of grapevine microRNA172 family members during flower development.

BACKGROUND: Grapevine (Vitis vinifera L.), which has important nutritional values and health benefits, is one of the most economically important fruit crops cultivated worldwide. Several studies showed a large number of microRNAs (VvmiRNAs) involved in the modulation of grape growth and development, and many VvmiRNA families have multiple members. However, the way by which various members from the same miRNA family work is unclear, particularly in grapes. RESULTS: In this study, an important conserved VvmiR172 family (VvmiR172s) and their targets were set as a good example for elucidating the interaction degree, mechanism, and spatio-temporal traits of diverse members from the same miRNA family. miR-RACE and Stem-loop RT-PCR were used to identify the spatio-temporal expressions of various members of VvmiR172s; together with RLM-RACE, PPM-RACE, Western blot, transgenic technologies, their interaction degree, and regulation mechanism were further validated. The expression of VvmiR172c was significantly higher than that of VvmiR172a, b, and d and showed a positive correlation with the abundance of VvAP2 cleavage products. These findings indicated that VvmiR172c might be one of the main action factors of the VvmiR172 family in flower development. The ability of VvmiR172c to cleave target genes differed due to divergence in complementary degree with VvAP2 and expression levels of various members. In VvmiR172 transgenic lines, we observed that 35S::VvmiR172c resulted in the earliest and abundant flowering, indicating the strong function of VvmiR172c. In contrast, the non-significant phenotypic changes were detected in the VvAP2 transgenic lines. The qRT-PCR and Western bolt results demonstrated that VvmiR172c plays a major role in targeting VvAP2. CONCLUSIONS: VvmiR172 up-regulated the expression of NtFT and decreased the expression of NtFLC. The up/down regulation of VvmiR172c was the most pronounced. The functions of four VvmiR172 members in grape differed, and miR172c had the strongest regulation on AP2.