Identification of VvAGL Genes Reveals Their Network's Involvement in the Modulation of Seed Abortion via Responding Multi-Hormone Signals in Grapevines.

The formation of seedless traits is regulated by multiple factors. AGLs, which belong to the MADS-box family, were reported to be important regulators in this process; however, the underlying mechanism remains elusive. Here, we identified the VvAGL sub-family genes during the seed abortion process in seedless grapevine cv. 'JingkeJing' and found 40 differentially expressed VvAGL members and 1069 interacting proteins in this process. Interestingly, almost all members and their interacting proteins involved in the tryptophan metabolic pathway (K14486) and participated in the phytohormone signalling (KO04075) pathway, including the growth hormone (IAA), salicylic acid (SA), abscisic acid (ABA), cytokinin (CTK), and ethylene signalling pathways. The promoters of AGL sub-family genes contain cis-elements in response to hormones such as IAA, ABA, CTK, SA, and ETH, implying that they might respond to multi-hormone signals and involve in hormone signal transductions. Further expression analysis revealed VvAGL6-2, VvAGL11, VvAGL62-11, and VvAGL15 had the highest expression at the critical period of seed abortion, and there were positive correlations between ETH-VvAGL15-VvAGL6-2, ABA-VvAGL80, and SA-VvAGL62 in promoting seed abortion but negative feedback between IAA-VvAGL15-VvAGL6-2 and CTK-VvAGL11. Furthermore, many genes in the IAA, ABA, SA, CTK, and ETH pathways had a special expressional pattern in the seed, whereby we developed a regulatory network mediated by VvAGLs by responding to multihormonal crosstalk during grape seed abortion. Our findings provide new insights into the regulatory network of VvAGLs in multi-hormone signalling to regulate grape seed abortion, which could be helpful in the molecular breeding of high-quality seedless grapes.

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.

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.

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.

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.

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.

Geographical location influence 'Cabernet Franc' fruit quality in Shandong province.

Grape quality is a key factor in determining wine quality, and it depends not only on management skills, but also on the geographic location of the producing area. In China, Shandong is the province with the largest wine production, and 'Cabernet Franc' is widely planted. This study evaluated the 'Cabernet Franc' fruit quality in relation to geographical conditions in five 'Cabernet Franc' producing districts of Shandong province, China, including Dezhou Aodeman Winery (DZ), Tai'an Zhongqingsongshi Winery (TA), Penglai Longhu Winery (PL), Rushan Taiyihu Winery (RS), and Rizhao Taiyangcheng Winery (RZ). At the time of veraison and maturity, fruit was harvested from five areas, and compared for cosmetic and internal fruit quality. The soluble sugar content in the Rizhao area was rich, and the weight and volume of single fruit were relatively large. The titratable acid of the berries in Tai'an area was high. RNA-seq analysis showed that the number of genes in the véraison stage was 19,571-20,750, and the number of genes in the mature stage was 19,176-20,735. The analysis found that areas with multiple high-quality characteristics tended to have more DEGs (differential expressed genes). And the DEGs in different areas were mainly distributed on chromosome 7, and at least on chromosome 15. DEGs in 5 areas were enriched on 855 GO terms and 116 KEGG pathways during berries development. Among them, it was found that the up/down-regulation of DEGs was related to the formation of berry quality, which helps to explain the impact of environment on grape quality components. In summary, this study is helpful to understand the influence of cultivation location on the quality of 'Cabernet Franc' in different production areas in Shandong province, and further provide a reference for the production of high-quality wine grapes and winemaking.

Characterization and regulatory mechanism analysis of VvmiR156a-VvAGL80 pair during grapevine flowering and parthenocarpy process induced by gibberellin.

MicroRNA156 (miR156) is an important conserved miRNA family in plants. Recently, we revealed VvmiR156a could involve in the modulation of gibberellin (GA)-mediated flower and berry development process of grapevine (Vitis vinifera L.). However, how to manipulate this process is unclear. For this, we used the GA-induced grapevine parthenocarpy system to investigate the regulatory roles of VvmiR156a during this process. Here, we cloned the mature and precursor sequences of VvmiR156a in Wink grape and identified its potential target gene VvAGL80, which belongs to the MADS-box gene family. Moreover, using RNA ligase-mediated 5' rapid amplification of cDNA ends (RLM-RACE) and poly(A)polymerase-mediated 3' rapid amplification of cDNA (PPM-RACE) technologies, it confirmed that VvAGL80 was the true target gene of VvmiR159a. Analysis of promoter cis-elements and ß-glucuronidase (GUS) staining showed that both VvmiR156a and VvAGL80 contained GA-responsive elements and could respond to GA treatments. Quantitative real-time-polymerase chain reaction (qRT-PCR) analysis exhibited the VvmiR156a and VvAGL80 showed opposite expression trends during grapevine flower and berry development, indicating that VvmiR156a negatively regulated the expression of VvAGL80 during this process. After GA treatment, the expression of miR156 in flowers was downregulated significantly, while that of VvAGL80 was upregulated, thereby accelerating grapevine flowering. Furthermore, GA treatment enhanced the negative regulation of VvmiR156a on VvAGL80 in seed, especially at the seed-coat hardening stage, which was the key period of seed growth and development. Our findings enriched the knowledge of the regulatory mechanism of the miRNA-mediated grapevine parthenocarpy process.

Characterization and Action Mechanism Analysis of VvmiR156b/c/d-VvSPL9 Module Responding to Multiple-Hormone Signals in the Modulation of Grape Berry Color Formation.

In recent years, more and more reports have shown that the miR156-SPL module can participate in the regulation of anthocyanin synthesis in plants. However, little is known about how this module responds to hormonal signals manipulating this process in grapes. In this study, exogenous GA, ABA, MeJA, and NAA were used to treat the 'Wink' grape berries before color conversion, anthocyanin and other related quality physiological indexes (such as sugar, aroma) were determined, and spatio-temporal expression patterns of related genes were analyzed. The results showed that the expression levels of VvmiR156b/c/d showed a gradually rising trend with the ripening and color formation of grape berries, and the highest expression levels were detected at day 28 after treatment, while the expression level of VvSPL9 exhibited an opposite trend as a whole, which further verifies that VvmiR156b/c/d can negatively regulate VvSPL9. Besides, VvmiR156b/c/d was positively correlated with anthocyanin content and related genes levels, while the expression pattern of VvSPL9 showed a negative correlation. Analysis of promoter cis-elements and GUS staining showed that VvmiR156b/c/d contained a large number of hormone response cis-elements (ABA, GA, SA, MeJA, and NAA) and were involved in hormone regulation. Exogenous ABA and MeJA treatments significantly upregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early stage of color conversion and made grape berries quickly colored. Interestingly, GA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes in the early color-change period, but significantly upregulated in the middle color-change and ripening stages, therefore GA mainly modulated grape berry coloring in the middle- and late-ripening stages. Furthermore, NAA treatment downregulated the expression levels of VvmiR156b/c/d and anthocyanin structural genes and delayed the peak expression of genes. Meanwhile, to further recognize the potential functions of VvmiR156b/c/d, the mature tomato transient trangenetic system was utilized in this work. Results showed that transient overexpression of VvmiR156b/c/d in tomato promoted fruit coloring and overexpression of VvSPL9 inhibited fruit coloration. Finally, a regulatory network of the VvmiR156b/c/d-VvSPL9 module responsive to hormones modulating anthocyanin synthesis was developed. In conclusion, VvmiR156b/c/d-mediated VvSPL9 participated in the formation of grape color in response to multi-hormone signals.