VvWRKY5 positively regulates wounding-induced anthocyanin accumulation in grape by interplaying with VvMYBA1 and promoting jasmonic acid biosynthesis.

Wounding stress induces the biosynthesis of various secondary metabolites in plants, including anthocyanin. However, the underlying molecular mechanism remains elusive. Here, we reported that a transcription factor, VvWRKY5, promotes wounding-induced anthocyanin accumulation in grape (Vitis vinifera). Biochemical and molecular analyses demonstrated that wounding stress significantly increased anthocyanin content, and VvMYBA1 plays an essential role in this process. VvWRKY5 could interact with VvMYBA1 and amplify the activation effect of VvMYBA1 on its target gene VvUFGT. The transcript level of VvWRKY5 was notably induced by wounding treatment. Moreover, our data demonstrated that VvWRKY5 could promote the synthesis of jasmonic acid (JA), a phytohormone that acts as a positive modulator in anthocyanin accumulation, by directly binding to the W-box element in the promoter of the JA biosynthesis-related gene VvLOX and enhancing its activities, and this activation was greatly enhanced by the VvWRKY5-VvMYBA1 protein complex. Collectively, our findings show that VvWRKY5 plays crucial roles in wounding-induced anthocyanin synthesis in grape and elucidates the transcriptional regulatory mechanism of wounding-induced anthocyanin accumulation.

Berry texture QTL and candidate gene analysis in grape (Vitis vinifera L.).

Berry texture is a noteworthy economic trait for grape; however, the genetic bases and the complex gene expression and regulatory mechanism for the diverse changes in berry texture are still poorly understood. In this study, the results suggest that it is difficult to obtain high-mesocarp firmness (MesF) and high-pericarp puncture hardness (PPH) grape cultivars with high pericarp brittleness (PerB). The high-density linkage map was constructed using whole-genome resequencing based on 151 F1 individuals originating from intraspecific hybridization between the firm-flesh cultivar 'Red Globe' and soft-flesh cultivar 'Muscat Hamburg'. The total length of the consensus map was 1613.17 cM, with a mean genetic distance between adjacent bin markers of 0.59 cM. Twenty-seven quantitative trait loci (QTLs) for berry MesF, PPH, and PerB were identified in linkage groups (LGs) 1, 3, 4, 6, 8, 9, 10, 11, 14, 16, and 17, including twelve QTLs that were firstly detected in LGs 6, 11, and 14. Fourteen promising candidate genes were identified from the stable QTL regions in LGs 10, 11, 14, and 17. In particular, VvWARK2 and VvWARK8 refer to chromosome 17 and are two promising candidate genes for MesF and PPH, as the VvWARK8 gene may increase pectin residue binding with WARK for high berry firmness maintenance and the allele for VvWARK2 carrying the 'CC' and 'GA' genotypes at Chr17:1836764 and Chr17:1836770 may be associated with non-hard texture grape cultivars. In addition, real-time quantitative polymerase chain reaction (RT-qPCR) verification revealed that the promising candidate transcription factor genes VvMYB4-like, VvERF113, VvWRKY31, VvWRKY1, and VvNAC83 may regulate cell wall metabolism candidate gene expression for grape berry texture changes.

VvWRKY5 enhances white rot resistance in grape by promoting the jasmonic acid pathway.

Grape white rot is a disease caused by Coniella diplodiella (Speg.) Sacc. (Cd) can drastically reduce the production and quality of grape (Vitis vinifera). WRKY transcription factors play a vital role in the regulation of plant resistance to pathogens, but their functions in grape white rot need to be further explored. Here, we found that the expression of the WRKY IIe subfamily member VvWRKY5 was highly induced by Cd infection and jasmonic acid (JA) treatment. Transient injection and stable overexpression (in grape calli and Arabidopsis) demonstrated that VvWRKY5 positively regulated grape resistance to white rot. We also determined that VvWRKY5 regulated the JA response by directly binding to the promoters of VvJAZ2 (a JA signaling suppressor) and VvMYC2 (a JA signaling activator), thereby inhibiting and activating the transcription of VvJAZ2 and VvMYC2, respectively. Furthermore, the interaction between VvJAZ2 and VvWRKY5 enhanced the suppression and promotion of VvJAZ2 and VvMYC2 activities by VvWRKY5, respectively. When VvWRKY5 was overexpressed in grape, JA content was also increased. Overall, our results suggested that VvWRKY5 played a key role in regulating JA biosynthesis and signal transduction as well as enhancing white rot resistance in grape. Our results also provide theoretical guidance for the development of elite grape cultivars with enhanced pathogen resistance.

PD98059 protects SH-SY5Y cells against oxidative stress in oxygen-glucose deprivation/reperfusion.

Mitochondria play a key role in the cerebral ischemia-reperfusion injury. Although the extracellular signal-regulated kinase 1/2 inhibitor PD98059 (PD) is a selective and reversible flavonoid that can protect the mitochondria in a rat model of cardiac arrest/cardiopulmonary resuscitation, its role requires further confirmation. In this study, we investigated whether PD could maintain mitochondrial homeostasis and decrease reactive oxygen species (ROS) production in neuroblastoma (SH-SY5Y) cells exposed to oxygen-glucose deprivation/reperfusion (OGD/R). PD improved the mitochondrial morphology and function, reversed the increase in ROS production and cell apoptosis, and reduced total-superoxide dismutase and Mn-superoxide dismutase activities induced by OGD/R. PD decreases ROS production and improves mitochondrial morphology and function, protecting SH-SY5Y cells against OGD/R-induced injury.

Candidate gene discovery of Botrytis cinerea resistance in grapevine based on QTL mapping and RNA-seq.

Grape gray mold disease (Botrytis cinerea) is widespread during grape production especially in Vitis vinifera and causes enormous losses to the grape industry. In nature, the grapevine cultivar 'Beta ' (Vitis riparia × Vitis labrusca) showed high resistance to grape gray mold. Until now, the candidate genes and their mechanism of gray mold resistance were poorly understood. In this study, we firstly conducted quantitative trait locus (QTL) mapping for grape gray mold resistance based on two hybrid offspring populations that showed wide separation in gray mold resistance. Notably, two stable QTL related to gray mold resistance were detected and located on linkage groups LG2 and LG7. The phenotypic variance ranged from 6.86% to 13.70% on LG2 and 4.40% to 11.40% on LG7. Combined with RNA sequencing (RNA-seq), one structural gene VlEDR2 (Vitvi02g00982) and three transcription factors VlERF039 (Vitvi00g00859), VlNAC047 (Vitvi08g01843), and VlWRKY51 (Vitvi07g01847) that may be involved in VlEDR2 expression and grape gray mold resistance were selected. This discovery of candidate gray mold resistance genes will provide an important theoretical reference for grape gray mold resistance mechanisms, research, and gray mold-resistant grape cultivar breeding in the future.

VqMYB154 promotes polygene expression and enhances resistance to pathogens in Chinese wild grapevine.

Resveratrol plays a crucial phytoalexin role in the grapevine and is beneficial to human health. However, the molecular mechanism of resveratrol accumulation in the enhancement of disease resistance is unclear. Here, we report that the transcription factor VqMYB154 from Vitis quinquangularis accession Danfeng-2 is strongly expressed under artificial inoculation with Uncinula necator and regulates resveratrol accumulation. Unlike its homolog, VqMYB154 has a pathogen-induced promoter and responds to stimulation by U. necator, Pseudomonas syringae, and other treatments. Yeast one-hybrid and GUS activity assays confirmed that VqMYB154 can activate the stilbene synthase genes VqSTS9, VqSTS32, and VqSTS42 by directly binding to their promoters. Overexpression of VqMYB154 in grape leaves resulted in activation of the stilbene pathway, upregulation of STS genes, and accumulation of stilbenoids. In addition, heterologous overexpression of VqMYB154 in Arabidopsis activated resistance-related genes and resulted in greater programmed cell death and accumulation of reactive oxygen species, which led to resistance against P. syringae. These results suggest that the transcription factor VqMYB154 from V. quinquangularis accession Danfeng-2 participates in the regulatory mechanism that improves the biosynthesis and accumulation of stilbenes and enhances resistance to disease in grapevine.

The WRKY53 transcription factor enhances stilbene synthesis and disease resistance by interacting with MYB14 and MYB15 in Chinese wild grape.

Resveratrol is notable not only for its functions in disease resistance in plants but also for its health benefits when it forms part of the human diet. Identification of new transcription factors helps to reveal the regulatory mechanisms of stilbene synthesis. Here, the WRKY53 transcription factor was isolated from the Chinese wild grape, Vitis quinquangularis. Vqwrky53 was expressed in a variety of tissues and responded to powdery mildew infection and to exogenous hormone application. VqWRKY53 was located in the nucleus and had transcriptional activation activity in yeast. A yeast two-hybrid assay and a bimolecular fluorescence complementation assay confirmed that VqWRKY53 interacted physically with VqMYB14 and VqMYB15, which have previously been reported to regulate stilbene synthesis. When Vqwrky53 was overexpressed in grape leaves, the expression of VqSTS32 and VqSTS41 and the content of stilbenes were increased. A yeast one-hybrid assay demonstrated that VqWRKY53 could bind directly to the promoters of STS genes. Overexpression of Vqwrky53 activated ß-glucuronidase expression, driven by STS promoters, and co-expressing Vqwrky53 with VqMYB14 and VqMYB15 showed stronger regulatory functions. Heterologous overexpression of Vqwrky53 in Arabidopsis accelerated leaf senescence and disease resistance to PstDC3000.

Expression of stilbene synthase VqSTS6 from wild Chinese Vitis quinquangularis in grapevine enhances resveratrol production and powdery mildew resistance.

MAIN CONCLUSION: In grape (Vitis), stilbene phytoalexins can either be in situ synthesized or transported to the site of response during powdery mildew infection, enhancing disease resistance. Resveratrol is a phytoprotective stilbenoid compound that is synthesized by stilbene synthase (STS) in response to biotic and abiotic stresses, and is also known to have health benefits in the human diet. We have previously shown that transgenic Vitis vinifera cv. Thompson Seedless plants overexpressing a stilbene synthase gene, VqSTS6, from wild Chinese Vitis quinquangularis had a higher stilbenoid content, leading to an enhanced resistance to powdery mildew (Uncinula necator (Schw.) Burr). However, the biosynthesis and transportation in the plant tissue under powdery mildew infection are still unclear. Here, inhibitor and micro-grafting technologies were used to study the accumulation of resveratrol following powdery mildew infection. We observed that the levels of STS expression and stilbenoids increased in response to powdery mildew infection. Powdery mildew and inhibitor treatment on detached grape branches showed that resveratrol was in situ synthesized. Experiments with grafted plantlets showed that the abundance of stilbenoid compounds increased in the shoot during VqSTS6 overexpression in the root, while VqSTS6-Flag fusion was not tranported to the scions and only expressed in the transgenic rootstocks. Compared with wild-type Thompson Seedless plants, the non-transgenic/VqSTS6 transgenic (scion/rootstock) grafted Thompson Seedless plantlets exhibited increased resistance to powdery mildew. In addition, overexpression of VqSTS6 in roots led to increased levels of stilbenoid compounds in five other European grape varieties (V. vinifera cvs. Chardonnay, Perlette, Cabernet Sauvignon, Riesling and Muscat Hamburg). In conclusion, stilbenoid compounds can be either in situ synthesized or transported to the site of powdery mildew infection, and overexpression of VqSTS6 in the root promotes stilbenoids accumulation and disease resistance in European grapevine varieties.

VqbZIP1 isolated from Chinese wild Vitis quinquangularis is involved in the ABA signaling pathway and regulates stilbene synthesis.

Resveratrol is an important phytoalexin in grapevine. Not only does it confer increased disease resistance and but as a food component it offers significant benefits in human health. Abscisic acid (ABA) is an important phytohormone involved in many biological processes in plants and can also promote the accumulation of stilbenes. Stilbene synthase (STS) is an important enzyme which catalyzes the last step of resveratrol synthesis. Our study characterizes a basic leucine zipper (bZIP) transcription factor, VqbZIP1, isolated from Chinese wild Vitis quinquangularis accession Danfeng-2. The results show that VqbZIP1 encodes 299 amino acids and belongs to the Group A subfamily of the bZIP family. VqbZIP1 showed transcriptional activation activity in yeast and is predicted to be located in the nucleus. The yeast two-hybrid assay and bimolecular fluorescence complementation (BiFC) assay together show that VqbZIP1 interacts with VqSnRK2.4 and VqSnRK2.6. VqbZIP1, the STS genes, VqSnRK2.4 and VqSnRK2.6 can all be induced by ABA treatment. A GUS activity experiment indicates VqbZIP1 can activate the GUS reporter gene driven by STS promoters. Further studies show that co-expression of VqbZIP1 with VqSnRK2.4 or VqSnRK2.6 can confer higher efficiency than expression of VqbZIP1 alone in activating the STS promoters. Overexpression of VqbZIP1 in grape leaves promoted the transcript level of the STS genes and the accumulation of stilbenes. Overexpression of VqbZIP1 in Arabidopsis thaliana can confer ABA sensitivity. In summary, our results suggest VqbZIP1 participates in the ABA signaling pathway and regulates stilbene synthesis.

VqMAPKKK38 is essential for stilbene accumulation in grapevine.

Vitis species, including grapevine, produce a class of secondary metabolites called stilbenes that are important for plant disease resistance and can have positive effects on human health. Mitogen-activated protein kinase (MAPK) signaling cascades not only play key roles in plant defense responses but also contribute to stilbene biosynthesis in grapevine. MAPKKKs function at the upper level of the MAPK network and initiate signaling through this pathway. In this study, a Raf-like MAPKKK gene, VqMAPKKK38, was identified and functionally characterized from the Chinese wild grapevine V. quinquangularis accession 'Danfeng-2'. We observed that VqMAPKKK38 transcript levels were elevated by powdery mildew infection, high salinity conditions and chilling stresses, as well as in response to treatments by the hormones salicylic acid (SA), methyl jasmonate (MeJA), ethylene (Eth) and abscisic acid (ABA). In addition, based on both transient overexpression and gene suppression of VqMAPKKK38 in grapevine leaves, we found that VqMAPKKK38 positively regulates stilbene synthase transcription and stilbene accumulation probably by mediating the activation of the transcription factor MYB14. In addition, both hydrogen peroxide (H2O2) and calcium influx activated VqMAPKKK38 expression and stilbene biosynthesis, which suggests that VqMAPKKK38 may be involved in the calcium signaling and ROS signaling pathways.

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.