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

Abstract 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.


Introduction
Anthocyanins, a group of secondary metabolites distributed widely in the plant kingdom, play essential roles in plant growth and development, including coloration [1], alluring pollinators and seed dispersers [2], and acting as antioxidants or signaling molecules to protect plants from abiotic and biotic stresses [3][4][5].Because of these multiple biological functions, anthocyanins are often referred to as 'nature's Swiss army knife' [6].
In addition to internal factors, anthocyanin production is also mediated by diverse environmental stimuli, including temperature, drought, pathogens, nutrients, and wounding.It has been previously proven that cold and drought stresses can boost the accumulation of anthocyanins to reduce damage [34,35].It has also been reported that grapevine VabHLH137 contributes to defense-induced proanthocyanidin and anthocyanin synthesis to enhance tolerance to Colletotrichum gloeosporioides [36].Other studies have shown that double-knockout mutant pap1-D/fls1 plants accumulate large amounts of anthocyanins and exhibit improved resistance to osmotic stress [37].Moreover, the anthocyanin content was greatly increased in rust-infected symptomatic apple tissue, and the transcription levels of McCHS,McDFR,McANS,McFLS,McMYB10,McNCED,and McLOX were also upregulated [38].Furthermore, cold stress strongly induces anthocyanin production in purple head Chinese cabbage by triggering the upregulation of BrMYB2 and BrTT8, which are crucial for the co-activation of the expression of anthocyanin structural genes [39].However, there are few reports on wounding-regulated anthocyanin biosynthesis.
WRKY proteins are plant-specific TFs containing one highly conserved WRKY DNA binding domain (WRKYGQK) in their Nterminal sequence.WRKY TFs can selectively identify and bind to the W-box (TTGACC/T) elements in the promoter region of downstream genes to regulate their transcription [40].The WRKY TF family is one of the largest families of regulatory proteins, and thus far, 70 members have been found in Arabidopsis [41], 104 members in rice [42], and 59 members in grapevine [43,44].WRKY TFs have been reported to play important roles in plant growth and metabolism, including seed germination [45], leaf senescence [46], stress responses [47], and anthocyanin biosynthesis [48,49].For instance, the WRKY proteins Arabidopsis TTG2 and Petunia PH3 can regulate f lavonoid synthesis through their interactions with the MBW complex [48,50], and VvWRKY26, a homologous gene of AtTTG2 and PhPH3, functions as a positive modulator in f lavonoid accumulation in grape berries [51].Arabidopsis AtWRKY75, apple MdWRKY41, and Brassica napus BnWRKY41-1 act as negative modulators in anthocyanin accumulation [49,52,53].PyWRKY26 is reported to interact with PybHLH3 to co-target the PyMYB114 promoter, thereby contributing to anthocyanin deposition in red-skinned pear [54].In addition, heterodimer formation between VvWRKY40 and VvMYB15 synergistically promoted anthocyanin biosynthesis in grape fruits by enhancing each other's activation on the VvF3 5 H and VvUFGT transcription [55].However, little has been reported on how WRKY TFs respond to abiotic stress-induced anthocyanin accumulation.
In the present study, we identified that VvWRKY5, a woundingresponsive gene, can greatly promote wounding-mediated anthocyanin synthesis in a VvMYBA1-dependent manner in grape.On the one hand, VvWRKY5 interacted with VvMYBA1, a key promoter of anthocyanin synthesis, and enhanced VvMYBA1 binding to its target genes, enhancing anthocyanin accumulation.On the other hand, VvWRKY5 could promote the biosynthesis of JA, a phytohormone that acts as a positive modulator of anthocyanin accumulation, by directly activating the transcription of VvLOX, and this activation was enhanced by the VvWRKY5-VvMYBA1 protein complex.Altogether, our findings provide novel insights into the functions of WRKY TFs in wounding-induced anthocyanin synthesis.

VvMYBA1 plays a positive role in wounding-induced anthocyanin production in grape
To evaluate the impact of wounding on anthocyanin production in grape fruits, we picked 'Red Globe' grapes at 110 days after full bloom (DAFB), produced wounds using a wooden stick, and incubated them under light conditions.After 5 days of wounding treatment, an even redder color was observed around the wounding sites (Fig. 1a).In addition, a higher anthocyanin content was detected in wounding-treated grape fruits than in the nonwounding treatment controls (Fig. 1b).This finding indicates that wounding stress promotes anthocyanin synthesis in grape fruits and is consistent with previous studies [5,56,57].
VvMYBA1 is well known to be a positive modulator of anthocyanin synthesis in grape, with a crucial role in grape berry skin coloring [18,58].To evaluate the possible contribution of VvMYBA1 to wounding-promoted anthocyanin accumulation, a VvMYBA1-TRV (virus-mediated VvMYBA1 inhibition) recombinant plasmid was constructed for Agrobacterium-mediated transient injection assays (Fig. S1, see online supplementary material), with the empty vector TRV serving as the control.After 5 days of wounding treatment, we discovered that woundinginduced anthocyanin production was dramatically reduced in the VvMYBA1-TRV grape fruits compared to the controls (Fig. 1c and d), demonstrating that VvMYBA1 plays a positive function in the anthocyanin accumulation induced by wounding treatment.

VvWRKY5 directly interacts with VvMYBA1
WRKY-MYB module plays a crucial role in anthocyanin synthesis [53,59].To search for the WRKY genes in response to wounding stress in grape, we examined the expression levels of 20 WRKY genes in wounded grape fruits by RT-qPCR analysis.The results showed that three WRKY genes (VvWRKY5, VvWRKY13, and VvWRKY24) were significantly induced (>3-fold) by wounding stress (Fig. S2, see online supplementary material).To explore whether these three WRKY genes are involved in VvMYBA1mediated anthocyanin accumulation under wounding treatment, we performed yeast two-hybrid (Y2H) assays.The VvMYBA1 gene was inserted into the pGADT7 vector, whereas the VvWRKY5 N (VvWRKY5 without the self-activating fragment), VvWRKY13, and VvWRKY24 were incorporated into the pGBKT7 vectors.Yeast cells were transformed with the recombinant plasmids and grown at 30 • C on SD (−T/−L) medium and SD (−T/−L/−H/−A) medium.According to the Y2H results, the yeast cells cotransformed with VvMYBA1-AD and VvWRKY5 N -BD exhibited normal growth on SD (−T/−L/−H/−A) medium (Fig. 2a), while yeast cells co-transformed with VvMYBA1-AD/VvWRKY13-BD and VvMYBA1-AD/VvWRKY24-BD could not grow normally (Fig. S3, see online supplementary material), indicating that VvWRKY5 interacts with VvMYBA1 in yeast cells.Subsequently, GST pull-down assays were performed using the VvWRKY5-HIS and VvMYBA1-GST fusion proteins.We found that the fusion protein VvMYBA1-GST could be pulled down by the VvWRKY5-HIS fusion protein, while the GST protein itself could not be pulled down (Fig. 2b), indicating that VvWRKY5 interacts with VvMYBA1 in vitro.Next, luciferase complementation imaging (LCI) experiments were carried out in Nicotiana benthamiana Control, 'Red Globe' grape fruits without wounding; Wounding, the equatorial side of 'Red Globe' grape fruits was treated with a wooden stick, and the fruit stalk was wrapped with absorbent cotton to reduce water loss.Scale bar, 1 cm.b Total anthocyanin content in wounded grape fruits.c Effect of silencing VvMYBA1 on wounding-induced anthocyanin accumulation.TRV: 'Red Globe' grape fruits were injected with an empty TRV before being treated with a wooden stick; VvMYBA1-TRV: 'Red Globe' grape fruits were injected with VvMYBA1-TRV before being treated with a wooden stick.Scale bar, 1 cm.d Total anthocyanin content in TRV and VvMYBA1-TRV 'Red Globe' grape fruits after wounding treatment.Data are means ± SDs of three separate experiments.Statistical significance at P < 0.01 ( * * ) was detected using Tukey's test.
to prove the interaction of VvWRKY5 and VvMYBA1.As the results show in Fig. 2c, the f luorescence signal produced by VvWRKY5-cLUC+VvMYBA1-nLUC co-transformation in N. benthamiana leaves was stronger compared with that produced by the controls (cLUC+nLUC, cLUC+VvMYBA1-nLUC, and VvWRKY5-cLUC+nLUC).Finally, in vivo bimolecular f luorescence complementation (BiFC) assays illustrated that the simultaneous expression of VvWRKY5-YFP N and VvMYBA1-YFP C generated a stable YFP f luorescence signal in the onion nucleus, while no YFP signal was observed in the controls (Fig. 2d), further confirming the interaction between VvWRKY5 and VvMYBA1.

VvWRKY5 is responsive to wounding stress
To understand how VvWRKY5 responds to wounding stress, we next monitored the sustained expression pattern of VvWRKY5 in grape fruits following wounding treatment.RT-qPCR analysis showed that VvWRKY5 expression was up-regulated upon wounding treatment, with peak transcription levels detected at 4 d, and subsequently maintained a high expression level (Fig. 3a).Interestingly, we discovered that the expression of VvMYBA1 and its target gene VvUFGT was also up-regulated after wounding stress (Fig. 3a).The anthocyanin content near the wounding site also continuously increased until the end of the experiment (Fig. 3b).In addition, the correlation analysis showed that VvWRKY5 expression was highly correlated with the total anthocyanin content (the correlation coefficient was 0.8582; Fig. 3c).Collectively, these findings imply that VvWRKY5 may be involved in wounding-induced anthocyanin synthesis.

VvWRKY5 contributes to wounding-induced anthocyanin synthesis in a VvMYBA1-dependent manner
Given that VvWRKY5 was responsive to wounding treatment and that its protein could interact with VvMYBA1, a positive modulator of wounding-promoted anthocyanin synthesis, we predicted that VvWRKY5 might be associated with wounding-promoted anthocyanin synthesis.To verify this hypothesis, transient injection assays were performed in 'Red Globe' grape fruits (110 DAFB).The VvWRKY5-pRI (VvWRKY5 overexpression) and VvWRKY5-TRV (virus-induced VvWRKY5 suppression) recombinant vectors were constructed and injected into grape fruits by Agrobacteriummediated genetic transformation before being treated with a wooden stick.Meanwhile, empty vectors pRI and TRV were utilized as controls.The findings revealed that VvWRKY5 overexpression significantly promoted wounding-induced anthocyanin biosynthesis; conversely, VvWRKY5 inhibition reduced wounding-induced anthocyanin synthesis (Fig. 4a and b), which suggested that VvWRKY5 plays a positive role in woundinginduced anthocyanin synthesis.In addition, we observed that the expressions of critical genes associated with anthocyanin synthesis (VvCHS, VvCHI, VvF3H, VvDFR, VvANS, and VvUFGT) were also up-regulated in grape fruits with VvWRKY5 overexpression and down-regulated expression in fruits with VvWRKY5 suppression (Fig. 4c), indicating that VvWRKY5 may contribute to wounding-induced anthocyanin deposition by modulating the expression levels of genes associated with anthocyanin biosynthesis.
Interestingly, we observed that the suppression of VvMYBA1 partially reduced the promotive effect of VvWRKY5 on woundinginduced anthocyanin deposition (Fig. 4a and b).In addition, the up-regulated transcription levels of anthocyanin biosynthesisrelated genes became less significant in grapes with simultaneous overexpression of VvWRKY5 and silencing of VvMYBA1 (Fig. 4c).
To further reveal the connection between VvMYBA1 and VvWRKY5 in anthocyanin synthesis, we also created stable transgenic grape calli with suppressed VvMYBA1 expression in the background of VvWRKY5 overexpressed calli (VvWRKY5-OE + VvMYBA1-Anti).Anthocyanin accumulation experiments revealed that suppression of VvMYBA1 significantly reduced the effect of VvWRKY5 overexpression-promoted anthocyanin accumulation (Fig. 4d and e).In addition, the expression levels of the genes associated with anthocyanin synthesis were all significantly inhibited in VvWRKY5-OE + VvMYBA1-Anti grape calli compared to VvWRKY5-OE grape calli (Fig. 4f).Collectively, these results suggest that VvWRKY5 may promote wounding-induced anthocyanin deposition in a VvMYBA1-dependent manner.

VvWRKY5 enhances the transcriptional activity of VvMYBA1
As mentioned above, overexpression of VvWRKY5 dramatically up-regulated the expressions of structural genes associated with anthocyanin synthesis and slightly increased the expression of VvMYBA1 (Fig. 4c and f).Previous research has demonstrated that VvMYBA1 directly activates the transcription of VvUFGT to promote anthocyanin synthesis [18,60].However, the Y1H experimental results in this study indicated that VvWRKY5 did not bind directly to the promoters of VvMYBA1 and VvUFGT (Fig. S4, see online supplementary material).Considering the interaction between VvWRKY5 and VvMYBA1, we hypothesized that VvWRKY5 may carry out its regulatory function by affecting VvMYBA1's binding capacity to its target gene VvUFGT.To verify this speculation, we conducted electrophoretic mobility shift assays (EMSAs) using the fusion proteins VvWRKY5-HIS and VvMYBA1-HIS as well as the biotin-labeled VvUFGT probe containing the MBS element.The EMSAs showed that VvMYBA1 can directly bind to the VvUFGT probe (Fig. 5a, lane 3), while empty HIS (Fig. 5a, lane 1) and VvWRKY5 (Fig. 5a, lane 2) cannot.When the VvUFGT probe was mutated with two nucleotides, the shifted band disappeared (Fig. 5a, lane 7).Importantly, as more amounts of the VvWRKY5-HIS fusion protein were added, the binding affinity of the VvMYBA1-HIS fusion protein and VvUFGT probe was gradually increased (Fig. 5a, lanes 4-6), indicating that VvWRKY5

JA promotes anthocyanin synthesis in grape
JA has been reported to be actively involved in the modulation of plant anthocyanin accumulation [61,62].To investigate the effect of JA on anthocyanin biosynthesis in grape, 100 μM MeJA (a stable JA derivative) was used to treat 'Red Globe' grape fruits (110 DAFB).The treated grape fruits were then incubated under light conditions for 7 days.Compared with the control grapes, MeJA treatment significantly induced grape berry coloring, with increased anthocyanin content (Fig. 6a and b).Similarly, we found that MeJA treatment could promote anthocyanin synthesis in grape calli (Fig. 6c and d).In conclusion, these findings imply that JA contributes to anthocyanin accumulation in grape.
Our previous studies revealed that overexpression of VvWRKY5 boosted endogenous JA and JA-Ile levels in grape calli [63].Therefore, we speculate that VvWRKY5 may also promote wounding-induced anthocyanin deposition by enhancing the increased endogenous JA content.Next, we looked at the expression of genes involved in JA biosynthesis (VvLOX, VvAOS, VvOPR3, VvOPCL1, and VvAOC) in WT control and VvWRKY5-OE calli.We found that compared with the WT calli, the mRNA levels of JA biosynthesis genes were all significantly elevated in VvWRKY5-OE grape calli, especially VvLOX and VvOPR3 (Fig. 6e).

VvWRKY5 binds to the VvLOX promoter to promote its transcription
To further investigate the molecular mechanism by which VvWRKY5 regulates JA synthesis, the interactions between VvWRKY5 and the promoters of VvLOX and VvOPR3 were analysed using yeast one-hybrid (Y1H) experiments.Our findings showed that Y187 yeast cells co-transformed with VvWRKY5-AD and proVvLOX-pHIS2 grew normally in SD (−T/−L/−H) medium containing 160 mM 3-AT (Fig. 7a), indicating that VvWRKY5 could interact with the promoter of VvLOX.However, VvWRKY5 could not bind to the promoter of VvOPR3 (Fig. S5, see online supplementary material).WRKY TFs specifically recognize W-box elements in the promoter sequences of their target genes [64].In this work, we identified only one W-box element (−540 bp) in the VvLOX promoter using PlantCARE analysis.Then, we designed EMSA probes using the promoter segment containing this W-box element.The results of EMSAs confirmed that VvWRKY5 could bind to the VvLOX promoter (Fig. 7b, lane 2), and this binding gradually weakened or even completely suppressed as the concentration of competitor probes increases (Fig. 7b, lanes 3-5).In addition, when the VvLOX probe was mutated with two nucleotides, the shifted band disappeared (Fig. 7b, lane 6).Subsequently, we performed chromatin immunoprecipitation PCR (ChIP-PCR) experiments to test the binding of VvWRKY5 to the VvLOX promoter in grape calli.According to the ChIP-PCR results, the promoter fragment of VvLOX was highly enriched in VvWRKY5-OE grape calli (Fig. 7c), ref lecting the in vivo interaction of VvWRKY5 to the VvLOX promoter.Collectively, these outcomes showed that VvWRKY5 specifically binds the W-box motif in the VvLOX promoter.

Discussion
Wounding is one of the most important stresses on fruits and can lead to deterioration of grape quality.In particular, hightemperature and high-humidity environments are more likely to cause bacterial invasion in the wound area.Solving the problem of grape fruit wounding damage can effectively extend the storage shelf life of grapes and improve their commodity value.Anthocyanins are a type of plant secondary metabolite that plays an important function in protecting plants from external stresses [65,66].Numerous studies have demonstrated that anthocyanin biosynthesis is greatly increased when plants are subjected to environmental stresses [34], including cold stress [67,68], salt stress [69,70], drought stress [71,72], and wounding stress [73,74].In apple, wounding treatment of apple fruits leads to more anthocyanin accumulation [59].In Rosa rugosa, anthocyanin content increased in wounded petals [5].In this study, we found that more anthocyanin accumulated around the wounding sites of grape fruits (Fig. 1a and b), indicating that wounding can induce anthocyanin biosynthesis in grape.However, the specific molecular mechanism underlying wounding-induced anthocyanin production is unclear.
The MBW protein complex is important for modulating anthocyanin synthesis [12,75].Many environmental factors that modulate anthocyanin accumulation depend on the MBW complex [13].In previous studies, VvMYBA1 has been confirmed as a positive modulator in anthocyanin production and fruit coloration in grapes through directly promoting the transcription of the structural gene VvUFGT [18,60].In the current study, we observed that VvMYBA1 expression was up-regulated under wounding treatment (Fig. 3a), and wounding promoted anthocyanin accumula-tion in grape in a VvMYBA1-dependent manner (Fig. 1), which further proved the essential role of VvMYBA1 in anthocyanin synthesis as well as in wounding-induced anthocyanin deposition.
The coordinated actions of multiple TFs are important for regulating plant growth and regulation, and MYB TFs can collaborate with other TFs to co-modulate downstream gene transcription.For example, in apple, MdMYB63 and MdERF106 interact to increase the promoter activity of MdSOS1, thereby improving salt tolerance [76].MdMYB10 and MdbHLH3 interact to co-modulate anthocyanin accumulation in fruit skin, and MdMYBPA1 and MdbHLH33 interact to enhance anthocyanin biosynthesis in low-temperature environments [14,77].In loquat, EjMYB1/EjMYB2 have been demonstrated to work together with EjAP2-1 to promote the synthesis of lignification [78].In our study, we verified that VvMYBA1 could interact with VvWRKY5 by four protein interaction assays (Fig. 2).In addition, the interplay between VvWRKY5 and VvMYBA1 synergistically increased the transcriptional activation of VvMYBA1 on its target gene VvUFGT (Fig. 5).Furthermore, expression analysis showed that VvWRKY5 transcription was dramatically induced under wounding treatment (Fig. 3a), which ultimately results in further increases of anthocyanin content in wounded grapes.Our observation of this WRKY-MYB complex is similar to previous findings in apple that MdWRKY40 and MdMYB1 collaborate to facilitate the binding of MdMYB1 to its targets MdDFR/MdUFGT promoters [59].It is worth noting that VvMYBA1 and VvMYBA2 are considered as a part of a single haplotype (allele), as they are densely packed in a single locus, which is known as the berry color locus [60].Therefore, whether VvMYBA2 is involved in wounding-induced anthocyanin production and whether the VvWRKY5-VvMYBAs regulatory module is also applicable to VvMYBA2 will be our next research focus.
WRKY TFs play pivotal roles in various plant physiological events, including stress responses and anthocyanin biosynthesis [79,80].In our previous studies, we demonstrated that overexpression of VvWRKY5 improved white rot resistance in grape fruits and calli [63].In this study, we found that VvWRKY5 also works as a positive modulator in wounding-promoted anthocyanin synthesis, which expands functional research on the WRKY5 protein in grape.
Given our previous findings that the JA and JA-Ile contents were dramatically enhanced in VvWRKY5-overexpressing gape calli relative to the WT [63], we speculate that VvWRKY5 may also modulate wounding-induced anthocyanin biosynthesis by promoting JA biosynthesis.Notably, we found that exogenous JA treatment contributes to anthocyanin accumulation in grape (Fig. 6), which corresponds with prior studies in Arabidopsis [62], apple [81], and pear [82].To date, there have been many reports on the molecular mechanism of JA signaling-modulated anthocyanin production.In Arabidopsis, the JA signaling receptor COI1 seems to be a critical factor in the expressions of the JA-mediated regulators (PAP1 and PAP2) and JA-induced anthocyanin synthesis.In addition, JAZ proteins are regarded as negative modulators of the JA response and anthocyanin biosynthesis.JA stimulates the degradation of JAZ proteins to release MYC2, thereby promoting anthocyanin biosynthesis [83].Although the molecular mechanisms for the induction of anthocyanin synthesis by JA signaling have made great progress, the molecular regulatory networks of JA biosynthesis, especially how JA biosynthesis can be induced by wounding, remain largely unclear.Our comprehensive biochemical assays proved that wounding can greatly induce the transcription of VvWRKY5 (Fig. 3a), and VvWRKY5 then binds directly to the VvLOX promoter to increase its activity, thereby promoting JA biosynthesis near the wounding site (Fig. 7).In addition, the interaction between VvWRKY5 and VvMYBA1 resulted in higher activation of the VvLOX promoter by VvWRKY5, indicating that the WRKY-MYB module is also critical in JA-induced anthocyanin biosynthesis.Interestingly, cis-element analysis revealed that both VvWRKY5 and VvMYBA1 promoters contained MeJA responsive elements (Fig. S6, see online supplementary material), and the expression levels of VvWRKY5 and VvMYBA1 were significantly induced after MeJA treatment (Fig. S7, see online supplementary material), indicating a possible feedback loop in wounding-induced anthocyanin.Wounding induced the biosynthesis of MeJA, and MeJA induced the expression of VvWRKY5 and VvMYBA1.Furthermore, VvWRKY5 also promote the biosynthesis of MeJA.
Our data provide new insight into the possible mechanism of wounding-induced anthocyanin accumulation (Fig. 8).In short, VvWRKY5 is a positive modulator of wounding-induced anthocyanin production.In the presence of wounding, the expression level of VvWRKY5 is elevated.On the one hand, VvWRKY5 interacts with VvMYBA1 and enhances VvMYBA1 activation on the VvUFGT promoter, thereby promoting anthocyanin synthesis.On the other hand, VvWRKY5 promotes the synthesis of JA, a phytohormone that functions as a positive modulator in the accumulation of grape anthocyanins, by directly linking to the VvLOX promoter to induce its expression, and this activation is also enhanced by VvMYBA1.Given the importance of anthocyanin in protecting plants from external stresses [65,66] and the extensive stress responses of VvWRKY5, we speculate that VvWRKY5 may also be involved in the modulation of other stress responses, which requires further investigation in the future.Collectively, our findings contribute to a better understanding of the function of VvWRKY5 in wounding-induced anthocyanin production in grape.

Conclusion
VvWRKY5 plays a positive regulatory role in grape woundinginduced anthocyanin synthesis.We found that VvWRKY5 can interact with VvMYBA1 to enhance VvMYBA1's activation of VvUFGT promoter, thereby promoting anthocyanin accumulation.In addition, VvWRKY5 can promote JA biosynthesis by directly binding to the VvLOX promoter to induce its expression, while VvMYBA1 can also enhance this activation.This study enriched the transcriptional regulation mechanism of wounding-induced anthocyanin accumulation.

Plant materials, growth conditions, and treatments
The grape cultivar 'Red Globe' fruits were harvested at 110 DAFB from an orchard at Shenyang Agricultural University, Shenyang city, Liaoning Province, China, and utilized for the wounding and MeJA treatments.For the wounding treatment, the equatorial side of 'Red Globe' grape fruits was treated with a wooden stick, the fruit stalk was wrapped with absorbent cotton to reduce water loss, and the wounding-treated grape fruits were incubated in an incubator under constant light conditions (3000 lux light intensity, 22 • C).Samples of the grape skins surrounding the wounds were taken for the anthocyanin determination and gene expression analysis.For the MeJA treatments, grape fruits were immersed with 100 μM MeJA solution for 1 min, dried naturally, and then incubated in an incubator with constant light.The anthocyanin determination and gene expression analysis were conducted after 7 days of MeJA treatment.
The tobacco (N.benthamiana) utilized for transient expression experiments was grown in an incubator at 25 • C with a period of 16 h light/8 h dark.

Grape fruit transient transformation
The coding sequence (CDS) of VvWRKY5 was ligated into the pRI101-AN vector to obtain the VvWRKY5-pRI overexpression vector, whilst the 393-bp VvWRK5 CDS fragment and 390-bp VvMYBA1 CDS fragment were cloned into the pTRV2 vectors to obtain the VvWRKY5-TRV and VvMYBA1-TRV suppression expression vectors.Agrobacterium tumefaciens EHA105 carrying the above recombinant constructs was used to inject 'Red Globe' grape fruits (110 DAFB) in different combinations.The infected grapes were incubated in an incubator under constant light conditions for 5 days at 22 • C. Each treatment had three biological replicates, and 20 injected grape berries were used as one biological replicate.The relevant primers are listed in Table S1 (see online supplementary material).

Stable genetic transformation of grape calli
VvWRKY5-OE grape calli were obtained as previously described [63].The VvWRKY5-pRI plasmid was transferred into A. tumefaciens EHA105 via the freeze-thaw method.Two-week-old grape calli were incubated with plasmid-transformed A. tumefaciens at room temperature for 30 min.Then, the infected calli was transferred to antibiotic-free B5 medium for 2 days in the dark at room temperature.Finally, they were plated on B5 medium containing 40 mg/L paromomycin sulphate and 300 mg/L cefotaxime sodium in the light to select for transformants.VvWRKY5-OE/VvMYBA1-Anti grape calli were obtained by introducing the VvMYBA1-pFGC1008 recombinant vector into VvWRKY5-OE grape calli.The positive transgenic grape calli were detected by RT-qPCR and cultured in a phytotron under constant light conditions.The relevant primers are listed in Table S1 (see online supplementary material).

Determination of total anthocyanin content
The methanol-HCl method was utilized for total anthocyanin extraction in grape [85].Powdered grape peel or calli samples (0.2 g) were combined with 5 mL methanol containing 0.1% (v/v) HCl overnight in darkness at 4 • C. The determination of total anthocyanin content in samples was accomplished using the pH differential method [86].The absorbance of extracts from the samples was recorded at 520 and 700 nm utilizing a spectrophotometer (UV-2450, Kyoto, Japan).The results were displayed as milligram cyanidin-3-glucoside equivalents/100 g FW using a molecular weight of 449.2 and a molar absorptivity of 26 900.All experiments were repeated in triplicate.

RT-qPCR assay
Total RNA was gathered from about 15 representative grape berries and reversed into single-stranded DNA as described previously [87].RT-qPCR was conducted with the UltraSYBR Mixture on a 7500 Real-Time PCR equipment (Applied Biosystems, Foster City, CA, USA).Three biological and technical replicates were used for every sample.VvActin (XM_002278316.4) was served as an endogenous standard.The 2 -ΔΔCt approach was utilized to determine the level of gene expression [88].The relevant primers utilized are provided in Table S2 (see online supplementary material).

Y2H assays
The VvMYBA1 coding region was ligated to the activation domain vector pGADT7, while the N-terminal of the VvWRKY5 coding region was inserted into the DNA-binding domain vector pGBKT7.The recombinant vectors VvMYBA1-AD and VvWRKY5 N -BD were co-transferred into the Y2H yeast cells.Transferred yeast strains were grown on SD selection medium (−T/−L/−H/−A) containing X-α-gal and cultured in a 30 • C incubator for 3 days to investigate their interactions.The relevant primers utilized are provided in Table S1 (see online supplementary material).

Pull-down assays
We fused the full-length VvWRKY5 and VvMYBA1 to the pET-32a and pGEX-4 T-1 vectors, respectively.The VvWRKY5-HISand VvMYBA1-GST-fused proteins were produced in BL21 cells (TransGen, Beijing, China).Proteins were purified employing a commercial kit (CWbio, Beijing, China) following the protocols provided by the manufacturer.Western blot (WB) tests were conducted to examine the pulled-down products with GST and HIS antibodies.Relevant primers utilized are provided in Table S1 (see online supplementary material).

LCI assays
For LCI assays, the VvMYBA1 CDS was ligated into the pCAMBIA 1300-nLUC vector, while the VvWRKY5 CDS was introduced into the pCAMBIA1300-cLUC vector.These recombinant constructions of VvWRKY5-cLUC and VvMYBA1-nLUC were translated into A. tumefaciens GV3101 cells.Then, N. benthamiana leaves were infiltrated with an Agrobacterium mixture (OD 600 = 1) carrying the recombinant plasmids.Two days after infiltration, luciferase signaling was detected by a Tanon-5200 plant visualization device (Shanghai, China).The relevant primers utilized are provided in Table S1 (see online supplementary material).

BiFC assays
The codon-free VvWRKY5 CDS was connected to the 35S-pSPYNEY-YFP vector, and the complete VvMYBA1 CDS was connected to the 35S-pSPYCEY-YFP vector.Thereafter, we introduced the recombinant plasmids VvWRKY5-YFP N and VvMYBA1-YFP C into A. tumefaciens strain GV3101.The onion epidermal cells were incubated with Agrobacterium suspension (OD 600 = 1) carrying

Figure 1 .
Figure 1.Role of VvMYBA1 in wounding-induced anthocyanin synthesis.a Effects of wounding treatment on anthocyanin production in grape fruits.Control, 'Red Globe' grape fruits without wounding; Wounding, the equatorial side of 'Red Globe' grape fruits was treated with a wooden stick, and the fruit stalk was wrapped with absorbent cotton to reduce water loss.Scale bar, 1 cm.b Total anthocyanin content in wounded grape fruits.c Effect of silencing VvMYBA1 on wounding-induced anthocyanin accumulation.TRV: 'Red Globe' grape fruits were injected with an empty TRV before being treated with a wooden stick; VvMYBA1-TRV: 'Red Globe' grape fruits were injected with VvMYBA1-TRV before being treated with a wooden stick.Scale bar, 1 cm.d Total anthocyanin content in TRV and VvMYBA1-TRV 'Red Globe' grape fruits after wounding treatment.Data are means ± SDs of three separate experiments.Statistical significance at P < 0.01 ( * * ) was detected using Tukey's test.

Figure 2 .
Figure 2. Interaction of VvWRKY5 with VvMYBA1.a Interaction of VvWRKY5 N (1-201 aa) with VvMYBA1 in Y2H assays.The negative control (NC) was the co-transformation of AD and BD.b Confirmation of the connection between VvWRKY5 and VvMYBA1 by pull-down assays.The protein band emphasized by the GST antibody illustrates that VvWRKY5-HIS pulled down VvMYBA1-GST.c Interaction of VvWRKY5 with VvMYBA1 in LCI assays.Agrobacterium strains GV3101 harboring VvWRKY5-cLUC and VvMYBA1-nLUC constructs were immersed into N. benthamiana leaves.After incubation in the dark for 3 days, the f luorescence signal was detected.d VvWRKY5 and VvMYBA1 interaction in a BiFC assay.The scale bar represents 20 μm.

Figure 3 .
Figure 3. Expression profiles of VvWRKY5 under wounding treatment.a RT-qPCR analysis of the expression patterns of VvWRKY5, VvMYBA1, and VvUFGT in grape fruits at 0, 1, 2, 3, 4, and 5 days after wounding treatment.VvActin was utilized as an internal control gene.Control, 'Red Globe' grape fruits without wounding.b Total anthocyanin content in grape fruits at 0, 1, 2, 3, 4, and 5 days after wounding treatment.c Correlation analysis of the VvWRKY5 expression level and total anthocyanin content in grape fruits at 0, 1, 2, 3, 4, and 5 days after wounding treatment.Data are means ± SDs of 3 separate experiments.Statistical significance at P < 0.05 ( * ) and P < 0.01 ( * * ) was detected using Tukey's test.

Figure 5 .
Figure 5. VvWRKY5 promotes VvUFGT transcriptional activation via VvMYBA1.a EMSAs demonstrated that VvWRKY5 enhances the binding capacity of VvMYBA1 to the VvUFGT promoter.The character '+' denotes presence, while '−' denotes absence.b Diagram of the reporter structure harboring the VvUFGT promoter and the effector structures harboring VvWRKY5 and VvMYBA1 CDSs.c Based on the results of dual-luciferase reporter experiments, the VvWRKY5-VvMYBA1 interaction enhanced the transcriptional activation of VvMYBA1 on the VvUFGT promoter.Data are means ± SDs of three separate experiments.Different letters indicate statistical significance at P < 0.05 detected using Tukey's test.

Figure 6 .
Figure 6.The impacts of exogenous MeJA treatment on grape anthocyanin synthesis.a, b Phenotypes (a) and total anthocyanin content (b) of 'Red Globe' grape fruits treated with 100 μM MeJA for 7 days.Control, grape fruits without MeJA treatment.Scale bar, 1 cm.c, d Phenotypes (c) and total anthocyanin content (d) of 'Gamay' grape calli cultured on a B5 media containing 100 μM MeJA.Control, grape calli without MeJA treatment.Scale bar, 1 cm.e Expressions of JA synthesis genes in VvWRKY5-OE grape calli.Data are means ± SDs of three separate experiments.Statistical significance at P < 0.01 ( * * ) was detected using Tukey's test.

Figure 7 .
Figure 7. VvWRKY5 interacts with the promoter of VvLOX and activates its transcription.a Y1H experiments demonstrated the interaction between VvWRKY5 and the VvLOX promoter.The AD vector served as the NC.The optimal concentration of 3-AT was 160 mM.b EMSAs revealing that VvWRKY5 binds to the W-box site in the VvLOX promoter.The characters '+' and '−' represent presence and absence, respectively.As the competitive probe, a 200-fold excess of the non-labeled probe was used.c ChIP-PCR results illustrated that VvWRKY5 interacted with the VvLOX promoter in vivo.Anti-GFP antibody was utilized to precipitate cross-linked chromatin samples obtained from VvWRKY5-GFP-overexpressed grape calli.The GFP-overexpressed grape calli served as the NC.d Diagram of the reporter structure harboring the VvLOX promoter and the effector structures harboring VvWRKY5 and VvMYBA1 CDSs.e Dual-luciferase reporter experiments suggested that VvWRKY5 promotes the promoter activity of VvLOX, and this activation was enhanced by VvMYBA1.Data are means ± SDs of three separate experiments.Different letters indicate statistical significance at P < 0.05 detected using Tukey's test.

Figure 8 .
Figure 8.A possible model for the function of VvWRKY5 in wounding-induced anthocyanin deposition in grape.On the one hand, wounding induces the transcription of VvWRKY5, which protein combines with VvMYBA1 to amplify the transcriptional activation of VvMYBA1 on the VvUFGT promoter, thereby facilitating wounding-mediated anthocyanin synthesis.On the other hand, VvWRKY5 directly activates the transcription of VvLOX to boost the synthesis of JA, a phytohormone that plays a promotive role in grape anthocyanin production.JA, jasmonic acid.