Identication of Ubiquitin Ligase From Grapevine Ring C3H2C3E3 and Characterization of Drought Resistance Function of VyRCHC114

Background: RING is one of the largest E3 ubiquitin ligase families and C3H2C3 type is the largest subfamily of RING, playing an important role in plants’ development and growth and their biotic and abiotic stress responses. Results: A total of 143 RING C3H2C3-type genes (RCHCs) were discovered from the grapevine genome and separated into groups (I-XI) according to their phylogenetic analysis, with these genes named according to their positions on chromosomes. Gene replication analysis showed that tandem duplications play a predominant role in the expansion of VyRCHCs family together. Structural analysis showed that most VyRCHCs(67.13%) had no more than 2 introns, while genes clustered together based on phylogenetic trees had similar motifs and evolutionarily conserved structures. Cis-acting element analysis showed the diversity of VyRCHCs regulation. The expression proles of eight DEGs in RNA-Seq after drought stress were similar to those in qRT-PCR analysis. The in vitro ubiquitin experiment showed that VyRCHC114 had E3 ubiquitin ligase activity, overexpression of VyRCHC114 in Arabidopsis improved drought tolerance, moreover, the transgenic plant survival rate increased by 30%, accompanied by changing of electrolyte leakage, chlorophyll content and the activities of SOD, POD, APX and CAT were changed. AtCOR15a, AtRD29A, AtERD15 and AtP5CS1 were expressed quantitatively, the results showed that they participated in the drought stress response may be regulated by the expression of VyRCHC114. Conclusions: Valuable new information on the evolution of grapevine RCHCs and its relevance for studying the functional characteristics of grapevine VyRCHC114 genes under drought stress emerged from this research.


Background
To survive in a changing environment, post-translational modi cation of proteins often occurs when plants perceive and transmit internal or external signals. The acetylation, methylation, phosphorylation, and ubiquitination of proteins are the main types of post-translational modi cation, which play a key role in different plant development stages and plant-environment interactions. The process of classifying intracellular proteins under the action of a variety of special enzymes, and speci cally modifying the screened target proteins, is called ubiquitination [1]. In eukaryotic cells, the ubiquitin system is complex and mainly involves ubiquitin (a small molecule protein), an intact 26S proteasome, and ubiquitinactivating enzyme (E1), ubiquitin-binding enzyme (E2), and ubiquitin-ligase (E3) [2]. The inactivated ubiquitin-dependent ATP is rst activated by E1 through the thioester bond formed between the C-terminal of ubiquitin and the cysteine residue of E1; then the ubiquitin signal connected to E1 is transferred to the acetylcysteine of E2. In the next step, the ubiquitin linked to E2 is transferred directly or indirectly to the lysine residue of the target protein via E3. It is noteworthy that E3 ubiquitin ligase is the main factor which determines the speci c protein binding during ubiquitination [3], in that it can repeatedly add ubiquitin to the substrate protein, so that eventually the target protein is degraded by the 26S protease [4].
In recent years, mounting research has shown that the RING E3 ligase gene also gure prominently in abiotic stress responses of plants [5]. SpRing is a RING-type E3 ubiquitin ligase located in endoplasmic reticulum and participates in salt stress signal transmission in wild tomato variety Solanum pimpinellifolium 'PI365967'. In addition, SpRing is silenced by virus-induced gene silencing, resulting in increased sensitivity of wild tomato to salt stress. Overexpression of Arabidopsis thaliana in spring can improve its salt tolerance [6]. SDIR1 (SALT-AND DROUGHT-INDUCED REALLY INTERESTING NEW GENE FINGER1) is a RING-type E3 ubiquitin ligase that regulates the salt stress response and ABA signaling in Arabidopsis by degrading the target protein SDIRIP1 (SDIR1-INTERACTING PROTEIN1). The downstream transcription factor ABI5 (ABA-INSENSITIVE5) is regulated by SDIRIP1, and overexpression of ABI5 increases salt tolerance [7]. The E3 ubiquitin ligase OsHTAS (Oryza sativa HEAT TOLERANCE AT SEEDLING STAGE), which regulates the stomatal opening state in the leaves by regulating ROS homeostasis, thus improving the basal heat resistance of the leaves. It involves two pathways, aba dependent and dst-mediated [8]. In Arabidopsis, CHYR1 (CHYZINC-FINGERANDRINGPROTEIN1) encodes the RING-type E3 ubiquitin ligase which interacts with a related protein kinase KINASE2 (SnRK2) and can be phosphorylated by SnRK2.6 on its Thr-178 residues. When mediated by ABA, CHYR1 promotes the production of reactive oxygen species (ROS), stomatal closure, and drought tolerance in plants [9]. The capsicum annular E3 ubiquitin ligase, CaAIRF1 (Capsicum annuum ADIP1 INTERACTING RING FINGER PROTEIN1), can interact with protein phosphatase CaADIP1 and positively regulate ABA signaling pathway to improve drought tolerance [10]. In Zea mays, ZmXerico1 encodes a RING-type E3 ligase, which can regulate the stability of ABA8'-hydroxylase protein and thereby enable control of the dynamic balance of ABA; hence, expression of ZmXerico1 endows maize plants with ABA sensitivity and improves their water use e ciency under drought stress [11]. Furthermore, Arabidopsis AtAIRP1, AtAIRP2, AtAIRP3 and Capsicum annuum CaAIR1 jointly encode a E3 ubiquitin ligase, by regulating ABA signaling transduction to regulate drought responses, the expression of these genes increases ABA-mediated stomatal closure [12][13][14][15]. Collectively, the above studies suggest E3 ligase is crucial for responding to abiotic stress.
Grapevine (Vitis vinifera L.) is a major cash crop, whose cultivated varieties have a total worldwide output of nearly 70 million tons of the fruit berries from more 7 million hectares of harvested land [16]. This plant is mainly grown to produce table grapes, fruit juices, and wine [17]. Most grapevine producing areas in the world incur seasonal droughts. According to global climate modeling, droughts will intensify in the near future. Drought can adversely affect the growth and development of grapevines, because under drought stress the concentration of cytokinin in their stems decreases, vegetative and reproductive growth is inhibited [18]. When a grapevine is in full bloom, drought stress will also affect its pollination process, which decreases the fruit setting rate and affects the size of the individual fruit berries produced [19]. With worsening water shortages, drought stress may well become a key factor impacting grapevine and wine production worldwide [20]. Therefore, it is of great signi cance to grapevine production and breeding to study the drought resistance of wild grapevine plants as this could uncover the molecular mechanisms enabling them to withstand drought effects. Vitis yeshanensis is a wild grapevine plant native to arid areas of China, whose morphological characteristics indicate adaptability to arid environments in many aspects [21]. Several studies have shown that wild Vitis yeshanensis has stronger drought resistance than other cultivars [22,23].
Although the RING-type gene family has been found in more and more plant species, and its importance for plants' stress responses and growth and development increasingly recognized, RING-type genes have yet to be fully identi ed in grapevine. The RING type E3 ubiquitin ligase is reportedly involved in grapevine's stress and growth, but too few studies have investigated E3 ubiquitin ligase's involvement in the regulation of grapevine response to drought stress. This study aimed to characterize the RING-type E3 ubiquitin ligase in grapevine's genome and its relevance for drought stress. To do this, genome-wide identi cation of C3H2C3 genes, the largest subfamily of grapevine RING-type, was carried out, coupled to their phylogenetic analysis, gene structure analysis, chromosome mapping, gene replication analysis, and cis-acting element analysis in gene promoter regions. We also quanti ed the expression levels of these genes under simulated drought treatment, and obtained a group of DEGs. The VyRCHC114 gene was con rmed by RT-qPCR, and then the ubiquitin ligase activity of this gene veri ed. The functioning of this gene under drought conditions was elucidated using Arabidopsis transgenic plants. Our study provides an important basis for ubiquitin regulation of drought stress in grapevine.

Results
Genome-wide identi cation of RING C3H2C3 type nger proteins in grapevine The results of the Hidden Markov Model (HMM) were analyzed, and the gene sequences were extracted and given to SMART, CDD, and Pfam for domain authentication. From this, 143 VyRCHC genes were obtained by comparing and screening genes with eight conservative metal ligands, and the alignment members were not abandoned. The physicochemical properties of each of the 143 VyRCHCs were identi ed ( Table 1). The number of amino acids encoded by the 143 VyRCHCs ranged from 70 (VyRCHC50) to 763 (VyRCHC98). For these genes, the molecular weights of their products varied from 7.83 kDa to 83.58 kDa, while their isoelectric points varied from 3.88 to 9.95.

Analysis of VyRCHCs in the C3H2C3 domain
The typical RING domain is considered to be an octahedral group of metal-bound cysteine and its residues, which can chelate two zinc ions in a spherical cross-supported structure, in which the metal ligands 1 and 3, and 2 and 4, each bind to one zinc ion. This structure, however, requires a certain distance between adjacent metal ligands, it being variable between ml2 ~ ml3 and ml6 ~ ml7. We calculated statistics for this distance between adjacent metal ligands (Table S2). It was found that, except those between ml2 ~ ml3 and ml6 ~ ml7, the distances between other metal ligands were constant, while those from ml2 to ml3 spanned 11 to 24 amino acids, and for ml6 ~ ml7 the distance varied from 8 to 14 amino acids. The 143 VyRCHCs C3H2C3 domains have two amino acids between ml1 ~ ml2 and ml5 ~ ml6, while ml3 ~ ml4 contains one amino acid, ml7 ~ ml8 contains two amino acids as does ml4 ~ ml5. To understand whether these RING C3H2C3 structural domains are conserved apart from their eight special metal ligands, their comparative analysis was conducted (Fig. S1). This revealed that some amino acids in the structural domain of RING C3H2C3 have a typical position bias (Fig. 1a). In the C3H2C3 type RING region, the ml2 located in front of amino acid residues is the most common Ile (I) or Val (V); likewise, the phenylalanine (Phe, F) residue is typically before ml5, the leucine residue (Leu, L) is always next to ml2, and the aspartic acid (Asp, D) residue is usually positioned after ml6, while the tryptophan residue (Trp, W) is usually the fourth following ml6. Notably, a very conservative proline (P) was found situated after ml7. According to the RING-type C3H2H3 domain schematic diagram, two pairs of metal ligands bind to a zinc ion (Fig. 1b). The total amino acid length of the C3H2C3 domain per VyRCHC gene and the corresponding number of different lengths were calculated: the vast majority of these were 41 and 42, accounting for 88.8% of all genes (Fig. 1c).

Phylogenetic analysis of VyRCHCs
To infer the evolutionary relationships of grapevine's VyRCHCs, Phylogenetic analysis of RCHC protein sequences of Arabidopsis, tomato, and grapevine were constructed (using the Maximum Likelihood method). According to the phylogenetic analysis, these 180 genes could be divided into 6 subgroups: ~ (Fig. 2). Group has the least number of members, only 12, and the group with the largest number of members is group , while the RCHC gene of Arabidopsis thaliana or tomato is found in each group. It is worth noting that more RCHC genes of Arabidopsis thaliana and tomato are gathered in group . Most of the RING-type C3H2C3 genes of grapevine display some homology to RCHC genes of Arabidopsis or tomato. In addition, in different groups, some gene pairs showed high similarity, which were con rmed in the distance of evolutionary relationship, the location of RING conserved domain and the length of protein sequence. For instance, SlATL33 and VyRCHC62, SlATL46 and VyRCHC108, SlATL51 and VyRCHC110, AtBRH1 and VyRCHC116, AtRHA1A and VyRCHC13, AtSDIR1 and VyRCHC97, AtRHC1A and VyRCHC59 etc. Next, a phylogenetic tree containing only 143 VyRCHC protein sequences was constructed (using the NJ method). To facilitate their study and analysis, the 143 members were divided into 11 groups (I ~ XI) according to the classi cation and phylogenetic analysis of Fig. S2, from which 27 pairs of genes with high homology were found. Based on their color-coded names, the VyRCHCs were then divided into six groups according to the number of conserved amino acids in their protein sequence.

Characterization of the motifs and gene structure of VyRCHCs
To further understand the diversity in motif composition between VyRCHCs, the MeMe analysis of VyRCHC proteins from groups I to XI was carried out. From this, 12 conserved motifs were identi ed in the VyRCHC protein, respectively named motif 1 to motif 12 (Fig. 3b), in which motif 1 and motif 2 is found in almost every VyRCHCs, this motif combines to form the eight most important metal ligand (Cys-Cys-Cys-His-His-Cys-Cys-Cys) structures of every VyRCHC gene. Importantly, there are 13 such structures in some genes, such as PA, CUE, DUF1117, zinc_ribbon_9, and zf-CHY, among others. These structures domain could be relevant for the function of VyRCHCs. The sequence information of motif 1 ~ 12 is presented in Table 2 (motif data). We next analyzed the exons, introns, and several key structures of VyRCHCs ( Fig. 3c). Most VyRCHCs(67.13%) had no more than 2 introns, with a maximum of 19 introns in VyRCHC29 and none intron in 57 VyRCHCs (Fig. S3). The longest intron length was found in VyRCHC141.
According to the phylogenetic analysis of VyRCHCs (Fig. 3a), 45 pairs of genes can be found in the evolutionary tree. The results of the MeMe and gene structure analyses of these gene pairs were also similar ( Fig. 3b and Fig. 3c). For example, the conserved motifs in the protein sequences of VyRCHC44/64 are highly similar, and the gene's structure type and length are also similar, such as for VyRCHC94/95, VyRCHC38/97, VyRCHC18/78, VyRCHC28/67 and VyRCHC11/107, to name a few. Unexpectedly, the MeMe analysis of VyRCHC22/23/24, VyRCHC55/127, VyRCHC105/133, and VyRCHC13/116 gene pairs gave near identical results to those from the gene structure analysis, revealing a remarkably similar protein sequence length, gene structure length and the intron number among them.
We thus speculate these four gene pairs may perform similar functions in grapevine plants.

Chromosomal localization and gene replication analysis of VyRCHCs
According to the location of VyRCHCs in the grapevine genome, 143 VyRCHCs were placed on 20 chromosomes (Fig. 4a), albeit unevenly distributed among them. Imprinting of the VyRCHCs was found in each chromosome of grapevine, but the number of genes on different chromosomes varied. The most found were 12 VyRCHCs on chromosome 11, the 11 VyRCHCs were identi ed on chromosome 1,7,13 and 18. Further, we also observed that these most of these VyRCHCs are likely distributed at both ends of the chromosome, leaving only a small portion of them in its middle part. Gene replication events include tandem replication and segmental replication, both of which are very vital for expanding the number of members of the gene family. To clarify the ampli cation mechanism of VyRCHCs during their evolution, we studied their potential repetitive events of VyRCHCs. According to the intraspeci c alignment of 143 VyRCHCs, 9 pairs of genes, 7 and 2, were respectively identi ed as associated with tandem or segmental replication events. Among the 9 pairs of gene events, the tandem repeat frequency between chromosomes 1 was the highest, there were six tandem replication events, moreover, one pair of genes on chromosomes 3 identi ed as tandem replication genes, These results suggested that the main replication event mode of grapevine VyRCHCs family is via tandem replication; hence, it could have played a crucial role in the ampli cation of VyRCHCs during their evolutionary history.
To explore the selection of grapevine VyRCHCs in terms of their repetition and differentiation, the nonsynonymous (Ka), synonymous (Ks), and Ka/Ks of each duplicated VyRCHCs were calculated. Among the 9 pairs of repetitive genes in grapevine, the Ka/Ks values of one pair were all less than 0.5, while the average Ka/Ks value was 0.325. It is worth noting that 8 pairs had Ka/Ks values less than 0.5, indicating that most of the repeated grapevine VyRCHCs were under negative selection during evolution (Table 3). Figure 4b shows that grapevine, Arabidopsis, and tomato all retained similar RCHC genes in their evolutionary history. It is worth noting the absence of homologous genes with VyRCHC29 in tomato, but their presence in Arabidopsis, which may have arisen from gene deletions in the process of evolution, given that the same genes are VyRCHC11, VyRCHC38, VyRCHC107, VyRCHC119, and VyRCHC137. Nonetheless, two or more RCHC genes in Arabidopsis and tomato were found homologous to one VyRCHC gene; for example, VyRCHC89 and Solyc07g053850.3/Solyc12g005470.2 and AT4G28370/AT2G20650, as well as those of VyRCHC1, VyRCHC32, VyRCHC97, VyRCHC104, VyRCHC118, and VyRCHC142. Hence, these genes may be parallel gene pairs and the putative source of ampli cations of RCHC genes during evolution.

Cis-acting element analysis in VyRCHCs promoter
To further investigate the transcriptional regulation of VyRCHCs, cis-acting elements in the 2000 bp region upstream of the VyRCHCs' codon were predicted. The predicted cis-acting elements can be divided into seven categories according to their functions: namely, light response (32), hormone response (11), growth and development response (9), stress response (6), enhanced promoter cis-acting (6), binding site cis-acting (6), and other functional cis-acting (2) elements. Most promoters of grapevine VyRCHCs contained the CAAT-box or TATA-box, which are involved in the enhanced promoter cis-acting elements. In addition, 127 VyRCHCs promoters harbored the stress response element ARE, more than half of the promoters of the VyRCHCs having the hormone response elements ABRE, TGACG-motif, CGTCA-motif, and over half of the VyRCHCs also featured the G-box, GT1-motif, and Box 4 in their promoters (Table  S3). In the 2000 kb region upstream of VvRCHCs, discovered very many different functions of cis element, in addition to the common cis element with light response and enhanced the promoter, also found that the more growth and adversity stress related cis element, this suggests that VyRCHCs may widely participating in various life activities of plant.
It is known that the RING gene can play a key role in plants' growth and response to abiotic stresses.
Accordingly, the cis-acting elements related to abiotic stress, growth and hormone regulation were focused upon here. The respective locations of the ve major acting elements associated with hormone response, binding sites, growth and development, and stress of our concern, on the promoter of the VyRCHCs (Fig. S4a) were determined. To accurately identify the stress-related elements, we focused on four kinds (anaerobic induction, injury response, low temperature, drought response ( Fig. S4b), low temperature response, defense and stress response), whose locations are also depicted. In addition, we counted the number of major elements related to stress, growth and development, and hormone responses in the VyRCHC gene promoter (Fig. S4b). Evidently, concerning growth and development, the number of O2-sites is the largest, there are 5 promoters of VyRCHC6 and 4 promoters of VyRCHC40. In terms of stress, the number of ARE is very large, found in 89% of the VyRCHCs promoters, moreover, 5 of the most promoters of VyRCHC14 and VyRCHC81 occurred. In terms of hormone response, the number of ABRE is dominant, found in 64% of the VyRCHCs promoters, moreover, 9 of the most promoters of VyRCHC3 and VyRCHC16 occurred. Surprisingly, 22 of the VyRCHC74 gene promoters were found and 11 of the VyRCHC128 gene promoters were found. These results suggested that VyRCHCs may be associated with cis-acting elements of different functions; in other words, these genes may be regulated by these elements and thereby in uence related plant life activities.
Expression analysis of VyRCHCs in roots of two grapevine rootstocks with different drought sensitivity To investigate differential VyRCHCs' expression between plants having contrasting drought-resistant genes (101.14 vs. M4) under drought stress and their potential functioning, the grapevine RNA-Seq transcriptome database of the published dataset was used [24]. We checked the expression of 143 VyRCHCs; of them, 7 VyRCHCs were not expressed at any time, so we excluded them.
To understand the expression of these VyRCHCs under the drought treatment, we used the ratio of WS (Water Stress) to WW (Well-Watered) gene expression of the two genotypes to draw an expression heat map, expression values are reported as log2 of the fold change (WS/WW) fold change. (Fig. 5a), the differential multiple matrixes of these VyRCHCs is recorded in Table S4. However, more than 60% of the VyRCHCs in the two genotypes were highly expressed under the imposed drought. To screen out the key genes, in each time period of the treatment, the gene that conforms to |log2 (WS/WW)| > 1 is considered a differential gene, and the Venn diagram was made using the differentially screened genes of the droughttolerant genotype M4 at different times (Fig. 5b). By looking at the different genes in each period, there are nally 8 genes that are different in three periods. To robustly verify the gene expression levels, we quanti ed the expression levels of these 8 key genes (Fig. 6), whose pattern basically conformed to the trend shown in Fig. 5c. That is, the VyRCHC114 gene was signi cantly down-regulated at 2 days, with a strong downward trend through 4, 7, and 10 days of the drought treatment. The VyRCHC66, VyRCHC68, VyRCHC69 and VyRCHC95 genes had a similar expression trend, being slightly up-regulated at 2 days of drought, but strongly down-regulated at 4, 7, and 10 days thereafter. These results suggested eight key genes are probably involved in regulating the plant response to drought.
Identi cation of E3 ubiquitin ligase activity of VyRCHC114 To clarify whether VyRCHC114 has E3 ubiquitin ligase activity, we conducted an in vitro ubiquitin activity assay, achieved by using puri ed MBP-VyRCHC114 fusion protein mixed with ubiquitin, E1, and E2 and by western blotting with the MBP antibody. Ubiquitin molecules were detected on the fusion protein linked by MBP antibody (Fig. 7a). This same method was used to detect ubiquitin antibody tags. The VyRCHC114 protein was detected in the fusion protein linked by the ubiquitin antibody, which indicated it had E3 ligase activity.
We knew the RING-C3H2C3 type protein can form a RING structure for ubiquitin regulation, but this process depends on the interaction between the eight conserved metal ligands. To further illustrate whether and how E3 ligase activity of VyRCHC114 depends on these conserved metal ligands, as shown in Fig. 7c, we selected four different amino acid sites for mutation (two key conservative and two nonconservative metal ligand sites). Four corresponding proteins (C320S, C328S, H341A, N355A) were obtained, and their ubiquitin activity in vitro was tested by the same method. After the immuno-blotting analysis of MBP antibody and ubiquitin antibody, evidently the two mutant proteins C320S and H341A lost their E3 ubiquitin ligase activity due to mutations at key sites, but the two mutant proteins C328S and N355A maintained theirs (Fig. 7b). These results indicated these conserved metal ligand sites are crucial factors for demonstrating the VyRCHC114's ligase activity.
Overexpression of VyRCHC114 enhances Arabidopsis drought tolerance To clarify the effects of VyRCHC114's role in plant responses to drought, we selected transgenic Arabidopsis (OE #2, #5, #13) with high expression levels of the VyRCHC114 gene for subsequent experiments (Fig. 8b). After 15 days of drought imposed upon wild plants and transgenic plants, followed by normal watering for 6 days, phenotype observations revealed plants overexpressing VyRCHC114 had signi cantly improved the drought tolerance (Fig. 8a). Further, on average, more than 70% of the plants overexpressing VyRCHC114 survived the drought stress, which was signi cantly higher than the 30% survival of the EV-transformed group (Fig. 8c).
To understand the relationship between plant growth and drought resistance, electrolyte leakage rate ( Fig. 9a) and chlorophyll content (Fig. 9b) were both measured. These were similar between VyRCHC114overexpressed and EV-transformed plants in the non-stress treatment, but after 8 days of drought stress, the electrolyte permeability of the former was signi cantly lower than the latter's, while the chlorophyll content was signi cantly higher in overexpressing than EV-transformed plants. Additionally, the changes in photosynthesis under drought stress were further analyzed by measuring potential photosynthetic e ciency (Fig. 9c) and capacity storage capacity (Fig. 9d). Each was not signi cantly different between EV-transformed and VyRCHC114 overexpression plants when they were non-stressed; however, Fv/Fm was signi cantly higher in the latter than the former at 4 days, and especially at 7 days, of drought stress. At 4 days, energy storage capacity of VyRCHC114-overexpressed plants was not signi cantly different from that of EV-transformed plants, but at 7 days of drought stress, that of the former exceeded the latter.
Hence, these results suggested VyRCHC114 could enhance the drought resistance of plants by participating in the regulation of photosynthesis.
Moreover, much research has shown that antioxidant enzymes can in uence plants' drought tolerance. Common antioxidant enzymes are ascorbate peroxidase (APX), superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), so we examined their activity. As Fig. 10 shows, under non-stress conditions, the activity of these antioxidant enzymes was similar between the plants, whereas when drought stressed for 4 and 7 days, the activities of APX (Fig. 10a), SOD (Fig. 10b), POD (Fig. 10c) and CAT (Fig. 10d) were signi cantly higher in plants overexpressing VyRCHC114 than those EV-transformed. Taken together, these data indicate VyRCHC114 may also improve drought tolerance by elevating antioxidant enzyme activity.
AtCOR15a, AtERD15, AtP5CS1, and AtRD29A are known to be key genes for regulating plant responses to drought stress, so we quanti ed their expression under imposed drought. As expected, when non-stressed, there was no signi cant difference between plants overexpressing VyRCHC114 overexpression and those EV-transformed. By contrast, under drought stress, all four genes were signi cantly higher in VyRCHC114overexpressed plants than in those EV-transformed (Fig. 11).

Discussion
The RING C3H2C3 gene family has since been identi ed in many plant species [25][26][27][28]. Related studies have shown that RING genes are involved in a variety of biological processes, growth and development and hormonal responses, as well as plant responses to abiotic stresses [29]. However, for grapevine, the RING C3H2C3 gene had not yet been identi ed in its whole genome, with few reports available on its relevance for grapevine's growth and developmental regulation or response to abiotic stress. In our study, we analyzed the whole genome of grapevine for the RING C3H2C3 gene family members. Using the criteria of whether the eight conserved metal ligands are present, a total of 143 non-redundant RING C3H2C3 genes were thus identi ed. Studies have shown that grapevine's genome size is about 0.5 times that of tomato, containing 0.75 times as many genes as tomato [30,31]. According to the known RING C3H2C3genes in tomato, the genes account for 0.58 in grapevine, which lies between the multiples of genome length and the number of genes [26].
Many RING C3H2C3 of E3 ubiquitin ligases belong to the ATL gene family [32]. According to Arabidopsis and tomato RING C3H2C3 genes. we divided grapevine's RING C3H2C3 genes into six categories ( ~ ) (Fig. 2). Each group has Arabidopsis or tomato in the same branch. This shows that grapevine genes have sequence similarity with Arabidopsis thaliana and tomato genes. Our evolutionary analysis of grapevine, Arabidopsis, and tomato provided evidence that the replication events of these genes occurred after their retention during the differentiation from common ancestors before species differentiation. Gene replication can arise from fragment replication, tandem replication, transposable events and even whole genome replication, which not only provide the evolutionary potential for species to produce new functional traits but also are a main driving force for species differentiation [33,34]. In the identi cation of gene families from many species, gene replication events have proven instrumental in their expansion [35]. We observed that some grapevine VyRCHCs correspond to one or more intraspeci c homologous genes, thus indicating VyRCHC gene family's ampli cation in this plant may have been caused by gene duplication. Studies have shown that tandem replication often occurs in widely and fast evolving gene families, a good example being Nucleotide Binding Sites Leucine Rich Repeat (NBS-LRR) resistance families [36]. Segmental replication is more common in slow evolving gene families, like the MYB gene family [36]. We found 7 of 2 pairs of RING-type C3H2C3 genes participated in segmental replication (Fig. 4). Therefore, a nding consistent with other grapevine gene families, like the WRKY family genes in the autopolyploid Saccharum spontaneum [37]. We calculated the Ka/Ks values of 9 pairs of repetitive genes in grapevine (Table 3), nding that repeated VyRCHCs were in a strong state of puri cation and selection in their evolution, with an average Ka/ Ks of 0.395.
Cis-acting elements in gene promoter regions may be critical for gene regulation. The DELLA protein and its interacting RING nger protein inhibit the gibberellin response, by binding to the promoter of a subset of the gibberellin response gene in Arabidopsis [38]. Analysis of cis-acting elements in grapevine's VyRCHCs revealed the existence of different types of cis-acting elements upstream of the C3H2C3 genes. Except for a large number of components related to optical responses, plant hormone regulation, growth and development, and stress were also common upstream of different VyRCHCs. This situation is in fact rather common in RING genes of all species [25][26][27][28]. Ubiquitin ligase SDIR1 regulates stress-responsive abscisic acid signal by interacting with ABRE abscisic acid response element [39]. ABA, GA, ethylene, trauma, drought, heat stress, and pathogen response elements are present in the promoter region of OsRING genes of rice plants, for which pathogen infection, SA, ABA, JA, and ethephon (ET) treatments could induce target genes expression to different degrees [40]. A similar analysis of RING C3H2C3 gene mRHCP1 was recently done in maize [41]. Therefore, we speculate that these VyRCHCs may be involved in a variety of different regulatory mechanisms through these cis-acting elements.
According to the analysis of RNG-Seq data set (Fig. 5a), more than 60% of VyRCHCs were signi cantly up-regulated or down-regulated under drought stress, indicating those genes may play a key role in how grapevine responds to drought. Studies have revealed the molecular mechanism of many circular genes involved in the drought stress. For example, in Arabidopsis, XERICO, SDIR1, AtAIRP1, AtAIRP2, AtAIRP3 and AtAIRP4 has been found to play a key role in the drought response of plants. In addition, an E3 ubiquitin ligase atrzf1 mutation increased the proline content of Arabidopsis and improved this plant's drought tolerance [42]. Similarly GpDSR7 encodes an E3 ubiquitin ligase, which when overexpressed in Arabidopsis increased its tolerance to drought stress [43]. In our study, and according to previous screening methods [44], we focused on genes which were signi cantly up-regulated or down-regulated at four time periods during the drought treatment, as they more likely to play a key role in grapevine's drought stress response ( Fig. 5b and 5c). Comparing the RNA-Seq dataset with the RT-qPCR data (Fig. 6), it was found that the VyRCHC114 gene was signi cantly down-regulated within 2 days of experiencing drought, which continued to decline strongly through 10 days after this treatment. Hence, we postulated the VyRCHC114 gene may possess E3 ubiquitin ligase activity enabling it to participate in the regulation of grapevine drought stress responses. Verifying this, we detected that VyRCHC114 has E3 ubiquitin ligase activity and the VyRCHC114 gene is overexpressed in Arabidopsis. Some related indexes of Arabidopsis plant tness under drought stress were observed. That experiment demonstrated that the survival rate of overexpressed plants and the activities of many antioxidant enzymes were signi cantly increased. Drought stress signi cantly inhibited the greater electrolyte permeability of overexpressed plants; correspondingly, the decreasing trend of chlorophyll content was eliminated, while reductions in photosynthetic e ciency and energy storage capacity were signi cantly inhibited.
Drought stress greatly impacts the photosynthesis of plants, by affecting their photosynthetic rates and carbon metabolic pathways [45]. A lowered rate of photosynthesis can lead to excessive accumulation of reactive oxygen species (ROS), leading to cytotoxicity, membrane lipid peroxidation, and even cell death [46,47] which can be countered by antioxidant enzymes as a form of plant defense. The overexpression of maize E3 ubiquitin ligase gene in transgenic tobacco can reportedly improve the drought resistance of tobacco [48]. Not only that, other abiotic stresses may also be regulated by photosynthesis, thus enabling plants to adapt to stress conditions [49]. According to our results, VyRCHC114 overexpressing plants maintained a strong photosynthetic rate and energy storage capacity while under drought stress. The reason for this may be an increase of their chloroplast content, pointing to VyRCHC114's possible involvement in the regulation of chlorophyll biosynthesis pathway as an E3 ubiquitin ligase. Nonetheless, we also examined the expression of genes known to play a major role in drought stress responses of plants [50][51][52]. Our results revealed that the expression levels of these genes were signi cantly higher in VyRCHC114-overexpressed than EV-transformed Arabidopsis plants. Moreover, antioxidant system may be involved in plant abiotic stress tolerance mediated by the E3 ubiquitin ligase. Here, we provide physiological evidence that VyRCHC114 heterologous expression enhances drought resistance by increasing the activity of antioxidant enzymes, which can scavenge for and eliminate ROS to indirectly reduce membrane damage.

Conclusions
VyRCHC may act as an E3 ligase to mediate substrate degradation through the ubiquitin proteasome mechanism. This interaction may cause the target protein to be labeled by ubiquitin signaling, which leads to proteasome degradation. Since VyRCHC114 likely represents a new class of positive /negative regulatory factors in the drought signal pathway, however, positive/negative depends on the regulatory characteristics of the target protein, but we think the degraded protein is a positive regulator of drought signaling, so that more of this substance may activate drought signaling. Therefore, VyRCHC114 may improve the water retention ability and antioxidant defense of plants by regulating their chlorophyll content and antioxidant system, thus participating in drought stress response. So far, however, the target protein of the plant VyRCHC114 gene has not been determined, nor is the mechanism of augmented SOD, POD, APX and CAT activities clearly understood. In future work, we will focus on the identi cation of VyRCHC114 target proteins under drought stress and activation mechanisms of the antioxidant system in VyRCHC114-transgenic plants.

Plant materials
The grapevine variety Vitis yeshanensis was sampled from the eld, located in the grape germplasm resource garden of Northwest A&F University. Annual plants are selected for treatment, and the treatment method is the same [53]. Root samples were taken at 0d, 2d, 4d, 6d, 8d and 10d respectively for subsequent experiments. Transgenic and wild type (WT) plants of Arabidopsis thaliana ecotype Columbia (Col-0) plants were grown in vermiculite: perlite (1:1, v/v) mix in plastic pots in a growth chamber. Arabidopsis plants were grown in a soil mix of peat moss, perlite, and vermiculite (3:1:1, v/v/v) under a 12-h/12-h day/night cycle at 25℃ with 60% relative humidity. For the drought stress treatment, plants were transformed with an empty vector (EV) or to overexpress VyRCHC114 (OE#2, OE#5, or OE#13 lines) [54], all of which were grown on individual MS medium plates for 7 days before transplantation into soil, there were 5 strains in the control and three transgenic lines. This experiment mainly followed previous research methods, albeit slightly modi ed [55]. After 3 weeks, these plants received a 12-day drought stress treatment (no water provided), after which they were re-watered and their survival recorded 6 days later. All experiments were repeated three times.
Identi cation of RING-type C3-H2-C3 genes in the grapevine genome To identify the C3H2H3 type of RING, the most recent grapevine genome le in the Ensembl Plants Database (http://plants.ensembl.org/index.html) was downloaded and used. The grapevine RING C3H2C3 candidates were identi ed based on the HMM pro les (PF13639 and PF12678) with an e-value cutoff of 0.01. The screened proteins screened were given to Pfam (http://pfam.xfam.org/search/) and SMART ( ttp://smart.embl-heidelberg.de/), e value less than 0.01. Finally, 143 genes were con rmed as RING C3H2C3 genes, after manually checking whether their protein sequences had eight de nite metal ligands (Cys-Cys-Cys-His-His-Cys-Cys-Cys). The physicochemical properties of each RING-type C3H2C3 protein were predicted using the Protparam online tool (https://web.expasy.org/protparam/). The 143 VyRCHCs were named according to their positional information on the chromosomes.
Bioinformatics analysis of VyRCHCs family CLUSTALX 2.0 software was used to perform a multiple-sequence alignment of the 143 grapevine genes and the 20 tomato and 17 Arabidopsis RCHC protein sequences; it was also used to manually remove any untrusted gaps at both sequence ends. A phylogenetic tree was generated in MEGA 7.0 using the ML (maximum likelihood) method and bootstrapping with n = 1000 replicates, with all other settings set to their default values; the online EVOLVIEW (https://www.evolgenius.info/evolview/#login/) tool carried out the tree's visualization. The online program Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn/) was used to identify the genetic structure the VyRCHCs. Using the MEME online program (http://MEME.nbcr.net/meme/introduction.html), the VyRCHC protein sequences could be analyzed under these parameters: an optimal motif width of 6 ~ 35 and a maximum number of motifs of 12. According to the annotated positions in grapevine genome data, the 143 grapevine VyRCHCs were located on 20 chromosomes. By referring to previous studies, BLASTN was used to compare the CDS sequences of VyRCHCs in grapevine and tomato (e-value = 1 × 10 − 10 , homology > 75%). The tandem repeat gene pairs and segment repeat gene pairs of VyRCHCs were also identi ed [56,57]. Further, the Ka/Ks ratio between repetitive genes pairs can be used to infer the selection pressure in the process of genome evolution. Next, the MCScanX program was used to detect the collinear region between VyRCHCs in grapevine and tomato/Arabidopsis; any collinear gene pair of VyRCHCs was marked with red and green lines. The cis-elements were identi ed from the upstream 2-kb promoter sequences of the VyRCHCs after submitting them to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) [58], to obtain their image display, the resulting XML le was uploaded to TBtools [59].

Expression analysis of VyRCHCs in grapevine under drought stress
To analyze the grapevine RCHC genes' expression levels under drought stress, we obtained from the NCBI database (registration number: SRA110531) two different drought resistance genes (101. 14 (Table S4). The R package 'pheatmap' was used to produce a heatmap for this data.
RNA extraction and quantitative real-time PCR (qRT-PCR) The qRT-PCR primers were designed using Primer Premier 5 software. The RNA from Arabidopsis and grapevine (Vitis yeshanensis) leaves was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, Beijing, China), after which reverse transcription of RNA into cDNA was done using the Prime Script RT Reagent Kit (Takara, Dalian, China). The qRT-PCR was performed in an IQ 5 real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA) with SYBR Premium EX Taq II (Takara, Dalian, China). The reaction volume was 25 µl. The relative expression level corresponding to β-TUB4 gene was calculated by using the 2 −ΔΔCt method [60]; each reaction was prepared in triplicate and repeated three times. Primer sequence information in Table S1.

E3 ubiquitin ligase activity assay
The open reading frame (ORF) of VyRCHC114 and the different site mutants C320S, H341A, C328S, and N355A were separately cloned into the SalI/KpnI site of the pMAL-c5X vector (New England Biolabs UK Ltd, Hitchin, UK). According to the manufacturer's instructions, the pMAL protein fusion and puri cation system (New England Biolabs) was used to purify the fusion protein. Ubiquitination activity was then measured that according to the method described above [61], albeit with the following modi cations made: 250 ng of puri ed E3 (MBP-VyRCHC114, C320S, H341A, C328S, and N355A) in the ubiquitination buffer (50 mM Tris-HCl (pH 7.5), while the other reagents and steps used were the same. Primer sequence information in Table S1.

Physiological analysis of drought stress response of transgenic Arabidopsis
To determine the water loss rate, 10 leaves were detached from 3-week-old transgenic and WT plants and immediately weighed. The samples were then placed on dry lter paper at a relative humidity of 40-45% at room temperature and weighed over a time course. Leaves were sampled after dehydration to detect cell death, electrolyte leakage, malondialdehyde, antioxidant enzyme activity. The leaves collected before dehydration were used as a negative control.
For chlorophyll content measurements, approximately 0.05 g of fresh leaf material was placed in 5 ml of 96% ethanol and incubated at 4•C in the dark overnight. The absorbance of the extracted pigments was measured at 665 and 649 nm using a spectrophotometer (Hitachi Limited, Tokyo, Japan) and the chlorophyll content was calculated as previously described [53].
Relative electrolyte leakage was measured as previously described [62], as was MDA content [61]. In addition, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) enzyme activities were extracted from 0.5 g leaves from abiotic stress treated plants as well as control plants, and measured as described by [63].  Figure S1 Schematic diagram of C3H2C3 conserved protein sequence alignment of VyRCHCs. Figure S2 Phylogenetic analysis of RCHC protein in grapevine. Figure S3 Number of introns in VyRCHCs. Figure S4 Analysis of cis-acting elements in the VyRCHCs. Table S1 The sequences of the primers used in these experiments.

Competing interests
The authors have no con ict of interest to report.  Tables   Due to technical limitations, table 1       Expression of 8 candidate genes were screened in qRT-PCR for plants under drought stress and control conditions. The x-axis represents the different days during the treatment and the y-axis the relative levels of a gene's expression. Each treatment group had three biological repeats whose averages are plotted with the standard deviation. The asterisks indicate the signi cant level (* P < 0.05, ** P < 0.01).  OEs and EV-transformed groups were signi cantly different at P < 0.05 and P < 0.01 (Student's t-test).   Relative expression levels of drought resistance genes in transgenic and EV-transformed Arabidopsis after drought stress. a AtCOR15a, b AtERD15, c AtP5CS1, d AtRD29A. Data are the mean ± SD (standard deviation). The asterisk, (*) and (**), indicates that overexpressed (OEs) and EV-transformed groups of plants were signi cantly different at P < 0.05 and P < 0.01 (Student's t-test).

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