Genome-wide identification of RING C3H2C3 type finger 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 identified (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: Ⅰ ~ Ⅵ (Fig. 2). Group Ⅰ has the least number of members, only 12, and the group with the largest number of members is group Ⅲ, 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 Ⅵ. 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 confirmed 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 classification 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 identified 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 identified 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 amplification mechanism of VyRCHCs during their evolution, we studied their potential repetitive events of VyRCHCs. According to the intraspecific alignment of 143 VyRCHCs, 9 pairs of genes, 7 and 2, were respectively identified 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 identified 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 amplification of VyRCHCs during their evolutionary history.
To explore the selection of grapevine VyRCHCs in terms of their repetition and differentiation, the non-synonymous (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 amplifications 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 five 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 influence 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 . 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 drought-tolerant genotype M4 at different times (Fig. 5b). By looking at the different genes in each period, there are finally 8 genes that are different in three periods. To robustly verify the gene expression levels, we quantified 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 significantly 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.
Identification 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 purified 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 non-conservative 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 significantly improved the drought tolerance (Fig. 8a). Further, on average, more than 70% of the plants overexpressing VyRCHC114 survived the drought stress, which was significantly 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 VyRCHC114-overexpressed and EV-transformed plants in the non-stress treatment, but after 8 days of drought stress, the electrolyte permeability of the former was significantly lower than the latter’s, while the chlorophyll content was significantly higher in overexpressing than EV-transformed plants. Additionally, the changes in photosynthesis under drought stress were further analyzed by measuring potential photosynthetic efficiency (Fig. 9c) and capacity storage capacity (Fig. 9d). Each was not significantly different between EV-transformed and VyRCHC114 overexpression plants when they were non-stressed; however, Fv/Fm was significantly 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 significantly 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 influence 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 significantly 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 quantified their expression under imposed drought. As expected, when non-stressed, there was no significant difference between plants overexpressing VyRCHC114 overexpression and those EV-transformed. By contrast, under drought stress, all four genes were significantly higher in VyRCHC114-overexpressed plants than in those EV-transformed (Fig. 11).