Identification of mTERF genes in grape genome
MTERF genes in the grape genome were identified by BLASTP with HMMER 3.0 [26] searching key domain mTERF PFAM file (PF02636). A total of 25 grape mTERF genes were identified, which were named as VvmTERF1-VvmTERF25 according to sequence of their chromosomal locations (Table 1). A high conserved mTERF domain was found in all the VvmTERF proteins.
Phylogenetic analysis and classification of grape mTERF genes
In order to evaluate the evolution relationship of VvmTERF gene family, a total of 95 mTERF genes from Arabidopsis (35), maize (35) and grape (25) genomes were collected for a phylogenetic tree construction using MEGAX software. Detailed sequence information of Arabidopsis and maize mTERF genes were obtained from a previous study [40]. The tree topology result demonstrated that nine groups (CladeI–IX) were classified based on their conversed domains (Figure 1). Of the 25 VvmTERF genes, Clade Ⅷ contained 7 genes, the most among all the clades, while other clades had 1 to 5 members, respectively. Only one grape mTERF gene, VvmTERF24, belonged to Clade I where 4 members were identified in Arabidopsis and in maize, respectively [19, 40]. It is worth noting that the well functional characterized mTERF genes from Arabidopsis, such as SOLDAT10 (AtmTERF1, AT2G03050), BSM/RUG2 (AtmTERF4, AT4G02990), and SHOT1 (AtmTERF18, AT3G60400) were distributed in group II, IV and VI, respectively. Meanwhile, a certain of grape mTERF genes belong to these groups, indicating their close evolutionary relationships with Arabidopsis mTERF genes from the same group.
Exon–intron structure analysis of VvmTERF genes
Structures analysis on the exon and intron boundaries of the VvmTERF genes will provide important clues as they played significant roles in evolution of various gene families. The number of exons per grape mTERF gene ranged from 1 to 22 (Figure 2). Among them, VvmTERF20 had the most exons of 22, followed by VvmTERF14 (10), VvmTERF16 (7), VvmTERF18 (6), VvmTERF9 (6), VvmTERF24 (6) and VvmTERF4 (6),while VvmTERF3, VvmTERF8, VvmTERF11-13 and VvmTERF21 had only one exon each. These results indicated that during the long evolution of VvmTERF gene family, both exon loss and gain have occurred, which might lead to diversified function among the otherwise closely related mTERF genes. In clade Ⅳ, for example, the number of exons was quite large, ranging from three to ten, while the genes in clade I and Ⅸ had a relatively smaller number, ranging from one to six exons. It is worth noted that VvmTERF genes from same clades had similar exon/intro structures while genes from different groups normally showed distinct structures. This difference in exon/intro patterns might be resulted from a series of gene replication events.
VvmTERF conserved motifs analysis
Searching for known conserved motifs in grape mTERF proteins was conducted via Pfam [25] and SMART [29] databases. In order to predict the potential motifs in the putative grape mTERF family gene sequences, the MEME (Multiple Em for Motif Elicitation) program [30] was used and 15 mTERF motifs in grape were identified and clustered (Figure 2 and Table 2) using the ClustalW 2.0 program [27]. Grape mTERF sequences had 1–15 mTERF motifs. For example, all class Ⅷ sequences had more than 10 mTERF motifs, and clade IX mTERF sequences had 5–8 mTERF motifs. Identified in human mTERF proteins previously[12], conversed mTERF motifs containing repeats of leucine zipper-like heptad X3LX3 structure was also found in grape mTERF motifs (Table 2), suggesting that fundamental structures and functions of mTERF proteins in Vitis might be similar to human mTERF proteins.
Aiming to find predicted motifs shared among related proteins within the grape mTERF gene family, the MEME database program [30] was performed. As shown in Figure 2, a total of 15 motifs, designated as motifs 1–15, were discovered among these 25 proteins. Among them, motifs 2 and 8 were found in most grape mTERF proteins. Motif sequences comparison with PFAM mTERF domain alignment revealed that motifs 1, 4 and 5 partly covered the PFAM mTERF domain (PF02536), and motif 5 belonged to specific organelle-targeting mTERF proteins, such as the group Ⅳ grape mTERF proteins (Figure 2). It is probably that group-specific motifs lead to characteristic functions in various life activities.
Synteny analysis of VvmTERF and AtmTERF genes
Arabidopsis is a well-studied model species which can provide available genomic information to a less-studied species through genomic comparison method [38, 41]. As showed in Figure 3, a large-scale syntenies study containing 6 pairs of grape and Arabidopsis mTERF genes were recognized. Grape orthologues including VvmTERF2, VvmTERF6, VvmTERF13, VvmTERF15, VvmTERF24 and VvmTERF25 displayed synteny location with Arabidopsis mTERF genes AtmTERF6, AtmTERF4, AtmTERF19, AtmTERF10, AtmTERF9 and AtmTERF17, respectively (Table S2). The number of synteny results indicated that several mTERF genes might arise before the divergence of Arabidopsis and grape lineages, and also suggested that partial deletion of the grape genes might occur in specific syntenic locations during genome evolution.
Cis-element analysis of grape mTERF gene promoters
To understand the possible regulatory mechanism of VvmTERF genes in multiple stress responses and functions in chloroplast and mitochondrion, a 2-kb sequence upstream of the translational initiation site of each VvmTERF gene was analyzed by the PlantCARE database. Meanwhile, Actin1 was chosen in grape genome as the housekeeping gene (Figure 4). The sequences of VvmTERF gene promoters were found to contain various hormone regulation-related cis-elements such as those responsive to auxin, MeJA (Methyl Jasmonate), gibberellin, abscisic acid and salicylic acid. In addition, various defense and stress-related elements were also observed. These elements included light and wound responsive elements, osmotic stress-related elements, and low temperature and drought responsive elements.
Analysis of expression profiles among the grape mTERF genes in different tissues and organs
To discover the potential function of VvmTERF proteins during different stages of grape development, the tissue/organ-specific expression profiles of VvmTERF genes were analyzed in the V. vinifera cv. Corvina global gene expression atlas from the GEO DataSet (GSE36128). This dataset contained expression information of 54 sample tissues and organs in different developmental phases acquired by microarray database (Figure 5). The results showed that some VvmTERF genes such as VvmTERF6, 9, 11 and 23 displayed similar expression patterns in different tissues and organs, while other VvmTERF genes like VvmTERF1, 3, 10 and 16 demonstrated tissue/organ-specific expression profiles, suggesting multiple roles played by these VvmTERF genes in grapevine.
Expression patterns of VvmTERF genes under different exogenous hormone treatments
To explore potential stress-related genes characterized in this research, plant hormones ABA, MeJA, SA and ethylene were chosen because of their well-known functions in regulating plant signaling networks [42]. Interestingly, almost all these VvmTERF gene expressions were influenced by exogenous hormone treatments (Figure 6). For example, after the ABA treatment, a total of 13 VvmTERF genes displayed multiple degrees of up regulation while 8 genes were down regulated. MeJA treatment led to the expression increase of 17 VvmTERF genes and decrease on 7 genes. However, the expression patterns under SA and Eth treatments were different from those regulated by ABA and MeJA as more down regulated genes were observed. A total of 5 VvmTERF genes were up regulated and 12 were down regulated by SA, while 7 were up regulated and 14 were down regulated by exogenous Eth hormone treatment. According to the semi-quantitative RT-PCR result, VvmTERF2, VvmTERF6, VvmTERF16, VvmTERF23 and VvmTERF26, which were downregulated by the ETH treatment, displayed obvious upregulation by the MeJA treatment, indicating an existence of different regulatory networks among these phytohormones.
Expression profiles of VvmTERF genes in response to biotic infections
In order to adapt to changing environments, the ability of tolerating to diverse stresses becomes a significant trait in plant. Identification and functional analysis of genes involved in biological signal transduction pathways is of great significance in providing a fundamental information for plant development and stress responses. To investigate their role in responding to biotic stress, express analysis of the 25 VvmTERF genes were conducted in potted ‘Thompson Seedless’ grapevines in greenhouse after inoculating with powdery mildew (PM) and downy mildew (DM) pathogens. As shown in Figure 6, most VvmTERF genes demonstrated a tendency of downward expression after the inoculation. For example, the expression of clade Ⅷ genes-VvmTERF5, 7-12-decreased in both E. necator and P. viticola treatments, while VvmTERF7 and VvmTERF10 genes have slightly decreased after P. viticola inoculation (Fig 6). Besides, the expression level of VvmTERF6, VvmTERF14 and VvmTERF19 held steady in both biotic treatments. On the other hand, VvmTERF11, VvmTERF17 and VvmTERF21 displayed an increasing trend in both PM and DM treatments in comparison with the control. Based on semi RT-PCR analysis, three grape mTERF genes (VvmTERF2, VvmTERF4 and VvmTERF20) were chosen for further detailed analysis using real-time qPCR. The qPCR results were consistent with the those obtained by semi RT-PCR.