Genome-wide characterization and expression analysis of the Dof gene family related to abiotic stress in watermelon

Genome-wide identification and protein properties of Dof family in watermelon

To identify the watermelon Dof family genes, HHM profile of the Dof domain (PF02701) was used as a query to perform an HMMER search against the watermelon proteome, which was downloaded in watermelon (97103) v1 genome from the cucurbit genomics database (CuGenDB; http://cucurbitgenomics.org). A comprehensive search was also performed by using the amino acid sequences of Arabidopsis and rice Dof proteins from a previous study (Lijavetzky, Carbonero & Vicente-Carbajosa, 2003), which were obtained from the TIGR database (https://rice.plantbiology.msu.edu/) and the TAIR database (https://www.arabidopsis.org/), respectively. The putative sequences were submitted to Pfam (http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg.de/) for checking the presence of the Dof domain.

Sequence analyses and phylogenetic tree construction

The biochemical features including molecular weight (MW) and isoelectric point (pI) of all Dof proteins were determined by ProtParam server (http://web.expasy.org/protparam/). The subcellular localizations of the watermelon Dof proteins were predicted with CELLO v2.5 tool (http://cello.life.nctu.edu.tw/). The MEME tool (http://meme-suite.org/tools/meme) was used to predict and analyze the conserved motifs of watermelon Dof proteins with the maximum number of motifs being set as 10, and other parameters were set as default. The predicted motifs were further confirmed by searching against InterProScan (http://www.ebi.ac.uk/interpro/search/sequence-search/), and structure schematic diagrams were illustrated using the TBtools software (Chen et al., 2018). The coding region sequences (CDS) and genomic DNA (gDNA) sequences of ClDof genes were downloaded from watermelon (97103) v1 genome database (http://cucurbitgenomics.org/organism/1), and then the exon–intron structures of ClDof genes were displayed by the GSDS tool (Gene Structure Display Server, http://gsds.cbi.pku.edu.cn/) based on the alignment of CDSs with the corresponding gDNA sequences. For gene ontology (GO) analysis, the annotations of ClDof genes were obtained from watermelon (97103) v1 genome database and visualized with the WEGO program (http://wego.genomics.org.cn/). For promoter analysis, we determined the putative promoter sequence for each ClDof gene, which was defined as the upstream 1,500 bp region of the transcription start site (ATG), and analyzed the stress-related and hormone-related cis-elements using the PlantCARE tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). For phylogenetic tree construction, the Dof proteins of watermelon, cucumber, rice and Arabidopsis were aligned by Clustal Omega with default parameters. The Dof protein IDs of above species were listed in Table S1. Then, the MEGA program (v7.0) was used to construct a Neighbor-Joining tree with parameters of 1,000 bootstrap replicates and pairwise deletion.

Chromosomal location, gene duplication, and synteny analysis

The chromosomal location information of watermelon Dof genes was obtained from the watermelon genome database, and MapChart was used to display the physical positions of all ClDof genes along each chromosome. Gene duplication and synteny of Dof genes from watermelon and cucumber were examined using multiple collinear scanning toolkits (MCScanX) software with default parameters as previously reported (You et al., 2018).

Plant materials and treatments

Seeds of the watermelon cultivar “Xinong 8” (Citrullus lanatus L.) were first sterilized and germinated in an incubator (28 °C). Then, the germinated seeds were sown in pots and cultivated under a 12 h day/12 h night cycle (25 °C/19 °C, day/night temperature cycle) until the seedlings developed to four leaves. Uniformly developed four-leaf-stage watermelon plants were then exposed to NaCl (200 mm) and ABA (100 µm) treatments for 0 h, 1 h, 3 h, 9 h and 24 h. All leaves from watermelon plants were collected and rapidly frozen in liquid nitrogen and stored at –80 °C until RNA extraction.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was isolated using the total RNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer’s protocol. Then, RNase-free DNase I was added in RNA solution to remove any contaminated gDNA. First-strand cDNA synthesis was carried out following the manufacturer’s procedure (ReverTra Ace qPCR-RT Kit, Toyobo, Japan). Primers were designed using Primer Premier 5.0 software (Table S2). The qRT-PCR was performed on an CFX96 instrument (Bio-Rad, Alfred Nobel Drive Hercules, CA, USA) using SYBR Green qPCR kits (TaKaRa, Japan). The watermelon constitutive actin gene (Cla007792) was used as the endogenous control (Zhou et al., 2018b). The PCR amplification conditions included an initial heat-denaturing step at 95 °C for 3 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 1 min at 72 °C. Relative expression levels were calculated using the 2^−ΔΔCt method (Livak & Schmittgen, 2001), and each treatment included three independent biological replicates and three technical replicates. Data were statistically analyzed by one-way ANOVA using SPSS 19.0 software, and Tukey’s multiple range tests were used to detect significant treatment differences (P < 0.05).

Genome-wide identification of Dof family genes in watermelon

A total of 36 Dof genes were identified and named as ClDof1–36 according to their order on the chromosomes. Detailed information including the CDS length, protein length, predicted MW and pI of each gene is listed in Table 1. The amino acid sequences and gene sequences of ClDof members are listed in Tables S3–S5. These genes had CDS lengths ranging from 492 bp (ClDof1) to 1575 bp (ClDof33), and encoded proteins ranging from 163 to 524 amino acid residues, with the predicted MW varying from 17.64 to 56.71 kDa. The pIs of the ClDof proteins ranged from 5.00 (ClDof30) to 9.95 (ClDof13). The CELLO v2.5 tool was used to analyze the subcellular localization of ClDof proteins. The results showed that nearly all ClDof proteins were localized in the nucleus, with the exception of ClDof6, which possibly had a nuclear and extracellular localization (Table 1). The GO annotation results indicated that ClDof proteins were assigned into three major categories and 18 subcategories (Table S6; Fig. S1).

Phylogenetic characterization of the watermelon Dof gene family

To study the evolutionary relationship of Dof family genes between watermelon and other plants, a phylogenetic tree based on multiple sequence alignment was constructed by using the amino acid sequences of ClDofs together with those from cucumber (CsDofs) (Wen et al., 2016), rice (OsDofs) and Arabidopsis (AtDofs) (Lijavetzky, Carbonero & Vicente-Carbajosa, 2003). The phylogenetic tree showed that these Dof proteins could be classified into nine groups, namely A, B1, B2, C1, C2.1, C2.2, C3, D1 and D2, with well-supported bootstrap values (Fig. 1). Nearly all groups included ClDofs, CsDofs, OsDofs and AtDofs, with the exception of group C3, which comprised only dicotyledonous Dofs (ClDofs, CsDofs, and AtDofs). Besides, the numbers of ClDofs in groups A, B1, B2, C1, C2.1, C2.2, C3, D1 and D2 were 3, 7, 3, 3, 5, 2, 1, 8 and 4, respectively (Fig. 1).

Conserved motif analysis of ClDofs

By using the MEME program, a total of 10 conserved motifs were identified (Fig. 2). Amongst them, motif 1 was annotated as a Dof domain, which was widely present in all ClDof proteins, with the exception of ClDof4. Some other motifs were specifically present in individual groups. For example, motif 3, 4, 6, 7 and 10 were exclusively present in the ClDofs in group D1, while motif 2 was present in all ClDofs of group B1. Besides motif 2, nearly all group B1 ClDofs included motif 9 (except for ClDof20). In addition, motif 8 was present in all group C1 ClDofs, as well as some ClDofs in groups C2.1 and C2.2 (Fig. 2). It is worth noting that besides motif 1 and 8, three motif 5 and one motif 9 were also present in ClDof5 of group C1.

To better understand the structural features of Dof domain, multiple sequence alignment of the Dof domain sequences of ClDofs was carried out. As a result, the Dof domain of ClDofs was highly conserved, and nearly all ClDof proteins harbored the four Cys residues associated with zinc finger structure, with the exception of ClDof4 (Fig. 3), which may result in the divergence of its function from that of other ClDofs.

Exon–intron arrangement analysis of Dof family genes in watermelon

The CDS and gDNA sequences of the 36 ClDof genes were used to examine the distribution of exons and introns. As a result, most of the ClDof genes (20 out of 36) contained no introns, 11 ClDof genes (ClDof5, ClDof10, ClDof15, ClDof23, ClDof27, ClDof28, ClDof21, ClDof24, ClDof32, ClDof33 and ClDof34) had one intron each, whereas five ClDof genes (ClDof4, ClDof11, ClDof13, ClDof20 and ClDof36) possessed two introns (Fig. 4).

Chromosome distribution, gene duplication and synteny analysis of ClDof genes

Using the MapInspect program, a total of 34 ClDof genes were mapped on 10 of the 12 chromosomes in watermelon genome, while ClDof1 and ClDof2 were located on chromosome 0 (Fig. 5). In detail, there were 10, 2, 5, 2, 3, 3, 2, 2, 1 and 4 ClDof genes on chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10, respectively. Moreover, the gene duplication events were analyzed using the MCScanX program, and a total of 20 ClDof genes exhibited segmental duplication, which made up 21 pairs of segmental duplication genes (Fig. 5).

To reveal the orthologous relationships of Dof genes on chromosomes between watermelon and cucumber genomes, a comparative analysis was performed between Dof genes in watermelon and cucumber by MCScanX. A total of 31 orthologous gene pairs were identified between watermelon and cucumber (Fig. 6), indicating close relationships between ClDof and CsDof genes.

Promoter cis-elements of the ClDof genes

To assess the transcriptional regulation and potential functions of the ClDof genes, the cis-elements in the promoter regions of the ClDof genes were investigated and described in Fig. 7. Five kinds of stress-related and nine kinds of hormone-related cis-elements were identified in this study (Fig. 7). The ARE element was the most abundant among the stress-related cis-elements, and the promoter regions of 28 ClDof genes harbored the ARE element (Fig. 7), which is essential for the anaerobic induction. Other four stress-related cis-elements, including W-box, WUN-motif, MBS and TC-rich repeats, were found in 17, 16, 13 and 10 promoter regions of ClDof genes, respectively. Furthermore, various hormone-related cis-elements were also identified among the promoters of ClDof genes (except for ClDof4), including abscisic acid (ABA)-responsive element (ABRE), ethylene-responsive element (ERE), salicylic acid (SA)-responsive element (TCA-element), methyl jasmonate (MeJA)-responsive element (CGTCA-motif), auxin-responsive elements (AuxRR-core and TGA-element), and gibberellin-responsive elements (P-box, GARE-motif and TATC-box) (Fig. 7). These findings indicated that ClDof genes might play certain roles in various stress responses and hormone signaling.

Tissue-specific expression profiles of the ClDof genes

To evaluate the functions of ClDof genes in the growth and development of watermelon, the expression of nine selected ClDof genes in different tissues (mature and expanding leaves, roots, stems, stem apexes, flowers and fruits) was examined with qRT-PCR. Most ClDof genes were highly expressed in flowers and/or fruits, such as ClDof11, ClDof21, ClDof27, ClDof29, ClDof35 and ClDof36 (Fig. 8; Table S7), suggesting that they may function in flower and fruit development of watermelon. In addition, ClDof2, ClDof5, ClDof8, ClDof21 and ClDof35 displayed the highest expression in leaves, and relatively lower expression in other tissues, especially roots, stems, and tendrils (Fig. 8). Besides expanding leaves, ClDof5 also showed relatively higher expression in fruits as compared with other tissues, while its expression was extremely low in flowers. Finally, nearly all ClDof genes exhibited moderate transcript abundance in stem apexes (Fig. 8), implying their possible roles in stem apex development of watermelon.

Expression profiles of ClDof genes in response to salt stress and ABA treatment

To reveal the possible roles of ClDof genes in response to abiotic stress, we determined the expression levels of 12 selected ClDof genes under salt stress and ABA treatments using qRT-PCR. Under salt stress, nine ClDof genes (ClDof3, ClDof5, ClDof8, ClDof20, ClDof22, ClDof27, ClDof29, ClDof35 and ClDof36) were up-regulated at certain time points (Fig. 9; Table S7). Amongst them, ClDof5 was induced gradually and reached the highest transcript abundance at 24 h, while the expression of ClDof36 showed a decrease at early time point (1 h) and then gradually increased until 24 h (Fig. 9). In addition, three ClDof genes (ClDof2, ClDof11 and ClDof21) were down-regulated across all time points under salt stress, indicating their negative roles in response to salt stress. It should be noted that the expression levels of ClDof3, ClDof8, ClDof20, ClDof22, ClDof27 and ClDof35 were significantly decreased at some time points (Fig. 9). We also determined whether these ClDof genes are regulated by ABA. As shown in Fig. 10 (Table S7), the expression of all detected ClDof genes was significantly altered by ABA treatment, and the expression of five ClDof genes (ClDof2, ClDof11, ClDof21, ClDof27 and ClDof36) showed a decreasing tendency at early time points (1 h and 3 h) and finally increased at the late time points (24 h). It is worth noting that the expression of four ClDof genes (ClDof3, ClDof5, ClDof20 and ClDof22) was dramatically induced at 1 h, followed by sharp decreases thereafter. These results indicated that the ClDof genes may play crucial roles in stress responses.