Comprehensive Analysis of TIFY Transcription Factors and Their Expression Profiles under Jasmonic Acid and Abiotic Stresses in Watermelon

The TIFY gene family is plant-specific and encodes proteins involved in the regulation of multiple biological processes. Here, we identified 15 TIFY genes in the watermelon genome, which were divided into four subfamilies (eight JAZs, four ZMLs, two TIFYs, and one PPD) in the phylogenetic tree. The ClTIFY genes were unevenly located on eight chromosomes, and three segmental duplication events and one tandem duplication event were identified, suggesting that gene duplication plays a vital role in the expansion of the TIFY gene family in watermelon. Further analysis of the protein architectures, conserved domains, and gene structures provided additional clues for understanding the putative functions of the TIFY family members. Analysis of qRT-PCR and RNA-seq data revealed that the detected ClTIFY genes had preferential expression in specific tissues. qRT-PCR analysis revealed that nine selected TIFY genes were responsive to jasmonic acid (JA) and abiotic stresses including salt and drought. JA activated eight genes and suppressed one gene, among which ClJAZ1 and ClJAZ7 were the most significantly induced. Salt and drought stress activated nearly all the detected genes to different degrees. These results lay a foundation for further functional characterization of TIFY family genes in Citrullus lanatus.


Introduction
TIFY transcription factors are plant-specific transcriptional regulators characterized by the presence of a highly conserved motif (TIF(F/Y)XG) in the TIFY domain (previously called ZIM domain) with a length of approximately 36 amino acids (aa) [1,2]. According to their domain architectures, the TIFY proteins can be divided into four subfamilies, including TIFY, ZIM-like (ZML), jasmonate ZIM-domain (JAZ), and PEAPOD (PPD). Except for the TIFY subfamily, which only harbors the TIFY domain, members of other three subfamilies possess some additional domains [3]. For example, the ZML subfamily proteins possess a GATA zinc-finger DNAbinding domain and a CCT domain (CONSTANS, CO-like, and TOC1) [4,5]. The JAZ subfamily proteins have a conserved jasmonic acid-(JA-) associated domain (Jas, also known as CCT-2 motif) with a SLX 2 FX 2 KRX 2 RX 5 PY (X represents any amino acid) consensus sequence, while the PPD subfamily proteins contain a typical PPD domain in the N-terminus and a truncated Jas motif lacking the conserved P and Y residues [6][7][8].
AtTIFY1/AtZIM is the first identified TIFY gene in plants, and overexpression of AtTIFY1/AtZIM promotes the elongation of the petiole and hypocotyl, which is independent of gibberellin and brassinosteroids [4,23]. AtZML1/AtTIFY2b and AtZML2/AtTIFY2a are important components of the CRYPTOCHROME1-(cry1-) mediated response to excess light [24]. In tomato, SlJAZ2 was described as an important regulator of the transition from vegetative growth to reproductive growth [18]. In addition, many JAZ genes were found to play key roles in JA signal transduction and participate in the regulation of various developmental processes and responses to biotic and abiotic stresses in plants. For example, OsTIFY3/OsJAZ1 can regulate floral development and root elongation by interacting with OsMYC2 and OsCOI1b in rice, and substitution or deletion of the core segments of OsJAZ1 can affect the specificity and sensitivity of JA signaling during flower and root development [13,25]. In addition, OsJAZ1 was identified as a negative regulator of rice drought resistance, partially by regulating the JA and ABA signaling pathways [14]. Another rice JAZ member, OsJAZ8, can confer resistance to rice bacterial blight by regulating JAresponsive volatile compounds [26]. Additionally, rice plants expressing OsJAZ8 and OsJAZ8ΔC (C-terminal truncated) under the control of the salt-inducible ZOS3-11 promoter exhibited higher tolerance to salt stress in early stages [27].
In this study, we predicted and classified the TIFY family genes of watermelon and analyzed their distribution patterns, phylogenetic relationships, and protein and gene structures. In addition, we examined the expression profiles of several ClTIFY genes in various tissues during fruit development of watermelon as well as under diverse abiotic stresses. Our findings lay a solid foundation for further understanding the role of TIFY genes in the growth and development of watermelon.

Genome-Wide Identification of TIFY Genes in
Watermelon. To identify the TIFY family members in watermelon, the hidden Markov model (HMM) profiles of the TIFY domain (PF06200), Jas domain (PF09425), and CCT domain (PF06203) were obtained from the Pfam database (http://pfam.sanger.ac.uk/), and these domains were used as queries to search the watermelon genome database (http:// cucurbitgenomics.org/organism/1) using HMMER 3.0 software (http://hmmer.org/) with an E value cutoff of 1e −5 . Arabidopsis and rice TIFY protein sequences were also used as queries to obtain the watermelon TIFYs by BlastP through searching the watermelon genome database with a cutoff E value of 1e −5 . According to previous reports [2,37], Arabidopsis and rice TIFY protein sequences were downloaded from the Arabidopsis genome database at TAIR (The Arabidopsis Information Resource, https://www .arabidopsis.org/) and from the rice genome database at RGAP (Rice Genome Annotation Project, http://rice .plantbiology.msu.edu/), respectively. The nonredundant sequences were subsequently confirmed with the Pfam database and the SMART database (http://smart.emblheidelberg.de/).

Protein Properties, Sequence Analyses, and Phylogenetic
Tree Construction. The biochemical features including isoelectric point (pI) and molecular weight (MW) of watermelon TIFY proteins were calculated with the ProtParam tool (http://web.expasy.org/protparam/). Conserved motifs were identified using the MEME tool (http://meme-suite .org/tools/meme) with the maximum number of motifs set as 10. The exon-intron structures of watermelon TIFY genes were displayed by the GSDS tool (http://gsds.cbi.pku.edu.cn) based on the alignment of coding region sequences (CDS) with the corresponding genomic sequences. For phylogenetic tree construction, TIFY protein sequences from watermelon, tomato, rice, and Arabidopsis were aligned by Clustal Omega with default parameters. Then, the alignments of protein sequences were used to construct the phylogenetic tree by MEGA 7.0 using the Neighbor-Joining (NJ) method, with parameters of 1,000 bootstrap replicates and pairwise deletion.

Chromosomal Location and Duplication
Analysis. The genetic positions of watermelon TIFY genes on chromosomes were obtained from the watermelon genome database, and the MapChart software was used to present the chromosomal positions and relative distance of ClTIFY genes on the basis of their ascending order of physical position (bp). Gene duplications were conducted using multiple collinear scanning toolkit (MCScanX) software with the default parameters as previously reported [38].

In Silico Expression
Analysis of ClTIFY Genes. The inbred line 97103 strand-specific RNA-seq of both the flesh and rind at four pivotal stages of fruit development (10 days after pollination, 10 DAP; 18 DAP; 26 DAP; and 34 DAP) was analyzed [39]. Fragments per kilobase of exon model per million mapped (FPKM) values were log 2 -transformed and heat maps with hierarchical clustering were plotted using the OmicShare tools (http://www.omicshare.com/tools). . At the four-leaf stage, watermelon seedlings grown in hydroponics with Hoagland's solution were treated with JA and abiotic stresses. For JA treatment, the leaves were sprayed with 100 μM methyl jasmonate (MeJA) solution. For abiotic stress treatments, the plants were exposed to 200 mM NaCl solution for 24 h (salt stress), or under 20% PEG-6000 (w/v) for 24 h (drought stress) under the same photoperiod and light conditions. Untreated seedlings were used as the controls. The leaves were sampled at different time points (0 h, 1 h, 3 h, 9 h, and 24 h) after treatment. For the analysis of the transcripts of TIFY genes in different tissues of watermelon, the roots, stems, mature leaves, stem apexes, and fruits were sampled separately from 8-week-old watermelon plants. All samples were collected and immediately frozen in liquid nitrogen and then stored in -80°C prior to RNA extraction.
2.6. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted using the total RNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) according to the manufacturer's instructions. Single-stranded cDNAs were synthesized following the manufacturer's instructions (ReverTra Ace qPCR RT Kit, Toyobo, Japan). The gene-specific primers are shown in Supplementary Table S1. qRT-PCR was performed with the iCycler iQ™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR was run 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. The watermelon Actin gene (Cla007792) was used as an internal control. Each treatment was performed with three independent biological replicates and three technical replicates. Relative expression levels were calculated as described previously [40].

Statistical Analysis.
All statistical analyses were conducted using the SPSS18 statistical package (Chicago, IL, USA). The data were subjected to one-way analysis of variance (ANOVA), and means were compared by Tukey's multiple comparisons test. P < 0 05 was accepted as significant, and the differences between treatment means are indicated by different letters.

Identification and Characterization of TIFY Family
Members in Watermelon. A total of 15 ClTIFY family genes were identified in the watermelon genome, which were further examined using SMART and PFAM to confirm that their coding protein sequences contain the TIFY, Jas, ZIM, and PPD domains. The results revealed that these 15 ClTIFY genes consisted of eight ClJAZs, four ClZMLs, two ClTIFYs, and one ClPPD. These genes were named according to their chromosomal positions and the domains that they contained as described in other plant species [6,34,35]. The amino acid sequences of TIFY family members varied from 118 aa (ClJAZ1) to 450 aa (ClTIFY2) in length, from 13.05 kDa (ClJAZ1) to 47.43 kDa (ClTIFY2) in theoretical MW, and from 4.73 (ClZML1) to 10.56 (ClJAZ5) in pI values (Table 1).

Phylogenetic Characterization of Watermelon TIFY Gene
Family. To study the evolutionary relationship of the watermelon TIFY gene family, a phylogenetic tree was created from the 72 TIFY protein sequences, including 18 in Arabidopsis, 20 in rice, 19 in tomato, and 15 in watermelon. As a result, these TIFY proteins could be classified into four subfamilies: JAZ, ZML, TIFY, and PPD ( Figure 1), among which the JAZ subfamily was the largest and could be further divided into five groups (JAZ I-V). It is noteworthy that the JAZ group I was close to the ZML subfamily, which was composed of ZIM and ZML proteins including four in watermelon, four in rice, three in Arabidopsis, and three in tomato, respectively. Three JAZ groups (I, III, and V) comprised JAZ proteins from Arabidopsis, rice, tomato, and watermelon; JAZ group IV included JAZ proteins from Arabidopsis, rice, and tomato; whereas JAZ group II only contained OsJAZs ( Figure 1). The PPD proteins constituted a unique clade, including two members from Arabidopsis (AtPPD1 and AtPPD2), two from tomato (SlPPD1 and SlPPD2), and only one from watermelon (ClPPD1). The TIFY subfamily consisted of two TIFY proteins from watermelon, and one TIFY protein from each of Arabidopsis, rice, and tomato. It should be noted that the watermelon ClTIFY proteins were more closely related to the TIFY proteins from Arabidopsis and tomato than to those from rice ( Figure 1).  Figure S1). In addition, thirteen of these 15 watermelon TIFY proteins (except for ClTIFY1 and ClTIFY2) possessed the Jas domain (PF09425). The conserved sequences of ClJAZs and ClZMLs were SLXRF(L/F)(E/Q)KRKXRX 5 PY and SLXRFR(E/Q)KRKXRX 7 Y, while ClPPD contained a truncated Jas motif lacking the conserved P and Y residues ( Figure S1). Moreover, the GATA zinc-finger (ZnF_GATA, PF00320) domain was represented by CX 2 CX 20 CX 2 C in all the four ClZML proteins ( Figure S1), which was also described in Arabidopsis [23] and pigeon pea [31].
To further explore the diversity of the conserved domains of watermelon TIFY proteins, the MEME online tool was employed, and 10 conserved motifs were identified and named as motifs 1-10. Among them, motifs 1 and 5 or motifs 1 and 10 composed the TIFY domain, and motif 2 was annotated as the Jas domain ( Figure 2). Nearly all watermelon Cl JA Z6 SlJAZ 6

AtJAZ4
AtJ AZ3 Sl JA Z8 S lP P D 1 S l P P D 2 C l P P D 1 A t P P D 1 TIFY proteins contained motifs 1 and 2, with the exception of ClTIFY1 and ClTIFY2, both of which lacked motif 2 but harbored an additional motif 6. In addition, motif 3 was annotated as the ZnF_GATA domain, motifs 2 and 4 made up the CCT domain, and both domains were only present in the ClZML proteins. In addition, both ClJAZ1 and ClJAZ2 contained motif 9 at their N-terminus, which was associated with a classical EAR motif (LXLXL) (Figure 2; Table S2).
Moreover, some watermelon TIFY proteins had unique motifs. For example, motif 8 was observed in ClJAZ7, ClJAZ8, and ClPPD1 but was absent in other watermelon TIFY proteins (Figure 2). with the CDS sequences and corresponding genomic DNA sequences. The results revealed that the ClTIFY genes contained 1-9 introns, and the number of introns in each subfamily varied. For example, in the JAZ subfamily, ClJAZ4 and ClJAZ6 had six introns; ClJAZ3, ClJAZ5, and ClJAZ8 harbored four introns; ClJAZ2 and ClJAZ7 contained two introns; while ClJAZ1 possessed only one intron (Figure 3). It can be observed that all ZML subfamily genes have longer genomic sequences than the TIFY genes of other subfamilies, and the intron number of ClZMLs was 4-9. However, some members in the same subfamily shared similar intron numbers but with different intron lengths, such as ClJAZ4 and ClJAZ6, ClJAZ3 and ClJAZ5, and ClTIFY1 and ClTIFY2 (Figure 3).

Chromosome Distribution and Duplication Analysis of ClTIFY Genes. The 15
ClTIFY genes were distributed among eight chromosomes, with chromosomes 8 and 9 containing the most genes (three genes for each), followed by chromosomes 3, 6, and 7 (two genes for each), while chromosomes 1, 4, and 5 contained the fewest ClTIFY genes (one gene for each) (Figure 4). The segmental and tandem duplication events among the ClTIFY genes were further determined. As a result, three paralogous gene pairs (ClJAZ1/ClJAZ2, ClJAZ3/ClJAZ8, and ClJAZ7/ClJAZ8) were found to be related to segmental duplication events, while only one tandem duplication event was identified on chromosome 8 (ClZML1/ClZML2).

Tissue-Specific Expression Profiles of the ClTIFY Genes.
To gain insights into the expression profiles of the ClTIFY genes in various tissues, qRT-PCR analysis was carried out to examine the expression of nine selected ClTIFY genes in leaves, roots, fruits, flowers, and stem apexes. The qRT-PCR data revealed the preferential expression of these ClTIFY genes ( Figure 5). Among them, ClJAZ1, ClJAZ4, and ClJAZ7 were highly and preferentially expressed in flowers, leaves, and fruits, respectively. In addition, ClJAZ3, ClZML1, and ClZML2 had higher expression in fruits than in other tissues. Interestingly, two TIFY subfamily members, ClTIFY1 and ClTIFY2, were highly expressed in roots and stem apexes ( Figure 5). Additionally, ClPPD1 expression was also high in stem apexes, but relatively lower in fruits, flowers, and other tissues. The results showed that the ClTIFY genes had overlapping but spatially varying expres-sion, indicating that they may play important roles in specific tissues.

Characterization of the Expression of ClTIFYs during Watermelon Fruit Development. The expression profiles of
ClTIFYs during fruit development in watermelon were analyzed according to the transcriptome data from a previous study [39]. The results showed that four ClTIFY genes were differentially expressed during watermelon flesh and rind development ( Figure 6). During flesh development, ClZML1 showed low expression all the time, while ClJAZ4 showed an observable accumulation of transcripts. During the development of rind, ClJAZ4 and ClJAZ8 exhibited specifically higher expression at some time points, while ClJAZ5 showed lower transcripts at all stages of rind development ( Figure 6). These findings indicated that these genes might play a role in the fruit development of watermelon.

Expression
Profiles of ClTIFY Genes in response to Salt, Drought, and JA Treatments. To gain more insights into the roles of ClTIFY genes in response to hormone and various stresses, the expression of nine selected ClTIFY genes was  (Figure 7).  It was observed that nearly all the detected ClTIFY genes were highly induced by salt treatment, with the exception of ClJAZ4, which seemed to be insensitive to salt treatment ( Figure 8). Notably, the transcripts of ClJAZ1, ClJAZ3, and ClJAZ7 displayed more dramatic increases at 1 h than at other time points after treatment (Figure 8), implying that they play vital roles in the response of watermelon to salt stress in earlier periods.
Upon JA treatment, eight out of the nine selected ClTIFY genes exhibited significantly upregulated expression, except for ClPPD1, whose expression was significantly reduced at all time points (Figure 9). Among the upregulated ClTIFY genes, ClJAZ1 and ClJAZ7 were the most significantly induced ones, exhibiting 533-and 77-fold changes, respectively.

Discussion
In the present study, we systematically identified 15 TIFY family genes from the watermelon genome, including eight ClJAZs, four ClZMLs, two ClTIFYs, and one ClPPD. Since only one PPD gene was present in the watermelon genome, the number of TIFY genes in watermelon was smaller than that in other plant species, whose numbers of TIFY genes range from 18 to 54, such as Arabidopsis (18) [37], rice (20) [2], Brachypodium distachyon (21) [28], pear (21) [35], poplar (24) [6,34], maize (30) [29], Brassica rapa (36) [5], wheat (49) [16], Gossypium hirsutum (50), and G. barbadense (54) [33]. This difference might be attributed to gene duplication events, including large segmental duplications and small-scale tandem duplications, which have been proven to play a key role in the expansion and function diversification of genes in TIFY gene family [2,28,41]. In this study, only three segmental duplications and one tandem duplication were observed (Figure 4), and the number is much smaller than that in other plants, indicating that the identified TIFY genes are indispensable for the growth and development of watermelon.
Phylogenetic analysis showed that watermelon TIFY proteins can be divided into four subfamilies (JAZ, ZML, TIFY, and PPD), and the JAZ subfamily can be further classified into five groups (Figure 1). It was observed that some JAZ proteins were clustered in monocot-or eudicot-specific patterns, suggesting that these JAZ proteins might have predated the divergence between monocotyledonous and dicotyledonous plants (Figure 1). Multiple sequence alignments showed that the Jas motif of ClJAZs is strikingly similar to the special consensus sequence of SLX 2 FX 2 KRX 2 RX 5 PY in other plants [3,5], which has been reported to participate in the formation of the JA-Ile-COI1-JAZ complex [42]. However, the sequence of conserved Jas domains from ClZML proteins was SLXRFR(E/Q)KRKXRX 7 Y, which is slightly different from the characteristic sequence ( Figure S1), suggesting that ClJAZs and ClZMLs might play different roles in regulating jasmonate responses in watermelon. We further detected 10 conserved motifs by MEME, and some conserved motifs were unique to specific subfamilies in watermelon TIFY proteins (Figure 2), which may contribute to the function diversification of TIFY proteins.
Considering that the exon/intron organization can provide additional clues for understanding the evolutionary relationships among gene families [43], the gene structures of ClTIFY genes were determined in this study. Similar to the case in other plants [1,31], ClZML genes have longer structures and a larger number of introns than other genes ( Figure 3). Besides, ClZML genes exhibited a relatively similar pattern of exon/intron organization but had different intron numbers, indicating that gain or loss of introns occurred during the evolution of ClZML genes, which may lead to the functional divergence [43,44].
Moreover, the JAZ subfamily genes exhibited the highest variability in exon/intron organization, while ClJAZ4 and ClJAZ6 had the same number of introns and similar lengths of exons (Figure 3), suggesting that these two genes are highly conserved during evolution and may have similar functions.
The signal molecule JA plays vital roles in plant growth, development, and responses to environmental stresses. Previous studies have revealed that the TIFY genes play vital roles in various biological processes of plants, such as petiole and hypocotyl elongation [4], lamina size and curvature [9,11], flower development [13,25,45], and seed germination [46]. TIFYs may regulate plant development through the JA signaling pathway. For example, some JAZ proteins can interact with the WD-Repeat/bHLH/MYB complexes to repress JA-mediated trichome initiation and anthocyanin accumulation in Arabidopsis [47]. In Arabidopsis, JAZ4 and JAZ8 competitively interact with WRKY57 to mediate JA-induced leaf senescence [48]. In this study, all the selected ClTIFY genes were regulated by JA and exhibited preferential expression in specific tissues ( Figures 5 and 9), suggesting that these genes play specific roles in regulating the normal development of plants probably through the JA signaling pathway. It is noteworthy that a majority of ClTIFY genes were highly expressed in flowers and fruits ( Figure 5), and four ClTIFY genes might play crucial roles in watermelon fruit development (Figure 6), suggesting that ClTIFY genes may function in the development of the flower and fruit of watermelon. It should be noted that abiotic stress can increase the JA content, and TIFY genes were also shown to play vital roles in response to abiotic stresses through the JA signaling pathway. For example, overexpression of apple MdJAZ2 in Arabidopsis decreased JA sensitivity and increased the tolerance to salt and drought stresses during seedling development [49]. Moss PnJAZ1 acts as a repressor to mediate the crosstalk between JA and ABA signaling pathways, and thus increases tolerance to salt stress [21]. In this study, the vast majority of the detected ClTIFYs exhibited differential accumulations under salt and drought stresses. Similar results were also reported in other plants, such as apple [41] and B. rapa [5]. In addition, nearly all the detected ClTI-FYs were upregulated by JA treatment, with the exception of ClPPD1, whose expression was downregulated ( Figure 9). It is worth noting that ClJAZ1 and ClJAZ7 showed the most remarkable increases in expression under JA and drought treatments (Figures 7 and 9), indicating that they may play essential roles in regulating the response to drought stress by the JA-mediated signaling pathway. ClJAZ1 was clustered together with AtJAZ7 in the JAZ V group (Figure 1), and overexpression of AtJAZ7 was found to confer drought tolerance in Arabidopsis [50]. Under salt treatment, ClJAZ7 exhibited the highest expression among the detected ClTIFYs (Figure 8), demonstrating that it may play a major role in salt stress response. Similarly, overexpression of OsTIFY11a/Os-JAZ9 in rice resulted in significantly enhanced tolerance to salt and dehydration stresses, and suppression of OsJAZ9 resulted in reduced salt tolerance through the regulation of JA signaling [2,15]. Further studies of these ClTIFY genes are needed to unravel their regulatory roles in the development of watermelon and in abiotic stress response through the JA signaling pathway.

Conclusions
In this study, a total of 15 TIFY genes were identified in the watermelon genome, including eight ClJAZs, four ClZMLs, two ClTIFYs, and one ClPPD. The analysis of qRT-PCR and RNA-seq data revealed that some TIFY genes play organ-specific roles. Expression patterns of the nine selected TIFY genes in response to JA and abiotic stress indicated that they may be involved in abiotic stress response by the JA signaling pathway. Our findings lay a foundation for a further functional characterization of the TIFY family genes in watermelon and a clarification of how TIFY genes can be utilized for the improvement of watermelon via biotechnological strategies.

Data Availability
The original data of the TIFY family genes are available from the watermelon genome database (http://cucurbitgenomics .org/organism/1).