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Title page Genomic Survey and Expression Profiling of MYB Gene Family in Watermelon (Citrullus lanatus) Qing Xu1, 2*, Jie He1*, Xiao-Jin Hou3, Xian Zhang1** 1 College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, China 2 Institute of Food Crops, Hubei Academy of Agriculture Sciences, Wuhan, 430064 , China 3 College of Horticulture & Forest, Huazhong Agriculture University, Wuhan, 430000, China *These authors contribute equally to this paper **Corresponding author: Xian Zhang Address: College of Horticulture, Northwest A&F University, Yangling, Shaanxi, 712100, China Tel: +86 29 8703 2098 E-mail: [email protected] Fax: +86 29 8708 2613 1 Abstract MYB proteins constituted one of the largest transcription factors (TFs) family in plants, and functionally diverse in regulating plant development, metabolism, and multiple stress responses. However, the function of the watermelon MYB proteins remains elusive to date. Here, we describe the identification and classification of the 162 watermelon MYB transcription factors in terms of their gene structure, chromosomal distribution, gene duplication, conserved protein motif, phylogenetic relationship, and expression pattern under abiotic stress. According to these analyses, the watermelon MYB genes were categorized into three groups (1R-MYB, 2R-MYB, and 3R-MYB). Gene structure analysis revealed that 1R-MYB genes contain relatively more introns than 2R-, 3R-MYB genes. Amino acids alignments for all the MYB-motif of ClaMYBs demonstrated high conservation of these genes. Meanwhile, it was found that the MYB-repeats in 2R-MYB located mostly at the N-terminus of the protein, whereas the MYB-repeats in 1R-MYB located more divergently across the gene. Investigation of their chromosomal localization revealed that these ClaMYB genes are distributed across the eleven watermelon chromosomes. Gene duplication analyses showed that tandem duplication events contribute predominantly to the expansion of the MYB gene family in watermelon genome. Phylogenetic comparison of the MYB proteins 2 from Arabidopsis and watermelon revealed that some watermelon MYBs clustered into the functional clades of Arabidopsis MYB proteins. Expression analysis under different abiotic stress conditions identified a group of watermelon MYB proteins implicated in the plant stress responses. The comprehensive investigations of watermelon MYB genes in this study provide a useful reference for the future cloning and functional analysis of watermelon MYB proteins. Keywords Watermelon (Citrullus lanatus); MYB transcription factor; abiotic stress; expression profile. 3 Introduction Plants have evolved intricate mechanisms to adapt and respond to the constantly changing environment. Gene expression regulation at the transcriptional level by transcription factors is a key regulation step in all organisms. Data from the Arabidopsis genome project suggested that approximately 5% of the genes encode transcription factors, which multiply the complexity of transcriptional regulation (Riechmann and Ratcliffe 2000). The majority of the transcription factors can be grouped into multiple gene families according to the type of DNA binding domain that they encode (Pabo and Sauer 1992). Among them, MYB proteins constitute one of the largest transcription factor families, and are widely found in eukaryotes (Lipsick 1996; Riechmann and Ratcliffe 2000). The MYB family of transcription factors is typically featured by the presence of one to four imperfect MYB repeats of about 51-53 amino acids (Sakura, et al. 1989). Each repeat encodes three α helices, with helices 2 and 3 form helix-turn-helix structure, and helix 3 was found to be a recognition helix that bind directly to the major groove of DNA (Ogata, et al. 1992; Ogata, et al. 1994). Three conserved and regularly spaced (18-19 amino acid) tryptophan in each repeat formed a hydrophobic core for maintaining the helix-turn-helix structure (Ogata, et al. 1992; Ogata, et al. 1995). According to the number of repeats that the MYB domain contains, the MYB protein family can be classified into 4 four subfamilies, which are R2R3-MYB (two repeats), 3R-MYB (3 repeats), 4R-MYB (4 repeats), and MYB-related (MYB domain contains single repeat or partial MYB repeat) (Dubos, et al. 2010). Since the discovery of the first MYB gene of oncogene v-MYB of avian myeloblastosis virus in chicken, three closely related MYB proteins were identified in vertebrates (A-Myb, B-Myb, and c-Myb), and all of them have three MYB-repeats (Ito 2005; Klempnauer, et al. 1982). Loss-of-function mutants analysis of the MYB proteins revealed their specific roles in regulating cell cycle (Oh and Reddy 1999). In contrast, the most abundant type in plants is R2R3-MYB subfamily (Dubos, et al. 2010; Lipsick 1996; Martin and Paz-Ares 1997; Riechmann and Ratcliffe 2000; Rosinski and Atchley 1998). To date, a large number of MYB proteins have been rapidly identified and characterized in different plant species, such as in rice, maize, soybean, poplar, grape, and cucumber (Du, et al. 2012; Du, et al. 2012; Katiyar, et al. 2012; Li, et al. 2012; Matus, et al. 2008; Wilkins, et al. 2009). Functional studies for these MYB proteins revealed their roles in multiple plant-specific processes, such as secondary metabolism (Feng, et al. 2004; Gonzalez, et al. 2008; Jin, et al. 2000; Stracke, et al. 2007), organ development (Phan, et al. 2011; Preston, et al. 2004; Zhou, et al. 2009; Zhu, et al. 2010), signal transduction (Cheng, et al. 2009), stress responses (Agarwal, et al. 2006; Cominelli, et 5 al. 2005; Seo and Park 2010; Seo, et al. 2009), and circadian rhythms (Lu, et al. 2009). Watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai] is one of the most important economical crops worldwide, and accounts for 7% of the total world vegetable production (FAO, 2009)(Guo, et al. 2012). During the long term domestication, the modern watermelon cultivar is quite susceptible to different kinds of biotic and/or abiotic stresses (Ren, et al. 2012). Even though the broad involvement of MYB proteins in response to various stresses has been reported in different plant species, the functions of this gene family are poorly understood in watermelon. Given the recent release of the whole genome sequence, we had the opportunity to analyze the MYB gene family in watermelon. In the present study, 162 members of watermelon MYB TFs were indentified, and a comprehensive analysis in terms of gene structure, chromosome location, gene duplication, conserved MYB domain, phylogenetic analysis and expression profiles of the MYB genes under different abiotic stress conditions were performed. These investigations provided the basic information about watermelon MYB gene family, and formed a fundamental clue for future cloning and functional studies of this protein family, especially in the resistant breeding in Citrullus lanatus. Materials and Methods 6 Identification of MYB genes in watermelon and protein property analysis The watermelon genome sequences were downloaded from Cucurbit Genomics Database (http://www.icugi.org/cgi-bin/ICuGI/index.cgi). To identify the maximum number of MYB domain-containing sequences, two steps were conducted. First, the 198 Arabidopsis MYB-containing proteins were used as queries to search for MYB-containing genes across the whole genome (Dubos, et al. 2010). Second, HMM profile (PF00249) of MYB DNA-binding domain from the Pfam database was used to confirm whether each of the candidate MYB genes was a member of the MYB family in watermelon. To gain more information about the nature of the MYB proteins, the Molecular weight (kDa) and Isoelectric point (PI) of each protein were calculated by online ExPASy program (http://www.expasy.org/tools/). The sub-cellular localization of MYB proteins were predicted with online protein localization prediction server WoLF PSORT, ESLpred, ProtComp 9.0. Conservation analysis of R2R3-MYB proteins 7 Sequences of R2 and R3 repeat of 89 watermelon R2R3-MYB proteins were aligned with the ClustalX, and the sequence logo of R2 and R3 repeats were generated by submitting the multiple sequences to the website http://weblogo.berkeley.edu (Crooks, et al. 2004). Gene structure, chromosomal location and gene duplication analysis of MYB genes The DNA and cDNA sequences corresponding to each predicted MYB gene were downloaded from watermelon genome database of CUGI. The intron/exon structure and splicing phase were analyzed using the web-based bioinformatics tool GSDS (http://gsds.cbi.pku.edu.cn/) (Guo, et al. 2007). Chromosome locations of the watermelon MYB genes were drawn by Map-Inspect (http://mapinspect.software.informer.com/1.0/). For gene duplication events analysis, the criteria from previous reports (Hu, et al. 2012) were adopted with minor revisions. The segmental duplication was defined as a 200kb flanking region that contained six or more homologous pairs with fewer than 25 non-homologous gene interventions. A gene pair was considered as tandem duplication if they shared ≥40% amino acid sequence similarity and located within less than 200kb region. Protein similarity was analyzed by NCBI BlastP program. 8 Phylogenetic tree construction To examine the phylogenetic relationship and evolutionary history of MYB gene family between Watermelon and Arabidopsis, the neighbor joining phylogenetic tree was built from the alignment of 162 ClaMYB proteins and 198 Arabidopsis proteins using Mega v4.0 software (Tamura, et al. 2007). Tree nodes were evaluated by bootstrap analysis for 1000 replicates. Sequences and functional annotations of all Arabidopsis MYB proteins were obtained from TAIR (http://www.arabidopsis.org/) database. Plant materials and treatments The watermelon inbred line 134(C. lanatus (Thunb.) Matsum. & Nakai var. lanatus) were used as the material for expression pattern analysis in this study. The seeds were first sterilized with 1.5% sodium hypochlorite, soaked in distilled water for 5-6 hours, and then maintained at 30℃ for germination. The germinated seedlings were planted into the peat-perlite substrate (2:1, v/v) , and then transferred into the greenhouse with temperature 28℃ day/18 ℃ night at Northwest A&F University. Seedlings with 3-true-leaves were subjected to the following different stress treatments. 9 For cold treatment, the seedlings were transferred to 4℃ chamber, and maintained for 1, 4, and 12 hours. The leaves were sampled at each time points. The non-treated seedlings were used as negative control. The ABA treatment was carried out by spraying leaves with the freshly prepared 100μM ABA solution, then the leaves were sampled at 1, 4, and 12 hours post treatment, respectively. The seedlings sprayed with sterile water were taken as control. High salt treatment was performed by soaking the seedlings into 250 mM NaCl solution, followed by sampling leaves at 1, 4, and 12 hours after the treatment, respectively. The seedlings soaked with sterile water were used as control. For each treatment, three leaves from three different plants were mixed to form one sample, and all the treatments were performed in triplicates. The above mentioned samples were frozen immediately in liquid nitrogen, and stored at -80℃ for further analysis. Expression profile analysis Total RNA from plant tissues were extracted with TRiZol simple kit (TIANGEN, Germany). Quality of total RNA was checked by Nanodrop spectrometer (JASCO, Japan) and agrose gel electrophoresis. First-strand complementary DNA was synthesized from 2μg of DNase treated total 10 RNA using Superscript First-strand synthesis Reagent Kit (Thermo, USA) followed by the product’s manual. Gene-specific primers used for real-time PCR were designed by Premier 5.0 software, and detailed in Supplemental Table S1. Quantitative real-time PCR was performed with a thermal cycler IQ5 (Bio-Rad, USA) using Premix ExTaq TMII SYBR Green Master Mix (Takara, Japan). Reactions were performed using a total volume of 10μL, which contained 100ng of cDNA, 0.3μM each primer, and 1x SYBR Green Master-mix. The PCR cycling conditions were set as the following: initial de-naturation at 95℃ for 5min, followed by 35 cycles of 95℃ for 30s, 55℃for 30s, 72℃for 30s, and final extension of 10min at 72℃. The melting curve was recorded after 32 cycles to verify the primer specificity. The watermelonβ-actin gene was used as internal reference gene (Kong, et al. 2014). Transcripts were quantified using 2^-delta delta Ct method (Livak and Schmittgen 2001). Results Identification and classification of MYB genes in watermelon To identify the complete members of MYB genes, the entire chromosomes and scaffold sequences in the Watermelon Genome 11 Database were searched using the MYB proteins from Arabidopsis (AtMYB) as queries. Meanwhile, the Hidden Markov Model (HMM) profile (PF00249) of the MYB DNA-binding domain from the pfam database (http://pfam.sanger.ac.uk/) was adopted to confirm the presence of the MYB domains in these genes, and only the hits of E-values <0.01 were considered as MYB genes (Fin, 2006). As a result, a total of 162 genes were identified as proteins with typical MYB repeats. According to the number of MYB repeats in each gene, the identified 162 genes were assigned into three MYB subfamilies, which including 1 R1R2R3-MYB member (3 repeats), 89 R2R3-MYB members (2 repeats), and 72 MYB-related members (1 repeat). These MYB-containing genes were subsequently designated as ClaMYB1-89 for the 89 R2R3-MYB genes, ClaMYB3R1 for the single one R1R2R3-MYB gene and ClaMYB1R1-72 for the 72 genes that containing just 1 MYB repeat (Supplemental Table S1). Gene structure of ClaMYB genes To gain insight into the gene structures, the exon-intron organizations of ClaMYB genes were obtained by comparing the cDNA sequences with the corresponding genomic DNA sequences. The results showed that the closely related genes were generally more similar in gene structures, 12 differing only in the lengths of introns and exons (Fig. 1). The number of introns varied between different MYB genes, with a maximum intron number of 15 and a minimum of 0 (supplemental table S1). It was worth noting that a large number of 2R-MYB genes (60, 67%) have a conserved splicing pattern gene structure with three exons and two introns (Fig. 1). On the contrary, gene structure of the 1R-MYB genes showed more variable organizations. More than a half (39; 54%) of the 1R-MYB genes contains at least 4 introns. Intronless genes were detected for both 1Rand 2R-MYB genes as well. Four 2R-MYB and ten 1R-MYB intronless genes were found for each subclass. In addition, it was found that the MYB-repeats for most of the 2R-MYB genes located at the N-terminal of the gene, whereas the single MYB-motif in MYB-related genes was more divergently distributed across the gene (Fig. 1). Analysis of MYB protein properties and sub-cellular localization To further understand the characteristics of the ClaMYB proteins, their physiochemical properties were analyzed. The identified 89 R2R3-MYB genes encoded proteins ranging from 126 (ClaMYB37) to 877 (ClaMYB36) amino acids in length. The predicted molecular weight of these proteins varied from 14.6 to 99.7 KDa, with an average of 33.8 KDa (Table 1). Length of the 72 identified MYB-related proteins varied 13 from 61 (ClaMYB1R16, 59) to 1662 (ClaMYB1R46) amino acids, and the range of the predicted molecular weight was 6.8-181 KDa (Table 1). The predicted PI (theoretical isoelectric points) of R2R3-MYB proteins ranged from 4.8 (ClaMYB54) to 9.93 (ClaMYB82), which was smaller than that of the MYB-related proteins 4.8 (ClaMYB1R6) - 10.7 (ClaMYB1R72) (Table 1). The only 3R-MYB protein had 977 amino acids (108.4 KDa), and the predicted PI of this protein was 5.4 (Table 1). In addition, sub-cellular localization of the ClaMYB proteins were also analyzed by using several protein localization prediction softwares, such as WoLF PSORT, ESLpred, ProtComp 9.0 (Bhasin, et al. 2005; Horton, et al. 2007). The consensus outcome revealed that the 92% ClaMYB proteins localized in the nucleus and the remaining proteins were found in chloroplast, mitochondria, and cytoplasm (Supplemental Table S2). Conserved sequence analysis of watermelon R2R3-MYB proteins It was well documented that the R2R3-MYB subfamily proteins shared high degree of sequence similarity with the MYB domain region (Ogata, et al. 1995; Stracke, et al. 2001). Therefore, the sequence logo was produced to determine the level of the conservation for the R2 and R3 repeat of ClaR2R3-MYB proteins within each residue position (Fig. 2). The results revealed that R2 and R3 MYB repeats of the ClaR2R3-MYB 14 members showed characteristic amino acids, such as the regularly spaced tryptophan (W) residues. As shown in figure 2a, the R2 repeat contained three highly conserved tryptophan residues. However, only the second and the third tryptophan residue were conserved in R3 repeat, while the first tryptophan was substituted with phenylalanine (F) (Fig. 2b). Besides, alternative highly conserved residues were observed in other positions with more than 90% of the ClaR2R3-MYB proteins, which including Glu-7, Asp-8, Arg-39, Cys-44, Arg-45, Arg-47, and Asn-50 in the R2 repeat and Glu-7, Gly-19, Ile-26, Ala-27, Arg-34, Thr-35, Asp-36, and Asn-37 in the R3 repeat (Fig. 2a and b). Together, these results confirmed the highly conserved nature of the R2R3-MYB proteins. Phylogenetic and functional analysis of the ClaMYB proteins To determine the phylogenetic relationship and assess the evolutionary history of ClaMYB proteins, an unrooted neighbor-joining phylogenetic tree was constructed using the full length MYB protein sequences from watermelon and Arabidopsis (Fig. 3a and b). Previous studies indicated that the MYB-related subfamily had more expanded differentiation and evolution than 2R-MYB and 3R-MYB proteins in plants (Jiang, et al. 2004; Yanhui, et al. 2006). Therefore, phylogenetic trees for the MYB-related subfamily and 2R-, 3R-MYB subfamily were constructed. 15 The Arabidopsis MYB proteins sequences (AtMYBs) downloaded from the TAIR database were taken as reference for the phylogenetic analysis for a better classification. As a result, the 89 ClaR2R3-MYB and one Cla3R-MYB proteins together with the 126 R2R3-MYB and 5 of 3R-MYB from Arabidopsis were clustered into 39 clades (C1-C39, Fig. 3a). Two of the ClaR2R3-MYB proteins (ClaMYB15 and ClaMYB7) had not yet been assigned into any clades. This tree was validated for it showing the consistent functional clades of the AtMYBs which were characterized in previous studies (Dubos, et al. 2010; Matus, et al. 2008). Meanwhile, it was easy to find that most of the ClaMYB proteins can be grouped with the Arabidopsis counterparts (31 out of 39 groups). However, unequal representation for the watermelon and Arabidopsis MYB proteins within given clades was observed. For instance, clade C5, C8, C16, C20, and C26 included just one ClaMYB but two or more AtMYBs, suggesting that these MYB genes in Arabidopsis experienced a large expansion after the divergence from watermelon. Further, species-specific MYB proteins were also detected. For example, none of ClaMYBs were found in clade C1 and C2, whereas clades of C3, C4, C7, C15, C34, and C38 contained no ClaMYB proteins but members from Arabidopsis. These results indicated that MYB proteins in these clades might have particular roles that were either lost or 16 acquired after the divergence from their last common ancestor between watermelon and Arabidopsis during evolution. The phylogenetic tree of the 72 ClaMYB-related proteins were constructed together with the 67 AtMYB-related proteins (60 MYB-related and 7 atypical MYB proteins)(Yanhui, et al. 2006). 18 clades were finally generated by this phylogenetic tree construction (Cr1-Cr18, Fig. 3b). Four MYB proteins (ClaMYB1R6, ClaMYB1R30, ClaMYB1R56, and ClaMYB1R61) did not fit into any clades. Approximately half of the ClaMYB-related proteins (30 out of 72) were clustered with AtMYB-related proteins into 9 clades (Cr2, Cr3, Cr4, Cr5, Cr6, Cr8, Cr10, Cr11, Cr12). However, it was found that MYB members in 9 clades showed a species-specific manner. None of AtMYB-related proteins were found in Cr13-Cr18, whereas only AtMYB-related proteins were observed in Cr1, Cr7, and Cr9. Based on the phylogenetic analysis and referring to the classification by Chen (2006), this family of proteins can also be divided into five subfamilies: CCA1-like, CPC-like, TBP-like, I-box-binding-like, and R-R-type. CCA1-like was composed of 3 clades (Cr8, Cr9, and Cr12) in the phylogenetic tree analysis. In accordance with the fact that the CCA1-like was the largest subfamily of the five (Yanhui, et al. 2006), there were 10 (33%) ClaMYB-related proteins were classified into this subfamily. The CPC-like subfamily was divided into 2 clades (Cr3 and Cr6) in this study, whereas the I-box-like proteins mainly 17 clustered in Cr10. Seven ClaMYB-related proteins were grouped into the two subfamilies of CPC-like and the I-box-like respectively. TBP-like subfamily was composed of 3 clades (Cr1, Cr2, and Cr4). R-R-type subfamily contained one clade (Cr11), and there was just one ClaMYB-related protein belonged to this subfamily. Chromosome distribution and gene duplication of the ClaMYB genes The position of all 162 ClaMYBs was mapped on Watermelon chromosome available from the watermelon annotation project (Cucurbit Genomics Database). As shown in figure 4, all of the 89 R2R3-MYB genes and 72 MYB-related genes were scattered throughout the eleven chromosomes of watermelon genome, suggesting that ClaMYB genes underwent a broad expansion across the whole watermelon genome. However, uneven distribution of the ClaMYB genes was also found in the genome. Chromosome regions with a higher density of watermelon MYB genes were on chromosome 1, 2, 5 and 10, which accounted for 14.6%, 13.5%, 13.5%, 13.5% of the total R2R3-MYB genes and 16.7%, 16.7%, 13.9%, 9.7% of the overall MYB-related genes (Fig. 4). In contrast, chromosome 3, 8, 11 contained the fewest number of the R2R3-MYB and MYB-related genes (Fig. 4). The single 3R-MYB gene identified was found on chromosome 3 (Fig. 4). In addition, one 18 R2R3-MYB gene (ClaMYB1) and two MYB-related genes (ClaMYB1R1 and ClaMYB1R2) were not assigned to any chromosomes due to gaps of the physical map for watermelon genome. (Fig. 4: Chr0). In order to understand the evolution history of MYB gene family in watermelon, the gene duplication events were investigated with consideration into the physical position (<200kb) and the sequence similarity of the proteins (>40%) (Holub 2001; Hu, et al. 2012). According to this criteria, six pairs of R2R3-ClaMYBs were found to form physical clusters on different chromosomes (Chr1:ClaMYB13 and ClaMYB14; Chr2: ClaMYB16 and ClaMYB17; Chr3: ClaMYB29 and ClaMYB30; Chr7: ClaMYB62 and ClaMYB63; Chr9: ClaMYB69 and ClaMYB70; Chr10: ClaMYB84 and ClaMYB85) (Fig. 5). Meanwhile, three clusters were observed for ClaMYB-related genes for showing high protein sequence similarities and close physical position on chromosome 1 and chromosome 2 (Chr1: ClaMYB1R12 and ClaMYB1R13; Chr2: ClaMYB1R21, 22, 23, 24, 25; ClaMYB1R17 and ClaMYB1R18) (Fig. 5). However, genome survey revealed no segmental duplication exists for watermelon MYB genes. These findings suggested that the tandem duplication contribute to the increasing watermelon MYB genes. Expression profiles for ClaMYB genes under different stress conditions 19 To identify ClaMYB proteins with a possible role in response to abiotic stress, the expression pattern of 25 candidate genes (18 R2R3-MYB and 7 MYB-related) was investigated by the three treatments at 0, 1, 4, 12 hours by real-time polymerase chain reaction (PCR). These genes were selected based on the phylogenetic analysis for either they fell into the clades of Arabidopsis MYB genes with known function in the above mentioned stress responses or fell into the clades of members with unknown functions characterized yet. As shown in figure 6, the expression profiles of the selected genes revealed that most of the MYB genes showed up or down-regulation under different stress conditions. In cold treatment, 4 genes (ClaMYB2, ClaMYB75, ClaMYB1R2, and ClaMYB1R11) showed significantly repressed expression whereas 3 genes (ClaMYB54, ClaMYB40, and ClaMYB1R7) showed induced expression upon all examined time points, which suggesting that they might play negative or positive roles in cold signaling pathway. The remaining genes showed either induced or repressed expression only under certain conditions, which suggesting the temporal regulation of these genes for their specific roles. Interestingly, it was found that 52% of the selected genes were induced to different extent after high salt treatments (13 out of 25). In contrast, only 4 genes (ClaMYB81, ClaMYB70, ClaMYB1R7, and ClaMYB1R18) demonstrated reduced transcripts under the same conditions. Similarly, 6 genes (ClaMYB37, ClaMYB39, ClaMYB1R63, 20 ClaMYB5, ClaMYB38, and ClaMYB1R32) showed strong induction with more than three times after the exogenous ABA treatment, and 4 genes (ClaMYB70, ClaMYB32, ClaMYB1R7, and ClaMYB1R18) presented repression under the same conditions. The diverse expression pattern revealed by the results indicated that the selected genes played vital roles in response to different stress treatments, thus providing a useful resource for future gene expression and functional analysis. Discussion The MYB transcription factor gene family has been defined as the largest transcription factor family in plants, and involved in numerous signaling pathways (Riechmann and Ratcliffe 2000). So far, more than 100 members of MYB gene family have been identified in different plant species, with 198 in Arabidopsis, 183 in rice, and 197 in soybean (Yanhui, et al. 2006)). However, the MYB protein family in watermelon remains unknown. In this study, a total of 162 putative ClaMYB proteins were identified by watermelon genome search. Among that, half of them (55%) were defined as R2R3-MYBs, which the ratio was similar to that of the R2R3-MYB proteins identified in rice genome (56.77%) (Katiyar, et al. 2012). Meanwhile, it was worth noting that there were more R2R3-MYBs in watermelon (89) than in cucumber (55 R2R3-MYBs). This was 21 probably due to the smaller genome size of cucumber or R2R3-MYB genes underwent a more extensive expansion in watermelon (Li, et al. 2012). Despite the 3R-MYB subfamily proteins were prevalent in vertebrate, few was found in plants, only 5 in Arabidopsis, 4 in rice (Katiyar, et al. 2012; Wilkins, et al. 2009). In this study, just one 3R-MYB protein was found, suggesting that 3R-MYB family proteins were more conserved in plant and animals, but might be play less pronounced roles in comparison with that of R2R3-MYB proteins in plants. The MYB-related proteins were the second largest subfamily of MYB proteins (44%) identified, which was the similar fraction to that of MYB-related proteins in rice (40%) (Katiyar, et al. 2012). However, the 4R MYB genes were not found in this study. It was probably because of the 4R-MYB proteins had no specific roles during the evolution of watermelon. Regularly spaced tryptophan residues were considered as playing significant roles in sequence-specific DNA binding (Ogata, et al. 1995). Conservation analysis of the R2 and R3 repeat within the R2R3-type MYB proteins revealed a high degree of conservation of the residues in different positions, including the three and two highly conserved tryptophan residues in the R2 and R3 repeats. However, the first tryptophan was replaced by phenylalanine in the R3 repeat. This observation is in agreement with the conservation analysis for the 22 R2R3-MYB proteins in other plants like in Arabidopsis, cucumber, and wheat (Li, et al. 2012; Stracke, et al. 2001; Zhang, et al. 2012). Despite recent events of diversification, gene duplications are of paramount importance for organism evolution (Yang, et al. 2008). Gene duplication analysis illustrated that tandem duplication events rather than segmental duplication contributed mainly to the expansion of the ClaMYB genes in this study. Meanwhile, the fact that the ClaMYB genes distributed throughout the genome of eleven watermelon chromosomes suggested that R2R3-MYB and MYB-related genes underwent a thorough expansion during the evolution. Phylogenetic analysis and evolutionary relationship of ClaMYB gene family has been systemically studied among different species. It is generally believed that the members fall in a given group or clade may undergo recent common evolutionary origins and harbor conserved functions. In the present study, comparative analysis of MYB genes from Arabidopsis and watermelon suggested conservation but also expansions for watermelon MYB proteins. Even though majority of the clades contained MYB members from both watermelon and Arabidopsis (31 out of 39 clades), species-specific clades were detected (figure 3a). For example, ClaMYBs in clade C1 and C2 did not cluster with any of the AtMYBs, which suggesting that these proteins were acquired after the divergence from the common ancestor with Arabidopsis, and might play 23 crucial roles in watermelon development for specific adaption. Furthermore, no ClaMYBs were found to cluster with the Arabidopsis counterparts in C3, C4, C7, C15, C34, C38, possibly due to the loss of these MYB genes during the evolution of watermelon genome or acquired after the divergence from the last common ancestor. Similar results were also observed for the phylogenetic analysis of MYB-related proteins. However, more watermelon MYB-related proteins were found not to cluster with the Arabidopsis counterparts (40 out of 72 members), which indicating that MYB-related proteins underwent a more rapid expansion after the divergence with Arabidopsis lineage. phylogenetic analysis revealed that the ClaMYB proteins were clustered into the functional clades of Arabidopsis MYBs, which provided an excellent reference to elucidate the functions of the watermelon MYB proteins (Fig. 3a). For example, the ClaMYB1 was grouped together with AtMYB117 and AtMYB105 in clade 8, which were implicated in lateral organ separation and axillary meristem formation (Lee, et al. 2009). The ClaMYB47/60 were clustered with the sperm cell differentiation protein AtMYB125/DUO1 in clade 9 (Borg, et al. 2011). The ClaMYB62 was clustered into C18, sharing a high sequence similarity with Arabidopsis protein AtMYB123/TT2, which had a role in the biosynthesis of proanthocyanidins (PAs) and tannins (Nesi, et al. 2001). The ClaMYB63 was grouped into C20 with AtMYB26, referring to the regulation of 24 anther development (Steiner-lange, et al. 2003). Similar function was also found for ClaMYB55 and ClaMYB75 in C30, C31 for clustering with the pollen regulators of AtMYB35/80, respectively (Phan, et al. 2012). Additionally, ClaMYB9/42 were assembled together with AtMYB103/17 in C22 and C29, which representing the functional clades of regulation of cell wall thickening and early inflorescence development (Higginson, et al. 2003; Zhang, et al. 2009). In other clades of C6, C10, C25, C28, and C39, many ClaMYB proteins were widely found to cluster with AtMYBs that implicated in abiotic or/and biotic stresses (Fig. 3a). AtMYB41/74/102 in clade 39 were involved in drought stress (Denekamp and Smeekens 2003; Kranz, et al. 1998). AtMYB30 encodes a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack in C28 (Vailleau, et al. 2002). AtMYB13/15 from C25 were involved in ABA-mediated responses to environmental signals (Reyes and Chua 2007). The ClaMYB65/76 from C10 were grouped with three GA signaling components of AtMYB33/65/101 (Gocal, et al. 2001). AtMYB44 from C6 regulated ABA-mediated stomatal closure in response to abiotic stresses, and three other members of AtMYB77/73/70 were likely to be associated with stress responses as well (Jung, et al. 2008). In this study, 25 ClaMYB genes from the clades with AtMYB members involved in different stress stumuli or clades with unknown functions 25 characterized yet were selected to clarify their functions in different stress response pathways (low temperature, high salt, and exogenous ABA application). The results of the expression profile analysis demonstrated that all the 25 genes respond to at least one treatment. More genes (12 out of 25) were found to be repressed than induced (3 out of 25: ClaMYB54, ClaMYB40, and ClaMYB1R7) under cold conditions. Interestingly, common genes were also found to be significantly induced or repressed under both high salt and ABA treatment conditions. For example, the expression of ClaMYB5, ClaMYB37 was constitutively up-regulated under high salinity and exogenous ABA conditions. Conversely, the ClaMYB70, ClaMYB81, ClaMYB1R7, ClaMYB1R18 showed dramatic reduction for their transcript level under the same conditions. These results suggested that these proteins might be involved in the crosstalk of different signaling pathways. Additionally, temporal expression patterns for some ClaMYB genes were also observed. For example, ClaMYB15 was significantly repressed only at 4 hours of cold inducement. ClaMYB54 showed increased transcripts immediately after the salt treatment. These results collectively implied that ClaMYBs formed a complex signaling network to precisely regulate plant growth and development under stress conditions and/or possibly a connection point with other signaling pathways. 26 Author contribution statement QX and XZ conceived and designed the research. QX and JH performed the bioinformatic analysis and experiments. 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R2R3-MYB 89 126 877 300 14.6 99.7 33.8 4.8 9.93 6.9 MYB-related 72 61 1662 321 6.8 181 35.6 4.8 10.7 7.6 R1R2R3-MYB 1 977 108 5.4 Figure. 1 The phylogenetic relationship and intron-exon organization of ClaMYBs. Neighbor-joining tree on the left representing phylogenetic relationships among the 89 R2R3-MYB proteins, which were clustered into 14 subgroups (S1-S14). The tree on the right included the 72 MYB-related proteins, which were divided into 11 phylogenetic subgroups (Sr1-Sr11). The gene structure for each watermelon MYB was presented by green exon(s), red MYB domain(s), and spaces between the boxes are the intron(s). The sizes of the exons and introns can be estimated by the scale lines on the bottom. Figure. 2 The R2 and R3 MYB repeats are highly conserved across all watermelon R2R3-type MYB proteins. Sequence logos for the R2 (a) and R3 (b) MYB repeats are based on the sequence alignments of the R2 and R3 domain of all watermelon R2R3-MYB proteins. Conservation of residues across all proteins is shown by height of each letter. The bits score indicates the information content for each position in the sequence. Asterisk indicates the five conserved tryptophan (W) residues and one phenylalanine (F) residue in R2 and R3 domain. Figure. 3 Phylogenetic relationships. a, phylogenic tree of watermelon, Arabidopsis 2R-, 3R-MYB proteins. The complete amino acid sequences of the 90 watermelon, 32 131 Arabidopsis R2R3-MYB and 3R-MYB proteins were aligned by ClustalW, and the unrooted tree was generated using the MEGA v4.0 program with neighbor-joining (NJ) method. b, Phylogenetic tree of watermelon, Arabidopsis MYB-related proteins. The complete amino acid sequences of the 72 watermelon, 60 Arabidopsis MYB-related proteins and 7 Arabidopsis atypical-MYB proteins were aligned by ClustalW, and the unrooted tree was generated using the MEGA v4.0 program with neighbor-joining (NJ) method. In both cases, red dots indicate the watermelon MYB proteins. Green dots are Arabidopsis MYB proteins. Asterisk indicates watermelon proteins that did not fit into any clades. Functions of some clades were annotated. Figure. 4 Distribution of the 162 ClaMYB genes on watermelon chromosomes. The percentage of watermelon R2R3-MYB, MYB-related, and R1R2R3-MYB genes distributed on the eleven watermelon chromosomes were indicated. Figure. 5 Physical location and gene duplication of ClaMYB genes. The 159 MYB genes were positioned on eleven chromosomes (Chr1-11). The chromosome number was indicated at the top of each chromosome. Genes in black on the left side of each chromosome indicated the R2R3-MYB genes, red color represented the MYB-related genes, and the blue one is the 3R-MYB gene. The six R2R3-MYB gene clusters were shaded in light green on chromosome 1, 2, 3, 7, 9 and 10; the three MYB-related gene clusters were highlighted with light purple on chromosome 1 and 2. Figure. 6 The expression analysis of ClaMYB genes in response to cold, ABA, and salt treatments. Total RNA was extracted from the watermelon seedlings at 0, 1, 4, 12 hours after exposure to cold, ABA, and NaCl treatment, respectively. Transcript levels of each gene were calculated relative to the expression of Actin. Error bar represented the SD of three repeats. 33 Supplemental tables Table S1: Nomenclature and classification of ClaMYB genes; Table S2: Predicted subcellular localization of ClaMYB proteins; Table S3: Gene-specific primers used for the expression analysis; 34 35