Genome‑wide analysis of the MYB gene family in pumpkin

The MYB gene family exerts signiﬁcant inﬂuence over various biological processes and stress responses in plants. Despite this, a comprehensive analysis of this gene family in pumpkin remains absent. In this study, the MYB genes of Cucurbita moschata were identiﬁed and clustered into 33 groups (C1-33), with members of each group being highly conserved in terms of their motif composition. Furthermore, the distribution of 175 CmoMYB genes across all 20 chromosomes was found to be non-uniform. Examination of the promoter regions of these genes revealed the presence of cis-acting elements associated with phytohormone responses and abiotic/biotic stress. Utilizing quantitative real-time polymerase chain reaction (qRT-PCR), the expression patterns of 13 selected CmoMYB genes were validated, particularly in response to exogenous phytohormone exposure and various abiotic stressors, including ABA, SA, MeJA, and drought treatments. Expression analysis in diﬀerent tissues showed that CmoMYB genes are expressed at diﬀerent levels in diﬀerent tissues, suggesting that they are functionally divergent in regulating growth and abiotic stresses. These results provide a basis for future studies to characterize the function of the MYB gene family under abiotic stresses in pumpkins.

Previous studies have established the pivotal role of the MYB gene family in cell development, primary and secondary metabolism, and stress responses [5,19,20].Notably, the overexpression of SpMYB (from Solanum pimpinellifolium L3708) demonstrated heightened resistance to necrotrophic pathogens and enhanced salt and drought stress tolerance in tobacco [21,22].
Similarly, transgenic rice plants overexpressing OsMYB2P-1 exhibited increased tolerance to inorganic phosphate starvation through the promotion of primary root elongation [23].In Arabidopsis, AtMYB60 and AtMYB61 have been implicated in root development and stomatal aperture [24,25], while AtMYB44, AtMYB52, and AtMYB96 have been associated with drought stress response via ABA-mediated pathways [26][27][28].Despite extant studies on the MYB gene family, a comprehensive genome-wide characterization of this family remains absent in C.

moschata.
Pumpkins, recognized for their escalating economic importance due to bioactive compounds such as carotenoids, phenolic compounds, and flavonoids, also serve as rootstocks for other cucurbit crops, contributing to enhanced tolerance against soil-borne diseases and abiotic stresses [29,30].In recent years, water scarcity has become increasingly important for crops because of global climate issues.The yield and quality of pumpkin can be severely affected by drought stress, so it is important to identify candidate genes responsible for drought stress tolerance.Highlighting the crucial role of the MYB gene family in stress responses, this research specifically concentrates on the comprehensive genome-wide identification and expression analysis of MYB transcription factors (TFs) in pumpkins under abiotic stress conditions.The investigation encompasses the construction of phylogenetic relationships among CmoMYB proteins, along with an exploration of chromosome localization, genomic structure, and motif protein composition.Subsequently, thirteen selected MYB TFs undergo scrutiny, and their expression patterns under abiotic stress are meticulously analyzed using qRT-PCR.A notable pattern of regulation emerged, with 10 genes exhibiting up-regulation and 1 gene demonstrating down-regulation following ABA treatment at MYB protein phylogenetic trees were constructed and CmoMYB proteins were grouped to explore the evolutionary relationships between CmoMYB and AtMYB proteins.Full-length CmoMYB and AtMYB protein sequences were implemented for multiple sequence alignment using the ClustalW Program in the software MEGA, and the self-help method of phylogenetic experiments (following parameters: p-distance, partial deletion, and bootstrap=1000) was used to construct the neighbor-joining (NJ) phylogenetic tree.The tree was visualized and optimized through the ChiPlot (https://www.chiplot.online/#Phylogenetic-Tree).
For the duplication analysis of the pumpkin MYB genome, we employed BLAST to compare the genomic sequences and retrieved essential genomic features such as chromosome length, position, inter-chromosomal linkage, and gene annotations from the GFF3 files.The integration of these files, along with the gene link format file, GFF file format file, and Chr-Layout format file was performed using the File Merge function of the MCScanX package in TBtools (v1.108) to investigate MYB gene duplication events in pumpkin.

Chromosomal location, genetic structure, conserved motifs, and cis-acting regulating element prediction
To investigate the genetic structure and chromosomal localization of the MYB gene, gff3 files about C. moschata were acquired from the Cucurbitaceae database.Subsequently, the conserved motifs within CmoMYB proteins were delineated utilizing the Multiple Em for Motif Elicitation (MEME) Program (https://meme-suite.org/meme/tools/meme.cgi), with a specified parameter setting of 10 as the maximum number of conserved motifs to be identified.Furthermore, the 2 kbp promoter sequences associated with CmoMYB genes were obtained.The PlantCARE database website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/)served as the platform for the prediction of cis-acting elements.TBtools software (v1.108) was used to visualize the above results.

Plant materials, abiotic stress, and hormones treatment
The expression levels of CmoMYBs were investigated under three hormones and abiotic stress, including ABA, SA, JA, and simulated drought.The 'TianMiyihao' was used as the experimental material.Seeds were sterilized with 75% ethanol, then rinsed five times with sterile water and germinated on Petri dishes.After three days, seedlings with good growth conditions were cultured with Hoagland's nutrient solution in a greenhouse (28℃, 70-80% humidity, 16 h light/8 h dark).The roots, stems, and leaves of pumpkin were harvested to analyze the expression Manuscript to be reviewed levels of CmoMYB genes.Meanwhile, uniformly sized three-leaf seedlings were treated with simulated drought treatment (20% PEG6000) and different hormones (100 µM JA, 10 µM ABA, and 100 µM SA).Leaves from each group were obtained after 0, 3, and 6 h of treatment, respectively.The samples were snap-frozen in liquid nitrogen and stored at −80℃.Each sample consisted of three different plant leaves and three experiments were performed.

RNA extraction, cDNA synthesis, qRT-PCR, and Expression analysis
Total RNA from each sample was extracted by using Trizol reagent (Takara, Beijing, China), and the cDNA was synthesized by using the First-Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).The specific primers for the ComMYB genes were designed using Primer Premier 5 software (Table S2) for qRT-PCR analysis.The reaction consisted of 10 µL AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China), 0.2 µmol L -1 upstream and downstream primers, 2.0 µL cDNA, and up to 20 µL with ddH 2 O.The qRT-PCR process was set as follow steps: 95 ℃ for 3 min, followed by 40 cycles of 95 ℃ for 10 s, 60 ℃ for 30 s, and 60 ℃ for 30 s.The Cmoβactin gene was used as the normalization reference gene.The relative expression level of genes was calculated by the 2 −∆∆Ct method.Three experimental replicates were performed for each sample.For tissue-specific expression analysis of 175 CmoMYB genes, the expression data for four tissues (leaves, roots, stem, and fruit) were obtained from the Cucurbitaceae database [31].
The TBtools software was used to construct a heatmap for specific expression analysis.

Identification of the CmoMYB gene family
To identify members of the CmoMYB gene family, we conducted a comprehensive search of the entire set of Cucurbita moschata protein sequences to identify proteins harboring either MYB or MYB-like domain sequences, which served as initial candidate proteins.Subsequently, candidate proteins underwent further validation through the HMMER website, with proteins containing divergent conserved domains being excluded from the analysis.A total of 175 CmoMYB proteins were successfully identified and individually designated.Detailed information regarding the CmoMYB genes is provided in supplementary Table S1.As shown in Table S1, a total of 175 CmoMYB proteins were successfully identified (see Table S1  Manuscript to be reviewed spanning from 82 (CmoMYB171, CmoMYB172) to 1,038 (CmoMYB150) amino acids, with an average length of 343 amino acids.Furthermore, the calculated molecular weights (Mw) of the CmoMYB proteins ranged from 9.80 to 114.86 kilodaltons (kDa), while the theoretical isoelectric points (pI) ranged from 4.61 (CmoMYB30) to 11 (CmoMYB163).In addition to being distributed in the nucleus, some proteins were also distributed in the cytoplasm (Table S1).
To explore the orthologous relationship between CmoMYB proteins and their counterparts in Arabidopsis thaliana (AtMYB proteins), we employed the neighbor-joining method to construct a phylogenetic tree (Figure 1).Analysis of the phylogenetic tree revealed the classification of MYB proteins from both species into 33 distinct categories.Among these, 126 AtMYB proteins were allocated into 25 subgroups.Notably, the majority of CmoMYB proteins (depicted by purple circles in Figure 1) clustered together with their homologous AtMYB proteins (marked by green circles in Figure 1), indicating evolutionary conservation.However, exceptions were observed in clades C16 (comprising CmoMYB64, CmoMYB74, and CmoMYB159) and C24 (encompassing CmoMYB4, CmoMYB5, and CmoMYB6), which exclusively contained CmoMYB proteins.This observation suggests that MYB proteins may have undergone distinct evolutionary adaptations to environmental changes.
Several CmoMYBs are clustered in different specific locations, such as the top of chromosomes 1 and 14, and the bottom of chromosomes 4 and 11.For the selection of duplicated MYB gene pairs, MYB duplicates were scrutinized within the pumpkin genome, resulting in the detection of 135 homologous MYB gene pairs distributed across 20 chromosomes (refer to Figure S2 and Table S3).Among these, 142 genes exhibited one to four homologous MYB counterparts.Such as CmoMYB8 and CmoMYB50 both have four homologous MYB genes.Manuscript to be reviewed
Employing the MEME tool facilitated the identification of conserved motifs, thus enhancing the comprehension of CmoMYB protein diversity within the pumpkin genome.The findings, illustrated in Table 1, delineated the presence of 10 conserved motifs.Figure 2 depicted that numerous CmoMYBs predominantly comprised motifs 1, 2, 3, 4, 5, 6, and 7/8.While motifs typically occur singularly, exceptions were noted; for instance, motif 3 was repeated in CmoMYB115 and CmoMYB173 but was absent in CmoMYB151.Similarly, motif 4 exhibited duplication in CmoMYB30 but was not detected in CmoMYB159.Moreover, closely related CmoMYB proteins tended to exhibit analogous motifs, suggesting functional similarities within specific subgroups.Notably, certain CmoMYBs displayed unique motifs; for instance, motif 9 exclusively appeared in the C12 and C13 subfamilies.This underscores the potential involvement of specific motifs in executing distinct functions within MYB proteins.

Cis-acting element prediction of CmoMYB gene promoters
To explore the transcriptional regulatory properties of CmoMYB genes, cis-acting elements were predicted by using PlantCARE online software (Figure 3).The analysis unveiled a pervasive distribution of stress-and hormone-responsive elements within the promoters of CmoMYB genes, in addition to numerous core cis-elements.Enumeration of distinct cis-elements revealed that abscisic acid-responsive elements (ABRE, 557) were the most prevalent in the CmoMYBs' promoters, succeeded by MeJA-responsive elements (TGACG-motif and CGTCA-motif, 547) and salicylic acid-responsive elements (SARE and TCA-element, 124).Furthermore, these promoters encompassed auxin-responsive elements (TGA, AUXRE), drought-induced response elements (MBS), flavonoid biosynthetic regulation (MBSI), low temperature-responsive elements (LTR), defense and stress-response elements (TC-rich repeats), and various other cis-acting elements.Manuscript to be reviewed

Expression analysis of the CmoMYB genes
Utilizing expression data extracted from the Cucurbitaceae genome database, a heat map delineating the expression patterns of CmoMYB genes across four distinct tissues was generated using TBtools (Figure S3).Approximately 90 of these 175 (51.4%)CmoMYBs showed the highest expression level in root, 59 (34%) in stem, 37 (21%) in leaf, and 34(19%) in fruit.Besides, a total of 13 CmoMYB genes from different subgroups (Figure 1) were selected for expression analysis by qRT-PCR.The expression levels of 13 CmoMYBs were performed in three tissues (root, stem, and leaf) at the three-leaf seedlings stage (Figure 4).A variety of expressions of these CmoMYBs were found in the three tissues.Most CmoMYBs including CmoMYB99, CmoMYB142, CmoMYB154, CmoMYB144, CmoMYB116, CmoMYB70, CmoMYB46, CmoMYB3, and CmoMYB9 were significantly up-regulated in the roots, several CmoMYB genes including CmoMYB165, CmoMYB142, CmoMYB59, and CmoMYB29 were highly expressed in the leaves, while CmoMYB64 and CmoMYB59 genes were highly expressed in the stem, indicating that these CmoMYBs might be involved in the various biological processes in the different tissues.

The expression levels of CmoMYB genes under hormones and abiotic stresses
The expression levels of many CmoMYB genes were significantly changed after ABA treatment (Figure 5).ABA treatment for 3 h induced the expression levels of 10 CmoMYBs at a degree from 1.23-fold to 65.33-fold, while CmoMYB99 was not affected, and reduced the expression of CmoMYB165 and CmoMYB154 to 77.3% and 63.9%, respectively.The CmoMYB genes were significantly regulated after 6 h after ABA treatment (10 up-regulated and 1 downregulated), except for CmoMYB64 and CmoMYB59, which returned to initial values.As shown in Figure 6, 11 CmoMYB genes were significantly regulated at 3 h under JA treatment (9 up-regulated and 2 down-regulated), except for CmoMYB59 (which started to decrease at 6 h) and CmoMYB3 (which started to increase at 6 h).The transcription levels of 7 CmoMYB genes were less significant at 6 h than at 3 h, such as, the expression levels of CmoMYB99 decreased from 50.70-fold to 11.93fold, CmoMYB116 decreased from 15.94-fold to 7.79-fold, and even CmoMYB142 and CmoMYB29 did not change significantly at 6 h.After SA treatment, all CmoMYB genes were significantly regulated at 3 h (11 up-regulated and 2 down-regulated) (Figure 7).After 6 h of treatment, the expression level of 12 CmoMYB genes started to decrease, except for CmoMYB144, which was up-regulated.Compared with 0 h, 8 CmoMYB genes had no significant change, only 3 CmoMYB genes had a significant change, and 2 CmoMYB genes were still continuously down- CmoMYB29 decreased to 77%, and CmoMYB64 and CmoMYB154 did not change significantly (Figure 8).Interestingly, 7 genes (CmoMYB165, 142, 154, 70, 46, 59, and 9) appeared to be downregulated at 3 h and significantly up-regulated at 6 h.

Discussion
The MYB gene family has been systematically characterized across diverse plant species, including Chinese pear, rice, Arabidopsis, and soybean [2,5,18,23].Despite extensive investigations in these species, the CmoMYB gene family in Cucurbita moschata remains relatively understudied, with its functional roles yet to be elucidated.This current investigation represents the foundational analysis of the MYB superfamily genes within C. moschata, leveraging the available genomic resources as detailed by Sun H et al. [31].A comprehensive survey revealed 175 proteins harboring MYB repeats.The Mw and pI of these proteins emerged as crucial parameters influencing their molecular and biochemical functionalities [48].Notably, our analysis unveiled substantial variations in both size and pI among CmoMYB proteins, a trend reminiscent of findings reported by Li et al. [32], implying potential context-dependent functional diversity among CmoMYB proteins.Typical TFs encompass four pivotal domains, including nuclear localization signal, DNA-binding, transcriptional regulatory sites, and oligomerization domains [2].Protein subcellular localization predictions show that all CmoMYB proteins are localized to the nucleus.The chromosomal distribution analysis revealed that CmoMYBs are dispersed across the 20 chromosomes of C. moschata.however, this distribution appears uneven.Chromosome 14 emerged as the locus harboring the highest number of CmoMYB genes, followed by chromosome 1, while chromosome 16 exhibited the fewest CmoMYB genes.Furthermore, numerous CmoMYBs tend to cluster in distinct genomic regions, displaying notably elevated densities towards the chromosomal telomeres (Figure S1).This pattern of ComMYB gene distribution bears resemblance to observations documented in prior studies across other species, such as chili peppers [33] and potatoes [17].
As part of this study, a phylogenetic tree was constructed for CmoMYBs and AtMYBs in order to further investigate their orthologous relationship (Figure 1).The resulting phylogenetic Manuscript to be reviewed tree revealed 33 distinct categories.Our findings align with previous research, indicating that members within the same branch share conserved functions, likely stemming from a common ancestor [34,35].Most of the CmoMYB proteins were clustered with AtMYB proteins.For example, the CmoMYBs of CmoMYB104, CmoMYB38, CmoMYB36, CmoMYB40, CmoMYB30, CmoMYB102, and CmoMYB26 grouped with AT3G62610, AT2G47460, and AT5G49330 to form clade C26 (The syntenic analysis between these proteins was shown in Figure S4), which may indicated that these CmoMYB genes might related to the biosynthesis of flavonoids [36][37][38]; clade C11 was consists of CmoMYB83, CmoMYB94, CmoMYB86, and CmoMYB96 of pumpkin and the reported proteins AT3G27810, AT5G40350, AT3G01530 (The syntenic analysis between these proteins was shown in Figure S5), which are involved in the control of PAL genes and the elongation of staminal filaments [39,40].Conversely, clades C16 and C24 lacked AtMYBs, indicating that certain CmoMYB genes may be unique to pumpkin.
Overall, the clustering patterns offer valuable insights into the roles of CmoMYB proteins.
Moreover, the analysis of gene structure and motif protein alignment strongly supports subgroup classification (Figure 2).Consistent with prior studies, our observations indicate that MYB genes within the same subgroup typically exhibit similar exon-intron structures, highlighting their high conservation across species [2,41].
MYB proteins feature a structurally dynamic region, responsible for regulatory activities, alongside a conserved MYB structural domain, which facilitates the recognition of target gene promoters [42].Analysis of the motif results showed that CmoMYB proteins belonging to the same subgroup shared the common motifs, suggesting that they might have similar functions.In addition, we analyzed the expression profile of CmoMYB genes in different tissues (Figure S3).90 CmoMYBs had the highest level of transcript accumulation in root tissue, 59 CmoMYBs in stem tissue, 37 CmoMYBs in leaf tissue, and 34 CmoMYBs in seed tissue.In the analysis of 13 genes, most CmoMYBs in roots were significantly up-regulated, and CmoMYB29 was expressed only in leaves.In contrast, CmoMYB64 and CmoMYB59 genes were highly expressed in stems, suggesting that these CmoMYBs may be involved in various biological processes in different tissues, such as root growth, stem elongation, and leaf development (Figure 4).
Cis-acting elements act as sites for specific binding of transcription factors and are involved in the regulation of gene expression [43].To explore whether the MYB gene responds to adversity stress through cis-acting elements, we delineated the cis-acting elements in the promoter of the Manuscript to be reviewed CmoMYBs (Figure 3).Prominently, abscisic acid-responsive elements predominated, succeeded by MeJA-responsive elements, salicylic acid-responsive elements, drought-induced response elements, as well as defense and stress-response elements, among others.The findings imply the potential involvement of the CmoMYB gene family in abiotic stress and hormone-mediated responses.Moreover, the differential transcriptional regulation observed among various CmoMYB gene types underscores the diverse functional roles played by CmoMYBs in cellular processes.
Guided by these predictions, a subset of 13 CmoMYB genes was scrutinized for their responsiveness to three distinct hormones and simulated drought stress conditions.Differential stress responses among these genes were observed, potentially attributed to the distinct repertoire of cis-acting elements harbored within their promoters, with notable regulation evident across most CmoMYB genes (Figures 3 and 5-8).Furthermore, our inquiry into the responsiveness of these genes to various stressors revealed a spectrum of regulatory patterns, possibly linked to the diversity and abundance of cis-acting elements present in their promoters.Noteworthy prior research highlights the significance of OsMYB1R1, TaMYB31, GaMYB85, and SsMYB113 in mediating drought stress responses [44][45][46][47].In our investigation, CmoMYB99, CmoMYB144, CmoMYB116, CmoMYB70, and CmoMYB3 exhibited heightened expression under drought stress, contrasting with the down-regulation of CmoMYB29.These findings underscore the nuanced transcriptional dynamics under varying stress contexts, indicative of intricate responses mediated by complex signaling pathways [48][49][50][51].Collectively, our results underscore the pivotal role played by the CmoMYB gene family in orchestrating responses to diverse stress stimuli.

Conclusions
The MYB superfamily, ubiquitously distributed across eukaryotes, assumes pivotal roles in governing plant growth and development.Notably, environmental stresses, exemplified by drought, constitute significant natural threats to crop plants.The discernment of genes responsive to abiotic stress emerges as a potentially efficacious strategy for augmenting resistance to such adversities in pumpkins.Within the purview of this investigation, a comprehensive analysis of the entire genome identified 175 MYB proteins.While these proteins exhibited a high degree of conservation in motif composition, their expression profiles varied across different tissues, indicative of the nuanced activity and diversity inherent in CmoMYBs, governing pumpkin growth.

Figure 1 .
Figure 1.The Neighbor-Joining phylogenetic tree of MYB proteins between C.moschata and Arabidopsis.

Figure 2 .
Figure 2. The conserved motifs and exon/intron structures of MYB genes.

Figure 3 .
Figure 3. Cis-acting elements in the promoter region of ComMYB genes.

Figure 4 .
Figure 4. Expression levels of the selected 13 CmoMYB genes in different tissues.

Table 1 (on next page)Table 1 .
Specific conserved motifs identified by MEME among CmoMYB proteins in C.