Genome-Wide Identification and Chilling Stress Analysis of the NF-Y Gene Family in Melon

The nuclear factor Y (NF-Y) transcription factor contains three subfamilies: NF-YA, NF-YB, and NF-YC. The NF-Y family have been reported to be key regulators in plant growth and stress responses. However, little attention has been given to these genes in melon (Cucumis melo L.). In this study, twenty-five NF-Ys were identified in the melon genome, including six CmNF-YAs, eleven CmNF-YBs, and eight CmNF-YCs. Their basic information (gene location, protein characteristics, and subcellular localization), conserved domains and motifs, and phylogeny and gene structure were subsequently analyzed. Results showed highly conserved motifs exist in each subfamily, which are distinct between subfamilies. Most CmNF-Ys were expressed in five tissues and exhibited distinct expression patterns. However, CmNF-YA6, CmNF-YB1/B2/B3/B8, and CmNF-YC6 were not expressed and might be pseudogenes. Twelve CmNF-Ys were induced by cold stress, indicating the NF-Y family plays a key role in melon cold tolerance. Taken together, our findings provide a comprehensive understanding of CmNF-Y genes in the development and stress response of melon and provide genetic resources for solving the practical problems of melon production.


Introduction
Nuclear factor Y (NF-Y), also known as heme activator protein (HAP) or CCAAT binding factor, is widely distributed and evolutionarily conserved in eukaryotes such as yeast, mammals, and plants [1]. NF-Y is composed of three subunits: NF-YA (CBF-B/HAP2), NF-YB (CBF-A/HAP3), and NF-YC (CBF-C/HAP5). Generally, NF-YB and NF-YC recognize each other and interact through the HFM domain to form the NF-YB/C heterodimer in the cytoplasm. Then, the dimer will enter the nucleus, recognize with specific NF-YA and conduct secondary assembly to form a complete NF-Y heterotrimer complex (NF-YA/B/C) [2] or form a heterotrimer with another factor (X) in the nucleus (NF-YB/C-X) [3]. The trimer complex can regulate target genes through the N-terminal of the NF-YA subunit or X factor, the C-terminal transcriptional activation domain of the NF-YC subunit (glutamine rich hydrophobic domain), or recruit other transcription factors to regulate target genes through protein interaction [4,5].

Motif Composition and Multiple Alignments of the CmNF-Ys
Combining MEME motif ( Figure 2) and multiple alignments (Figure 3), the results showed that there were significant differences in conserved regions among the three subfamilies. The CmNF-YA conserved domains consisted of two α-helices, while the CsNF-YB and CmNF-YC were composed of four α-helices ( Figure 3). CmNF-YAs were more conservative, mainly containing motif 2 (light blue), motif 4 (purple), and motif 10 (fluorescent green) excluding CmNF-YA6 ( Figure 2). These three motifs were the two core subdomains of NF-YA, one responsible for the NF-YB/C interaction and the other for DNA binding (Figure 3a) [36]. CmNF-YBs and CmNF-YCs harbored the HFM of the H2B and H2A, respectively, which were conserved regions for DNA binding and subgroup member interactions (Figure 3b,c) [36]. However, CmNF-YBs were the most conserved, and their conserved motifs were uniform, including motif 1 (red), motif 3 (light green), and motif 5 (orange) (Figure 2), while CmNF-YCs were the most diverse, motif1, motif 3, motif 6, motif 7, and motif 9 were unevenly distributed among the eight members, implying the diversity of their functions.

Phylogenetic Analysis of CmNF-Ys
The NF-Y family members of three plants, Arabidopsis thaliana (36), cucumber (27) and melon (25), were used to construct a maximum likelihood (ML) phylogenetic tree with
Dulce and Tam-Dew are climacteric and non-climacteric lines, respectively [37]. During fruit development, the expression of CmNF-Ys in two genotypes was basically the same, and nearly half of the genes were constantly and steadily expressed (Figure 7b). There were six CmNF-Ys (CmNF-YA6/B1/B2/B3/B8/C6) that were undetected during the whole process of fruit development (RPKM<1) (Figure 7b). Notably, CmNF-YC1 maintained the highest expression in the four stages and increased with fruit development and maturity; on the contrary, the expression of CmNF-YA3, CmNF-YB11, and CmNF-YC3 decreased continuously (Figure 7b). Intriguingly, the expression of CmNF-YA1 and CmNF-YB7 was basically stable within 30 DAA, and only decreased sharply at maturity, suggesting that they might be involved in the regulation of fruit ripening (Figure 7b). These results indicated that the six genes might regulate melon fruit ripening, among which CmNF-YC1 might be the principal gene.
Dulce and Tam-Dew are climacteric and non-climacteric lines, respectively [37]. During fruit development, the expression of CmNF-Ys in two genotypes was basically the same, and nearly half of the genes were constantly and steadily expressed (Figure 7b). There were six CmNF-Ys (CmNF-YA6/B1/B2/B3/B8/C6) that were undetected during the whole process of fruit development (RPKM<1) (Figure 7b). Notably, CmNF-YC1 maintained the highest expression in the four stages and increased with fruit development and maturity; on the contrary, the expression of CmNF-YA3, CmNF-YB11, and CmNF-YC3 decreased continuously (Figure 7b). Intriguingly, the expression of CmNF-YA1 and CmNF-YB7 was basically stable within 30 DAA, and only decreased sharply at maturity, suggesting that they might be involved in the regulation of fruit ripening (Figure 7b). These results indicated that the six genes might regulate melon fruit ripening, among which CmNF-YC1 might be the principal gene.

Expression Profiles of CmNF-Ys under Cold Stress
As shown in Figure 8a, low temperature caused melon to lose water and wilt gradually. To obtain candidate genes for cold tolerance in melon, we did RNA-seq and qRT-PCR analysis in melon seedling stage. There were 13 CmNF-Ys that were severely inhibited or not expressed under low temperature (blue areas). On the contrary, 12 CmNF-Ys were up-regulated (Figure 8b). Among them, CmNF-YA2/A3 and CmNF-YB4/B7 were up-regulated only at one or two stages during cold treatment (Figure 8b). While other genes, such as CmNF-YA1, CmNF-YB6/B10, and CmNF-YC1/C2/C5/C7/C8, were up-regulated throughout almost the whole process (Figure 8b). These eight genes were selected for qRT-PCR analysis of gene expression under cold stress. The results were generally consistent with the changes of transcriptome data (Figure 8c). These results showed that CmNF-Ys might play an important role in melon response to cold stress.

Expression Profiles of CmNF-Ys under Cold Stress
As shown in Figure 8a, low temperature caused melon to lose water and wilt gradually. To obtain candidate genes for cold tolerance in melon, we did RNA-seq and qRT-PCR analysis in melon seedling stage. There were 13 CmNF-Ys that were severely inhibited or not expressed under low temperature (blue areas). On the contrary, 12 CmNF-Ys were upregulated (Figure 8b). Among them, CmNF-YA2/A3 and CmNF-YB4/B7 were up-regulated only at one or two stages during cold treatment (Figure 8b). While other genes, such as CmNF-YA1, CmNF-YB6/B10, and CmNF-YC1/C2/C5/C7/C8, were up-regulated throughout almost the whole process (Figure 8b). These eight genes were selected for qRT-PCR analysis of gene expression under cold stress. The results were generally consistent with the changes of transcriptome data (Figure 8c). These results showed that CmNF-Ys might play an important role in melon response to cold stress. The seedlings were exposed to cold stress (15 °C/6 °C, day/night, LT) for 24 h. The third true leaves were sampled for RNA-Seq and qRT-PCR after 0, 6, 12, 18, and 24 h cold treatment. We calculated the relative value before and after cold treatment (LT/CK) and converted the data into log2 to draw the heat map. Error bars indicate the standard error of the mean. A one-way ANOVA was performed using Duncan's multiple-range test. Different lowercase letters represent significant differences at the 0.05 level.

Discussion
The NF-Y family is widely distributed in eukaryotic organisms, including yeast, plants, and mammals. In mammals and yeast, all three NF-Y subunits are encoded by a single gene [1]. However, NF-Y genes undergo replication and expansion in plants, and each subunit is encoded by a gene family. This provides more NF-YA/NF-YB/NF-YC trimer combinations, increasing the complexity of NF-Y function [3]. NF-Y transcription factors recognize and bind the CCAAT box, which exists in the promoter region of 20~30% of eukaryotic genes [36]. It is proven that the NF-Y family is widely involved in plant growth, fruit maturation, and stress response. To date, the NF-Y family has been identified in a variety of crops [7,17,26,28,38,39]. However, there are few reports about the melon NF-Y family. How NF-Y members participate in the growth, development, and stress response in melon is still unknown.
In this study, a total of 25 CmNF-Y genes were identified (Table 1), which was similar to the number in other cucurbits, such as cucumber [6] and watermelon [38]. However, Figure 8. Expression profiles of CmNF-Ys under cold stress. The seedlings were exposed to cold stress (15 • C/6 • C, day/night, LT) for 24 h. The third true leaves were sampled for RNA-Seq and qRT-PCR after 0, 6, 12, 18, and 24 h cold treatment. We calculated the relative value before and after cold treatment (LT/CK) and converted the data into log 2 to draw the heat map. Error bars indicate the standard error of the mean. A one-way ANOVA was performed using Duncan's multiple-range test. Different lowercase letters represent significant differences at the 0.05 level.

Discussion
The NF-Y family is widely distributed in eukaryotic organisms, including yeast, plants, and mammals. In mammals and yeast, all three NF-Y subunits are encoded by a single gene [1]. However, NF-Y genes undergo replication and expansion in plants, and each subunit is encoded by a gene family. This provides more NF-YA/NF-YB/NF-YC trimer combinations, increasing the complexity of NF-Y function [3]. NF-Y transcription factors recognize and bind the CCAAT box, which exists in the promoter region of 20~30% of eukaryotic genes [36]. It is proven that the NF-Y family is widely involved in plant growth, fruit maturation, and stress response. To date, the NF-Y family has been identified in a variety of crops [7,17,26,28,38,39]. However, there are few reports about the melon NF-Y family. How NF-Y members participate in the growth, development, and stress response in melon is still unknown.
In this study, a total of 25 CmNF-Y genes were identified (Table 1), which was similar to the number in other cucurbits, such as cucumber [6] and watermelon [38]. However, this is obviously less than that of other crops, such as the 36 in Arabidopsis [5], 59 in tomato [16], and 60 in alfalfa [39], indicating that the NF-Y genes amplification degree of cucurbits is relatively small. Alternatively, there may also be replication gene loss events in the evolution process [40,41]. Each member of the subgroup had the characteristic structure, conservative domain, and motif composition of the corresponding subunit (Table 1, Figures 2-4), which proved that melon NF-Ys were relatively conservative in evolution. However, NF-YA6, NF-YB1/B2/B3/B8, and NF-YC6 were almost not expressed in the tissues (roots, leaves, female flowers, male flowers, and fruits) (RPKM < 1) (Figure 7a) and have almost no effect on fruit development (Figure 7b) and cold tolerance (Figure 8). The commonness of these genes is that they differ greatly from the corresponding subgroups in structure, showing the lack of UTR regions and few or no introns (Figure 1). That is, there is a defect in the 5'-terminal promoter region, or there is a lack of intron sequence compared with the corresponding normal gene, which is in accord with the characteristics of processed pseudogenes [42]. Therefore, it is speculated that these genes might be pseudogenes. On the contrary, other genes might play multiple roles in different ways, in melon. NF-Y factors usually form heterotrimeric complexes to perform functions, mainly by directly binding to CCAAT cis-acting elements in the promoter region of target genes in the nucleus, and then regulating them or interacting with other factors to jointly activate or inhibit the expression of target genes [4,43]. The 25 CmNF-Ys in melon could form 528 trimer complexes, in theory. Even if the prediction threshold was set to the highest value, there were still 71 heterotrimers ( Figure 6), but the specific interaction results need to be verified by a yeast two-hybrid or three-hybrid test (Myers and Holt, 2018).
The role of NF-Ys under abiotic and biotic stress gradually emerged, such as the involvement of ZmNF-YB2/16 [21], OsNF-YA4/A7 [20], and PdNF-YB21 [10] in drought tolerance. AtNFY-C1 (AtHAP5A) positively regulates cold tolerance [18], and BnaMI169N-BnaNF-YA9 modulates responses to salt stress, drought, and ABA [48]. In melon, CmNF-YA1, CmNF-YB6/10, and CmNF-YC1/C2/C5/C7/C8 were continuously up-regulated under cold stress. On the whole, CmNF-YC members were more involved (Figure 8), which was different from that in Petunia hybrida. PhNF-YAs were mainly involved in cold tolerance response [27]. In addition, CmNF-YB4/B7 was only up-regulated after 6 h of cold treatment, which might play a role in the early cold response. Above all, the aforementioned genes might be candidate genes for cold tolerance in melon. A few studies have reported NF-Ys are involved in defending against pathogen infection [49][50][51]. For melon, powdery mildew (PM) and fusarium wilt (FOM) are the main limiting factors affecting melon production [52,53]. Preliminary analysis showed that CmNF-YB7/C3 ( Figure S1) and CmNF-YA4/A5/B5/C2/C7 ( Figure S2) played a role in defensing fusarium wilt and PM, respectively, implying their possible involvement in melon immune signaling. In conclusion, here, we provide an overview of melon NF-Y genes and a reference for further study on the functions of NF-Y in melon under biotic and abiotic stresses. This study is only the beginning of the research on NF-Ys in melon, and the functions of many candidate genes have not been verified yet. In the future, we will further elucidate the gene functions of these candidate genes in biotic and abiotic stresses, especially CmNF-YC1. Yeast two-hybrid or three-hybrid assays can be used to screen for reciprocal NF-YA, NF-YB, or other transcription factors and to deeply elucidate the molecular mechanisms involved in melon cold tolerance. In addition, the role of NF-Ys in melon growth, development, and fruit ripening can also be further investigated.

Plant Materials and Cold Treatment
Melon 'L5283' were used herein in accordance with our previous study [54]. Seedlings were cultured in a growth chamber by using the commercial medium (peat:vermiculite = 3:1). At the one-leaf stage, seedlings were transplanted into plastic pots (7 cm × 7 cm). The seedlings were grown under 28 • C/18 • C (day/night) and 60% relative humidity. At the three-leaf stage, the seedlings were exposed to cold stress (15 • C/6 • C, day/night) for 24 h. This temperature simulated the cold stress in winter and spring according to the actual practice [55]. The third true leaves (from the bottom) were sampled after 0, 6, 12, 18, and 24 h cold treatment. All samples were immediately frozen in liquid nitrogen and kept at −80 • C for total RNA extraction. At least three biological repeats were performed for each treatment.

Identification of NF-Y Family Members in Melon
Firstly, the key words 'nuclear factor Y' were searched in the melon genome database [Melon (DHL92) v3.6.1 Genome, http://cucurbitgenomics.org/organism/, accessed on 12 December 2022] [18]. Meanwhile, the amino acid sequences of all 36 Arabidopsis NF-Ys were downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/, accessed on 12 December 2022) for protein BLAST in melon database, the expected threshold of e-Value was set to 1e −2 . The candidate NF-Y members were obtained by merging the above two methods. Candidate NF-Y genes were confirmed if they contained at least one NF-Y domain (PF02045 and PF00808) according to the Hidden Markov Model (HMM) profile in the Pfam database (http://pfam.janelia.org/, accessed on 12 December 2022) and SMARAT (http://smart.embl-heidelberg.de/, accessed on 12 December 2022).

Multiple Sequence Alignment and Phylogenetic Analysis
For phylogenetic analysis, the protein sequences of NF-Ys from Arabidopsis, cucumber, and melon were used to generate a phylogenetic tree by MEGA 11 software (version 11.0.10). Parameter Settings were as follows: alignment: by muscle; phylogeny: maximum likelihood (ML) tree; test of phylogeny: bootstrap method, 1000 replications; and model: Poisson model. The EVOLVIEW online website (http://www.evolgenius.info/evolview, accessed on 25 December 2022) was used to beautify the evolutionary tree. Multiple sequence alignment of CmNF-Y and AtNF-Y conserved domain sequences by DNAMAN 7.0 software.

Genetic Structure and Conserved Motif Analyses
CmNF-Y protein sequences were submitted to the MEME Suite 5.4.1 to analyze the conserved motifs (https://meme-suite.org/meme/tools/meme, accessed on 1 January 2023). The parameters were set to classic mode, any number of repetitions (any), up to ten motifs, and others were set to default [58]. The gene structures of the CmNF-Ys were analyzed with the GSDS 2.0 program (http://gsds.cbi.pku.edu.cn/, accessed on 1 January 2023) [59].

RNA Isolation and Analysis of CmNF-Ys Expression Patterns
Total RNA was extracted with an ultrapure RNA Kit (Tiangen, Beijing, China) and template cDNA synthesis was performed using the PrimeScript™ RT Master Mix (Tiangen, Beijing, China) following the manufacturer's instructions. A part of the template cDNA was used for RNA-Seq. Meanwhile, the template cDNAs amplified with a 20 µL of reaction solution by using TOROGreen ® qPCR Master Mix (Toroivd, Shanghai, China). The qRT-PCR program consisted of a preliminary step of 60 s at 95 • C, followed by 40 cycles at 95 • C for 10 s, and 60 • C for 30 s. The Actin7 from melon was used as an internal control [55]. The relative gene expression level was calculated via the 2 −∆∆Ct method. Three biological and three technical replicates were used for each sample. All the primers used in this study are listed in Table S2. In addition, the raw transcriptome sequencing data was used to analyze the tissue specificity of NF-Y members and their roles in fruit ripening, including five melon plant tissues, including roots, leaves, male flowers, female flowers, and fruits (PRJNA383830) [62], as well as fruit development of green-fleshed (Dulce) and orangefleshed (Tam-Dew) at 10, 20, and 30 days after anthesis (DAA) and maturity, respectively (PRJNA286120) [37].

Statistical Analysis
Data analyses were performed with Microsoft Excel 2019 and SPSS 20.0 software (SPSS, Chicago, IL, USA). An ANOVA was performed using Duncan's multiple-range test at a level of p < 0.05.

Conclusions
In this study, a total of 25 CmNF-Ys were identified, including six CmNF-YAs, eleven CmNF-YBs, and eight CmNF-YCs. Specific information on the protein characteristics/ architecture, genomic structures, conserved domains/motifs, and phylogenetic analysis were presented. The members of each subgroup were conservative in evolution, among which CmNF-YA6, CmNF-YB1/B2/B3/B8, and CmNF-YC6 might be pseudogenes. Other genes played an important role in melon growth and development, fruit ripening, and low temperature (Figure 9). Our results provide a basis for further studies on the functions of genes in the CmNF-Y family.