Genome-Wide Identification and Drought Stress Response Pattern of the NF-Y Gene Family in Cymbidium sinense

Cymbidium sinense, a type of orchid plant, is more drought-resistant and ornamental than other terrestrial orchids. Research has shown that many members of the NUCLEAR FACTOR Y (NF-Y) transcription factor family are responsive to plant growth, development, and abiotic stress. However, the mechanism of the NF-Y gene family’s response to abiotic stress in orchids has not yet been reported. In this study, phylogenetic analysis allowed for 27 CsNF-Y genes to be identified (5 CsNF-YAs, 9 CsNF-YBs, and 13 CsNF-YC subunits), and the CsNF-Ys were homologous to those in Arabidopsis and Oryza. Protein structure analysis revealed that different subfamilies contained different motifs, but all of them contained Motif 2. Secondary and tertiary protein structure analysis indicated that the CsNF-YB and CsNF-YC subfamilies had a high content of alpha helix structures. Cis-element analysis showed that elements related to drought stress were mainly concentrated in the CsNF-YB and CsNF-YC subfamilies, with CsNF-YB3 and CsNF-YC12 having the highest content. The results of a transcriptome analysis showed that there was a trend of downregulation of almost all CsNF-Ys in leaves under drought stress, while in roots, most members of the CsNF-YB subfamily showed a trend of upregulation. Additionally, seven genes were selected for real-time reverse transcription quantitative PCR (qRT-PCR) experiments. The results were generally consistent with those of the transcriptome analysis. The regulatory roles of CsNF-YB 1, 2, and 4 were particularly evident in the roots. The findings of our study may make a great contribution to the understanding of the role of CsNF-Ys in stress-related metabolic processes.


Introduction
Transcription factors have a significant role in almost all developmental processes in plants.NUCLEAR FACTOR Y (NF-Y) represents a group of sequence-specific transcription factors that are located in the cell nucleus and bind to the CCAAT element in gene promoters [1].NF-Y is a heterotrimeric complex composed of three different subunits: NF-YA, NF-YB, and NF-YC.All three subunits possess conserved DNA-binding domains and subunit interaction domains [2].In the formation of the heterotrimeric complex, NF-YB and NF-YC, which have H2B and H2A structural domains, form a tight histone dimer in the cytoplasm; then, the dimer translocates into the nucleus and interacts with NF-YA to form the final heterotrimer.The CCAAT element is one of the most common cis-elements in eukaryotic promoters [3].Due to the specific binding of NF-YA to the CCAAT box, the heterotrimer functions as a transcription factor that regulates downstream genes containing CCAAT binding sites in the promoter region [4].NF-Y transcription factors have been extensively studied in plants.
The orchid family is one of the largest and most widely distributed families in the plant kingdom, with over 28,000 species [23].Recently, genomic sequencing was conducted for various Cymbidium species, providing valuable resources for our study [24,25], which helps in the systematic exploration of the NF-Y gene family in the orchid family.Cymbidium sinense is a plant species in the orchid family that has a high ornamental value.Its unique deep purple color makes it highly distinctive, and it blooms around the traditional Chinese Spring Festival.With global climate change, arid regions in China are gradually expanding, greatly impacting the environments in which orchids can survive and be cultivated.Drought stress is one of the bottleneck factors for plant growth and development [26,27].In epiphytic orchids, water stress is the most important abiotic factor limiting the growth and development of epiphytic orchids [28].Although C. sinense belongs to terrestrial orchids, its high ornamental and economic value makes the cultivation of drought-tolerant varieties of significant importance for the conservation of orchid germplasm resources and improvement in the orchid industry supply chain.Although NF-Y has been widely reported in model plants and major crops, its roles and functions in the floral development of orchid species are not well understood.This study investigated the response of the NF-Y gene to drought stress in C. sinense by using bioinformatics methods and provides a set of potential drought-resistant candidate genes.It is of great significance for the identifying of key regulatory factors for drought resistance in C. sinense.

Identification and Physicochemical Properties
All 27 of the CsNF-Ys were identified (Table S1) and are presented in a phylogenetic tree (Figure 1).To explore the phylogenetic relationship of CsNF-Ys in C. sinense, a neighborjoining (NJ) phylogenetic tree was performed using 27 CsNF-Ys and 36 AtNF-Ys.Based on the classification of NF-Y gene families in A. thaliana and the composition of conserved protein domains in CsNF-Ys, the 27 CsNF-Ys were divided into three subfamilies: CsNF-YA, CsNF-YB, and CsNF-YC.Among these subfamilies, CsNF-YA had the fewest members, as it had only 5, while CsNF-YC had the most members with 13 genes.Additionally, the CsNF-YB subfamily consisted of nine members.We presented the evolutionary distances between different proteins in Figure S1.
The results of the analysis of the protein physicochemical properties in CsNF-Ys revealed that the isoelectric point (pI) of the NF-Y proteins in C. sinense ranged from 4.63 (CsNF-YB6) to 9.95 (CsNF-YA3) (Table 1).The molecular weight ranged from 12.14 kDa (CsNF-YC11) to 32.87 kDa (CsNF-YA5).The number of amino acids ranged from 112 (CsNF-YC11) to 293 (CsNF-YA5), and the aliphatic index ranged from 48.15 to 105.8.The protein physicochemical properties showed that the average hydropathy index (GRAVY) of the CsNF-Y proteins was mostly negative, with a minimum of −0.805 (CsNF-YA5).Notably, CsNF-YC11 stood out with a positive hydropathy index of 0.014.Therefore, the CsNF-Y proteins were predominantly hydrophilic, albeit with varying degrees of hydrophilicity.Among these 27 CsNF-Y proteins, only CsNF-YB1 and CsNF-YC5 had instability coefficients that were less than 40, and those of the majority of the CsNF-Y proteins were greater than 50 (63%).The results of the analysis of the protein physicochemical properties in CsNF-Ys revealed that the isoelectric point (pI) of the NF-Y proteins in C. sinense ranged from 4.63 (CsNF-YB6) to 9.95 (CsNF-YA3) (Table 1).The molecular weight ranged from 12.14 kDa (CsNF-YC11) to 32.87 kDa (CsNF-YA5).The number of amino acids ranged from 112 (CsNF-YC11) to 293 (CsNF-YA5), and the aliphatic index ranged from 48.15 to 105.8.The protein physicochemical properties showed that the average hydropathy index (GRAVY) of the CsNF-Y proteins was mostly negative, with a minimum of −0.805 (CsNF-YA5).Notably, CsNF-YC11 stood out with a positive hydropathy index of 0.014.Therefore, the CsNF-Y proteins were predominantly hydrophilic, albeit with varying degrees of hydrophilicity.Among these 27 CsNF-Y proteins, only CsNF-YB1 and CsNF-YC5 had instability coefficients that were less than 40, and those of the majority of the CsNF-Y proteins were greater than 50 (63%).

Gene Conserved Motif Analysis
The conserved motifs of the CsNF-Y proteins were further characterized with the MEME software.The results revealed a total of ten conserved motifs (Figure 2).Among them, Motif 2 was detected in all 27 genes, while Motif 9 was only detected in 3 genes.In the members of the CsNF-YA subfamily, Motifs 2, 6, 7, 8, and 10 were detected, with Motifs 2, 7, and 10 being present in all members of the CsNF-YA subfamily.Motifs 6 and 8 were detected in CsNF-YA1 and CsNF-YA5.In the CsNF-YB subfamily, Motifs 1, 2, 3, and 4 were detected, with Motif 4 being the only shared motif.In the CsNF-YC subfamily, Motifs 1, 2, 3, 5, 6, 8, and 9 were detected.Motif 5 was detected in almost all members of the subfamily, while Motif 6 was present in CsNF-YC2, 3, 4, 6, 7, 9, and 10.Motifs 8 and 9 were only found in CsNF-YC7, 9, and 10.All motif logos are displayed in Figure 2C.

Analysis of Amino Acid Conserved Domains
The protein sequences of CsNF-YA, CsNF-YB, and CsNF-YC were subjected to multiple-sequence alignment analysis by using Phylosuite and ESPript 3.0 to identify and analyze the conserved domains of the CsNF-Y proteins.Each member of these three subfamilies contained a highly conserved DNA-binding domain, and the DNA-binding and subunit interaction domains were represented.The multiple-sequence alignment of the CsNF-YA proteins revealed a conserved region consisting of approximately 60 amino acids (Figure 3A).Similarly, the protein sequence alignments of CsNF-YB (approximately 90 amino acids) and CsNF-YC (approximately 75 amino acids) also identified two conserved regions each (Figure 3B,C).

Analysis of Amino Acid Conserved Domains
The protein sequences of CsNF-YA, CsNF-YB, and CsNF-YC were subjected to multiplesequence alignment analysis by using Phylosuite and ESPript 3.0 to identify and analyze the conserved domains of the CsNF-Y proteins.Each member of these three subfamilies contained a highly conserved DNA-binding domain, and the DNA-binding and subunit interaction domains were represented.The multiple-sequence alignment of the CsNF-YA proteins revealed a conserved region consisting of approximately 60 amino acids (Figure 3A).Similarly, the protein sequence alignments of CsNF-YB (approximately 90 amino acids) and CsNF-YC (approximately 75 amino acids) also identified two conserved regions each (Figure 3B,C).

Gene Structure and Characterization Analysis
Furthermore, with the aim of enhancing our understanding of the structural composition of genes, the genomic DNA sequences of the CsNF-Y genes were analyzed to compare the structures and quantities of introns, exons, coding regions, and non-coding regions (Figure 4).Most members of the CsNF-Y family either lacked introns or had only a few introns, with only five genes having more than five introns.Additionally, the number of introns in the members of the CsNF-YB subfamily were consistently less than 10, while the CsNF-YA and CsNF-YC subfamilies each had one gene with 22 introns.Interestingly, the CsNF-YA1 and CsNF-YC10 genes, which had 22 introns, also had the highest number of exons [23].

Gene Structure and Characterization Analysis
Furthermore, with the aim of enhancing our understanding of the structural composition of genes, the genomic DNA sequences of the CsNF-Y genes were analyzed to compare the structures and quantities of introns, exons, coding regions, and non-coding regions (Figure 4).Most members of the CsNF-Y family either lacked introns or had only a few introns, with only five genes having more than five introns.Additionally, the number of introns in the members of the CsNF-YB subfamily were consistently less than 10, while the CsNF-YA and CsNF-YC subfamilies each had one gene with 22 introns.Interestingly, the CsNF-YA1 and CsNF-YC10 genes, which had 22 introns, also had the highest number of exons [23].

Chromosome Distribution Analysis
In order to investigate the chromosomal distribution of the CsNF-Y genes, we analyzed the genome of C. sinense.The analysis revealed an uneven distribution of a total of 27 NF-Y genes across 13 chromosomes (Figure 5).Based on the subfamily classification and chromosome position information, we assigned names to these genes: CsNF-YA1-5, CsNF-YB1-9, and CsNF-YC1-13.The highest number of NF-Y transcription factors (5, 18.5%) was found on chromosome 9. Chromosome 11 contained four CsNF-Y genes (14.8%), while chromosomes 3, 4, 6, and 17 each had only one CsNF-Y gene.
The CsNF-YC subfamily was distributed on most chromosomes, while the CsNF-YA

Chromosome Distribution Analysis
In order to investigate the chromosomal distribution of the CsNF-Y genes, we analyzed the genome of C. sinense.The analysis revealed an uneven distribution of a total of 27 NF-Y genes across 13 chromosomes (Figure 5).Based on the subfamily classification and

cis-Elements Analysis
To speculate on the potential functions of CsNF-Y genes, an analysis of the prediction of cis-elements was conducted by using the promoters of these genes (Figure 6).Several categories of cis-elements were observed in the CsNF-Y genes, and they were roughly classified into four types: light-responsive elements, plant growth elements, stress-responsive elements, and hormone-responsive elements.The CsNF-YC subfamily was distributed on most chromosomes, while the CsNF-YA subfamily was found on chromosomes 8, 11, 17, 18, and 19.Chromosome 17 exclusively contained a member of the CsNF-YA subfamily, CsNF-YA3.The CsNF-YB subfamily was distributed on chromosomes 1, 2, 3, 6, 9, 11, 18, and 19, with chromosomes 3 and 6 only harboring members of the CsNF-YB subfamily.The members of the NF-YC subfamily were widely distributed, with chromosomes 4, 5, and 10 exclusively containing members of the CsNF-YC subfamily, and no members of the CsNF-YC subfamily were found on chromosomes 3, 6, 17, 18, and 19.Some CsNF-YC transcription factors with similar conserved structures were located on the same chromosome, such as the four members of the CsNF-YC subfamily and CsNF-YB5 on chromosome 9.

cis-Elements Analysis
To speculate on the potential functions of CsNF-Y genes, an analysis of the prediction of cis-elements was conducted by using the promoters of these genes (Figure 6).Several categories of cis-elements were observed in the CsNF-Y genes, and they were roughly classified into four types: light-responsive elements, plant growth elements, stress-responsive elements, and hormone-responsive elements.

Analysis of the Interaction Network and Secondary and Tertiary Structures of CsNF-Ys Proteins
Protein-protein interaction prediction was conducted to further understand the biological function and regulatory network of CsNF-Ys.A total of 57 protein-protein interactions were discovered, and apart from 17 CsNF-Y proteins, a total of 40 proteins interacted with CsNF-Ys (Figure 7A).The proteins interacting with CsNF-Ys have been functionally validated to be associated with drought stress, such as through the DR1 gene [29] and the BZIP gene family [30].Moreover, proteins such as QQS, which were involved in plant development or the response to abiotic stress [31,32], were predicted to interact with CsNF-Ys.

Proteins
Protein-protein interaction prediction was conducted to further understand the biological function and regulatory network of CsNF-Ys.A total of 57 protein-protein interactions were discovered, and apart from 17 CsNF-Y proteins, a total of 40 proteins interacted with CsNF-Ys (Figure 7A).The proteins interacting with CsNF-Ys have been functionally validated to be associated with drought stress, such as through the DR1 gene [29] and the BZIP gene family [30].Moreover, proteins such as QQS, which were involved in plant development or the response to abiotic stress [31,32], were predicted to interact with CsNF-Ys.The analysis of the secondary structure of the CsNF-Y proteins (Table S2) revealed that the alpha helix occupied the largest proportion of the protein structure, followed by the random coil and then the extended strand, with the smallest proportion belonging to the beta-turn.The proportion of alpha helix structures in the CsNF-YA and CsNF-YC subfamilies was significantly larger than the proportion of random coil structures, while this difference was less pronounced in CsNF-YB.The tertiary structure of the CsNF-Y proteins was predicted through an online analysis with SWISS-MODEL, and the results are displayed in Figure 7B.The analysis of the secondary structure of the CsNF-Y proteins (Table S2) revealed that the alpha helix occupied the largest proportion of the protein structure, followed by the random coil and then the extended strand, with the smallest proportion belonging to the beta-turn.The proportion of alpha helix structures in the CsNF-YA and CsNF-YC subfamilies was significantly larger than the proportion of random coil structures, while this difference was less pronounced in CsNF-YB.The tertiary structure of the CsNF-Y proteins was predicted through an online analysis with SWISS-MODEL, and the results are displayed in Figure 7B.

Analysis of Expression Patterns under Drought Stress in Leaves and Roots
By comparing the FPKM values of leaves and roots under three levels of drought stress, we detected the expression profiles of individual CsNF-Y genes to investigate the organ-specific expression patterns of the NF-Y gene family in C. sinense under different drought conditions (Figure 8).

Analysis of Expression Patterns under Drought Stress in Leaves and Roots
By comparing the FPKM values of leaves and roots under three levels of drought stress, we detected the expression profiles of individual CsNF-Y genes to investigate the organ-specific expression patterns of the NF-Y gene family in C. sinense under different drought conditions (Figure 8).S3.
Among the 27 CsNF-Y genes, most of the detected CsNF-Y genes (18/27) exhibited altered regulation under drought stress.Notably, CsNF-B9 and CsNF-C5 showed consistent expression trends in both roots and leaves across the drought levels.Intriguingly, many members in both organs showed a positive drought response through significant downregulation.No CsNF-Ys in the leaves continued their upregulation under both mild and severe drought stress; on the contrary, five CsNF-Ys in the roots were concurrently  S3.
Among the 27 CsNF-Y genes, most of the detected CsNF-Y genes (18/27) exhibited altered regulation under drought stress.Notably, CsNF-B9 and CsNF-C5 showed consistent expression trends in both roots and leaves across the drought levels.Intriguingly, many members in both organs showed a positive drought response through significant downregulation.No CsNF-Ys in the leaves continued their upregulation under both mild and severe drought stress; on the contrary, five CsNF-Ys in the roots were concurrently upregulated under both conditions, potentially indicating that there was a more active drought stress response in the roots through transcriptional activation.

Expression of CsNY-Fs in Response to Drought Stress
To validate the reliability of the transcriptome data of CsNF-Y genes in leaves and roots during drought stress, we selected seven genes (CsNF-YA1, CsNF-YA2, CsNF-YB1, CsNF-YB2, CsNF-YB3, CsNF-YB4, and CsNF-YC3) with high expression or significant changes based on the transcriptome data and conducted qRT-PCR experiments (Figure 9).The results showed that the expression levels of these seven genes varied during drought stress, and the trends were generally consistent with the changes observed in the transcriptome data.
Consistent with the trend of the transcriptome data, as the severity of drought stress increased, most genes in both leaves and roots showed a negative response.Members of the CsNF-YB subfamily in the root exhibited a positive response, and their expression levels increased with the deepening of drought stress, consistent with the trend observed in the transcriptome data.Further correlation analysis revealed a highly significant correlation among CsNF-YA1, CsNF-YB1, CsNF-YB2, CsNF-YB3, and CsNF-YB4 (Figure 9C).CsNF-YB2 showed a significant correlation with CsNF-YB4, indicating a possible association in the expression of these two genes.

Expression of CsNY-Fs in Response to Drought Stress
To validate the reliability of the transcriptome data of CsNF-Y genes in leaves and roots during drought stress, we selected seven genes (CsNF-YA1, CsNF-YA2, CsNF-YB1, CsNF-YB2, CsNF-YB3, CsNF-YB4, and CsNF-YC3) with high expression or significant changes based on the transcriptome data and conducted qRT-PCR experiments (Figure 9).The results showed that the expression levels of these seven genes varied during drought stress, and the trends were generally consistent with the changes observed in the transcriptome data.S4.
Consistent with the trend of the transcriptome data, as the severity of drought stress increased, most genes in both leaves and roots showed a negative response.Members of the CsNF-YB subfamily in the root exhibited a positive response, and their expression levels increased with the deepening of drought stress, consistent with the trend observed in the transcriptome data.Further correlation analysis revealed a highly significant correlation among CsNF-YA1, CsNF-YB1, CsNF-YB2, CsNF-YB3, and CsNF-YB4 (Figure 9C).CsNF-YB2 showed a significant correlation with CsNF-YB4, indicating a possible association in the expression of these two genes.

Discussion
The NF-Y transcription factor is crucial for plant growth and development at various stages.Abiotic stresses (temperature, salinity, and drought) significantly impact plant growth.In response to these stresses, plants have developed strategies for enhancing stress tolerance.NF-Y transcription factors have attracted considerable attention in the field of plant research due to their essential functions in plant-microbe interactions, root  S4.

Discussion
The NF-Y transcription factor is crucial for plant growth and development at various stages.Abiotic stresses (temperature, salinity, and drought) significantly impact plant growth.In response to these stresses, plants have developed strategies for enhancing stress tolerance.NF-Y transcription factors have attracted considerable attention in the field of plant research due to their essential functions in plant-microbe interactions, root development, and adaptation to water-related stress [33].In plants, multiple genes encode each subunit of NF-Y, and the number of genes involved may vary among different species [1].In A. thaliana, 36 NF-Y genes have been identified.Among them, the AtNF-YA subfamily consists of 10 members, while the AtNF-YB and AtNF-YC subfamilies each have 13 members [34].A total of 34 NF-Y genes were identified in O. sativa, with 11 members of OsNF-YA, 11 members of OsNF-YB, and 12 members of OsNF-YC [32].In this study, we identified 27 CsNF-Y genes: 5 CsNF-A, 9 CsNF-YB, and 13 CsNF-YC genes.The variations in family size across species suggests evolutionary changes influenced by factors such as the living environment.
Phylogenetic analysis indicated a close relationship between CsNF-Y and AtNF-Y in the phylogenetic tree.C. sinense divides CsNF-Y into three subgroups based on AtNF-Y, and functionally similar genes are grouped together.One can predict the functions of NF-Ys based on the known functions of AtNF-Ys [35].Earlier studies found that AtNF-YA1 and AtNF-YA9, which are located on the same branch, are crucial regulators of embryonic development and flowering time [7,36].This suggests that the CsNF-YA1 and CsNF-YA5 genes, which are on the same branch in the phylogenetic tree, may have similar functions.NF-YB3 from Picea wilsonii, which is transiently expressed in Arabidopsis, exhibited characteristics of salt and drought stress tolerance.In Phalaenopsis, the PhNF-YC7 gene and other NF-Y family genes may indirectly regulate plant development and flowering time in response to low temperatures.We speculate that the NF-YC subfamily in Cymbidium may also have similar functional responses [22].
Multiple-sequence alignment analysis showed that the best CsNF-Y proteins contained conserved regions as a result of subunit interactions and DNA binding (Figure 4).These regions are also present in other plants [37].Previous studies indicate that the DNA-binding structure of NF-Y can bind to the CCAAT site [38].In addition, conservative motif analysis of the 27 CsNF-Y proteins revealed that all members contained Motif 2. Interestingly, all members of the CsNF-YB subfamily had acquired Motif 4, which was not present in the other subfamilies.The reliability of the phylogenetic tree and clustering was further confirmed by the presence of a similar gene structure composition and conserved motifs within specific subfamilies.
Consistent with the prediction of the cis-elements of the NF-Y family in other species, CsNF-Ys contained 39 cis-elements.The CsNF-Y genes were regulated by cis-elements and played crucial roles in the stress response.Genes containing the drought-responsive element MBS (MYB binding site) were mainly members of the CsNF-YB and CsNF-YC subfamilies.The member CsNF-YB3 in the CsNF-YB subfamily, which contains the most MBS ciselements, showed consistent expression in both leaves and roots, with the transcriptome changes following a similar trend.However, the gene CsNF-YC12, which contains the most MBS cis-elements in the CsNF-YC subfamily, was not expressed in either the leaves or roots.Instead, genes CsNF-YC5 and CsNF-YC10, which contain fewer MBS cis-elements, were expressed in both leaves and roots with consistent expression patterns.Combined with the transcriptome heatmap, it was found that the expression levels of most CsNF-YB subfamily members in the root system gradually increased with the severity of drought stress.In contrast, most CsNF-YA and CsNF-YC subfamily members showed lower expression levels under severe drought compared to the control group.This suggested that the response of CsNF-YB to drought stress may be closely related not only to MBS but also to Motif 4. Intronexon structure analysis indicated that most CsNF-Y sequences (37%) consisted of 0 introns and 1 exon, while CsNF-YA1 and CsNF-YC10 contained 22 introns and 23 exons.This situation was rarely observed in the NF-Y family in other species.In Citrullus lanatus, an analysis of the gene structure of ClNF-Y exhibited that all genes consisted of 0-5 introns and 1-6 exons [39].The structural analysis of MaNF-Y genes indicated that all of them contained 0-4 introns, and they evidenced a relatively consistent intron-exon organization [40].The high diversity in gene structure suggests extensive differentiation during the formation and evolution of the CsNF-Y genome.
We also investigated the expression patterns of CsNF-Ys under varying levels of drought stress and in different tissues to understand their roles in the drought stress response in C. sinense.In addition, under severe drought stress (L2 and R2), compared to the control group (L0 and R0), most of the CsNF-Y genes were upregulated in the roots and downregulated in the leaves.Some TaNF-Y genes in wheat leaves exhibited a significant upregulation in response to drought stress-particularly TaNF-YB2 [41].Interestingly, in C. sinense, slight drought treatment caused a significant change in gene expression in the leaves, while severe drought treatment induced a significant change in gene expression in the roots.We also observed that as the severity of drought stress deepened, most genes in the leaves showed a trend of downregulation; the genes in the CsNF-YA and CsNF-YC subfamilies were mostly downregulated in the roots, while the genes in the CsNF-YB subfamily were upregulated.This result may imply that members of the CsNF-YB family with a specific Motif 4 binding structure have some particular stress response mechanisms.
These findings are crucial for identifying potential genes that enhance molecular breeding efficiency.Moreover, these discoveries contribute to the selection of varieties of C. sinense with strong resistance to adversity and can provide important reference value for the understanding of the action of NF-Y transcription factors under stress conditions.These results may contribute to the improvement in the tolerance of Cymbidium to abiotic stress.12 h/12 h, a temperature range of 22-26 • C, and a humidity of 40%.The soil moisture level was monitored daily during the drought stress period.Healthy C. sinense plants were selected, and seedlings were grown under well-watered conditions for four days.On the fourth day, control samples L0 and R0 were collected.Irrigation was then stopped to initiate the drought stress treatment.Mild-stress samples (L1 and R1) were collected on the third day after irrigation was stopped, and severe-stress samples (L2 and R2) were collected on the seventh day after irrigation was stopped.Three replicates were collected per group, and leaf samples were taken from young leaves that were shorter than ten centimeters, while root samples were taken from newly formed roots at the base of the plants.The samples were collected in liquid nitrogen and stored at −80 • C until RNA extraction.After a 7-day period of drought stress, there were no apparent physical changes in the plant's appearance that could be observed with the naked eye, such as leaf wilting or yellowing.Therefore, based on the above observations, the plants remained in a healthy state despite the drought stress.Hence, we believe that the impact of the 7-day drought and sampling on RNA can be considered negligible.

Isolation of RNA, cDNA Preparation, and Expression Analysis
The cetyltrimethylammonium bromide (CTAB) method was employed to isolate total RNA from different tissues.Three replicates were collected per tissue.The reagent kit used for preparing RNA was Vazyme's FastPure Plant Total RNA Isolation Kit (Polysaccharides& Polyphenolics-rich), and the specific preparation method followed the product manual.The reagent kit used for preparing cDNA was Yeasen's Hifair AdvanceFast One-step RT-gDNA Digestion SuperMix for qPCR, and the specific preparation method followed the product manual.Subsequently, transcriptome sequencing was performed on the MGI2000 sequencing platform.To ensure data quality, the SOAPnuke v1.4.0 software (BGI-Shenzhen, China) was utilized to eliminate low-quality reads, reads containing adapters, and reads with poly-N sequences (with more than 5% unknown bases) [45].The resulting clean reads were aligned to the nucleotide sequences of CsNF-Ys by using HISAT2 version 2.1.0(University of Texas Southwestern Medical Center, Dallas, TX, USA) [46].The expression levels of the CsNF-Ys were calculated for each exon isoform by using the FPKM (fragments per kilobase of exon per million fragments mapped) method implemented in RSEM v1.2.8 (University of Wisconsin-Madison, Madison, WI, USA) [47,48].Finally, TBtools-II (version v2.207) was utilized to visualize the expression profiles.

RNA Extraction and Real-Time Reverse Transcription Quantitative PCR (qRT-PCR) Analysis
To validate the transcriptome data, we selected seven candidate genes for qRT-PCR to confirm their transcriptomic profiles.A total of three biological replicates were collected from two different tissues in three distinct drought stress periods.The collected samples were immediately frozen in liquid nitrogen and stored at −80 • C until total RNA extraction.The stable actin gene (Mol 022529) from the same species was chosen as the reference gene [49].Primers for the stable actin gene and the seven candidate genes were designed using Primer Premier 5.0 [50], as listed in Table S4.For reverse transcription, 1 µL of RNA was subjected to cDNA using the Hifair AdvanceFast One-Step RT-gDNA Digestion SuperMix for qPCR (Beijing, China).The resulting cDNA from each sample was diluted to a concentration of 10 ng/mL, and 2 µL of the diluted cDNA was used as the template for qRT-PCR.The qRT-PCR experiments were performed on the Applied Biosystems ® QuantStudio ® 3 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with SYBR Green dye.The reaction system was 20 µL, and the reaction program was set as follows: 1 min at 95 • C, followed by 40 cycles of 10 s at 95 • C and 20 s at 60 • C. Fluorescence signals were collected at the end of the annealing and extension step.The product specificity was determined through a melting curve analysis, and the Ct values for each sample were obtained.The expression levels of each gene were normalized to the actin internal control gene, and the relative gene expression levels were calculated by using the 2 −∆∆CT method [51].The correlation analysis of the seven selected genes for qRT-PCR experiments was performed using CorrPlot with the OmicStudio tool (https://www.omicstudio.cn/tool,accessed on 19 December 2023).

Conclusions
This study examined the NF-Y gene family in C. sinense.A total of 27 CsNF-Ys were identified in C. sinense for the first time, and various aspects, including conserved motifs, exon-intron structures, and secondary/tertiary structures of proteins, were analyzed.The findings revealed a high degree of conservation among CsNF-Y genes.The identification of cis-elements related to drought response in the promoter region of CsNF-Ys helped expand the knowledge of the drought resistance pathway in C. sinense.This study explored the performance of CsNFYs in three stages of water deficiency and verified their expression patterns in the leaves and roots.The consistent results showed distinct expression patterns of CsNF-Ys in the leaves and roots.Three CsNF-Ys (CsNF-YB1, CsNF-YB2, and CsNF-YB4) may be potential drought-resistant candidate genes.This study may provide valuable information for the study of the stress response mechanisms of NF-Y genes in different tissues of orchids and other species.

Figure 1 .
Figure 1.The NJ phylogenetic tree of NF-Y proteins from C. sinense, A. thaliana, and O. sativa.Proteins from C. sinense are indicated by pink dots, proteins from A. thaliana are indicated by blue dots, and proteins from O. sativa are indicated by green dots.The purple, yellow, and red bands indicate the NF-YA, NF-YB, and NF-YC subfamilies, respectively.

Figure 1 .
Figure 1.The NJ phylogenetic tree of NF-Y proteins from C. sinense, A. thaliana, and O. sativa.Proteins from C. sinense are indicated by pink dots, proteins from A. thaliana are indicated by blue dots, and proteins from O. sativa are indicated by green dots.The purple, yellow, and red bands indicate the NF-YA, NF-YB, and NF-YC subfamilies, respectively.

Figure 2 .
Figure 2. The phylogenetic tree and architecture of conserved protein motifs of the CsNF-Y gene family in C. sinense.(A) The phylogenetic relationship of CsNF-Y proteins in C. sinense.Members of the CsNF-YA, CsNF-YB, and CsNF-YC subfamilies are indicated by blue, yellow, and green colors, respectively.(B) The motif patterns of CsNF-Y proteins.Different colors represent different motifs, which are numbered from 1 to 10.Protein lengths can be estimated based on the scale at the bottom right.(C) The sequence information for Motifs 1-10, respectively.Protein lengths can be estimated based on the scale at the bottom.

Figure 2 .
Figure 2. The phylogenetic tree and architecture of conserved protein motifs of the CsNF-Y gene family in C. sinense.(A) The phylogenetic relationship of CsNF-Y proteins in C. sinense.Members of the CsNF-YA, CsNF-YB, and CsNF-YC subfamilies are indicated by blue, yellow, and green colors, respectively.(B) The motif patterns of CsNF-Y proteins.Different colors represent different motifs, which are numbered from 1 to 10.Protein lengths can be estimated based on the scale at the bottom right.(C) The sequence information for Motifs 1-10, respectively.Protein lengths can be estimated based on the scale at the bottom.

Figure 3 .
Figure 3.The multiple-sequence alignment of CsNF-Y protein sequences.(A-C) The sequence alignment of highly conserved domains of CsNF-YA, CsNF-YB, and CsNF-YC proteins in C. sinense.

Figure 4 .
Figure 4.The numbers of exons, introns, coding sequences (CDSs), and untranslated regions (UTRs) in the CsNF-Y genes.(A) The proportion of members with different numbers of introns, exons, CDSs, and UTRs in each subfamily among the total subfamily members.Green indicates a low proportion, and blue indicates a high proportion.(B) The numbers of exons, introns, CDSs, and UTRs on each gene in the CsNF-Y gene family, with different colors indicating different structures.The quantity can be estimated based on the bottom axis.

Figure 4 .
Figure 4.The numbers of exons, introns, coding sequences (CDSs), and untranslated regions (UTRs) in the CsNF-Y genes.(A) The proportion of members with different numbers of introns, exons, CDSs, and UTRs in each subfamily among the total subfamily members.Green indicates a low proportion, and blue indicates a high proportion.(B) The numbers of exons, introns, CDSs, and UTRs on each gene in the CsNF-Y gene family, with different colors indicating different structures.The quantity can be estimated based on the bottom axis.

Figure 5 .
Figure 5. Chromosome distribution of NF-Y gene family of C. sinense.

Figure 5 .
Figure 5. Chromosome distribution of NF-Y gene family of C. sinense.

Figure 6 .
Figure 6.Analysis of cis-regulatory elements in CsNF-Ys.(A) Heatmap of the quantity of cis-elements.The color bar above and the values in the boxes represent the classification and quantity of cis-elements, with deep blue in the boxes indicating a high quantity and yellow indicating a low quantity.Below are four different color bars: black, yellow, light blue, and deep blue, representing cis-elements for light response, plant hormone response, stress response, and plant growth and development, respectively.(B) The sum of the four types of cis-elements in each gene is represented by a bar chart with different colors, with each color indicating a different response element.The total number of cis-elements on each gene was annotated at the end of each bar chart.

Figure 6 .
Figure 6.Analysis of cis-regulatory elements in CsNF-Ys.(A) Heatmap of the quantity of cis-elements.The color bar above and the values in the boxes represent the classification and quantity of ciselements, with deep blue in the boxes indicating a high quantity and yellow indicating a low quantity.Below are four different color bars: black, yellow, light blue, and deep blue, representing cis-elements for light response, plant hormone response, stress response, and plant growth and development, respectively.(B) The sum of the four types of cis-elements in each gene is represented by a bar chart with different colors, with each color indicating a different response element.The total number of cis-elements on each gene was annotated at the end of each bar chart.

Figure 7 .
Figure 7.The predicted protein-protein interaction network and protein tertiary structures of CsNF-Ys.(A) The predicted protein-protein interaction network of CsNF-Ys.Each green oval represents a protein, and the darker the green, the stronger the interaction between the proteins.The outer circle represents proteins interacting with CsNF-Ys, while the inner circle represents NF-Y proteins of C. sinense.The interactions between these proteins are indicated by gray lines.(B) The predicted protein tertiary structures of CsNF-Ys.

Figure 7 .
Figure 7.The predicted protein-protein interaction network and protein tertiary structures of CsNF-Ys.(A) The predicted protein-protein interaction network of CsNF-Ys.Each green oval represents a protein, and the darker the green, the stronger the interaction between the proteins.The outer circle represents proteins interacting with CsNF-Ys, while the inner circle represents NF-Y proteins of C. sinense.The interactions between these proteins are indicated by gray lines.(B) The predicted protein tertiary structures of CsNF-Ys.

Figure 8 .
Figure 8.The expression profiles of CsNF-Y genes under different levels of drought stress in leaves and roots.(A) L0, L1, and L2 represent the control group, mild drought treatment, and severe drought treatment in leaves, respectively.(B) R0, R1, and R2 represent the control group, mild drought treatment, and severe drought treatment in roots, respectively.The color bar below represents the normalized FPKM values: red, high expression level; yellow, low expression level; blue, no expression.The detailed FPKM values are listed in TableS3.

Figure 8 .
Figure 8.The expression profiles of CsNF-Y genes under different levels of drought stress in leaves and roots.(A) L0, L1, and L2 represent the control group, mild drought treatment, and severe drought treatment in leaves, respectively.(B) R0, R1, and R2 represent the control group, mild drought treatment, and severe drought treatment in roots, respectively.The color bar below represents the normalized FPKM values: red, high expression level; yellow, low expression level; blue, no expression.The detailed FPKM values are listed in TableS3.

Figure 9 .
Figure 9. Verification of the effect of CsNF-Ys during drought stress with real-time reverse transcription quantitative PCR (qRT-PCR).(A) The relative expression levels of each gene in leaves.(B) The relative expression levels of each gene in roots.The blue bars represent the relative expression during the control period, indicating no drought stress.The light blue bars represent the relative expression during mild drought stress, and the deep blue bars represent the relative expression during severe drought stress.Black asterisks indicate the p values from the significance test (* p < 0.05, ** p < 0.01).(C) The correlation analysis for these seven CsNF-Ys genes.Primers for qRT-PCR are shown in TableS4.

Figure 9 .
Figure 9. Verification of the effect of CsNF-Ys during drought stress with real-time reverse transcription quantitative PCR (qRT-PCR).(A) The relative expression levels of each gene in leaves.(B) The relative expression levels of each gene in roots.The blue bars represent the relative expression during the control period, indicating no drought stress.The light blue bars represent the relative expression during mild drought stress, and the deep blue bars represent the relative expression during severe drought stress.Black asterisks indicate the p values from the significance test (* p < 0.05, ** p < 0.01).(C) The correlation analysis for these seven CsNF-Ys genes.Primers for qRT-PCR are shown in TableS4.

Table 1 .
Protein information of the NF-Y gene family in Cymbidium sinense, including the gene ID, gene name, genomic position, and protein physicochemical properties.