Genome-Wide Identification, Characterization and Expression Analysis of Plant Nuclear Factor (NF-Y) Gene Family Transcription Factors in Saccharum spp.

Plant nuclear factor (NF-Y) is a transcriptional activating factor composed of three subfamilies: NF-YA, NF-YB, and NF-YC. These transcriptional factors are reported to function as activators, suppressors, and regulators under different developmental and stress conditions in plants. However, there is a lack of systematic research on the NF-Y gene subfamily in sugarcane. In this study, 51 NF-Y genes (ShNF-Y), composed of 9 NF-YA, 18 NF-YB, and 24 NF-YC genes, were identified in sugarcane (Saccharum spp.). Chromosomal distribution analysis of ShNF-Ys in a Saccharum hybrid located the NF-Y genes on all 10 chromosomes. Multiple sequence alignment (MSA) of ShNF-Y proteins revealed conservation of core functional domains. Sixteen orthologous gene pairs were identified between sugarcane and sorghum. Phylogenetic analysis of NF-Y subunits of sugarcane, sorghum, and Arabidopsis showed that ShNF-YA subunits were equidistant while ShNF-YB and ShNF-YC subunits clustered distinctly, forming closely related and divergent groups. Expression profiling under drought treatment showed that NF-Y gene members were involved in drought tolerance in a Saccharum hybrid and its drought-tolerant wild relative, Erianthus arundinaceus. ShNF-YA5 and ShNF-YB2 genes had significantly higher expression in the root and leaf tissues of both plant species. Similarly, ShNF-YC9 had elevated expression in the leaf and root of E. arundinaceus and in the leaf of a Saccharum hybrid. These results provide valuable genetic resources for further sugarcane crop improvement programs.


Introduction
Plant growth, development, and biomass production are severely affected by various biotic and abiotic stresses. Plants exert various stress-responsive mechanisms to combat these stresses. These defensive mechanisms are in turn regulated by various genes. Transcription factors are renowned for their regulatory role in modulating gene expression [1,2]. Regulation of gene expression by transcription factors assists in modulating downstream

Identification of NF-Y Gene Members in Sugarcane
The sugarcane genome was searched for NF-Y genes using conserved regions of Sorghum bicolor NF-Y gene sequences, which is a close relative of sugarcane. The obtained sequences were further used as queries to find NF-Ys in sugarcane "(https://sugarcanegenome.cirad.fr/ (accessed on 20 March 2022)". Retrieved sequences were checked for core NF-Y domains using InterProScan "https://www.ebi.ac.uk/interpro/search/sequence/ (accessed on 1 April 2022)", MotifSearch "https://www.genome.jp/tools/motif/ (accessed on 7 April 2022)", and NCBI's Conserved Domain Database (CDD) "https://www.ncbi. nlm.nih.gov/Structure/cdd/wrpsb.cgi (accessed on 14 April 2022)". Redundant and incomplete sequences were removed. Identified NF-Y sequences (ShNF-Y) were located on the chromosomes using BLAST and manually marked. Gene numbers were assigned for ShNF-Y genes based on their physical positions on the chromosome. ShNF-Y sequences were predicted for open reading frames (ORFs), coding sequences (cds), and proteins. A bioinformatic pipeline for the identification, in silico and expression analyses of NF-Y genes is given in Figure 1.

Multiple Sequence Alignment and Phylogenetic Analysis
Conserved core regions of ShNF-YA, ShNF-YB, and ShNF-YC proteins were aligned with conserved regions of sorghum and Arabidopsis NF-Y subunits using Unipro UGENE v46.0. Phylogenetic analysis was carried out using MEGA 10.0.5. Multiple sequence alignment was carried out using MUSCLE with parameters (Gap Open-2.90, Gap Extend 0.00, Hydrophobicity Multiplier 1.20, Max Memory in MB 2048, Max Iterations 16, Cluster Method UPGMA, and Min Diag Length (Lambda) 24). Phylogenetic analysis was carried out with 1000 bootstrap replicates and with the following parameters: Substitutions type-Amino acid, Model-Poisson model, Rates among sites-Uniform rates, Pattern among lineages-Same (homogeneous), Gaps/Missing data treatment-Pairwise deletion, Number of threads-7.

Multiple Sequence Alignment and Phylogenetic Analysis
Conserved core regions of ShNF-YA, ShNF-YB, and ShNF-YC proteins were aligned with conserved regions of sorghum and Arabidopsis NF-Y subunits using Unipro UGENE v46.0. Phylogenetic analysis was carried out using MEGA 10.0.5. Multiple sequence alignment was carried out using MUSCLE with parameters (Gap Open-2.90, Gap Extend 0.00, Hydrophobicity Multiplier 1.20, Max Memory in MB 2048, Max Iterations 16, Cluster Method UPGMA, and Min Diag Length (Lambda) 24). Phylogenetic analysis was carried out with 1000 bootstrap replicates and with the following parameters: Substitutions type-Amino acid, Model-Poisson model, Rates among sites-Uniform rates, Pattern among lineages-Same (homogeneous), Gaps/Missing data treatment-Pairwise deletion, Number of threads-7.

Identification of Conserved Motifs in ShNF-Y Genes and Proteins
Conserved motifs in ShNF-Y genes and proteins were determined using the MEME suite "http://meme-suite.org/tools/meme (accessed on 10 February 2023)". The site distribution was set to any number of repetitions and number of motifs to be identified was set to 10.

RNA Extraction and Real-Time Quantitative PCR
Saccharum commercial hybrid Co 86032 and E. arundinaceus were grown in pots under glasshouse conditions with the conditions mentioned elsewhere [27,28]. Drought stress was induced in 90-day-old plants by withdrawing irrigation for 10 days. Young leaf tissues of 90-day-old plants from drought-treated Saccharum commercial hybrid Co 86032 and E. arundinaceus were collected at 5-and 10-day intervals, frozen in liquid nitrogen immediately, and stored at −80 • C. Total RNA extraction from the young leaf and root tissues was conducted using a Qiagen kit (Plant RNeasy Kit, Qiagen, Hilden, Germany). DNase I (Thermo Fisher Scientific, Lenexa, KS, USA) treatment was given to remove genomic DNA contamination from the extracted total RNA. First-strand cDNA synthesis using Revert Aid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Lenexa, KS, USA) was carried out in a total reaction volume of 20 µL with Dnase I-treated RNA of 1000 ng and an oligo dT (18) primer.
Expression of arbitrarily selected ShNF-Ys and EaNF-Ys in leaf and root tissues under drought stress was analyzed using quantitative real-time reverse transcription (qRT)-PCR. Forward and reverse primers used for real-time quantification studies are given in Table S1. PCR reactions were carried out with a total volume of 20 µL in the StepOne real-time PCR system (Applied Biosystems, Burlington, ON, Canada) with the following temperature profile: 10 min of denaturation at 95 • C, followed by 40 cycles of 15 s of denaturation at 95 • C, 60 min of annealing at 60 • C, and an extension of 5 min at 72 • C. The gene encoding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Normalization of raw threshold values was performed against GAPDH. Relative expression of the NF-YB2 gene in E. arundinaceus and Co 86032 was determined using the 2 −∆∆Ct method [29]. Three technical and biological replicates were used for the expression analysis studies.

Identification, Characterization of ShNF-Y Transcription Factors, and Conserved Domain/Motif Analysis
In sugarcane, 9 NF-YA, 18 NF-YB, and 24 NF-YC genes were identified using a blast search against a mosaic monoploid reference sugarcane genome. Identified NF-Ys were predicted for ORFs, cds, and protein sequences (Table 1). Conserved functional domains were predicted for NF-Y protein sequences using InterProScan, pfam, NCBI's Conserved Domain Database, and MOTIF Search. All the identified ShNF-Ys had conserved core functional domains necessary for subunit interaction and DNA binding. ShNF-YA, ShNF-YB, and ShNF-YC proteins had the conserved CBF-B/HAP2/NF-YA, CBF-A/HAP3/NF-YB, and ShNF-CBF-C/HAP5/NF-YC domains, respectively (Figures 2-4). ShNF-Y genes were predicted to have conserved motifs ( Figure S1). Ten gene motifs were identified in all three ShNF-Y genes. ShNF-Y proteins were also predicted to have conserved motifs ( Figure 5).

Physiochemical Properties and Subcellular Localization Analysis
ShNF-YA, ShNF-YB, and ShNF-YC proteins were predicted for physiochemical properties (molecular weight, isoelectric point, and hydropathicity) using the ExPASy Protparam tool (Table 1). ShNF-Y proteins were predicted for subcellular localization and gene ontology (Table S2). ShNF-Y proteins were predicted to be localized in the nucleus. Most of the ShNF-Y members were annotated as CCAAT-binding factor complexes. The gene ontology annotation of the ShNF-Y members had thylakoid, vacuoles, and cytoplasm gene ontology identifiers.

Physiochemical Properties and Subcellular Localization Analysis
ShNF-YA, ShNF-YB, and ShNF-YC proteins were predicted for physiochemical properties (molecular weight, isoelectric point, and hydropathicity) using the ExPASy Protparam tool (Table 1). ShNF-Y proteins were predicted for subcellular localization and gene ontology (Table S2). ShNF-Y proteins were predicted to be localized in the nucleus. Most of the ShNF-Y members were annotated as CCAAT-binding factor complexes. The gene ontology annotation of the ShNF-Y members had thylakoid, vacuoles, and cytoplasm gene ontology identifiers.

Multiple Sequence Alignment and Phylogenetic Analysis of ShNF-Y Proteins
Multiple sequence alignment (MSA) of conserved regions of ShNF-Ys along with orthologous species of sorghum and Arabidopsis was carried out (Figures 2-4). Alignment of NFY conserved regions depicted the conservation of amino acids involved in DNA binding and subunits interaction.
Phylogenetic analysis of NF-Ys in sugarcane, sorghum and Arabidopsis have shown that ShNF-YA subunits were equidistant from each other (Figure 6a). However, ShNF-YB and ShNF-YC subunits clustered distinctly forming closely related and divergent groups (Figure 6b,c). Phylogentic tree of all ShNF-Y proteins showed five major clades (Figure 6d).

Gene Structure, Chromosomal Distribution, and Synteny Analysis of ShNF-Y Genes
Gene structures for ShNF-Y genes were predicted. ShNF-YA genes had an average of four introns and the intronic regions were longer (Figure 7a). Gene lengths for ShNF-YA genes ranged from 821 to 6739 base pairs (bp). In contrast to ShNF-YA genes, ShNF-YB genes had more CDS regions than intronic regions. Most of the ShNF-YB genes lacked introns (Figure 7b). The gene length of ShNF-YB genes ranged from 312 to 2121 bp. ShNF-YC genes also had fewer intronic regions when compared to ShNF-YA genes (Figure 7c). The gene length of ShN-YC genes ranged from 324 to 8370 bp.
Phylogenetic analysis of NF-Ys in sugarcane, sorghum and Arabidopsis have shown that ShNF-YA subunits were equidistant from each other (Figure 6a). However, ShNF-YB and ShNF-YC subunits clustered distinctly forming closely related and divergent groups (Figure 6b,c). Phylogentic tree of all ShNF-Y proteins showed five major clades ( Figure  6d).

Gene Structure, Chromosomal Distribution, and Synteny Analysis of ShNF-Y Genes
Gene structures for ShNF-Y genes were predicted. ShNF-YA genes had an average of four introns and the intronic regions were longer (Figure 7a). Gene lengths for ShNF-YA genes ranged from 821 to 6739 base pairs (bp). In contrast to ShNF-YA genes, ShNF-YB genes had more CDS regions than intronic regions. Most of the ShNF-YB genes lacked introns (Figure 7b). The gene length of ShNF-YB genes ranged from 312 to 2121 bp. ShNF-YC genes also had fewer intronic regions when compared to ShNF-YA genes (Figure 7c). The gene length of ShN-YC genes ranged from 324 to 8370 bp.

Three-Dimensional Structure Prediction of ShNF-Y Proteins
Three-dimensional structures were predicted for ShNF-Y protein sequences ( Figure S2). ShNF-Y protein structures were dominated by helix and loop regions.

Three-Dimensional Structure Prediction of ShNF-Y Proteins
Three-dimensional structures were predicted for ShNF-Y protein sequences ( Figure  S2). ShNF-Y protein structures were dominated by helix and loop regions.

Expression Analysis of NF-Y Genes in the Saccharum Complex under Drought Stress
Quantification of expression of arbitrarily selected NF-Y genes (4 NF-YA, 9 NF-YB, and 5 NF-YC) revealed differential regulation in leaf and root tissues of the Saccharum hybrid Co 86032 and drought-tolerant E. arundinaceus at the 10th day of drought stress ( Figure 10). NF-Y gene expression indicated a tissue-specific and drought-inducible expression profile in the Saccharum hybrid and E. arundinaceus. The qRT-PCR results correlated with those of the RNAseq DGE analysis, illustrating the reliability of the transcriptome profile data obtained. Among the 14 genes tested, a few were upregulated in both leaf and root tissues, some others exhibited downregulation, and the rest were not influenced by the stress conditions in the transcriptome data (Table S3). A similar trend was found in the qRT-PCR analysis, with differences in the fold expression levels.

Expression Analysis of NF-Y Genes in the Saccharum Complex under Drought Stress
Quantification of expression of arbitrarily selected NF-Y genes (4 NF-YA, 9 NF-YB, and 5 NF-YC) revealed differential regulation in leaf and root tissues of the Saccharum hybrid Co 86032 and drought-tolerant E. arundinaceus at the 10th day of drought stress ( Figure 10). NF-Y gene expression indicated a tissue-specific and drought-inducible expression profile in the Saccharum hybrid and E. arundinaceus. The qRT-PCR results correlated with those of the RNAseq DGE analysis, illustrating the reliability of the transcriptome profile data obtained. Among the 14 genes tested, a few were upregulated in both leaf and root tissues, some others exhibited downregulation, and the rest were not influenced by the stress conditions in the transcriptome data (Table S3). A similar trend was found in the qRT-PCR analysis, with differences in the fold expression levels.

Discussion
NF-Y subunits regulate stress-responsive signaling pathways and networks by modulating downstream targets [30]. The role of NF-Y subunits in modulating epigenetic mechanisms also implies that they are crucial players in stress-defensive responses. Many reports suggest that overexpression of the NF-Y subunits confers tolerance to various stresses, including drought and salinity, in plants [13,21,[30][31][32][33]. NF-Y subunits act both as complexes and as individual subunits in regulating gene expression and stress response. As complexes, NF-Y subunits modulate gene expression by binding to the CCAAT box in the promoter regions [34]. Some studies indicate NF-YB and NF-YC binding with other transcription factors in transcriptional activation mechanisms [35,36].
Owing to the importance of NF-Y transcription factors in various stress-responsive mechanisms and developmental processes, several studies have been carried out to identify NF-Y genes in different plant species. In soybean, 21 NF-YA , 32 NF-YB, and 15 NF-YC genes have been identified. Certain NF-Y gene members have been identified to have a vital role in specific stress responses and developmental processes [37]. In Triticum aestivum, 10 NF-YA, 11 NF-YB, and 14 NF-YC genes have been identified. Expression analysis of TaNF-Y genes revealed some NF-Y members have ubiquitous expression, while some are organ-specific and some are drought-responsive [38]. In S. bicolor, 8 NF-YA , 11 NF-YB, and 14 NF-YC genes were identified. In silico expression analysis under salt, drought, cold, and heat stresses revealed that certain NF-Y genes are stress-responsive. In Vitis vinifera, 8 NF-YA , 12 NF-YB, and 8 NF-YC gene members were identified. Expression analysis revealed a number of VvNF-Y gene members involved in various biotic and abiotic stresses, phytohormone regulation, and sugar metabolism [39]. The wide expansion of NF-Y gene members in the genome highlights the essential role of these plant nuclear factors in various functions in sugarcane [40].

Discussion
NF-Y subunits regulate stress-responsive signaling pathways and networks by modulating downstream targets [30]. The role of NF-Y subunits in modulating epigenetic mechanisms also implies that they are crucial players in stress-defensive responses. Many reports suggest that overexpression of the NF-Y subunits confers tolerance to various stresses, including drought and salinity, in plants [13,21,[30][31][32][33]. NF-Y subunits act both as complexes and as individual subunits in regulating gene expression and stress response. As complexes, NF-Y subunits modulate gene expression by binding to the CCAAT box in the promoter regions [34]. Some studies indicate NF-YB and NF-YC binding with other transcription factors in transcriptional activation mechanisms [35,36].
Owing to the importance of NF-Y transcription factors in various stress-responsive mechanisms and developmental processes, several studies have been carried out to identify NF-Y genes in different plant species. In soybean, 21 NF-YA, 32 NF-YB, and 15 NF-YC genes have been identified. Certain NF-Y gene members have been identified to have a vital role in specific stress responses and developmental processes [37]. In Triticum aestivum, 10 NF-YA, 11 NF-YB, and 14 NF-YC genes have been identified. Expression analysis of TaNF-Y genes revealed some NF-Y members have ubiquitous expression, while some are organ-specific and some are drought-responsive [38]. In S. bicolor, 8 NF-YA, 11 NF-YB, and 14 NF-YC genes were identified. In silico expression analysis under salt, drought, cold, and heat stresses revealed that certain NF-Y genes are stress-responsive. In Vitis vinifera, 8 NF-YA, 12 NF-YB, and 8 NF-YC gene members were identified. Expression analysis revealed a number of VvNF-Y gene members involved in various biotic and abiotic stresses, phytohormone regulation, and sugar metabolism [39]. The wide expansion of NF-Y gene members in the genome highlights the essential role of these plant nuclear factors in various functions in sugarcane [40].
In this study, a genome-wide analysis of ShNF-Ys was performed, and 9 ShNF-YA, 18 ShNF-YB, and 24 ShNF-YC gene members were identified. In silico and real-time expression analyses of ShNF-Ys were performed. Physiochemical parameters, domain analysis, phylogeny, and three-dimensional structure prediction assisted in gaining insights about the ShNF-Ys.
Subcellular localization predictions of proteins helped to identify their localization in the cellular compartments, thereby helping to understand their role in the cellular machinery. This study predicted that all ShNF-Y proteins would be localized in the nucleus. For instance, the NF-YB proteins of E. arundinaceus (EaNF-YB2) [41], Poplar (PdNF-YB7) [42], A. thaliana (HAP3b) [43], and Oryza sativa (OsHAP3H) [44] are localized at the nucleus. The localization of foxtail millet NF-YB8 showed a cell-wide distribution pattern [24]. Similarly, Arabidopsis NF-YB3 was translocated to the nucleus only during stress conditions [6]. This suggests that ShNF-Y members might also localize in various cellular regions other than the nucleus and play vital roles in different cellular processes. However, experimental studies have to be carried out to ascertain the localization mechanisms of ShNF-Y proteins.
Synteny analysis showed that NF-Y orthologs were conserved within the syntenic blocks. Syntenic gene pairs identified between sugarcane, sorghum, and Arabidopsis could be exploited to understand functional equivalence between these species. Notably, 15 ShNF-Ys members were found to be syntenic with the NF-Y members across monocot and dicot species, which indicated that these orthologous pairs are conserved and may have existed before the ancestral divergence [45,46]. However, the intersections of the syntenic NF-Ys members may be valuable for exploration in evolution studies. These syntenic NF-Y gene pairs identified among sugarcane, sorghum, and Arabidopsis might also share similar expression patterns with key functional properties.
NF-Ys exhibit differential expression patterns under biotic and abiotic stresses, specifically drought stress in crop plants [10,47]. The differential expression pattern of NF-Y genes signifies their involvement in drought-responsive stress mechanisms. Tissue-specific expression of NF-Ys was recorded in some of the plant species [47,48]. Real-time expression analysis of ShNF-Y genes investigated the differential expression pattern in Erianthus and sugarcane. NF-YA3, NF-YA5, NF-YB2, NF-YB8, NF-YB9, NF-YC7, NF-YC9, and NF-YC18 genes had significantly higher expression under drought, whereas all other NF-Ys were either downregulated or had non-significant expression in the Saccharum complex. NF-YA, NF-YA1, and NF-YA9 were downregulated in leaf and root tissues of the Saccharum hybrid and E. arundinaceus under drought conditions. NF-YA3 showed significantly higher expression only in the root of E. arundinaceus, with a fold change of 3.65. Higher expression of NF-YA5 was recorded in leaf tissues of both E. arundinaceus and the Saccharum hybrid, as well as in the root of E. arundinaceus. Among the investigated NF-YB genes, ubiquitous higher expression of NF-YB2 was recorded in both tissues (fold changes of 4.123, 6.123, 1.236, and 3.896 in ShL, EaL, ShR, and EaR, respectively) except in Saccharum hybrid root. This indicates the involvement of NF-YB2 in the stress response. Root-specific expression was observed with the ShNF-YB8 gene. Higher transcript levels of ShNF-YB8 in the root suggests its plausible role in drought response. ShNF-YB16 was universally downregulated in leaf and root tissues of both varieties. Considering NF-YC, ShNF-YC7, and ShNF-YC9 had higher expression in leaves (a fold change of 3.8 (ShL) and 3.9 (EaL)). NF-YC14 had downregulated expression invariably in the leaves and roots of the Saccharum complex. NF-YC18 was downregulated in ShL, EaL, and ShR and upregulated (a fold change of 5.32) in EaR. Higher expression in EaR implies that NF-YC18 is root-specific and stressresponsive in E. arundinaceus. NF-YC24 had lower expression in E. arundinaceus and was downregulated in the Saccharum hybrid. In the Saccharum hybrid, ShNF-YA5, ShNF-YB2, ShNF-YC7, and ShNF-YC9 were leaf-specific, and ShNF-YB8 and ShNF-YC18 were rootspecific. In E. arundinaceus, elevated expression of NF-Y genes was observed in leaves as well as in roots. EaNF-YC7 and EaNF-YC9 had elevated expression in leaves. EaNF-YA5 and EaNF-YB2 were upregulated in both leaf and root tissues. However, E. arundinaceus had more root-specific expression of NF-Y genes compared to the Saccharum hybrid. EaNF-YA5 gene expression was prominent in E. arundinaceus roots, suggesting its potential role in root modifications during drought stress. Besides, EaNF-YB2 had a significantly higher fold change of expression in roots, endorsing its crucial role in stress-adaptive regulatory mechanisms. EaNF-YA3, EaNF-YB8, and EaNF-YC18 also had higher expression in roots.
Higher transcript levels of root-specific NF-Y genes in Erianthus suggest that the root plays an essential role in drought tolerance in the wild relative. Tissue-specific expression of NF-Y genes highlights their role in tissue-specific responses to combat drought stress. The NF-Ys might function as individual components and/or in combination with other genes in specific tissues to impart drought tolerance in sugarcane.
Overexpression studies on NF-Y gene members in different plant species envisaged their role in tolerance to various types of stresses. Previous investigation on the overexpression of AtNF-YA5 in Arabidopsis showed reduction in water loss and improved tolerance to drought in transgenics [13]. Likewise, transgenic rice overexpressing NF-YA7 exhibited tolerance to drought in an ABA-independent manner [49]. NF-YB3 from Picea wilsonii, when overexpressed in Arabidopsis, conferred tolerance to osmotic, salinity, and drought stresses [31]. Overexpression of the NF-YC gene in Amaranthus hypochondriacus conferred resistance to water-deficit stress in Arabidopsis [32]. ShNF-YA3, which had higher expression in the root, showed an identity of 88.31% with the rice nuclear transcription factor OsHAP2E, which upon overexpression exhibited tolerance to salinity and drought stresses. ShNF-YA5 showed an identity of 97.29% with TaNFYA-B1 nuclear transcription factor from T. aestivum, which stimulated root development upon overexpression [33]. This implies the significance of ShNF-YA5 in root development, an essential trait for drought tolerance. Similarly, ShNF-YC18, which was root-specific, shared a 93.1% identity with the A. hypochondriacus NF-YC gene. Hence, overexpressing ShNF-YC18 may provide drought tolerance in sugarcane. Similarly, higher expression of EaNF-YB2 was observed in E. arundinaceus compared to the Saccharum hybrid under drought and salinity conditions [41], suggesting that overexpression of ShNF-YB2 might assist sugarcane in developing tolerance against drought and salinity stresses. Erianthus, which has a well-developed root system inherently [50], showed higher expression of root-specific EaNF-Y genes under drought. Upregulation of NF-Y genes in roots shows that NF-Y genes have a vital role in root adaptation mechanisms for drought tolerance [31,42]. These studies suggest that overexpressing NF-Y genes in plants would assist in combating drought stress. Differentially expressed NF-Y genes under drought stress in the Saccharum complex can act as potential candidate genes for crop improvement.

Conclusions
Comprehensive genomic analysis and characterization of NF-Y genes in the commercial sugarcane (Saccharum spp.) hybrid identified 9 NF-YA, 18 NF-YB, and 24 NF-YC genes, and also revealed differential expression patterns of ShNF-Y genes in leaves and roots under drought conditions. This provides the basis for exploiting stress-responsive NF-Y genes as potential candidates for sugarcane genetic improvement through conventional breeding techniques and modern biotechnological approaches.