GhNFYA16 was functionally observed positively responding to salt stress by genome-wide identification of NFYA gene family in cotton
Environmental Sciences Europe volume 34, Article number: 95 (2022)
Nuclear transcription factor Y subunit A (NFYA) plays an important role in plant growth, development, and response to abiotic stress.
This study systematically analyzed the NFYA gene family. Chromosome location analysis found that some NFYA genes in Gossypium hirsutum may have been lost during evolution. Collinearity analysis and selection pressure analysis indicated that the GhNFYA gene family underwent fragment duplication and whole genome duplication during evolution. At the same time, promoter cis-element analysis and gene interaction network analysis predicted that the expression of GhNFYA gene may be regulated by plant hormones and stress. To further explore the function of the gene, Gossypium hirsutum seedlings were treated with 4 °C, 37 °C, salt and PEG stress, respectively, found that the expression of NFYA is stimulated by multiple environments. By constructing a co-expression network, interactions between genes were found to defend against salt stress. Through virus-induced gene silencing experiments, it was found that plants that silenced the GhNFYA16 gene were significantly more sensitive to salt stress.
This study found the relationship between the structure and function of NFYA gene family, provided a basis for the biological identification and functional verification of NFYA family members, and provided clues to clarify the specific roles of different types of NFYA proteins under different abiotic stress.
Transcription factors, as known as trans-acting factors, have two modes of action: the first is to bind DNA, various cis-acting elements widely distributed in the promoter region of eukaryotic genes; The other is binding proteins, such as transcription factor proteins and other related proteins . In this way, transcription factors can promote or inhibit the expression of downstream-related genes at the transcriptional level under specific conditions . Plants grow under stress conditions, and stress signals will eventually stimulate the expression of transcription factors through a series of transmissions, and specifically regulate the transcription and expression of related response genes, respond to stress signals and adapt to the environment . The activation and expression of transcription factor genes are regulated by environmental signals, such as high salt, drought and plant hormones, as well as plant self-development signals . The activity of transcription factors when they function is influenced by post-translational modifications and their distribution in the cell. It is also affected by factors, such as the interaction between its own protein and other proteins .
Nuclear transcription factor Y (NF-Y) is a transcription factor widely existing in eukaryotes, which can specifically bind to CCAAT-box, and CCAAT-box, as a cis-element, exists in about 1/4 of eukaryotic gene promoters . NF-Y is a trimer, which includes NF-YA (CBF-B or HAP2), NF-YB (CBF-A or HAP3), NF-YC (CBF-C or HAP5). In mammals, NF-YB and NF-YC are tightly bound together through a histone folding domain, and then combine with NF-YA in the nucleus to form the final trimer transcription factor , NF-YA can slide on the chromatin to look for the 25 bp CCAAT-box element. Once NF-YA is found, it will bind to it and insert it into the small groove of the DNA double helix, making the DNA double helix in a relaxed state, thereby improving favorably recruit RNA polymerase or other transcription factors, and then regulate downstream target genes positively or negatively .
As a conserved transcription factor, NF-YA plays a very important role in plant growth and abiotic stress . Studies have found that NF-YB in Zea mays can increase the drought resistance of crops without affecting the yield reduction. Under drought conditions, compared with control plants, the yield of ZmNF-YB2 overexpression Zea mays increases ; The drought resistance of AtNF-YA5 transgenic plants is significantly higher than that of wild-type plants ; Overexpressed Glycine max NF-YA3 gene reduces water evaporation from Arabidopsis leaves, thereby improving drought resistance. Meanwhile, NF-YA3 responds to drought, high salinity, low temperature and ABA . Under drought conditions, BpNFYA5 can promote the accumulation of proline, enhance water retention capacity of the plant and improve the drought resistance of the Brassica pekinensis. In this process, BpNFYA5 may also regulate some of the chlorophyll synthesis-related gene expression ; Overexpression of GmNFYA3 can significantly improve the tolerance of GmNFYA3 transgenic Arabidopsis to drought . In addition, the SiNF-YA6 gene can significantly improve the resistance of transgenic SiNF-YA6 plants to low nitrogen by increasing the expression of nitrogen transport gene in Setaria italica. In Medicago sativa, the NFYA1 gene is only expressed in a part of the root tip and regulates the development of metaphase root nodules. In conclusion, the functions of plant NFYA transcription factors are very complex, involving the flowering process, stress resistance and the regulation of growth and development of plants, which are very important in plants.
To further investigate the versatility of NFYA genes in plant development and defense response, the NFYA gene family was systematically investigated. This study provides potential candidate genes for functional studies of Gossypium genes and provides some molecular basis for Gossypium breeding. This may help to clarify the evolutionary mechanism of NFYA gene family in Gossypium and also provide us with further insights into stress response genes in Gossypium, and provide valuable information for cultivating stress-resistant Gossypium.
Materials and methods
Identification of NFYA family members
Four cotton genome files were downloaded from Cotton FGD: G. hirsutum (NAU version)  and G. barbadense (HAU version) , G. arboreum (CRI version)  and G. raimondii (JGI version)  (https://cottonfgd.org/) . Through Pfam database analysis, it was found that the protein sequence conservative domain of Arabidopsis AT3G20910 was PF02045 (http://pfam.xfam.org/), and Hidden Markov Model (HMM) uses the local software HMMER to screen the gene with this conserved domain in four cotton genomes as the candidate gene of NFYA family genes. Used Phytozome  to search and download protein sequences of Theobroma cacao (Theobroma cacao v1.1), Arabidopsis thaliana (Arabidopsis thaliana Araport11), Zea mays (Zea mays B73 RefGen_v3), Populus trichocarpa (Populus trichocarpa v3.0), Vitis vinifera (Vitis vinifera) v2.1, Oryza sativa (Oryza sativa v7.0) and Glycine max (Glycine max Wm82.a4.v1). Gene sequences with incomplete domains were manually deleted using NCBI CD-Search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The physicochemical properties of the GhNFYAs genes were analyzed using the online website ExPASY (https://web.expasy.org/protparam/) . TBtools software was used to draw NFYA chromosome location maps of 4 Gossypium species, G. hirsutum, G. barbadense, G. arboretum and G. raimondii .
Phylogeny analysis and sequence alignment
To study the evolutionary relationship of NFYA among different species, homologous genes of 11 species were obtained based on PF02045. The protein sequences of NFYA family members of 11 species were entered into MEGAX to construct an unrooted phylogenetic tree. The maximum likelihood statistical method was used for the interspecific phylogenetic tree with a bootstrap duplication 1000, while the intraspecific phylogenetic tree used the neighbor-joining method with a parameter of bootstrap duplication 500 .
Collinearity analysis of NFYA genes in four Gossypium species
The NFYA genes of four Gossypium species were analyzed using TBtools software. Prepare gene ID, CDS sequence, chromosome length file and genome files and protein files for NFYA family genes. The MCScanX software  was used to collinearity analysis among the repetitive gene pairs of the four Gossypium species: G. hirsutum, G. arboreum, G. raimondii and G. barbadense, and TBtools software was used to visualize the results .
Calculation of duplicate gene pairs for selection pressure
The MEGAX comparison method was used to identify the selection pressure duplication gene pairs of G. arboreum, G. hirsutum, G. raimondii, and G. barbadense. The criteria for classification are as follows: the short sequence after alignment covers more than 80% of the long sequence, and the minimum homology of the alignment region is equal to or greater than 80% . Selection pressure was investigated by calculated the non-synonymous to synonymous ratio (Ka/Ks) of duplicated genes.
Analysis of conservative protein motifs and gene structure
Multiple EM for Motif Elicitation (MEME) website (http://meme-suite.org/) was used to identify conserved protein motifs . The MAST file of MEME website, the NEWICK (NWK) file of phylogenetic tree analysis and the GFF3 genome file of G. hirsutum were obtained, and then the gene structure of NFYA family members was analyzed by TBtools software .
Analysis of GhNFYA promoter region and differentially expressed genes
CottonFGD database (http://www.CottonFGD.org/) was used as a promoter to obtain a 2000 bp DNA sequence in the upstream region of GhNFYA . The PlantCare website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) was used to predict cis-regulatory elements in the promoter region of the GhNFYA gene. The cis-acting elements related to plant hormones, plant growth and development and abiotic stress were selected for further analysis. To study the expression patterns of the GhNFYA gene family, RNA-seq data (PRJNA490626) downloaded from the NCBI database of the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) was used to analyze the expression levels of these genes under cold, heat, salt and PEG stress , the phylogenetic tree, cis-acting elements and expression caloric maps under different stress treatments were drawn by TBtools software .
Analysis of tissue specificity of NFYA family genes and salt stress expression in G. hirsutum
The gene expression data of the G. hirsutum family were obtained from the Gossypium Resources and Network Database (GRAND) established by the Cotton Research Institute, and the material used was the G. hirsutum material TM-1. All NFYA family genes were selected for tissue-specific analysis, and then the expression values of high-expressed genes in leaves were analyzed for 4 time periods (1 h, 3 h, 6 h and 12 h) under salt stress.
G. hirsutum cv H177 grown on sandy soil was cultivated in a constant temperature incubator with light at 28 °C for 16 h and darkness at 25 °C for 8 h. Cotton was treated with 100 mM NaCl stress when it reached the three leaf one heart stage.
Co-expression network analysis
Co-expression network analysis of GhNFYA genes under salt stress to determine the expression relationship between family members. Cytoscape software (version 3.8.0) was used to construct co-expression regulatory networks of genes . Co-expression network plots were made with p > 0.95 as the threshold.
Subcellular localization and interaction network of NFYA in G. hirsutum
Using websites such as Wolf-PSORT  and ProtComp 9.0  to predict the subcellular location of GhNFYA. To determine the subcellular localization of GhNFYA16, a GhNFYA16-GFP vector was constructed, and the recombinant GFP fusion protein was transiently overexpressed in tobacco leaves. The STRING (https://cn.string-db.org/) was used to predict the protein interaction of GhNFYA16 in Arabidopsis orthologous genes. According to the results, the interaction between GhNFYA16 and these genes in cotton can be inferred.
GhNFY’s VIGS function verification
Total RNA was extracted using EASYspin Plus Plant RNA Kit . RNA was reverse transcribed to cDNA using TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) (AT341). Real-time quantitative PCR detection was performed on an ABIPrism7500 Fast instrument, and the selected genes were amplified by a two-step method. The specific method refers to ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech Co., Ltd).
The 300 bp purified fragment was inserted into the cut pYL156 vector with restriction sites: XmaI and XbaI. The recombinant plasmid was transformed into Agrobacterium GV3101, and the Agrobacterium carrying pYL156 (empty), pYL156-GhNFYA16, pYL156-CLA1 (positive control) and pYL192 (helper vector) were placed in the Agrobacterium containing 50 μg/mL kanamycin and 25 μg/mL rifampicin, cultured overnight at 28 °C, 200 rpm protected from light, shaken to OD600 = 1.2–1.5. Centrifuge at 6000 rpm for 15 min to collect the bacteria, and discard the supernatant. Adding an equal volume of sterile resuspension solution (10 mM MES, 200 μM AS, 10 mM MgCl2, pH is about 5.8) to resuspend the bacteria, and let it stand for 4 h at room temperature in the dark. Choose Gossypium seedlings with just flattened cotyledons and water them before infection. Used a sterile needle to make a small hole in the epidermis of the cotyledon, and use a syringe to inject the bacterial solution into the epidermis of the cotyledon until the bacterial solution fills the entire cotyledon. After the injection, the Gossypium seedlings were protected from light for 24 h, and then cultured normally. The plants injected with pYL156 null control and pYL156-GhNFYA16 were treated with drought stress after the albino phenotype appeared on pYL156-CLA1 Gossypium plants.
Identification of NFYA family genes
A total of 94 family members were identified in the four cotton species. G. hirsutum mainly includes GhNFYA1-30, with 30 members. There are 32 members (GbNFYA1-32) in G. barbadense. There are 16 in G. arboreum (GaNFYA1-16). G. raimondii has 16 GrNFYA1-16. Other species were also classified according to their chromosomal location information (Additional file 1: Table S1). The amount of NFYA in tetraploid cotton (G. hirsutum and G. barbadense) was almost twice that in diploid cotton (G. arboreum and G. raimondii). It is proved again that the allotetraploid of cotton is formed by the hybridization of subgenome A and subgenome D to double chromosomes . Analyzing the physicochemical properties of the genes of the NFYA family of G. hirsutum, the isoelectric point ranges from 6.84 (GhNFYA11) to 10.22 (GhNFYA3). The minimum number of amino acids is 175 (GhNFYA3) and the maximum is 999 amino acids (GhNFYA11). Molecular weights ranged from 19.40KDa (GhNFYA3) to 109.38KDa (GhNFYA11) (Additional file 1: Table S2).
Phylogenetic analysis of NFYA family genes
To study the evolutionary relationship of NFYA in plants, the protein sequences of 181 family members identified in 11 species were aligned and a phylogenetic tree was constructed. There are 30 genes in G. hirsutum, 32 in G. barbadense, 16 in G. arboreum, 16 genes in G. raimondii, 10 in Arabidopsis thaliana, 11 in Oryza sativa, 7 in Theobroma cacao, 18 in Zea mays, 13 in Populus trichocarpa, 21 in Glycine max and 7 Vitis vinifera (Additional file 1: Fig. S1). The identified NFYA family members were renamed according to their chromosomal location. Phylogenetic trees were constructed by maximum likelihood method using MEGAX. The unrooted phylogenetic tree was further beautified using the EvolView website (Fig. 1). Through the overall analysis of the phylogenetic tree, the NFYA family members are classified, divided into three classes A, B, and C, mainly by referring to the phylogenetic trees of Arabidopsis thaliana and four Gossypium species. Class A includes the AtNFYA1, AtNFYA4, AtNFYA7 and AtNFYA9 genes in Arabidopsis thaliana and the similar NFYA genes in cotton, Populus trichocarpa, Glycine max, Zea mays and other species. NFYA genes are classified according to maizeGDB (https://www.maizegdb.org/), TAIR (https://www.arabidopsis.org/index.jsp), JGI Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html) and other online sites. Class B includes the AtNFYA3, AtNFYA5, AtNFYA6 and AtNFYA8 genes of Arabidopsis thaliana, Oryza sativa, Theobroma cacao, and four Gossypium species with similar evolutionary relationships. Class C mainly includes AtNFYA2, AtNFYA10 genes, Glycine max, Zea mays, Oryza sativa and a few NFYA genes of four Gossypium species.
As shown in Fig. 1A, the NFYA family members have two branches in class A. In each branch, the NFYA genes of the four Gossypium species have the closest evolutionary relationship. Meanwhile, the evolutionary relationship between cotton and Theobroma cacao, Populus trichocarpa, Glycine max and Vitis vinifera is also relatively similar. The NFYA genes in Arabidopsis thaliana and Glycine max are most closely related in evolution. Similar evolutionary relationships were also found in class B, and NFYA genes were more closely related in Zea mays and Oryza sativa. Combined with class C, it can be concluded that Theobroma cacao, Populus trichocarpa and Glycine max are the most closely related to cotton NFYA gene evolution. Arabidopsis thaliana is closest to Glycine max NFYA family genes. Maize and rice NFYA family genes have a relatively close evolutionary relationship. The number of NFYA family genes in G. hirsutum is more than twice that of Arabidopsis thaliana, Oryza sativa and Theobroma cacao, indicating that Gossypium has undergone significant gene amplification during its evolution . The number of genes of NFYA family members in the four Gossypium species is 30 in G. hirsutum, 32 in G. barbadense, 16 in G. arboreum, and 16 family members in G. raimondii. During the evolution from diploid Gossypium to G. hirsutum, there are two genes are missing. By constructing the phylogenetic tree of NFYA family members of four cotton species, it is found that there are mainly three branches a, b and c (Fig. 1B).
Chromosome location analysis of NFYA family
To further analyze the characteristics of NFYA family genes, the chromosomes of NFYA family in four Gossypium species were mapped (Fig. 2). These genes were renamed according to their location on the chromosome. Both G. hirsutum and G. barbadense have two genes that are not located on chromosomes, and other genes of NFYA show uneven distribution on different chromosomes. The genes of G. hirsutum and G. barbadense have the same distribution on Chr03, Chr06, Chr07, Chr10, Chr11 and Chr13. One gene is distributed on Chr03 and Chr07. There are two genes distributed on Chr06 and Chr11 chromosomes. There are four genes located on Chr10 and Chr13 chromosomes. Meanwhile, the NFYA family genes are distributed in G. hirsutum with one more gene than G. barbadense GbAt-01 and GbDt-05. Upland cotton has one less gene than GbAt-02, GbDt-04, GbDt-08 and GbDt-09 on the corresponding chromosomes of sea island cotton. The results suggested that some NFYA genes of upland cotton may have been lost during evolution. Analyzing the distribution of G. arboreum chromosomes of this gene family, it is found that the distribution of the rest of the chromosomes tends to be the same on the A genome except for chromosome 2 which is more than that of the A genome of G. hirsutum. A large number of genes additions and deletions occurred in G. raimondii, and only the number of genes on chromosomes 3 and 13 is the same as that of G. hirsutum and G. barbadense D genome.
Collinearity analysis of NFYA family genes
Colinear analysis of NFYA genes in four cotton species was performed to understand the evolutionary relationship of NFYA gene family in cotton. The evolution of gene family generally goes through three processes, namely, fragment duplication, tandem duplication and whole genome duplication . A joint analysis of the NFYA genes of G. arboreum, G. raimondii, G. hirsutum and G. barbadense, analyzed the gene duplication and collinearity between them. The NFYA genes of G. arboreum (Ga) and G. raimondii (Gr) were duplicated in G. hirsutum (Gh) and G. barbadense (Gb). It was shown that the two tetraploid genomes of upland cotton and sea island cotton were generated from the diploid genome in the process of genetic transformation. According to the chromosomal distance, similarity and coverage of NFYA gene family members of diploid and tetraploid Gossypium species, tandem repeats and tandem repeats were identified, so as to identify the evolutionary relationship between NFYA family genes.
Genes linked together by collinear lines represent the same gene. In Fig. 3, it can be seen that many chromosomes in the GhAt/GhDt, GbAt/GbDt subgenomes and the GaA, GrD genomes are connected by lines of the same color. That is, the GhAt/GhDt and GbAt/GbDt subgenomes have NFYA homologous genes in the GaA and GrD genomes. It shows that these genomes/subgenomes are closely related in evolution, and most NFYA genes have been preserved in the evolution of polyploidy. Genes located in the same chromosome region (e value < 1e-5) are classified as tandem repeats, while the rest of the genes from the same genome are considered to be fragment repeats. In all collinearity analysis results, they come from 4 different Gossypium species. Genome and subgenome genes are usually classified as genome-wide duplication. Homologous/like homologous gene pairs were identified for the GhAt/GhDt and GbAt/GbDt subgenomes of two tetraploid Gossypium species. After homology analysis, it was found that several gene loci were highly conserved between the At and Dt subgenomes of the two tetraploid Gossypium species. As mentioned above, G. hirsutum and G. barbadense are derived from the ancestors of diploid G. arboreum and G. raimondii . No tandem duplications were found in the NFYA gene family by analysis, with 98 fragment duplications and 266 genome-wide duplications. Based on these results, it is speculated that closely related gene pairs are usually generated through genome-wide duplication and fragment duplication, especially fragment duplication is the most important factor in the evolutionary process (Fig. 3).
Ka/Ks analysis of selective pressure of NFYA family genes
The evolution of NFYA gene pairs in four cotton species was obtained by selection pressure analysis. It can be judged whether there is selective pressure acting on NFYA family genes. In the process of evolution, the duplicated gene pair may also diverge from its original function, which eventually leads to non-functionalization (loss of original function), sub-functionalization (division of original function), and new functionalization (acquisition of new function) . To determine the nature and degree of selection pressure of repeated gene pairs and explore whether Darwin's positive selection is related to the divergence of repeated NFYA genes, Non-synonymous (Ka) and Synonymous (Ks) values were calculated for 314 duplicated gene pairs from 10 combinations of 4 cotton species. These combinations include G. hirsutum VS G. hirsutum (Gh-Gh), G. hirsutum VS G. barbadense (Gh–Gb), G. hirsutum VS G. arboreum (Gh–Ga), G. hirsutum VS G. raimondii (Gh–Gr), G. barbadense VS G. barbadense (Gb–Gb), G. barbadense VS G. arboreum (Gb–Ga), G. barbadense VS G. raimondii (Gb–Gr), G. arboreum VS G. arboreum (Ga–Ga), G. arboreum VS G. raimondii (Ga–Gr) and G. raimondii VS G. raimondii (Gr–Gr). According to the ratio of Ka/Ks, the selection pressure of duplicated gene pairs can be inferred. It is generally believed that Ka/Ks = 1 means neutral selection (pseudogene), Ka/Ks < 1 means negative purifying selection, and Ka/Ks > 1 means positive selection effect .
There are 314 duplicate gene pairs in the NFYA family genes in four cotton species. Among them, there are 13 gene pairs with positive selection effect, and 301 gene pairs with purifying selection. It indicates that the NFYA family genes are relatively conserved in the evolutionary process. Positive selection gene pairs appeared in Ga–Gr, Ga–Gb, and Ga–Gh were 2 pairs, 3 pairs and 2 pairs, respectively. It indicated that some NFYA genes had beneficial mutations in the process of hybridization of diploid cotton into allotetraploid. Likewise, there are also 1, 2 and 3 gene pairs in Gb–Gb, Gb–Gr, and Gh–Gr, respectively. There were 13 gene pairs that had beneficial mutations during evolution. There are 12 and 5 gene pairs with Ka/Ks values ranging from 0.49 to 0 in the Ga–Ga, Gr–Gr repeat gene pairs, respectively. Indicates that they were selected for complete purification (100%). Similarly, the number of Gb–Gh, Gh–Gh repeated gene pairs with Ka/Ks values between 0.99 and 0.5 are 8 and 1, respectively; the number of repeated gene pairs with Ka/Ks between 0.49 and 0 are 27 and 34.
In conclusion, 314 pairs of duplicate genes from four Gossypium species (Gh, Gb, Ga and Gr) were found in selection pressure analysis. Among them, 301 pairs (95.86%) of repeated gene pairs have Ka/Ks values less than 1, including 261 pairs of genes with Ka/Ks values less than 0.5 and 40 pairs of genes with a Ka/Ks value between 0.5 and 0.99, showing purification selection. Only 13 pairs (4.14%) of repetitive gene pairs have a Ka/Ks value greater than 1. These gene pairs may have undergone rapid evolution after repetition and have experienced positive selection pressure. Since most of the Ka/Ks values were less than 1.0, it was speculated that the Gossypium NFYA family genes has underwent strong purifying selection pressure and limited functional differentiation after fragment duplication and genome-wide duplication (Additional file 1: Fig. S2).
Analysis of motif and gene structure of NFYA family
Through the joint analysis of the phylogenetic tree, gene structure and motifs of the NFYA gene family, the characteristics of NFYA family members and their relationships were further understood. The phylogenetic tree of four cotton species was constructed using MAGAX software. Combined with the motif files obtained from the MEME website, the TBtools software was used to display the structure and taxonomic information of the four cotton species NFYA family (Fig. 4).
There are 10 motifs in NFYA genes in four cotton species. According to the phylogenetic tree and motif type, the four cotton species NFYA gene families were divided into three groups: I, II, III. The motifs of each class tend to be consistent and have obvious structural features. Class I includes all motif structures, and they are arranged in the order of motif8, motif9, motif5, motif6, motif4, motif2, motif10, motif1, motif3 and motif7. However, most of class II genes lack motif8, and few genes lack motif9. Compared with the class I NFYA genes, it is possible that a certain function will be lost. Class III contains fewer motif structures, lacking motif8, motif9 and motif6. In general, NFYA family genes contain motif5, motif4, motif2, motif10, motif1, and motif3, indicating that motif largely determine the similarity of family gene function and structure. From the point of view of gene structure introns and exons, all genes contain exons and introns. Meanwhile, the gene structures of class I, class II and class III have their own consistent characteristics. The class I of the NFYA gene introns and exons are compact and uniform, and the length of the exons are shorter. The exons of NFYA gene in class II were scattered. The exons of GhNFYA11, GaNFYA13 and GhNFYA25 genes are more dispersed, including a longer intron and exon. For the genes of class III, the exons are relatively short and only some of them contain longer exons, but the whole is consistent. In conclusion, NFYA family members have unique characteristics and obvious structural differences, which are relatively conservative in the process of evolution.
Analysis of differentially expressed genes of NFYA family in G. hirsutum
The members of the NFYA family play important roles in various important physiological and biochemical processes of plants . In addition, NFYA is also involved in the response to various environmental stimuli . To determine the function of GhNFYA gene in different environments, upland cotton was subjected to low temperature, high temperature, high salt and PEG stress. The expression level of GhNFYA gene during growth and development and its response to phytohormones were analyzed (Fig. 5). The cis-acting element is located in the promoter region of the gene and can be used as a reference for tissue specificity and stress response in different environments. The cis-acting elements of the NFYA gene family mainly include the cis-acting regulatory elements involved in the methyl jasmonate (MeJA) response, the cis-acting regulatory elements necessary for anaerobic induction, the MYB binding site involved in drought induction, the cis-regulatory elements involved in meristem expression, cis-acting elements involved in low temperature response, cis-acting elements involved in defense and stress response, cis-acting elements involved in stress response, and phytohormone-related regulatory elements (salicylic acid, auxin, gibberellin and abscisic acid, etc.) (Additional file 2: Table S3). The number of cis-acting elements varied among genes, for example, GhNFYA16 contained a cis-acting element for abscisic acid, a MYB binding site involved in drought induced, anaerobically induced action element, a cis-regulatory element involved in meristem expression, and a cis-acting element involved in stress response. In general, the NFYA family of G. hirsutum mainly contains cis-acting elements related to plant hormones, growth and development and adversity. It can be inferred that this gene family is related to adversity to a certain extent.
Gene expression patterns can provide an important reference for gene function analysis. It is related to the biological functions controlled by cis-acting elements. To explore the expression patterns of GhNFYA in G. hirsutum under different stress environments, the gene expression levels of cotton under four abiotic stress conditions of salt, cold, heat and PEG (1 h, 3 h, 6 h and 12 h) were analyzed . The results showed that the genes of the NFYA family had different degrees of response to cold, heat, salt and PEG. It can be seen that the expression level was higher at 12 h of salt treatment, and the expression level of cold and heat stress also changed in a trend. Combining salt and PEG stress, it can be seen that GhNFYA16 is differentially expressed under salt and PEG stress treatments, and the expression patterns of each gene are slightly different under stress treatments. These results further prove that GhNFYAs participate in the stress response of plants. Overall, it concludes that NFYA gene family has been influenced to more evolutionary events and extended. Moreover, some point mutations in exon regions and regulatory region of new family members might affect the function and expression of new family members [38, 39].
Tissue specificity of NFYA family genes in G. hirsutum and analysis of differentially expressed genes under salt stress
The tissue-specific presentation of 30 genes in the GhNFYA family showed that there were certain differences in the expression of 30 genes among different tissues (Fig. 6A). The expression levels of GhNFYA5, GhNFYA20, GhNFYA23 and GhNFYA28 genes were the highest in roots. 11 genes had the highest expression levels in the stem: GhNFYA2, GhNFYA6, GhNFYA7, GhNFYA12, GhNFYA13, GhNFYA14, GhNFYA15, GhNFYA17, GhNFYA19, GhNFYA22 and GhNFYA27. The relative expression of 12 genes GhNFYA1, GhNFYA8, GhNFYA9, GhNFYA10, GhNFYA11, GhNFYA16, GhNFYA18, GhNFYA21, GhNFYA24, GhNFYA26, GhNFYA29 and GhNFYA30 were the highest in leaves, and the expression of GhNFYA4 in roots and stems were higher than that in leaves. The expression levels of GhNFYA3 and GhNFYA25 did not differ among different tissues.
The most intuitive phenotypic changes occur in leaves when subjected to abiotic stress. Therefore, 12 highly expressed gene in leaves were selected, and the expression values were analyzed in periods (1, 3, 6 and 12 h) of NaCl stress (Fig. 6B, Additional file 3: Table S4), which provided support for subsequent virus-induced gene silencing experiments. The expression values of GhNFYA1, GhNFYA18, GhNFYA29 and GhNFYA30 were the highest when treated with NaCl for 12 h. The expression of GhNFYA8 decreased significantly after NaCl treatment for 3, 6 and 12 h compared with that after treatment for 1 h. The expression of GhNFYA16 increased significantly after 6 and 12 h of NaCl treatment. The expression of GhNFYA21 decreased significantly at 3 h compared with 1 h after NaCl treatment, and increased again at 6 h and 12 h. GhNFYA26 was significantly decreased after NaCl treatment for 3 h and 6 h, while its expression level was significantly increased after NaCl treatment for 12 h. Obviously, some GhNFYA genes showed significant differential expression after NaCl treatment.
G. hirsutum was treated with 100 mM NaCl stress when it grew to the three leaf one heart stage. It was found that the cotyledons began to wilt and lose their luster after 6 h of treatment, and the wilting was more serious after 12 h of treatment. After 24 h of treatment, part of the cotyledons fell off, true leaves wilted, leaf edges curled, and new leaves wilted to death (Fig. 6C).
Co-expression network analysis of GhNFYA genes under salt stress
To further understand the role of GhNFYA genes in salt stress, the correlation network of family members based on Pearson correlation coefficients (PCCs) was analyzed . Expression network analysis of genes under salt stress showed positive or negative correlations. A total of 142 gene pairs were positively correlated and 137 gene pairs were negatively correlated under stress (Fig. 7). Except for GhNFYA1, GhNFYA11, GhNFYA24 and GhNFYA29, other genes showed complex and highly similar functional relationships. 273 gene pairs that interact with each other during salt stress are involved in resilience. In conclusion, the expression network studies showed that GhNFYAs genes were closely related to each other in salt stress.
Subcellular localization of GhNFYA
The Programs website predicts that GhNFYA is most likely to be located in the nucleus and cytoplasm in G. hirsutum. According to the prediction results of the WoLF–PSORT website, GhNFYA is mainly located in the nucleus, cytoplasm, vacuole and chloroplast. For example, on the WoLF–PSORT website, the GhNFYA16 predicts subcellular localization in the nucleus and chloroplasts, while using the Programs website to predict that GhNFYA16 is localized in the nucleus. In general, subcellular localization verification focuses on the nucleus. Most of the GhNFYA are located in the nucleus, which may be related to their role as transcription factors that combine with NF–YB and NF–YC in the nucleus to form a trimer to regulate downstream target genes.
Combined with the differential expression of GhNFYAs genes under various stresses and the real-time expression of genes in 4 time periods under NaCl treatment, GhNFYA16 was selected for subcellular localization verification. The expression vector of GhNFYA16-GFP fusion protein was constructed. The recombinant plasmid containing the expression vector was injected into the epidermis of tobacco. 3 days later, the observe under a focusing microscope. The results showed that the green fluorescence signal of the fusion protein showed that GhNFYA16 was located in the nucleus (Fig. 8).
Analysis of GhNFYA protein interaction network
GhNFYA16 is an orthologous gene of Arabidopsis thaliana NFYA1. In this protein interaction network, Arabidopsis NF-YA1 interacts with NF-YB1, NF-YB2, NF-YB3, NF-YB6, NF-YC1, NF-YC2, NF-YC3, NF-YC4, NF-YC9 and NF-YC12. It is speculated that GhNFYA16 is closely related to the corresponding protein in cotton. Many functions were enriched in this protein network, such as positive regulation of photomorphogenesis, abscisic acid-activated signaling pathway, positive regulation of nitrogen compound metabolic process and regulation of developmental process and other Go enrichment. The functional diversification of NF-Y evolution in plants enables plants to actively respond to different abiotic stresses (Fig. 9).
Silencing GhNFYA16 reduced tolerance to salt stress in cotton
According to the analysis of the differentially expressed genes of the GhNFYA family, a highly expressed gene GhD01G1179.1 (GhNFYA16) was screened out after NaCl stress for 6–12 h. To further study the function of the GhNFYA16 gene, a VIGS experiment was performed on GhNFYA16 with Gossypium cv H177 as the material. 2 weeks later, the Gossypium with pYL156: PDS showed albino phenomenon, indicating that VIGS silence was successful. The silencing effect of GhNFYA16 gene was examined by quantitative real-time PCR (qRT-PCR). The results showed that the expression of pYL156: GhNFYA16 was significantly lower than that of pYL156 in the control group. After salt treatment, the conductivity rate and chlorophyll content of the plants were measured. It was found that after silencing the GhNFYA16 gene, the conductivity rate increased and the chlorophyll content decreased compared with the control group. Therefore, it can be inferred that GhNFYA16 is involved in the adaptability of cotton to salt stress (Fig. 10).
As an important economic crop, G. hirsutum is widely cultivated around the world and faces severe biotic and abiotic stress. The NF-Y transcription factor is closely related to adversity. Some studies have shown that NF-YA transcription factor participates in drought resistance, high salinity resistance, nitrogen–starvation responses, symbiotic nodule development, regulation of flowering time and embryogenesis . CmNF-Yb8 in chrysanthemum regulates the expression of CmCIPK6 and CmSHN3, alters stomatal movement and cuticle thickness in leaf epidermis, thereby affecting plant drought resistance . TaNFYA-B1 plays a vital role in the development of wheat root system and the use of nitrogen and phosphorus . Therefore, genome-wide identification of NFYA family genes was performed, focusing on the analyzing the relationship between NFYA family genes and their expression patterns under abiotic stress. This study lays a foundation for further exploring the role of NFYA transcription factors on Gossypium stress-related effects.
Through the CottonFGD and JGI Phytozome websites, the genome sequences of the NFYA family in 11 species According to the number of members, it was again proved that the number of NFYA genes in tetraploid Gossypium was almost double that in diploid Gossypium. The analysis shows that the closest evolutionary relationship with the Gossypium NFYA family is Theobroma cacao, Glycine max and Arabidopsis thaliana. It can be seen that soybean and Arabidopsis can be used as a reference for the study of NF-Y transcription factors in cotton. At present, there are many studies on soybean nitrogen fixation and drought resistance. The information of Arabidopsis NF-Y is more detailed, which can be used as a good reference for the study of NF-Y in cotton. Among them, the NFYA genes in Gossypium and Theobroma cacao are closely related throughout the phylogenetic tree, which further validates the previous report that Theobroma cacao and Gossypium share a common ancestor .
After chromosomal mapping of the NFYA gene family in cotton, it was found that the gene of this family in G. hirsutum was lost or added on some chromosomes compared with sea island cotton. In the process of identifying the NFYA gene family, it was found that G. hirsutum had two less genes than G. barbadense, and the two missing genes were probably located on the chromosomes of GhAt-02, GhDt-04, GhDt-08 and GhDt-09. Meanwhile, the random and uneven distribution of genes further illustrates that gene loss may occur during evolution, or it may be the result of incomplete genome assembly .Gene duplication produces functional differences and is considered to be the most important factor in speciation and environmental adaptability . For gene duplication, the aligned sequence should include at least 70% of the same part, and should cover more than 80% of their total length . Two duplicate genes located on the same chromosome are probably due to tandem duplication, while the existence of the same subgenome on different chromosomes is mainly due to fragment duplication . During evolution, apart from small-scale tandem duplications, most segment duplications contribute to the generation of new genes, thereby contributing to the complexity of plant genomes . Our research has found 98 pairs of duplicated genes. Compared with the previous genes, the newly duplicated genes are functionally redundant, and this redundancy is considered to be the basic driving force for evolutionary innovation . During the amplification of family genes, the discovery of chromosomal interactions, polyploidy, evolutionary differences and the transfer of inheritance between genomes provides valuable information. Based on the above analysis, it is believed that the GhNFYA gene family has undergone fragment duplication and genome-wide duplication during the evolution process, which ultimately contributed to the expansion of the NFYA family genes.
To explore the effect of divergence after NFYA gene duplication and the extent of selective pressure on duplicating genes. By counting 314 duplicate gene pairs in four cotton species, 301 pairs (95.86%) of them were found to have a Ka/Ks ratio less than 1, indicating homozygous selection . Only 13 pairs (4.14%) of duplicate pairs were generated by positive selection, which indicates that they may have undergone relatively rapid evolution after duplication. It is speculated that the Gossypium NFYA gene family has undergone strong purification selection. In addition, after whole genome duplication, functional differentiation is limited . The functional differentiation of repetitive gene pairs is the source of plants generating new genes, injecting new impetus into the evolution of plant genomes. Changes in gene expression patterns are another important factor leading to functional differentiation .
Using the obtained NWK files and motif files, the gene structure analysis of NFYA family in four cotton species was carried out. It can be seen that the motif types contained in each class are almost identical, but individual genes are structurally distinct from other NFYA genes. Fragment duplication and whole genome duplication may lead to changes in gene structure. Importantly, motifs 5, 4, 2, 10, 1, and 3 are critical determinants of NFYA family gene function. During evolution, other motifs have been gradually added to make genes play different roles in plants.
The NFYA family genes include plant hormones, such as auxin, abscisic acid, gibberellin and other cis-acting elements related to stress response. The cis-acting elements contained in each gene vary. Some genes were found to have fewer cis-acting elements than others. Combined with gene structure analysis, it was found that GhNFYA13 and GhNFYA27 had fewer cis-acting elements and longer introns. It further verified that the gene structure determines the function of the gene to a certain extent. By comparing the cis-acting elements of the GhNFYAs gene with the gene differential expression heatmap under cold, heat, salt and PEG stresses, it was more verified that this gene family played an important role in adversity. For example, the GhNFYA16 gene was significantly differentially expressed under both salt stress and PEG treatment. Through the analysis of tissue specificity and differentially expressed genes under NaCl stress, the role of NFYA family genes in stress was further verified. By constructing the co-expression network of GhNFYAs genes under salt stress, it was found that most of the genes play a positive or negative regulatory role, and the interactions between the genes jointly resist salt stress. As previously reported, GhNFYA10 and GhNFYA23 positively regulate salt stress . VIGS experiments were performed on the GhNFYA16 gene using cv H177 in G. hirsutum. Silenced plants were subjected to salt stress treatment. The study found that after silencing the GhNFYA16 gene, the plants had obvious phenotypic changes. In addition, plants are more sensitive to salt stress. This study has provided potential candidate genes for the study of Gossypium gene function, and provided a certain molecular basis for Gossypium breeding. It helps us to deeply understand the biological and molecular functions of NFYA genes in Gossypium, as well as their antioxidant effects under various oxidative stresses.
In this study, for the first time, a comprehensive analysis of four Gossypium species NFYA transcription factors was performed, and a total of 30 G. hirsutum NFYA genes were identified. The NFYA gene is divided into three categories through the phylogenetic tree, and it is found that the Gossypium NFYA gene has a close evolutionary relationship with Theobroma cacao and Populus tomentosa. Chromosome location and gene duplication analysis showed that this gene family was amplified in Gossypium through fragment duplication and whole genome duplication. The analysis of gene conserved domains, promoters and differentially expressed genes further identified the role of the NFYA family in Gossypium, and found that the GhNFYA gene played a role in a variety of plant hormones and environmental stimuli. To further verify the role of NFYA family genes in Gossypium, tissue-specific and differentially expressed genes were analyzed under NaCl stress, and VIGS experiments were performed on the GhNFYA16 gene, followed by salt stress after silencing. The results showed that GhNFYA16 played a role in Gossypium under salt stress. The data provided in this study will further provide useful information for studying the function of Gossypium NFYA transcription factors.
Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Hidden Markov Model
Quantitative real-time PCR
Virus-induced gene silencing
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This work was supported by China Agriculture Research System of MOF and MARA, and supported by National Natural Science Foundation of China (No. 31901509).
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: Figure S1 Distribution of NFYA family members in 11 species. Figure S2 Analysis of Non-synonymous (Ka) to Synonymous (Ks) ratio. Table S1 NFYA family genes correspond to eleven species gene renames. Table S2 Analysis of the physical and chemical properties of NFYA genes in G.hirsutum.
: Table S3 Cis-acting elements of upland cotton NFYA family members.
: Table S4 Expression values of 12 selected genes under salt stress.
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Xu, N., Cui, Y., Zhang, Y. et al. GhNFYA16 was functionally observed positively responding to salt stress by genome-wide identification of NFYA gene family in cotton. Environ Sci Eur 34, 95 (2022). https://doi.org/10.1186/s12302-022-00674-4