Genome-Wide Characterization of SNAC Gene Family in Ten Cotton Species and Function Analysis of GhSNAC3D Under Cold Stress

Abstract

The SNAC (Stress-responsive NAC) subfamily, a key branch of the conserved NAC transcription factor family, plays a central role in regulating plant stress response. However, systematic characterization of the SNAC family in cotton (Gossypium spp.) remains unclear. Employing a genome-wide screening approach, this study characterized 75 distinct SNAC transcription factor genes across ten Gossypium species, with tetraploid cottons harboring twice as many as their diploid progenitors. Phylogenetic analysis categorized the genes into three subgroups, with members of the same subgroup exhibiting conserved motif compositions and gene structures. Chromosomal localization revealed a conserved distribution pattern of SNAC genes between the Dt and At subgenomes in tetraploid cotton. Genomic collinearity analysis suggested that the primary driver of SNAC family expansion was segmental duplication. Promoter analysis predicted 2974 cis-regulatory elements, including cold- and hormone-responsive motifs, indicating their potential involvement in stress regulation. These GhSNAC genes indicated significant induced expressions under stress conditions, and GhSNAC3D exhibited the most significant up-regulated expression under low temperature stress. Genetic function studies displayed that VIGS-mediated GhSNAC3D-silencing significantly reduced the cold tolerance in cotton. This study systematically analyzed the genomic characteristics of the cotton SNAC family and functionally validated the molecular mechanism of GhSNAC3D-mediated cryogenic response, which lays a foundation for subsequent research on cold resistance in cotton and stress-resistant breeding.

1. Introduction

Cotton (Gossypium) is a globally cultivated economic crop of great importance [1]. Cultivated cotton is now widely distributed in high-altitude areas such as the northwest of Asia [2]. In these regions, cotton exhibits a heightened sensitivity to cooler temperatures, which leads to a lower germination rate, delayed sprouting, and weakened seedling vitality. This adversely impacts the growth and development of the cotton plants, ultimately causing a marked decline in both quality and yield [3,4]. Therefore, improving the cold resistance of cotton is an increasingly important goal.

Low temperatures represent an environmental challenge that significantly hinders the growth, productivity, and survival of plants [5,6]. As global climate change exacerbating temperature fluctuations, elucidating the detailed molecular mechanisms that govern the response to cold stress becomes increasingly important [7]. This understanding not only enhances our comprehension of fundamental biological processes but also opens avenues for improving the cold resistance of cash crops. Cold stress presents a considerable obstacle to cotton cultivation, especially in temperate regions and during the initial planting phases [8,9]. Cold stress, by causing physiological and molecular damage, leads to a reduction in germination rate and impaired seedling development, seriously threatening these initial growth stages [10]. After being subjected to cold stress, notable alterations occur in various physiological and biochemical indicators and the morphology of plant organelles [11]. Under low-temperature stress, numerous physiological and biochemical processes in plant cells undergo changes—for instance, the leakage of intracellular ions and structural alterations to various cellular components [12,13,14].

The NAC (NAM, ATAF1/2, CUC2) transcription factor family comprises a group of transcription factors that are specific to plants and play a crucial role in the responses to stress tolerance. The naming of the NAC transcription factor family originated from the NAM (No Apical Meristem) gene of Pharbitis, ATAF1/2 (Arabidopsis Transcription Activator Factor 1/2), and CUC2 (Cup-Shaped Cotyledon 2) genes of Arabidopsis thaliana at first [15]. The NAC family’s members possess a highly conserved DNA-binding domain located at their N-terminus, consisting of a 150–160 amino acid residue region known as the NAM domain [16]. This domain exhibits high conservation during the evolutionary process. Studies have demonstrated that the NAC transcription factor family, unique to plants, is crucial for regulating growth and development processes, hormone responses, and resistance to stress [17,18,19,20]. For instance, the enhanced drought tolerance in rice is attributed to the overexpression of OsNAC10 [21]; similarly, SNAC1 and ONAC045 overexpression contributes to improves drought and salt resistance in rice [22]. Additionally, the overexpression of SNAC2, OsNAC5, and OsNAC6 enhances rice’s ability to resist cold, salt, drought, and hot stresses [23]. In A. thaliana, the overexpression of soybean GmSNAC49 upregulates genes related to drought and ABA (Abscisic Acid) signaling pathways, thus increasing drought stress tolerance [24]. Furthermore, the overexpression of grapevine VvNAC17 improves drought resistance in A. thaliana by upregulating genes associated with ABA and stress responses. The Miscanthus MLNAC10 exerts the function of antioxidant enzymes by regulating the ABA signaling pathway, alleviates ROS-induced damage, and serves as an important regulatory factor in the tolerance to salt and drought stress [25].

The SNAC (Stress-Responsive NAC) subfamily, a significant component of the NAC family, plays a crucial role in the stress responses of plants. At present, comprehensive genome-wide analyses of the SNAC have been completed in several plants, including A. thaliana, Populus trichocarpa, Casuarina equisetifolia, Musa acuminata, and Solanum lycopersicum [26,27,28]. However, systematic recognition of the SNAC transcription factor family in cotton and research on its cold resistance function remain insufficient. This study achieved identification of the SNAC family in cotton through a genome-wide approach. Utilizing comprehensive genomic data, the SNAC gene family members in cotton were successfully identified [29]; then, through phylogenetic analysis, the chromosomal localization, gene structure, and collinear relationships of cotton SNAC genes were clarified. Additionally, investigations were conducted on their subcellular localization characteristics, expression patterns across different tissues and under various stress treatments, and phenotypic changes under cold stress. These research outcomes will enhance our comprehension of the mechanism by which SNAC family members function in cotton cold stress resistance, reveal the characteristics and diversity of cotton SNAC genes, and provide dual support for elucidating the mechanism of cotton cold stress response with evolutionary and functional molecular evidence.

2. Results

2.1. Characterization and Physicochemical Properties of SNAC Genes in Cotton

Through BLAST (v2.210) analysis, 5, 5, 10, 10, 10, 11, 10, 4, 5, and 5 SNAC genes were derived from the genomes of G. herbaceum (Ghe), G. turneri (Gtu), G. barbadense (Gb), G. hirsutum (Gh), G. darwinii (Gd), G. mustelinum (Gm), G. tomentosum (Gt), G. thurberi (Gth), G. raimondii (Gr), and G. arboreum (Ga), respectively (see Table S1). The NAM domain was confirmed in all candidate genes using the InterProScan and SMART tools. Notably, the number of SNAC genes in the tetraploid species G. hirsutum (Gh) is approximately twice that in its diploid ancestors, G. raimondii (Gr) and G. arboreum (Ga), indicating that most SNAC genes were retained during the polyploidization of Ga and Gr. Table S1 provides detailed physicochemical parameters of all SNAC proteins across the 10 Gossypium species, including gene ID, renamed ID, protein length, and molecular weight. The length of amino acid sequence for SNAC proteins ranges from 157 residues in GthSNAC3 to 348 residues in GdSNAC1A. Their molecular weight ranges from 18.04 kDa for GthSNAC3 to 38.69 kDa for GdSNAC1A, averaging at 34.22 kDa. Additionally, the isoelectric point (pI) ranges from 5.52 in GtuSNAC4 to 9.72 in GhSNAC3D. The instability indices for all SNAC proteins range from 24 to 51, with an average grand average of hydropathicity (GRAVY) of less than 0. This suggests that these proteins are unstable, soluble, and hydrophilic (Table S1). These results reveal variations in molecular weight, the number of amino acids, and isoelectric point among SNAC family members, which may contribute to subtle functional variations.

2.2. Chromosomal Mapping of the SNAC Gene Family

In this study, SNAC family members were designated sequentially according to their chromosomal positions (Table S1). Analysis revealed that most SNAC genes are distributed across 5 chromosomes, predominantly on chromosomes 1, 4, 5, 6, and 12. Notably, SNAC genes in the Gth genome are restricted to 3 chromosomes, with GthSNAC1 and GthSNAC2 co-localized on chromosome Gth07 (Figure 1). In the Gm-A subgenome, SNAC genes are distributed across 6 chromosomes, which may result from gene duplication and deletion events during evolution. Interestingly, as shown in Figure 1B,C, in the Dt and At subgenomes of tetraploid cotton, SNAC genes are basically located at the same position on different chromosomes. This observation further supports the high evolutionary conservation of the SNAC gene family.

2.3. Evolutionary Analysis of SNACs Coupled with Investigation of Conserved Motifs, Conserved Domains, and Gene Structures

To further characterize the phylogenetic trajectory of SNAC proteins in Gossypium and other species, SNAC protein sequences from A. thaliana, T. cacao, and cotton were subjected to multiple sequence alignment using ClustalW and analyzed using MEGA11 software (v11.0). A phylogenetic tree was constructed via the Neighbor-Joining algorithm (Figure 2). Phylogenetic analysis revealed that SNAC proteins could be classified into three groups. Cotton SNAC genes in nearly all clades tended to cluster together, likely due to their relatively conserved functions. Additionally, cotton orthologs from the A genome and At subgenome, as well as from the D genome and Dt subgenome, predominantly formed orthologous pairs at evolutionary branch terminals, indicating closer evolutionary relationships between At-A and Dt-D orthologs in cotton.

We reconstructed phylogenetic trees from the SNAC protein sequences of 10 species of cotton and analyzed the structural features of the genes (Figure 3A). Consistent with Figure 2, Figure 3A shows that all SNAC proteins in Gossypium were divided into three groups. Figure 3B reveals that nearly all Gossypium SNAC proteins contain motif1-5, with exceptions: GthSNAC3 lacks motif3, and GheSNAC1 lacks motif1 and motif5. This indicates that SNAC proteins are relatively conserved during cotton evolution. Figure 3C demonstrates that all SNAC proteins possess a conserved NAM domain. As shown in Figure 3D, paralogous genes within the same cluster exhibit similar exon/intron distributions, and nearly all SNAC proteins have three CDS regions. This structural similarity provides strong support for exploring the relationship between gene function and evolution. Additionally, several structural variants with distinct exon/intron architectures were identified: GthSNAC1 contains an additional CDS region, while GthSNAC2 and GthSNAC3 have lost one CDS region. These variations may result from evolutionary changes in the SNAC family.

2.4. Profiling of Cis-Acting Motifs Within the SNAC Genes Promoter

Cis-acting elements located in the promoter region are essential for gene functionality [30]. Most SNAC family members exhibit significant differences in both the types and quantity of these elements, indicating a diversification of function within the family [31]. Additionally, members with closer evolutionary relationships share similar element types and quantitative characteristics, as exemplified by GtSNAC4A, GmSNAC5A, GaSNAC4, GhSNAC4A, GbSNAC4A, GdSNAC4A, GtuSNAC5, GheSNAC4, GrSNAC4, GmSNAC5D, GhSNAC5D, GbSNAC5D, GtSNAC5D and GdSNAC5D. This pattern primarily arises from evolutionary linkage [32]. As shown in Figure 4A,B, a total of 2974 cis-regulatory elements were discovered within the cotton SNAC gene family, classified as follows: Elements responsive to plant hormones: P-box, GARE-motif, AuxRE, TGACG-motif, TCA-element, TATC-element, CGTCA-motif, and ABRE; Abiotic stress-responsive elements: WUN-motif, LTR, GC-motif, TC-rich repeats, ARE, and MBS; Factors associated with growth and development: HD_Zip1, GCN4_motif, O2-site, RY-element, and CAT-box. Elements that respond to light are the most prevalent, accounting for over 49% of the total. Plant hormone-responsive elements constitute approximately 37%, and abiotic stress-responsive elements about 10%. Despite their relatively small number, abiotic stress-responsive elements are critical for the unique functions of certain genes, particularly in mediating plant resistance to cold stress. These findings indicate that specific SANC genes have unique functions in the growth and development of plants by responding to different abiotic stress elements. Overall, the presence of these factors in cotton SNAC genes underscores their importance in cold resistance.

2.5. Collinearity Analysis of the SNAC Gene Family

To further explore the evolutionary pattern of SNAC genes in upland cotton, we conducted a collinearity analysis comparing SNAC genes in upland cotton with those from nine other cotton species. The results revealed 19, 20, 19, 20, 22, 34, 35, 35, and 35 collinear gene pairs in Gth, Ga, Ghe, Gr, Gtu, Gm, Gt, Gb, and Gd, respectively. The widespread collinearity among most genes suggests that SNAC genes may be highly conserved in plants (Figure 5). Cross-species collinearity analysis across the 10 cotton species identified a total of 239 collinear pairs among G. hirsutum and nine other species. In cotton, the majority SNAC gene pairs have undergone segmental duplication occurrences, indicating that segmental duplication serve as the dominant mechanism for the growth of the SNAC gene family (Figure 5).

2.6. SNAC Genes Expression Pattern Analysis

Based on transcriptome data analysis (PRJNA248163), significant differences were observed in the expression trends of the SNAC transcription factor family across distinct development stages and tissues in cotton. Notably, distinct functional divergence exists among different genes within the same family, indicating that SNAC genes exhibit obvious tissue-specific expression patterns (Figure 6A). For individual genes, although their expression levels vary significantly across tissues, their expression abundance remains relatively consistent compared to other family members.

Expression analysis further revealed that cotton’s cold stress defense mechanism may rely on key regulatory genes, likely through the coordinated regulation of multiple genes throughout the plant’s growth and development cycle. During cold stress treatments at different times, the levels of expression for GhSNAC1A, GhSNAC1D, GhSNAC2A, GhSNAC2D, GhSNAC3D, GhSNAC4A, GhSNAC4D, and GhSNAC5D showed a continuous upward trend. Among these, GhSNAC3D exhibited the most dramatic expression change: after 3 h of cold stress, the level of expression was roughly 120 times greater than that of the control group, and this increase reached 250-fold after 12 h of stress (Figure 6B). This indicates that GhSNAC3D is essential for regulating role cold stress resistance in cotton.

Additionally, most SNAC genes were observed to be up-regulated in response to heat, salinity, and drought stress, indicating that the SNAC gene family may play a crucial role in cotton’s adaptation to multiple abiotic stressors.

2.7. Subcellular Localization of the GhSNAC3D

Transcription factors typically exert their functions within the nucleus. In order to investigate the subcellular localization of GhSNAC3D, the complete coding of the cloned GhSNAC3D gene, with the stop codon removed, was inserted into the pCAMBIA-1300-GFP vector, allowing GhSNAC3D to be fused with GFP during expression in tobacco leaves. The results confirmed that the empty pCAMBIA-1300-GFP vector was expressed throughout the cell, with predominant distribution in the nucleus and cell membrane. In contrast, the GhSNAC3D-GFP fusion protein was exclusively solely to the nucleus, suggesting that GhSNAC3D is both expressed and functions within that cellular compartment (Figure 7).

2.8. GhSNAC3D Silenced Cotton Plants Showed High Sensitivity to Cold Stress

In order to investigate the function of the GhSNAC3D gene in cotton cold stress resistance, we employed VIGS, which is a simple and efficient transient expression method, for functional verification in cotton. As shown in Figure 8C, cotton plants in the TRV:00 and TRV:GhSNAC3D groups showed similar growth, whereas leaves in the TRV:PDS group (positive control) exhibited an obvious albino phenotype (Figure 8A), confirming successful VIGS establishment. To validate the silencing efficiency, qRT-PCR analysis was conducted. Results in Figure 8B revealed that the level of expression for GhSNAC3D in the TRV:GhSNAC3D group was markedly lower than in the TRV:00 group, with a silencing efficiency of approximately 70% relative to the control, indicating effective gene silencing.

To evaluate the effect of GhSNAC3D silencing on resistance to cold stress resistance, plants transformed with TRV:00 and TRV:GhSNAC3D were subjected to cold stress at 4 °C under a cycle consisting of 16 h of light followed by 8 h of darkness for a duration of 12 h. Plants were subjected to cold stress at 4 °C for 12 h starting at the beginning of the 16 h light period [6]. As shown in Figure 8C, leaves of TRV:GhSNAC3D plants showed more severe wilting compared to TRV:00 plants. The finding indicates that GhSNAC3D has a positive regulatory function in enhancing cotton cold stress resistance: reduced GhSNAC3D expression leads to decreased cold tolerance in cotton.

3. Discussion

SNAC family is classified under the category of transcription factors that are specific to plants, which is found in Malus domestica, Zea mays, Medicago sativa, Casuarina equisetifolia, Oryza sativa, Solanum lycopersicum, Arabidopsis thaliana, Populus euphratica, Brachypodium distachyon and other plants [33,34,35,36]. Increasing studies have confirmed that SNAC transcription factor family orchestrates developmental processes and serves as a central regulator in biotic/abiotic stress responses by modulating downstream target genes [37]. However, comprehensive investigations into the genome-wide identification, structural characterization, and low-temperature stress response mechanisms of the cotton SNAC gene family remain limited. This study implemented an integrated genome-wide identification and expression profiling of the SNAC family in cotton, elucidating their structural features and evolutionary relationships while examining the expression dynamics of GhSNAC genes under abiotic stress conditions.

Genome-wide analysis successfully characterized the SNAC transcription factor family in cotton, with approximately 75 SNAC genes annotated in the cotton genome across multiple studies (Table S1). This quantitative distribution resembles the conserved nature of SNAC family members across plant species, there are 18 stress-responsive BdSNACs in Brachypodium distachyon [38], while Medicago sativa contains 14 MsSNACs in [39], and Malus domestica has 17 MdSNACs [40]. Phylogenetic analysis, based on a co-constructed tree with SNAC genes from A. thaliana and T. cacao, classified cotton SNACs into three evolutionary groups, with Group C containing the largest number of genes (Figure 2), consistent with the taxonomic pattern of A. thaliana SNACs [41]. Members of the same subgroup demonstrate remarkable conservation in sequence and structural features, indicating functional conservation within the subgroup (Figure 3); while distinct gene structures and conserved motifs between subgroups suggest that functional differentiation may be due to specific variations in evolutionary processes [42]. Physicochemical property analysis revealed that all cotton SNAC family proteins are hydrophilic (Table S1), consistent with findings in A. thaliana, Casuarina equisetifolia, and Malus domestica [28,41,43]. Collinearity analysis identified 239 collinear gene pairs in cotton (Figure 5), with most originating from segmental duplication events. This indicates that the expansion of the SNAC gene family during cotton evolution is predominantly driven by segmental duplication events, which dominated multiple whole-genome duplication episodes. Such a duplication pattern may enhance plant adaptability to environmental stress through functional redundancy while improving genomic plasticity, which aligns with the evolutionary strategy of plants coping with stress [44,45].

Regulation of transcriptional plays a vital role in enabling plants to cope with abiotic stress, as cis-acting elements in gene promoters can activate or silence the activity of transcription factors in response to varying abiotic stressors [46]. The type and number of cis-acting elements are crucial determinants in the regulation of gene expression and the transcriptional process [46]. This study found that the regulatory regions of cotton SNAC genes harbor numerous cis-elements responsive to stress as well as hormone-responsive elements. This indicates their potential significance in mediating responses to abiotic stress and in hormonal regulatory pathways within cotton (Figure 4). Notably, similar cis-element distribution patterns have been observed in the promoter regions of SNAC genes in other plant species, supporting conserved regulatory mechanisms [47]. This convergent evolution may have formed during the long-term adaptation of Gossypium species to various environmental cold stress pressures [48]. Transcriptome data from stress response analysis showed that several GhSNAC genes exhibit distinct expression changes under cold stress (4 °C) (Figure 6). Further analysis revealed that most stress-responsive SNAC genes cluster in the same subgroup in the phylogenetic tree, consistent with findings in A. thaliana and Oryza sativa [49]. Among them, GhSNAC3D was particularly noteworthy: not only was it the most strongly induced by cold stress, but it also exhibited the highest expression level among all GhSNAC genes, which strongly suggested that GhSNAC3D has a special function in the regulation of the response to cold stress in cotton. To validate its function, GhSNAC3D-silenced cotton plants were generated, and cold tolerance assays were performed. Phenotypic analysis showed that, compared with wild-type (WT) plants, GhSNAC3D-silenced plants exhibited significantly reduced cold tolerance (Figure 6).

This research conducted a comprehensive analysis of the evolutionary traits and functional diversity of the SNAC in cotton, and combined with VIGS technology verification, highlighted the pivotal function of GhSNAC3D in the response to cold stress. In the future, transgenic lines can be constructed by transgenic technology to further explore the functional redundancy and synergistic mechanism of members of different subgroups. Simultaneously, it offers a theoretical foundation for the breeding of crops with resistance to stress.

4. Materials and Methods

4.1. Plant Materials and Experimental Treatments

The plant material utilized in this experiment is G. hirsutum cv. TM-1 and the seeds were purchased from the Institute of Cotton Research, Chinese Academy of Agricultural Sciences. Cotton seeds were sulfuric acid delinting and sown in nutrient bowl filled with nutrient soil: vermiculite: perlite = 3:1:1. Plant was maintained in controlled environment chambers with thermostatic regulation at 25 °C, 16 h light/8 h dark, and 70% relative humidity. Nutrient management was carried out using Hoagland nutrient solution (watered once a week until 3 days before stress treatment, and purchased from Tiangen Biotech BeiJing Co., Ltd. (Beijing, China)). Fifteen-day-old vegetative-stage plantlets were exposed to 4 °C treatment in growth chambers maintaining standardized photoperiodic parameters. Samples of leaves were collected at various intervals (0 h, 1 h, 3 h, 6 h and 12 h following stress induction) after treatment. Then freeze-fix immediately and maintain RNA integrity at −80 °C until nucleic acid extraction.

4.2. Characterization of Members of the SNAC Family

Genome datasets for ten Gossypium species (G. herbaceum, WHU, A1; G. arboretum, CRI, A2; G. thurberi, ISU, D1; G. raimondii, HAU, D5; G. turneri, NSF, D10; G. barbadense, ZJU, AD2; G. mustelinum, JGI, AD4; G. tomentosum, HGS, AD3; G. hirsutum, JGI, AD1; G. darwinii, HGS, AD5) were obtained from the Cotton Multiomics Database [50]. The homologous SNAC protein sequences from Arabidopsis were retrieved from the TAIR database [51]. The SNAC protein sequences from Arabidopsis as query sequences (the sequences listed in Supplementary Table S4), the BLAST program package within TBtools (v2.210) was utilized to conduct BLASTP alignment searches on the completed local whole—genome databases of 10 cotton species [52]. The E-value threshold was set to 1.0 × 10⁻²⁰ to reduce false positives and obtain the dataset of SNAC proteins. Only candidate genes with ≥60% homology to SNAC subfamily genes [53]. Predict and analyze the conserved domains of the preliminarily identified candidate homologous gene pairs in the InterProScan database and SMART database [54,55]. Eliminate gene sequences that either lack or do not include the conserved domains typical of the SNAC family, and further screen to obtain the final family members. In addition, using the pI/Mw tool from ExPASY was utilized to determine the physicochemical properties of each gene product, such as molecular weight (MW) and isoelectric point (pI).

4.3. Phylogenetic Investigation

The MEGA 11 (Molecular Evolutionary Genetics Analysis software, Version 11.0) software is used for phylogenetic research. The ClustalW method is employed to align the protein sequences of SNAC among T. cacao, A. thaliana, and ten Gossypium species. Then, these protein sequences are utilized to create a phylogenetic tree through the Neighbor-Joining (NJ) algorithm, with parameters set to 1000 bootstrap replicates and genetic distance calculated using the Poisson correction model. The generated tree is visualized using the iTOL website [56].

4.4. Chromosomal Distribution of SNAC Gene

Spatial distributions of these genes across ten Gossypium species were visualized on chromosomes using the MG2C (online tool for Multiple Sequence Alignment and Phylogenetic Tree Construction, Version 2.0) platform [57].

4.5. Genomic Architecture, Conserved Motifs, and Protein Domains Analyses

Conserved motifs within protein sequences were predicted through the use of the online MEME database [58], and conserved domains were predicted via the online CD-Search database. The genomic architecture, conserved motifs, and protein domains were visualized via TBtools.

4.6. Investigation of Cis-Acting Elements in the Promoter Region Upstream of the Land Cotton SNAC Gene

Cis-regulatory elements located in the 2000 bp upstream promoter regions of Gossypium SNAC family were predicted and examined using the PlantCARE database with visualization performed via TBtools [59].

4.7. Collinearity Analysis

Collinearity analysis of ten Gossypium SNAC genes was performed using the One Step MCScanX module in TBtools.

4.8. GhSNAC Expression Pattern Analysis

Transcriptomic datasets encompassing multiple tissues and abiotic stress conditions of G hirsutum were analyzed to profile SNAC gene expression patterns [60]. The transcriptomic dataset analyzed in this study was obtained from a public database with a login number of PRJNA248163 obtained from the NCBI SRA database. Heatmaps were generated using the Heatmap module in TBtools, with transcriptomic data standardized by Log₂(FPKM+1).

4.9. Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) Analysis

Total RNA was isolated from cotton leaves and purified using the RNAprep Pure Polysaccharide Polyphenol Plant Total RNA Extraction Kit (TIANGEN lot: A0516A). GhSNACs expression in these tissues was analyzed via quantitative real-time PCR (RT-qPCR), with cotton GhUBQ6 serving as internal controls [61]. Briefly, cDNA was reverse-transcribed from total RNA and utilized as template for RT-qPCR with gene-specific primers. Reactions were conducted using SYBR-Green real-time PCR premix following the manufacturer’s protocol, and relative expression levels were calculated via the 2^−ΔΔCt method [62].

4.10. Subcellular Localization

GhSNAC3D was amplified via PCR and cloned into the pCAMBIA1300-GFP (35S:GFP) expression vector through homologous recombination, yielding a pCAMBIA1300-GFP-GhSNAC3D (35S:GhSNAC3D-GFP) fusion construct (primers listed in Supplementary Table S2). The constructed vector was introduced into Agrobacterium tumefaciens GV3101 and subsequently infiltrated into the leaves of 4-week-old Nicotiana benthamiana, followed by dark incubation for 48 h. Fluorescence emissions from the leaf epidermis of N. benthamiana were visualized via a confocal microscope (Nikon Corporation, Tokyo, Japan). The study employed pCAMBIA1300-35S-mCherry-NLS (Puint, Xianning, China) as a nuclear marker for the cells.

4.11. Construction and Transformation of Cotton Vectors

To construct cotton silencing vectors, specific fragments (300 bp in length) of the GhSNAC3D and GhPDS genes were selected to avoid high homology with homologous genes (primers listed in Supplementary Table S2). These fragments were cloned into the pTRV2 vector. The constructed silencing vectors were then introduced into Agrobacterium LBA4404 via electroporation, and positive strains were verified by PCR [63]. The transformed Agrobacterium was cultured to the logarithmic growth phase, and the infection concentration was adjusted to an appropriate level (OD₆₀₀ = 1.5). The pTRV2 and pTRV1 empty vectors, positive control, and Ptrv:GhSNAC3D were mixed at a 1:1 ratio and infiltrated into cotton leaves by injection. Infected cotton plants were incubated in the dark overnight and subsequently transferred to a cotton cultivation room at a temperature of 25 °C, with a light cycle of 16 h on and 8 h off for cultivation.

4.12. Data Statistics

Statistical evaluations were conducted utilizing GraphPad Prism (v10.1.2). Duncan’s multiple-range test was employed to assess differences between measurements across time points or treatment groups. Significance thresholds were defined as follows: p > 0.05 (not significant), p < 0.01 (highly significant), and p < 0.05 (statistically significant).

5. Conclusions

This study systematically identified members of the cotton SNAC family and clarified the core role of GhSNAC3D in cold stress response, thus providing candidate genes for cotton stress-resistant molecular breeding. At present, only the phenotypic effects of the gene have been verified, and GhSNAC3D’s downstream target genes and specific molecular mechanisms still need to be further analyzed through experiments such as yeast one-hybrid and ChIP-seq. In the future, we will obtain stably overexpressed plants through genetic transformation, analyze their agronomic traits under field stress conditions, and explore the interaction network between GhSNAC3D and other stress-resistant genes.