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Unveiling Integrative Computational Analysis and Functional Profiling of GhSOS1 gene family in response to dual stresses Drought and Salinity in Gossypium hirsutum L.LARAIB IQRA1*, Muhammad Naveed Shahid1, Umar Akram2, Usman Arif 3
1 Department of Botany, Division of science and technology, University of Education, Lahore
2 MNS- University of Agriculture, Multan, Pakistan
3 Centre of excellence in molecular biology, University of the Punjab, Lahore
Corresponding Author email: laraibiqra841@gmail.com 
Abstract
Background
The global production of upland cotton (Gossypium hirsutum) has declined in recent years due to increasing drought and salinity stress. SOS1 gene family plays a key role in maintaining ion homeostasis and enhancing stress resilience.
Methods
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A genome-wide study was conducted to identify SOS1 genes, including analyses of gene structure, motifs, chromosomal distribution, phylogenetic relationships, Ka/Ks ratios, cis-regulatory elements, protein–protein interactions, 3D structures, and phosphorylation sites. Fifteen paralogs of G. hirsutum were identified and functionally characterized. For the validation of in-silico findings, expression of 15 genes was profiled during salt and drought stress by RNA-seq data that was available publicly and finally it led to selection of five highly responsive species. After exposing cotton seedlings to 400mM NaCl and 20% PEG, quantitative Realtime PCR was performed with different time intervals (0h, 3h, 6h, and 12 h).Results
The presence of cis-regulatory elements, including ABRE, DRE/CRT, MYB sites, and other transcription factor motifs, further confirmed their stress response role. Ka/Ks analysis indicated strong conservation among gene paralogs, with most under purifying selection. Protein–protein interaction analysis revealed SOS1 association with antiporters, facilitating Na⁺ efflux across the plasma membrane to reduce drought and salinity effects. Prominent transcriptional changes induced by stress was confirmed by qPCR results. The results showed consistency with in-silico predictions by up-regulation of GhSOS1-5 and GhSOS1-11.
Conclusions
Collectively, this is a comprehensive study of the SOS1 family in G. hirsutism which highlights its roles in conferring abiotic stress tolerance. These findings will serve as a valuable resource for molecular breeding.
Keywords:
Insilico characterization of SOS1
Cis-regulatory elements
phylogenetic analysis
qPCR validation of GhSOS1
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1. Introduction
Cotton is one of the most economically significant crops, serving as the primary source of natural fiber for the global textile industry. However, its productivity is increasingly threatened by abiotic stressors such as drought and salinity, which cause substantial yield losses. Disruption of ion homeostasis in plants is a key physiological indicator of salinity stress and drought [1].
A major limiting factor in productivity and growth of upland cotton (Gossypium hirsutum) is drought. [2]. Under drought conditions, cotton plants undergo a cascade of physiological changes, including reduced stomatal conductance and a decline in photosynthetic efficiency [3]. Another hallmark of drought stress is the excessive accumulation of reactive oxygen species (ROS), which can cause severe cellular damage [4]. In addition, drought alters carbohydrate concentrations in leaves and triggers osmotic adjustment mechanisms to maintain cellular homeostasis in G. hirsutum [5].
Salt stress is another major challenge to sustainable agriculture, directly reducing yield along with the growth of upland cotton (Gossypium hirsutum) [6]. Globally, nearly 20% of the agricultural land is affected by salinity [7]. Upland cotton, along with staple crops such as wheat and rice, is particularly vulnerable to saline irrigation water [8], necessitating the development of tolerance mechanisms and mitigation strategies [9]. Under salt stress, cotton plants typically show an increase in root length, whereas other physiological parameters, including stem diameter, leaf area, and chlorophyll a and b contents, tend to decline [10].
Traditional practices to mitigate soil salinity, such as soil drainage, soil leaching and strip cropping have shown only limited success [11]. Salinity tolerance, on the other hand, is an intricate phenomenon regulated by diverse biochemical, physiological and molecular responses [12]. Plants employ different mechanisms to tolerate salinity stress such as ion compartmentalization, maintenance of ion homeostasis and reactive oxygen species (ROS) detoxification [13]. Among these mechanism salt overly sensitive (SOS) signaling pathway plays a vital role in maintaining ion equilibrium, thereby mitigating the adverse effects of both salinity and drought stress [14]. This pathway was first characterized in Arabidopsis thaliana and comprises three core components: SOS1, SOS2, and SOS3. SOS1 encodes vacuole-localized Na+/H + exchanger responsible for pumping excessive sodium ions out of the cell. Additionally, SOS1 may contribute indirectly to high-temperature stress tolerance by maintaining ionic balance.
A crucial pathway of ion homeostasis in cotton under salinity and drought stresses is SOS (salt overly sensitive) pathway [15]. The SOS gene family is an evolutionarily conserved group of genes involved in maintaining ion homeostasis, particularly in response to salt stress [16]. Although SOS1 gene family has been widely studied in many other plants like Arabidopsis thaliana, Zea maize and Oryza sativa [17], genome-wide investigations in Gossypium hirsutum remain limited.
In recent years, genome-wide studies have emerged as powerful approaches for analyzing gene families and elucidating their functional roles in abiotic stress responses [18]. Therefore, the present study was undertaken to investigate the GhSOS1 gene family through comprehensive genome-wide characterization, including analyses of gene structure, conserved domains, motifs, protein homology and phylogeny. To validate insilico results expression profiling of GhSOS1 gene family qPCR was performed on candidate genes and expression level was noticed. Considering the economic importance of cotton, this study provides valuable insights into the functional characterization of the Salt Overly Sensitive 1 (SOS1) gene family, thereby contributing to the development of stress-resilient cultivars.
2. Materials and Methods
2.1 Identification of SOS1 Genes in G. hirsutum
Genome of Gossypium hirsutum was downloaded from Phytozome v.13 (https://phytozome-next.jgi.doe.gov/), a publicly available database. Candidate genes were shortlisted by BLASTP (https://phytozome-next.jgi.doe.gov/blast-search) search against queries with standard BLAST parameters e-value 1e-5 and identity more than 90%. Query sequences of SOS1 were retrieved from model plant Arabidopsis thaliana protein; sequences downloaded from its databases TAIR 10 (https://www.arabidopsis.org/). The identified protein sequences were subjected to NCBI CDD-batch search for identifying the conserved domains present in GhSOS1 using standard parameters (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and verified by Pfam database (http://pfam.xfam.org/) and SMART (http://smart.embl-heidelberg.de/) using default setting. All the protein sequences that do not contain Na+/H+ domain were discarded.
2.2 Construction of Phylogenetic Tree and selection pressure Analysis
To infer the evolutionary relationship of identified candidate GhSOS1 proteins with other plant species having same proteins, a phylogenetic tree was constructed by neighbor-joining method with 1000 bootstrap values using MEGA V11.0.13 software (https://www.megasoftware.net/), and results were saved as newick format and exported to online tool itol (https://itol.embl.de/upload.cgi) for constructing a graphic representation. To infer the selection pressure between candidate genes paralogs were determined by MEGA V11.0.13. Ka/Ks (non-synonymous/synonymous) ratio for the duplicated genes were determined to figure out the diversity in function and duplication events in gene pairs.
2.3 Gene Structure and Conserved Domain and motif Analysis
Gene structure was graphically determined by using TBtools v2.313 with the help of Gossypium hirsutum Gff annotation file method. Conserved domains were analyzed via Pfam and then motifs were determined by MEME Suite (https://meme-suite.org/) in candidate genes .
2.4 Physicochemical Properties and Subcellular Localization of GhSOS1 protein
In-silico characterization like molecular weight (KDa), CDS length (bp) and isoelectric point (pI) and GAVY of identified 15 proteins was measured by using ProtParam expasy (https://web.expasy.org/protparam/). To predict the sub-cellular localization of the candidate proteins CELLO V2.5 (http://cello.life.nctu.edu.tw/) and WOLPSORT (https://wolfpsort.hgc.jp/) were used .
2.5 Chromosomal Distribution
Chromosomal distribution and gene locus of identified 15 SOS1 genes was manually displayed by using online tool Mapchart (https://www.wur.nl/en/show/Mapchart.htm) .
2.6 Cis-Regulatory Element Analysis
PlantCARE database was used (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) in promoter sequences (2kb upstream) of these 15 proteins for determination of cis-regulatory elements and stress-responsive transcriptions factors.
2.7 Protein-Protein Interaction (PPI) Network Prediction
To see the regulatory Protein-protein interaction of 15 shortlisted hirsutum proteins online software STRING (https://string-db.org/) was used .
2.8 Prediction of Phosphorylation Sites and Transmembrane Domain
Phosphorylation sites were identified using online software Net Phos 3.1a (https://services.healthtech.dtu.dk/services/NetPhos-3.1/) in GhSOS1 and a bioinformatics tool TMHMM − 2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/) was used for determining the transmembrane domain.
2.9 Protein 3D structure
Protein tertiary structures of the identified GhSOS1 proteins was determined by SWIS-MODEL (https://swissmodel.expasy.org/) Suitable template structures were selected automatically based on highest GMQE.
2.10 In-Silico Expression Profiling and Candidate Gene Selection
Transcript abundance of the SOS1 gene family was analyzed under abiotic stress by using RNA-seq datasets of Gossypium hirsutum (upland cotton). CottonGVD database (https://db.cngb.org/cottonGVD/tool/pop_expression) was used for retrieval of publicly available RNA-seq datasets. Comparison between stress treatments and tissues was enabled by using normalized expression values (FPKM) of all 15 identified GhSOS1 genes. TB tools were used for compiling and visualizing these values as heatmap. Five GhSOS1genes which showed the substantial induction and consistent differential expression patterns in salt and drought conditions, were selected for experimental validation using quantitative real-time PCR (qPCR).
2.11 RNA Extraction and cDNA Synthesis
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Specific controlled conditions (28 ± 2°C Day/night temperature and a 16-h photoperiod) were used for growing upland cotton (Gossypium hirsutum L., till the tree-leaf stage in a greenhouse. For implementation of abiotic stress like drought stress, seedlings were exposed to heavy salt stress 400 mM NaCl and other set was exposed to 20% (w/v) polyethylene glycol-6000 (PEG). After treatment at time intervals of 0 h (control), 3 h, 6 h, and 12 h, leaves were harvested having three biological replicants for each time point. Total RNA was extracted from 100mg of fresh cotton leaves using the TRIzol reagent method (Invitrogen, USA) followed by a manual protocol [19]. RNA was precipitated by using 500 µL of isopropanol after the aqueous phase, and the pellet was air dried in RNase-free water [20]. Nanodrop spectrophotometer was used to assess the purity and concentration of RNA, and integrity was confirmed by 1% agarose gel electrophoresis [21]. Thermoscientific RevertAid First Strand cDNA Synthesis Kit was used to synthesize cDNA and to ensure its reverse transcription; oligo(dT) primers and random hexamers were used as a mix. Diluted cDNA was stored at − 80°C for qPCR analysis.2.12 Gene Expression Analysis (qPCR) of GhSOS1
Gene expression of selective GhSOS1 genes was studied by using SYBR Green ReadyMix - Fast SYBR Green qPCR system. Amplification protocol was followed stepwise denaturation at 95°C for 3 minutes, thermocycle 40 cycles of 95°C followed by annealing at 55oC for 30 sec, and extension [22]. Gene expression levels were calculated by 2-∆∆CT method [23]. Ubiquitin (GhUBQ) gene was used as housekeeping gene as stabilizer. Primers used are given in table (S1). Three biological replicates which were harvested at different time intervals were used for reaction along-with three technical replicates each individually. One-way analysis of variance (ANOVA) was used for assessing statistical significance of expression differences among treatments and time points. After ANOVA Tukey's post-hoc test (p < 0.05) was also performed and finally results were presented in bar graphs as mean ± standard error by using R software.
3. Results
3.1 Selection Pressure Analysis and Phylogenetic Tree
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To observe the evolutionary relationship of Gossypium hirsutum SOS1 gene with other plant species a phylogenetic tree was constructed by neighbor-joining method in MEGA V11.0.13. The results showed different clades and sub-groups. In clade-I marked as red includes majority of GhSOS1 members clustered with other SOS1 members of other plants i.e. rice OsNHX5. Clade-ll colored as blue also elucidated ancestral relationship of several GhSOS1 members with Arabidopsis thaliana. The highest bootstrap value present in Clade-III depicted strong evolutionary relationship of GhSOS1 gene family members with Arabidopsis AtNHX2, AtNHX4 and Vitis vinifera VvNHX2-1. Orange colored clade-IV showed cross species conservation between Gossypium hirsutum and other crops like Glycine max, Brassica napus and few members of Arabidopsis. GhSOS1-9 and GhSOS1-10 were observed in distinct clade-V showed these genes functionally divergent (Fig. 1). Ka/Ks ratio indicated most of the GhSOS1 gene pairs had strong purifying selection pressure, but few had diversifying type of evolution. Most of the GhSOS1 gene members had functional conservation but few had shown diversity under different evolutionary selection pressure. Segmental duplication event were observed in most of GhSOS1 genes while few conserved tandem duplication events (Table 1).Fig. 1
Phylogenetic tree of SOS1 proteins in Gossypium hirsutum and other plant species. The evolutionary history was inferred using the Neighbor-Joining method. The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the p-distance method and are in the units of the number of amino acid differences per site. This analysis involved 31 amino acid sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There were a total of 493 positions in the final dataset. Evolutionary analyses were conducted in MEGA11.
3.2Analysis of conserved domain, motif and gene structure of GhSOS1 Proteins
There was substantial variation in structures of these 15 GhSOS1 proteins. All genes contained Na+/H + and few contained K + flux domain in addition. All genes had different length and number of exons and introns present among entire sequences (Fig. 2). Conserved domain is predicted via NCBI CDD search domain and results revealed that all 15 GhSOS1 proteins possessed Na⁺/H⁺ exchanger domains (Fig. 3). Sequence conservation and similar functionalities among identified genes were determined by analyzing conserved motif in SOS1 gene family of G.hirsutum genes. Among 10 identified conserved motifs of all GhSOS1 genes, Motif 1, 2 and 9 were the most prevalent. The presence and a specific alignment of these motifs across GhSOS1 genes showcased functional conservation (Fig. 4).
Fig. 2
Exon-intron structure of GhSOS1 genes. Number of exons and introns are shown by using Tbtools-ll v.2.019 showing different numbers of axons introns across length of every protein. All containing conserved domains.
Fig. 3
Distribution of conserved motifs in GhSOS1 proteins. Conserved motifs were identified using MEME Suite. Motifs common to all proteins are linked to Na⁺/H⁺ exchanger domains, crucial for salinity response.
Fig. 4
Conserve motif analysis among 15 GhSOS1 proteins. Conserved motif architecture of GhSOS1 proteins in Gossypium hirsutum as identified by MEME suite. A total of 10 conserved motifs (Motif 1–Motif 10) were detected across all GhSOS1 protein sequences, each represented by a distinct colored box. The motifs were distributed in varying patterns, with Motif 1, 2, and 9 being the most prevalent across all family members. These motifs were primarily associated with the Na+/H < + exchanger domain, indicating their potential involvement in ion transport and salt stress response.
3.3 In-silico Physicochemical Properties of GhSOS1 Proteins
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Molecular weight of GhSOS1 proteins ranged between 56.8–90.5 k Da and isoelectric point varied between 5.35–8.99 that induces change in solubility of proteins. Amino acid counts ranges from 520–827 AA. The CDS length ranged from 774–1800 bps (Table 2). Significant variations among CDS length, molecular weight and isoelectric point elucidated diversity among protein orthologs.3.4 Subcellular Localization of GhSOS1 Proteins
Most of the GhSOS1 protein members were localized in plasma membrane (Fig. 5).
Fig. 5
Predicted subcellular localization of GhSOS1 proteins. Heatmap showing predicted subcellular localization of GhSOS1 proteins across different cellular compartments. The majority of proteins are predicted to localize to the plasma membrane, with few showing possible dual localization to vacuoles and cytoplasm. Color intensity represents prediction confidence.
3.5 Chromosomal Distribution and Gene Duplication
Chromosomal mapping via Mapchart tool showed non-random distribution of GhSOS1 genes across the A and D subgenomes, indicating the occurrence of segmental duplication events that contributed to the expansion of this gene family (Fig. 6).
Fig. 6
Chromosomal location of GhSOS1 genes in the G.hirsutum genome. Chromosomal distribution across nine different chromosome is determined by using Mapchart tool.
3.6 Promoter Analysis and Cis-Regulatory Elements
Multiple Cis-regulatory elements such as Auxin responsive elements, MYB-binding site transcription factors and drought resistant elements in promotor region were present in promotor region of GhSOS1 proteins actively work in drought and salinity stress tolerance in cotton (Fig. 7).
Fig. 7
Cis-regulatory elements detected in the promoter regions of GhSOS1 genes revealed multiple stress- and hormone-responsive motifs. These elements suggest transcriptional regulation of GhSOS1 genes under drought and salinity stresses, supporting their functional role in abiotic stress tolerance.
3.7 Protein-Protein Interaction and Functional Implications
PPI analysis revealed relationship of GhSOS1 show high confidence bond with sodium hydrogen exchanger proteins, HKT1 and few kinases (Fig. 8).
Fig. 8
Protein-protein interaction network of GhSOS1 proteins. Predicted using STRING and visualized in Cystoscope, SOS1 interacts with HKT1, NHX1, and CIPK family members.
3.8 Prediction of Phosphorylation Sites and Transmembrane Domain Prediction of GhSOS1
Phosphorylation sites were identified using Net Phos 3.1a which revealed phosphorylation of multiple potential serine (S), threonine (T), and tyrosine (Y) residues. Multiple phosphorylation sites indicate a versatile regulatory mechanism which potentially govern the function of GhSOS1 in presence of external signals especially drought and salinity stress. Phosphorylation sites were not uniformly distributed across protein chain, while threonine and tyrosine showing less higher frequency as compared to serine residue which exhibits highest one (Fig. 9a, b, c). Transmembrane topology of GhSOS1 was predicted using the TMHMM tool, which revealed multiple transmembrane helices distributed across the protein sequence. The analysis confirmed that GhSOS1 functions as an integral membrane protein, with alternating regions predicted to be embedded within the membrane (transmembrane domains), cytoplasmic (inside) or outside (Fig. 10a, b).
Fig. 9
Predicted phosphorylation sites in GhSOS1 proteins predicted using Net Phos 3.1. Most of the proteins have high level of serine threonine content indicating their potential role in phosphorylation.
Fig. 10
Predicted transmembrane domains in GhSOS1-1 protein. TMHMM prediction shows multiple helices indicating GhSOS1- nature as a membrane transporter.
3.9 Protein 3D Structure Prediction of GhSOS1
To dive into structural conformation and functional domains of GhSOS1, SWISS-MODEL was used to predict tertiary structure of this protein. The model revealed its role in ion transport by gaining insights into its structural conformation like defined transmembrane topology, Na⁺/H⁺ antiporters along with multiple α-helices spanning the membrane (Fig. 11). Preserved domains were identified, critical for cation exchange and their alignment with already known SOS1 homologs from other species of plants.
Fig. 11
3D structure prediction of GhSOS1-1 protein. Built via SWISS-MODEL, the structure reveals transmembrane helices and functionally important regions aligned with Na⁺/H⁺ antiporter activity.
3.10 In-Silico Expression Profiling and Candidate Gene Selection
Under salt and drought stress expression of all 15 GhSOS1 genes were profiled by using the RNA-seq data which was retrieved form the cotton transcriptome datasets available publicly. After treatment expression values at 0, 3, 6, 12, and 24 h were normalized and visualized as heatmap. As a result, different members from gene family were highlighted which showed different expression as compared to control. Genes which showed consistent and stress response expression patterns under salt and salinity conditions are GhSOS1-4, GhSOS1-5, GhSOS1-10, GhSOS1-11, and GhSOS1-13. Because of their expression consistency these genes were chosen for experimental validation (Fig. 12).
Fig. 12
Heatmap of 15 GhSOS1 genes showing log₂-normalized RNA-seq expression under salt and drought stress (0–24 h). Genes GhSOS1-4, GhSOS1-5, GhSOS1-10, GhSOS1-11, and GhSOS1-13 displayed consistent stress-responsive patterns selected for qPCR validation.
3.11 Gene Expression Analysis (qPCR) of GhSOS1 Genes
Distinct temporal responses were observed to both 400 mM NaCl and 20% PEG treatments after performing quantitative real-time PCR. Overall, up-regulation at early (3 h) and mid (6 h) time points was observed for GhSOS1-11 and GhSOS1-5 under salt stress. Similarly, a well-maintained increased expression was observed for GhSOS1-11 under drought. However, GhSOS1-13 showcased an average down-regulation at different time intervals and similarly a significant lowered transcript abundance under salinity was observed in GhSOS1-4. Meanwhile, GhSOS1-10 showed no remarkable changes and remained stable throughout treatments and time points. All of the reactions were performed in triplex and finally the data of gene expression was analyzed by using ANOVA and Tukey's post-hoc test (p < 0.05). Bar graphs show mean ± standard error and asterisks (*p < 0.05; **p < 0.01) above each bar display significance and shows treatment- and time-specific differences (Fig. 13a, b ,c ,d ,e).
Fig. 13
Relative expression of selected GhSOS1 genes under drought (20% PEG) and salt (400 mM NaCl) at 0, 3, 6 and 12 h. Expression was normalized to GhUBQ and calculated by the 2^−ΔΔCt method. Bars represent mean ± SE of three biological replicates (each with three technical replicates). Different letters above bars indicate significant differences at p < 0.05 (one-way ANOVA, Tukey’s HSD).
4. Discussion
Close relationship of GhSOS1 members with rice OsNHX5 suggested that both plants have functional conservation [24]. This diversification of SOS1 genes under different phylogenetic groups reflect the adaptive evolution under abiotic stress tolerance [25]. Phylogenetic relationship of SOS1 protein among different plants depicted possible functional evolutionary convergence of GhSOS1 [26]. The SOS1 motifs were found in association with the Na+_H + exchanger domain which plays a pivotal role in transport of sodium ion under drought and salt stress tolerance [27]. Such conserved motif have been reported in other plants for SOS1 genes and a bit of variation may highlight gene-specific roles in response to abiotic stress factors [28]. Na+/H + exchanger which plays a key role in ion-homeostasis[29]. Presence of sodium hydrogen and potassium hydrogen domains confirmed the functional role of SOS1 in G.hirsutum under drought and salinity stress [30]. This superfamily is known for its role to unload sodium ion from xylem and maintain ion balance under drought and salinity. Presence of these domains in GhSOS1 proteins clearly predicted that cotton is structurally equipped with sodium ion regulator protein to enhance the salt tolerance at genetic level [31]. By physiochemical properties hydrophilic nature and intracellular stability of protein is ensured. Low GAVY values in Areca palm determined low hydrophilicity among orthologs [33]. It is consistent with the evidence that SOS1 acts as exchanger protein across membrane to maintain ion balance under drought and salt stress [34]. SOS1 is protein showed involvement in stress-receptor signaling functions [35]. Distribution of same gene family over different set of genome depicts the functional divergence among their functions [36]. Segmental duplication, therefore, plays a key role in driving diversification of gene functions across chromosomes [37]. Presence of stress responsive cis-regulatory elements in promotor region illustrated the importance of insect resistance gene in meta-analysis study of barley [38]. Transcription factors present in promotor region regulates drought responsive genes [39]. Interaction between antiporters increases the significance of these proteins and their role in ion transport [40]. All known proteins in the network interacting with GhSOS1 have active role in stress signaling [41]. Phosphorylation plays a vital role under abiotic conditions in regulation of protein function and signaling pathways [42]. Several phosphorylation sites indicated crucial role in regulation of SOS1 activity and significance in post-translational modifications [43]. GhSOS1 as membrane bounded transporter which plays its presumed role in ion homeostasis and stress tolerance in G.hirsutum is strongly justified by presence of various transmembrane helices [44]. Transmembrane domains are connected because of intracellular and extracellular loops and may play a vital role in regulatory interactions [45]. These results go parallel with the previous findings about SOS1 proteins from other plants illustrating a typical structural and functional role [46]. 3D structure provided a base for molecular docking and functional characterization [47]. This report was aligned with the previously genome-wide surveys which shows that SOS1 transporters are crucial Na⁺/H⁺ antiporters in regulating the cytosolic ion balance [48].
A
These results align with the in-silico predictions by indicating GhSOS1-5 and GhSOS1-11 as the most salt and drought responsive members among GhSOS1 family. Quantitative real time PCR confirmed that under salt and drought stress, GhSOS1-5 and GhSOS1-11 show up-regulation particularly at early (3 h) and mid (6 h) stage, which suggest a signaling role for Na⁺ extrusion and osmotic adjustment. Similarly, in Arabidopsis thaliana and Oryza sativa an early induction of SOS1 orthologs has been observed, where increased SOS1 activity is associated with high salt tolerance by maintaining K⁺/Na⁺ ratio[49, 50]. The stable expression of GhSOS1-10 throughout treatment shows tissue specific and housekeeping functions which are not related to extreme level of stress and a consistency with cotton SOS1 paralogs showing constitutive expression. Interestingly, a remarkable down-regulation was observed in GhSOS1-4 which indicates negative regulation or different response towards osmotic stress. Certain SOS1 members show down-regulation under high salinity like in tomato and barley and it indicates feedback mechanism to tune ion flux for preventing overconsumption of energy[51]. However, GhSOS1-13 displayed mixed regulation, which suggest context dependent expression and influenced by post-translational controls or intervention of other signaling pathways like ABA or calcium signaling [52].5.Conclusion
This study presents the first genome-wide identification and functional characterization of the GhSOS1 gene family in Gossypium hirsutum. The findings highlight the pivotal role of GhSOS1 genes in drought and salt stress tolerance, while also uncovering their structural diversity and evolutionary conservation. Comprehensive in-silico analyses provided strong evidence of their function, including the presence of stress-responsive cis-regulatory elements, conserved Na⁺/H⁺ exchanger domains, and evolutionary relationships with SOS1 genes from other plant species, confirming functional conservation. Motif analysis and protein homology modeling further predicted strong functional similarity of GhSOS1 proteins with sodium/hydrogen exchanger porters. Most GhSOS1 proteins were localized to the plasma membrane, suggesting their direct involvement in ion transport and regulation of the SOS signaling pathway under abiotic stress. Substantially, five candidate genes were shortlisted based on FPKM values retrieved from publicly available RNA-seq data and tested for qPCR different intervals under 400Mm Salt and 20% drought. Subsequently GhSOS1-5 and GhSOS1-11 showed upregulation but GhSOS1-4 was downregulated under both stresses meanwhile GhSOS1-10 showed stability. These expression levels were supported by statistical analysis corroborating with in-silico predictions emphasized GhSOS1-5 and GhSOS1-11 as key regulators for ionic homeostasis. Collectively, these results demonstrate the critical role of GhSOS1 genes in enabling cotton to adapt to drought and salinity stress, and provide a valuable foundation for their targeted utilization in future molecular breeding programs aimed at developing stress-resilient cotton cultivars.
Electronic Supplementary Material
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Table legends
Table 1: Ka/Ks ratio selection pressure analysis among GhSOS1 genes
Table 2: Physiochemical properties of GhSOS1 proteins
Supplementary File
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Table S1: The list of primers used for qPCR analysisA
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Ethics approval
Not applicable.
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Data Availability
The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.
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Author Contribution
Laraib Iqra: Conceptualization, Methodology, Investigation, Writing - original draft. Muhammad Naveed Shahid: Supervision, Resources, Validation. Umar Akram: Data analysis, Software. Usman Arif: Formal analysis, Review & editing. All authors read and approved the final manuscript.
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