Characterization of Strubbelig-Receptor Family (SRF) Related to Drought and Heat Stress Tolerance in Upland Cotton (Gossypium hirsutum L.)

Abstract

Cotton is one of the world’s leading fiber crops, but climate change, drought, heat, and salinity have significantly decreased its production, consequently affecting the textile industries globally. To acclimate to these environmental challenges, a number of gene families involved in various molecular, physiological, and hormonal mechanisms play crucial roles in improving plants response to various abiotic stresses. One such gene family is the GhSRF, a Strubbelig-Receptor family (SRF), and member of the leucine-rich repeat (LRR-V) group. This family encodes leucine-rich repeat transmembrane receptor-like kinases (LRR-RLKs) and have not yet been explored in cotton. Arabidopsis thaliana Strubbelig-Receptor gene sequences were used as queries to identify the homologs in cotton, with subsequent support from the literature and functional prediction through online data. In the current study, a comprehensive genome-wide analysis of cotton was conducted, identifying 22 SRF putative proteins encoded by 22 genes. We performed the detailed analysis of these proteins, including phylogeny, motif and gene structure characterization, promoter analysis, gene mapping on chromosomes, gene duplication events, and chromosomal sub-cellular localization. Expression analysis of putative genes was performed under drought and heat stress conditions using publicly available RNAseq data. The qRT-PCR results showed elevated expression of GhSRF2, GhSRF3, GhSRF4, GhSRF10, and GhSRF22 under drought and heat stress. So, it could be speculated that these genes may play a role in drought and heat tolerance in cotton. These findings could be helpful in cotton breeding programs for the development of climate-resilient cultivars.

1. Introduction

Upland cotton belongs to the Gossypium genus, which is divided into two groups: “Old World cotton” and “New World cotton”. The Old World cotton group includes two diploid cultivated species, G. arboreum and G. herbaceum. The New World cotton group includes tetraploid cultivated species such as G. hirsutum (AD1) and G. barbadense (AD2). Other tetraploid species, which are wild types, contain G. tomentosum (AD3), G. mustelinum s (AD4), G. darwinii (AD5), and G. ekmanianum (AD6). Diploid species are further classified into eight genomes based on geographical distribution [1]. Climate change is the biggest challenge of the 21st century and significantly hampering agricultural yields [2,3]. The unprecedented and abrupt patterns of climatic changes have severely impacted the sustainability of major field crops [4]. Abiotic stresses, intensified by the changing climate, impose severe adverse effects on the natural environment of plant species [5]. The high temperature, one of the key abiotic factors, evidently reduced the production of major crops worldwide due to increases ranging from 2.6 to 4 °C [6,7]. Heat and drought stresses have significantly influenced the seed cotton, whether these factors occurred separately, alternately, or simultaneously [8]. Heat stress accompanied by the droughts may reduce crop yields up to 60% [9].

Plant species research differs considerably in the 21st century as compared with the 20th century. Genome-based studies had been revolutionized after the whole-genome sequencing of Arabidopsis thaliana [10]. Utilizing genome-wide predictions has provided a comprehensive understanding of gene functions across various biological processes [11]. For sustainable cotton production, advanced fast-track breeding tools integrated with genomic selections are indispensable in changing climate [12]. The response of the cotton genome to abiotic stresses is highly complex and genome-wide analyses had assured the identification of multiple genes in cotton [13,14]. To cope with the adverse conditions and to survive in changing climate, genome-wide characterization is crucial, ultimately indicating the role of different genes [15].

The regulation of many aspects of plant life cycle is carried out through receptor-like kinases (RLKs), a distinct group of surface receptors [16]. The Strubbelig-Receptor family (SRF) belongs to the leucine-rich repeat (LRR-V) group, encoding leucine-rich repeat transmembrane receptor-like kinases (LRR-RLKs). These kinases play crucial roles in plant development, including the formation of different plant parts such as carpels, petals, ovules, and root hairs [17,18,19]. According to a study, the role of the SRF4 gene was reported as a positive regulator of leaf size [16]. In plants, apical meristems and organ development patterns were influenced by intercellular signaling. In Arabidopsis, the role of leucine-rich repeat receptor-like Strubbelig proteins had been shown to regulate floral shape, ovule integument morphogenesis, cell division, and root hair patterning, indicating their presence as a typical kinase with Strubbelig members [20,21]. Variations in the Strubbelig Receptor Kinase 3 (SRF3) gene had resulted incompatibilities among various accessions of Arabidopsis thaliana due to different immune responses [22]. Silencing of the SCRAMBLED/STRUBBELIG (SCM/SUB) gene resulted in decreased cell proliferation, compromised cell expansion, and clear changes in auxin distribution patterns [23]. Mutation in the sub-2 allele of the Scrambled/Strubbelig (SCM/SUB) Receptor-like Kinase gene resulted in slow development of leaf blades with irregular shape [24]. A study on root architecture traits resulted in Strubbelig-Receptor Family3 (SRF3/SUB) as a candidate gene in canola [25]. This SUB gene was involved in signal transduction, ultimately regulating the synthesis of the cellulose, which is a central carbohydrate component of the cell wall. The signaling transduction of the SUB gene is further involved in the production of reactive oxygen species (ROS), which further influences stress-related gene activation mechanisms [26].

The SRF family has been studied in model plants and a few studies are reported in other plant species. As members of the leucine-rich repeat (LRR) class involved in membrane signal transduction, this family has been relatively underexplored for its role in conferring abiotic stress tolerance in plants. Therefore, we characterized this family for drought and heat tolerance in upland cotton.

2. Materials and Methods

2.1. Sequence Retrieval of SRF Protein Family Members

SRF genes were downloaded from cotton database CottonFGD [27] and Phytozome13 [28] using the AtSRF (AT1G11130, AT1G53730, AT1G78980, AT2G20850, AT3G13065, AT3G14350, AT4G03390, AT4G22130, AT5G06820) query sequences. A Hidden Markov Model (HMM) confirmed the mined GhSRF proteins in Gossypium hirsutum to avoid the omission of useful genes. The targeted cotton species included G. arboreum, G. raimondii, G. tomentosum, G. barbadense, G. mustelinum, G. hirsutum, and G. darwinii. Arabidopsis thaliana was selected as a model organism. Theobroma cacao species was selected as close relatives of upland cotton. Oryza sativa was selected from monocots to compare the evolutionary process of the SRF gene family. Organisms like Selaginella moellendorffii, Chlamydomonas reinhardtii, and Physcomitrium patens were selected as primitive organisms to compare the evolution of this gene family in cotton and other primitive and model organisms of diverse groups and further verified using SMART [29] and InterProScan5 [30]. The GhSRF genes were further examined thoroughly for biophysical properties using CottonFGD, EnsemblPlants [31], and ExPASy [32]. These properties were molecular protein length (A.A), protein molecular weight (KDa), charge, isoelectric point (pI), and grand average of hydropathy. Sub-cellular localization of the SRF gene was investigated using Cello V.2.0 [33].

2.2. Construction of Phylostratum, and Phylogeny Tree

The NCBI database (https://www.ncbi.nlm.nih.gov/ (accessed on 25 March 2024)) was used to create the phylip file and visualized in iTOL [34] to generate the phyla-stratum tree of all studied species. The ClustalW program in MEGA V.11 [35] was used for multiple sequence alignment (MSA) of the retrieved sequences from all targeted species. The phylogenetic tree using the neighbor-joining method with bootstrap values of 1000 replications was performed.

2.3. Sequence Logos and Conserved Amino Acid Analysis

Protein sequences from upland cotton species including G. hirsutum, G. arboruem, and G. raimondii were used to develop sequence logos using online WebLogo tool [36] and further visualized in TBtools [37]. The same region of all three species was used to perform this analysis.

2.4. Promoter, Protein Motifs, and Gene Structure Analyses

To investigate the cis-element from the promoter region of GhSRF genes, the 2000 bp upstream region of each gene was utilized. PlantCARE [38] was used to conduct promoter analysis, which was visualized in TBtools. Motifs present among the proteins were explored using MEME Suite [39]. All the default parameters were ensured except a few, like (i) occurrence of motif value of 1 for each sequence, (ii) residue range selected as 12–65 s in the motif width, (iii) motif number of 10, (iv) minimum sites in motif of 5. After setting default parameters, the MEME.xml or mast.xml file was retrieved. The further processing of these files to draw a motif structure was performed in TBtools. Gene structure display analysis was performed using Gene Structure Display Server 2.0 [40].

2.5. Sub-Cellular Localization, Gene Mapping on Chromosomes, Gene Duplication, Collinearity and Ratios of Ka/Ks

Sub-cellular localization was predicted using CELLO [41] and WOLFPSORT [42] and a heatmap of the studied genes was created with TBtools. MapChart v2.32 [43] was utilized to find the position of different genes on all the concerned chromosomes of the cotton. The codon alignment and duplication of genes was ensured using the CLUSTAL X 2.0 [44] PAL2NAL program [45]. The CODMEL program and PAML package [46] were the best-suited options used to evaluate the non-synonym (Ka) and synonym (Ks) values, as an indicator of duplication events. TBtools was used for the visualization of the results on the provided values of Ka/Ks. The circus-based tool “Circoletto” [47] was used to draw the sequence similarity and duplications in the Arabidopsis and upland cotton species. The peptide FASTA sequences of G. hirsutum were used as query sequences against peptide sequences of other cotton species. The similarity was provided in the form of a circle on the basis of bit-score values.

2.6. In Silico Expression Analysis of GhSRF Genes

RNA-seq data of GhSRF under drought and high temperature were retrieved from CottonFGD under the project available at the NCBI Geodataset (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA490626 accessed on 4 April 2024). The G. hirsutum line named Texas Marker 1 (TM-1) was used as experimental material. The expression of all genes under drought and heat stress conditions was presented through heat maps.

2.7. Expression Analysis and Validation of Candidate Genes

To perform the gene expression studies, the most tolerant line (VH-402) and most susceptible line (CIM-443) from previous investigations were used along with a standard line TM-1 (Texas Marker-1). The qRT-PCR was performed to investigate the relative expression patterns of selected candidate GhSRFs under drought and high-temperature conditions. Initially, the twenty-day-old seedlings of cotton genotypes were maintained under normal conditions with a temperature range of 30 °C during the day and 24 °C during the night, light intensity of 400–600 µmol m⁻² s⁻¹, and 16/8 h light/dark in a growth chamber. The humidity was kept to 50–60% under normal conditions. For drought stress, (PEG-17% mass fraction) was used to induce the drought effects on twenty-day-old cotton seedlings. For heat stress induction, seedlings were placed in another growth chamber with a day temperature of 45 °C, night temperature of 35 °C, and humidity of 60 to 70%, with the same light intensity of 400–600 µmol m⁻² s⁻¹ and the same photoperiods as the normal conditions. The experiment, designed as a completely randomized design with three replications, was maintained under normal and stress conditions for 72 h, after which leaf samples were collected for RNA extraction. The complementary DNA (cDNA) was synthesized from extracted RNA and Ghactin7 (NCBI-AY305729) was used as internal reference during the expression studies, and the experiment was independently repeated three times. The 2^−ΔΔCT formula was used to determine the relative gene expression [48]. The primers used in this study are presented in (Supplementary Table S1).

3. Results

3.1. Identification and Physiochemical Properties of SRF Genes in Cotton

Thirteen (13) SRF family genes from G. arboreum, eleven from G. raimondii, twenty-two from G. hirsutum, twenty-five from G. barbadense, twelve from G. tomentosum, fifteen from G. mustelinum, sixteen from G. darwinii, eight from A. thaliana. Fifteen from O. sativa, eight from T. cacao, four from S. moellendorffii, one from C. reinhardtii, and six from P. patents gene were retrieved (Supplementary Table S2). Biophysical properties like coordinates, CDS length, strand, protein length (aa), molecular weight (kDa), charge, isoelectric point, and grand average of hydropathy of the studied GhSRF members from the G. hirsutum L. are presented in Supplementary Table S3.

3.2. Sequence Alignment, Phylostratum, and Phylogenetic Tree

The phylostratum analysis revealed the evolutionary relationships among the targeted species at the whole-genome level (Figure 1A). The phylogenetic analysis identified the origins of the SRF genes and provided knowledge of their emergence and diversification. The phylogenetic analysis further elucidated the evolutionary history of SRF proteins in cotton, highlighting their relationships with SRF gene members in other organisms (Figure 1B). A total of 156 SRF family genes from different organisms were used to construct the phylogenetic tree, which divided SRF members into five major groups viz. a, b, c, d, and e. The group (SRF-d) was the largest group, with 39 (39 proteins) members, and all belonged to the Gossypium genus, along with some members from A. thaliana, O. sativa, and T. cacao, indicating a closer evolutionary relationship and shared ancestral traits or recent common ancestry. The group (SRF-d) was followed by the group (SFR-c), exhibiting the same patterns for genes and their evolution. The groups (SRF-a and SRF-e) included 26 (26 proteins) members each, and most of the members were from the Gossypium species. SRF group a, c, d, and e contained most of the members from the Gossypium genus, including G. arboreum, G. raimondii, G. hirsutum, G. barbadense, G. mustelinum, G. darwinii, G. tomentosum, and only a few members from A. thaliana, O. sativa, and T. cacao. The group (SRF-b) represented the members from all cotton species, model plants, monocots, and most primitive organisms. The presence of SRF members in Gossypium species and primitive organisms (SRF-b) indicated the conserved behavior of SRF genes during the evolutionary process. The phylogenetic analysis indicated the evolutionary linkage of SRF genes in different primitive and higher organisms.

3.3. Finding of Conserved Amino Acid Patterns

Sequence logos were generated to analyze the conserved patterns of SRF family proteins in G. hirsutum (AADD genome), G. arboreum (AA), and G. raimondii (DD). Sequence logos indicated that the SRF family is conserved among these species across the N and C termini, such as N (1), F (3), G (5), P (8), L (17), L (22), N (25), L (27), D (33), F (34), L (36), L (39), D (43), L (44), S (45), N (47), L (53), P (54), S (56), and F (57) (Figure 2). The patterns of C and N termini served as indicators to demonstrate the pattern of SRF protein sequence conservation in other organisms.

3.4. Analysis of Cis-Element in Promoter, Motif Finding and Gene Structure

GhSRF contained different cis-elements, such as MeJARE, ABRE, AUX, AUX-R, MYB-DI, GARE, SARE, WUN, BEJARE, and DSRE (Figure 3 and Supplementary Table S4). These elements are important for plant growth, development, and response to biotic and abiotic stresses. Moreover, similar patterns of motif were found in all members of this gene family among all studied genes of the SRF family indicating that this gene family is conserved in cotton (Figure 4 and Supplementary Table S5).

To demonstrate the event of the evolutionary development among all GhSRF genes, we constructed the gene structures. Out of 22 GhSRF genes, 11 members contained only exons. Among all, GhSRF12 was the smallest gene, with only four exons, and GhSRF5 was the largest, with sixteen exons but without any intron (Figure 4).

3.5. Gene Mapping on Chromosomes, Ratio of Ka/Ks, Gene Duplication, and Sub-Cellular Localization

The gene locus linkage confirmed the paralogous gene pairs in G. hirsutum among GhSRF genes (Figure 5). The GhSRF genes were present in five color groups on different chromosomes. The SRF genes like GhSRF1, GhSRF10, GhSRF13, and GhSRF19 belonged to Group-I and are present on chromosomes A-3, A-10, D-2, and D-10. Group-II enclosed the SRF genes, including GhSRF6, GhSRF9, GhSRF18, and GhSRF20, which occupied positions on chromosomes A-6, A-10, D-6, and D-10. Similarly, Group-III involved the SRF genes like GhSRF2, GhSRF4, GhSRF14, and GhSRF16, situated on chromosomes A-5, A-6, D-5, and D-6. Group-IV was the largest group with five members, including GhSRF3, GhSRF5, GhSRF11, GhSRF15, and GhSRF22 located at A-5, A-6, A-10, D-5, and D-6. The last group, Group-V, also had four SRF genes entitled GhSRF7, GhSRF8, GhSRF12, and GhSRF19, positioned at chromosomes A-9, A-9, D-2, and D-9. Among all twenty-two genes, most of these were present on the extreme terminal position and some were present near to the terminal position on both ends and only were present at the central position of the chromosomes. Most of the genes were localized on the terminals of the chromosome, with chromosome A05 and D05 having the maximum number of genes. Chromosomes A-6 and A-8 contained the largest length but the lowest number of genes on them. A set of 10 gene pairs in G. hirsutum were predicted through the analysis, and all experienced segmental duplication events. To further estimate the magnitude and type of selection as per Darwinian theory, we generated Ka/Ks ratios through the events of duplication in genes. It was observed that all 10 duplicated gene pairs showed Ka/Ks values < 1, indicating that the patterns of negative selection ultimately led to purifying selection (Supplementary Table S6). The peptide similarity analysis was performed in Circoletto by providing the peptide sequence of G. hirsutum as query; all other cotton species sequences were set as targets to see the duplications. The sequences presented in red showed more than 75 percent similarity with each other. The single blue color of GhSRF12 in G. hirsutum and G. barbadense confirmed its singular presence with no duplication, as predicted by Ka and Ks ratios (Figure 6A). The sub-cellular location results indicated that most of the SRF genes are present in the plasma membrane (Figure 6B and Supplementary Table S7).

3.6. Gene Expression Analysis of GhSRF Genes Under Drought and Heat Stress Conditions

The FPKM values of cotton accession (TM-1) for GhSRF genes were retrieved from the CottonFGD database and expression graphs of cotton SRF genes were developed under abiotic stress conditions of drought and high-temperature stress (Figure 7 and Supplementary Table S8). Extracted data showed that GhSRF3, GhSRF4, GhSRF10, and GhSRF22 were highly expressed under drought stress conditions. The expression of GhSRF2, GhSRF3, GhSRF4, GhSRF10, and GhSRF22 was high under high-temperature conditions. In general, GhSRF2, GhSRF3, GhSRF4, and GhSRF22 were highly expressed in different conditions of drought and heat. Based on FPKM expressions, highly expressed genes under abiotic stress conditions were selected to confirm the results through qRT-PCR. Relative gene expression results showed that GhSRF2, GhSRF4, and GhSRF10 have more transcript abundance of these genes under drought conditions, while GhSRF3 and GHSRF22 showed better transcript production under heat conditions (Figure 8). Further, the production of more transcripts in cotton accession VH-402 as compared to CIM-443 confirmed the higher tolerance in former accession under drought and heat conditions as compared to the latter.

4. Discussion

Genome-wide approaches has provided comprehensive understanding of the roles of gene families in various biological processes [11]. Genome-wide identification has facilitated the rapid identification of hundreds of novel genes in cotton research, accelerating the gene detection process [13,14]. The advancement in sequencing technologies, particularly in plant species such as “Gossypium” species, made the gene identification process much easier as compared to earlier times [49]. Members of the SRF protein family belong to the leucine-rich repeat (LRR-V) group, contributing in adaptation to environmental stresses. Several investigations have explored the role of SRF genes in various plant species [16,18,19,20,21,22]. In the current work, SRF genes in cotton (G. hirsutum) were explored for taxonomical classification, sequence similarities and duplication, gene mapping on chromosomes, motif, promoter, and expression analysis [50,51].

The SRF family is an expanded gene family across the plant kingdom due to their prints in multiple plant groups, including monocots (O. sativa), dicots (A. thaliana and T. cacao), tracheophytes (S. moellendorffii), embryophytes (P. patens), and chlorophytes (C. reinhardtii). In G. hirsutum, we identified 22 GhSRF genes, most of which present in pairs, except GhSRF7 and GhSRF12, identified as singletons without any pairing. The diversity of these SRF genes inferred their involvement in environmental and physiological adaptation in G. hirsutum. Gene duplication can lead to functional diversification, where duplicated genes evolve new functions (neofunctionalization) or subdivide the original function (subfunctionalization). Additionally, gene structure variation can result in alternative splicing, producing different protein isoforms from the same gene [52].

Phylogenetic analysis is usually performed to explore the lineage history of species [53]. We aligned the conserved peptide sequences of SRF’s in multiples species ranging from primitive to higher organisms and identified the evolutionary connections of SRF genes among these species [54]. The phylogenetic analysis divided the GhSRF family members into five different groups. Group-b included the genes from all species including mosses, ferns, model organisms, monocots, and dicots, indicating the origin of SRF genes earlier than divergence of monocots and eudicots. Group b represented the most primitive members of SRF genes, widely diversified among other species. In contrast, Groups a, c, d, and e contained a smaller number of GhSRF genes from ancient plants as compared to other species. The presence of SRF genes in Chlamydomonas reinhardtii (chlorophyte) and Selaginella moellendorffii (lycophyte) clearly indicated their lineage with the oldest known organisms. The phylostratum analyses suggested that SRF genes originated from early land plants, with potential orthologs of SRF genes existing throughout the kingdom plantae. Both G. hirsutum and G. barbadense relatively contained a higher number of SRF genes as compared to other studied plant species. Our results of sequence logos of cotton (G. hirsutum) with its progenitor species (G. arboreum and G. raimondii) displayed the conserved behavior of SRF genes on C and N terminals. Mutations during the evolution process of genes and gene families can lead to function-based divergence, with genes being added or deleted over time [55]. The gene length and structure varied due to duplication or deletion events, being observed across 22 GhSRF genes, depicting the role of variation in crop breeding [56,57]. Motif analysis provided the list of motifs present in each gene, highlighting the consistent patterns of gene functions within the same subfamily or family [58]. Chromosomal localization analysis is a fundamental approach to predict the position of genes on a chromosome. Further, it involves mapping identified genes to their respective chromosomes. It provides the crucial insight on duplication events, genome evolution, and complex regulation mechanisms [59].

The Ka/Ks ratios depicted the minor segmental duplication in SRF genes, indicating minor functional divergence. Generally, the results of the Ka/Ks ratio are interpreted in three ways: values of Ka/Ks is < 1 indicate purifying selection, Ka/Ks > 1 indicates positive selection, and Ka/Ks = 1 indicates neutral evolution [60]. GhSRF genes were found to be evenly distributed on both of the cotton genomes (A and D). Gene expression variation on different time scales provides essential understandings of gene functions and promoter diversity [61]. Cis-acting regulatory elements within the promoter are crucial for regulating the gene function under severe environments [62]. Various stress-related cis-regulatory elements were identified in SRF genes, including MeJARE, ABRE, MBS, AUX, SARE, GARE, WUN, BeJARE, and MYB-DI, with varying numbers across different SRF genes. Several studies have highlighted the significance of these regulatory elements in growth, development, and stress resilience [63]. The online expression was used to elucidate the role and transcriptional variation of these genes, which was further validated by qRT-PCR analysis. The candidate genes showed a clear difference in transcript abundance under different conditions. GhSRF2, GhSRF3, GhSRF4, GhSRF10, and GhSRF22 were selected as SRF candidate genes with a potential role under drought and heat stress conditions. The GhSRF2, GhSRF3, and GhSRF22 were expressed more under heat conditions, while GhSRF4 and GhSRF10 were expressed under drought conditions.

Future research should focus on knockout or overexpression studies of GhSRF genes to validate their roles in drought and heat tolerance mechanisms. Investigating interactions with other regulatory proteins and identifying downstream targets will enhance our understanding of their signaling pathways. Utilizing marker-assisted selection and gene editing technologies like CRISPR/Cas9 to develop cotton varieties with enhanced abiotic stress tolerance holds significant potential for improving yield and resilience. Field trials and breeding programs will be essential to evaluate the performance of these genetically modified plants under real-world conditions. This study lays a robust foundation for future research on the Strubbelig gene family, paving the way for innovative strategies to enhance cotton’s adaptability to environmental challenges.

5. Conclusions

In conclusion, this study has significantly advanced our understanding of the Strubbelig gene family (GhSRF) in cotton by elucidating their roles in drought and heat tolerance through comprehensive sequence and expression analyses. The identification of conserved motifs and regulatory elements, coupled with insights from gene duplication and evolutionary pressure analyses, highlights the genetic robustness and potential functional importance of GhSRF genes. Our findings suggest that GhSRF genes are integral to stress response mechanisms, providing a valuable resource for future genetic and biotechnological interventions. This work not only enriches the existing knowledge base but also offers practical implications for breeding programs aimed at developing more resilient cotton varieties.