Genome-Wide Characterization and Expression Profiling of the LSD Gene Family in Cotton (Gossypium hirsutum) Under Drought and Salt Stress

Genome-Wide Characterization and Expression Profiling of the LSD Gene Family in Cotton (Gossypium hirsutum) Under Drought and Salt Stress | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Characterization and Expression Profiling of the LSD Gene Family in Cotton (Gossypium hirsutum) Under Drought and Salt Stress Ayşe Arucan, Cüneyt Uçarlı This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9441383/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Cotton ( Gossypium hirsutum) is a major natural fiber crop whose productivity is severely constrained by drought and salinity stress. Understanding the molecular mechanisms underlying stress responses is critical for developing stress-resilient cultivars. The Lesion Simulating Disease (LSD) transcription factor family plays a crucial role in regulating programmed cell death and stress signaling pathways in plants. In this study, a genome-wide analysis identified eleven GhLSD genes in G. hirsutum , which were unevenly distributed across chromosomes and contained conserved LSD-type zinc finger domains, suggesting their involvement in stress-responsive regulatory networks. Expression profiling of six selected GhLSD genes under drought and salinity conditions revealed complex, tissue-specific, and time-dependent expression patterns. Under drought stress, GhLSD1, GhLSD4 , and GhLSD6 were significantly upregulated, indicating their potential roles as positive regulators of stress tolerance, whereas GhLSD2 was consistently downregulated, suggesting a possible negative regulatory function. Similarly, under salinity stress, GhLSD4, GhLSD5 , and GhLSD6 exhibited strong and sustained induction, highlighting their importance in long-term and tissue-specific salt stress responses. In contrast, the persistent downregulation of GhLSD2 further supports its potential role as a negative regulator in salinity stress adaptation. Overall, these findings demonstrate that the GhLSD gene family functions in a coordinated and dynamic manner in response to abiotic stress, with GhLSD4, GhLSD5 , and GhLSD6 emerging as promising candidate genes for improving drought and salinity tolerance in cotton through molecular breeding approaches. Lesion Simulating Disease abiotic stress salinity transcription factors stress tolerance zinc finger proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Introduction Cotton ( Gossypium spp .) is an economically important crop that supplies nearly 35% of the world’s natural fiber and also serves as a source of vegetable oil and bioenergy (Abdelraheem et al. 2019 ; Liu et al. 2021 ). In the 2024–2025 growing season, global cotton production reached around 121.07 million 480-lb bales, with China, India, Brazil, the United States, Australia, Pakistan, and Türkiye as the main producers (USDA, 2025 ). Türkiye ranks seventh globally, contributing about 3% of world production—mainly from the Southeastern Anatolia and Aegean regions—with an average yield of 1,799 kg/ha between 2020/2021 and 2024/2025 (Najib et al. 2022 ; USDA, 2025 ). Cotton plays an important role in studying polyploid evolution, fiber development, and stress adaptation (Chen et al. 2020 ). Gossypium represents a genus of the Malvaceae family encompesses about 50 species—45 diploid and 5 allotetraploid —with G. hirsutum (AD₁) and G. barbadense (AD₂) arising from hybridization between G. arboreum (A₂) and G. raimondii (D₅) followed by chromosome doubling (Li et al. 2014 ; Chen et al. 2020 ). G. hirsutum accounts for nearly 95% of global production, while G. arboreum and G. raimondii remain significant genetic resources for plant toleranse in drought and salinity and genomic researches ( Zhu et al. 2013; Yan et al. 2016 ; Shi et al. 2019 ). Global climate change has intensified abiotic stresses such as drought and salinity, which negatively affect fiber quality, plant development, and overall cotton productivity (Khan et al. 2025 ). Although cotton exhibits moderate salt tolerance (≈ 7.7 dS m⁻¹), severe stress can reduce yield by up to 67%, varying with stress severity and genotype (Maas and Hoffman 1977 ; Abdelraheem et al. 2021 ). These conditions impair photosynthesis, root development, and nutrient uptake, resulting in major agronomic and economic losses (Bano et al. 2022 ). Salinity is a critical condition that disrupts plant development by inducing osmotic stress, ionic toxicity, and nutrient imbalance (Ahmad et al. 2022 ). In cotton, excessive Na⁺ and Cl⁻ accumulation impairs water uptake, damages cellular membranes, and inhibits enzymatic activity, leading to oxidative stress and alleviate fiber characteristics (Lu et al. 2023 ; Chaudhary et al. 2024 ). Plants counteract these effects through ion homeostasis, antioxidant activation, and transcriptional regulation of genes associated with stress tolerance (Uçarlı 2020 ; Zhang et al. 2021 ). Drought is a major environmental problem which constrain growth of cotton seeds and fiber quality while promoting soil degradation and erosion (Xiao et al. 2020 ). Plants employ avoidance, tolerance, escape, and recovery strategies to withstand drought, involving mechanisms such as leaf rolling, stomatal closure, osmotic setting, and metabolic stabilization (Kneebone et al. 1992 ; Uçarlı 2025 ). Root system architecture is a key determinant of water uptake and signaling under deficit conditions (Zhang et al. 2024). At the molecular level, plant adaptation to water scarcity and salinity are governed by complex networks of transcription factors (TFs) that bind cis-regulatory elements to control gene expression (Latchman, 1997 ; Wärnmark et al. 2003 ). These TFs contain distinct DNA-binding and activation domains that regulate transcriptional activity and integrate environmental cues into adaptive responses (Nakano et al. 2006 ; Hrmova et al. 2021; Dhatterwal et al. 2024 ). In adverse condition regulation and secondary metabolism, key TF families are bZIP, MYB, WRKY, NAC, AP2/ERF, and MADS-box (Dubos et al. 2010 ; Rushton et al. 2010 ; Puranik et al. 2012; Licausi et al. 2013; Yu et al. 2020 ; Chowdhary et al. 2023 ; Gao & Dubos, 2024 ). The Lesion Simulating Disease ( LSD ) gene encodes a C2C2-type zinc finger protein that functions as a central regulator of programmed cell death (PCD) and stress acclimation (Dietrich et al. 1997 ; Takatsuji, 1998 ; Coll et al. 2011 ). In Arabidopsis thaliana , LSD negatively regulates PCD, with lsd1 mutants exhibiting runaway cell death and hypersensitivity to oxidative and pathogen-induced stresses due to disrupted ROS homeostasis (Bernacki et al. 2019 ). Functioning within the LSD–EDS1–PAD4 regulatory hub, LSD integrates redox and hormonal signaling to coordinate defense activation and stress adaptation under environmental extremes (Mühlenbock et al. 2008; Wituszyńska et al. 2013). However, its molecular functions in cotton remain largely unexplored, and elucidating the evolution and expression of LSD1 homologs in Gossypium could advance understanding of stress tolerance and facilitate breeding of resilient cultivars (Dietrich et al. 1997 ; Bernacki et al. 2019 ). In this study, we aimed to characterize and identify the structural features and conserved domains of GhLSDs through in silico analysis; also analyze the expression profile of the LSD gene under arid conditions and salinity in upland cotton ( Gossypium hirsutum ). Overall, this study provides new insights into the structural and functional attributes of GhLSDs and its potential role in cotton’s adaptive response to environmental stresses including salinity and drought. Materials and Methods In Silico Analysis of LSD Genes and Proteins in Cotton ( Gossypium hirsutum ) Members of the LSD (“Lesion Simulating Disease 1”) gene family and their corresponding protein sequences were identified in the Gossypium hirsutum genome using the CottonGen database ( https://www.cottongen.org/ ), NCBI ( https://www.ncbi.nlm.nih.gov/ ), and the Plant Transcription Factor Database (PlantTFDB v4.0 and v5.0; https://planttfdb.gao-lab.org/ ). LSD protein sequences from A. thaliana , G. arboreum , G. raimondii , and G. hirsutum were retrieved and confirmed via BLASTP and BLASTN searches ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). Gene characteristics, including nucleotide and protein length, chromosomal location, and exon-intron organization, were obtained from NCBI. The physicochemical properties of GhLSD proteins—molecular weight, isoelectric point (pI), hydropathicity (GRAVY), and sequence length were computed using ProtParam ( https://web.expasy.org/protparam/ ), while subcellular localization was estimated through DeepLoc-2.0 ( https://services.healthtech.dtu.dk/services/DeepLoc-2.0/ ). Conserved domains were validated with CDD ( https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml ) and SMART ( http://smart.embl.de/ ), and secondary structure components (α-helix, β-turn, extended strand, random coil) were analyzed with PSIPRED ( https://bioinf.cs.ucl.ac.uk/psipred/ ). Tertiary structures of GhLSD proteins were modeled using AlphaFold3 (Abramson et al. 2024 ). The structural organization of the genes was illustrated via GSDS 2.0 ( http://gsds.cbi.pku.edu.cn/ ), and conserved motifs were identified using MEME ( https://meme-suite.org/tools/meme ) with maximum of 10 motifs was allowed, with motif lengths ranging from 6 to 50 amino acids. Cis-acting regulatory elements within 2 kb upstream promoter regions were extracted from NCBI and analyzed using the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Multiple sequence alignments were performed with ClustalW, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replicates in MEGA-X ( https://www.megasoftware.net/ ), then visualized and annotated with iTOL ( https://itol.embl.de/ ). Plant Materials, Growth Conditions, and Treatments Cotton ( Gossypium hirsutum cv. Harem 2) seeds were obtained from the Republic of Türkiye Ministry of Agriculture and Forestry, Eastern Mediterranean Agricultural Research Institute. Seeds were surface-sterilized with 70% ethanol for 1 min, followed by 10% H₂O₂ for 2 h, and rinsed three times with sterile distilled water. The seed coats were carefully removed, and embryos were transferred onto Murashige and Skoog (MS) medium (Murashige & Skoog, 1962 ). Germination was carried out at 24°C under controlled growth chamber conditions for 3–4 days. For salt stress, 10-day-old seedlings were transferred to MS medium supplemented with 200 mM NaCl and sampled after 0, 2, 4, 8, 24, and 48 h. For drought stress, seedlings were transferred to glass jars containing MS medium infused with 25% polyethylene glycol (PEG6000) following the PEG overlay method described by (Pawar & Veena, 2020 ), and exposed to stress for the same time intervals. Leaf and root tissues were collected at each time point, immediately frozen in liquid nitrogen, and stored at − 80°C until RNA extraction and gene expression analysis. Total RNA Isolation and cDNA Synthesis Total RNA was isolated from the 10 day-old leaf and root samples using the FastPure Universal Plant Total RNA isolation kit (Vazyme- Nanjing, China), which is suitable for plant species rich in polysaccharides or polyphenols. The concentration and purity of total RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The A260/A280 ratio between 1.8 and 2.1 indicated acceptable purity, while an A260/A230 ratio above 1.8 confirmed the absence of major contaminants. RNA integrity was verified by 1% agarose gel electrophoresis, where distinct 28S and 18S rRNA bands indicated high-quality, intact RNA suitable for downstream applications. First-strand complementary DNA (cDNA) was synthesized from 1 µg of total RNA using the qScript cDNA Synthesis Kit (Quanta Biosciences, Germany) according to the manufacturer’s protocol. The synthesized cDNA was diluted 1:10 with nuclease-free water and stored at − 20°C until quantitative PCR (qRT-PCR) analysis. Quantitative Real-Time PCR (qRT-PCR) Analysis Gene-specific primers for six candidate GhLSD genes ( GhLSD1–GhLSD6 ) were designed using the Primer3 software ( https://primer3.ut.ee/ ). The ubq7 gene was used as an internal reference for normalization (Wang et al., 2013 ). qRT-PCR reactions were performed in 20 µL volumes containing 1 µL of cDNA (100 ng), 0.5 µL each of forward and reverse primers ( 10 µM), 10 µL of 2× SYBR Green Master Mix, and nuclease-free water to volume. Amplification was conducted on an A.B.T. (Atlas Biotechnology) real-time PCR system using the following thermal profile: 95°C for 5 min, followed by 40 cycles of 95°C for 15 s, 57°C for 20 s, and 72°C for 20 s , with a final melting curve analysis to verify amplification specificity. Each reaction included three biological replicates and two technical replicates. Relative gene expression was assessed following the 2⁻ ΔΔCT method (Livak and Schmittgen, 2001 ). Statistical Analyses The qRT-PCR data were analyzed using one-way ANOVA with the least significant difference Tukey HSD test via the SPSS21 statistical program (IBM). All analyses were performed using three independent biological replicates, each analyzed with two technical replicates. Results Identification of GhLSD Family Members Eleven LSD genes were identified in the tetraploid cotton species G. hirsutum genome, and these genes were distributed across the chromosomes of G. hirsutum (2n = 4x = 52). Eleven GhLSD genes were located on 5 out of the 13 chromosomes (chromosomes 5, 6, 8, 10, and 12) in both the A and D subgenomes (Table 1 ). In GhLSD genes, the coding sequence (CDS) length varied from 294 bp to 513 bp, while the number of exons ranged from four to eight. Notably, a correlation between exon number and CDS length was observed. Among the GhLSD genes, five are oriented on the positive strand, whereas six are positioned on the negative strand. Table 1 Summary of the GhLSD genes identified in the cotton ( Gossypium hirsutum L.) genome. Gene Name Accession No Chromosome Gene Location (5'-3') Strand CDS (bp) Exon No GhLSD1 XM_016842666.2 A05 NC_053428.1_16065629–16067143 Plus 294 4 GhLSD2 XM_016896396.2 A06 NC_053429.1_ 128108232–128112426 Minus 510 8 GhLSD3 XM_016848346.2 A08 NC_053431.1_ 112468123–112470334 Minus 444 7 GhLSD4 XM_016855484.2 A10 NC_053433.1_ 426067–430393 Plus 513 8 GhLSD5 XM_041083378.1 A12 NC_053435.1_ 102589789–102592455 Minus 441 7 GhLSD6 XM_041093586.1 D05 NC_053441.1_ 9470745–9476686 Plus 504 8 GhLSD7 XM_016832533.2 D05 NC_053441.1_ 14672500–14674115 Plus 294 4 GhLSD8 XM_041096384.1 D06 NC_053442.1_ 66551319–66555576 Minus 543 8 GhLSD9 XM_016865838 D08 NC_053444.1_ 56483411–56485604 Minus 444 6 GhLSD10 XM_016862004.2 D10 NC_053446.1_ 415235–419837 Plus 513 8 GhLSD11 XM_016881184 D12 NC_053448.1 _ 56748242–56750893 Minus 441 6 Chromosomal localization and Gene structure Upland cotton Gossypium hirsutum is allotetraploid (2n = 4X = 52) with subgenomes A and D. All of the A chromosomes are relatively larger than homoeologous D chromosomes. Eleven GhLSD genes were found to located on terminal parts of ten chromosomes among 26 chromosomes of G. hirsutum (Fig. 1 ). While 5 GhLSD genes were located on chromosomes A (A5, A6, A8, A10, A12), 6 GhLSD genes were located on D chromosomes (D5, D6, D8, D10, D12) Among these chromosomes, ChrD5 harbored two GhLSD genes (GhLSD6 and GhLSD7). In order to determine the differences in gene structure, CDS and intron structures of 11 GhLSD genes were compared and illustrated. The intron count of GhLSD genes varied from 3 to 7 (Fig. 2 ). Characterization of cotton LSD1 proteins The characterization of cotton LSD transcription factors was performed using different bioinformatics tools. The findings obtained are shown in Table 3.2. It was found that the length of the proteins ranged from 97 to 180 amino acids. GHLSD1 is the lightest LSD protein with a weight of 10.6 kDa, while the heaviest is GhLSD8 with a weight of 18.7 kDa. It was found that the theoretical isoelectric points (pI) are above 7, and GhLSD proteins have a basic structure. According to GRAVY values, GhLSD1 and GhLSD7 proteins were found to have hydrophilic properties, while other GhLSD proteins were found to have hydrophobic structure (GRAVY > 0). The aliphatic index (AI) values of cotton LSD proteins range from 68.25 (GhLS1) to 82.28 (GhLSD8), which indicates that LSD proteins have a thermostable structure. Subcellular localization analysis predicted that GhLSD proteins are localized in both the nucleus and the cytoplasm. According to the Instability Index (II) analysis results, it was determined that LSD proteins were generally stable in structure, while GhLSD1, GhLSD6 and GhLSD7 proteins (II values > 40) were determined to be unstable in structure (Table 2 ). Table 2 Characterization of Cotton LSD1 proteins Protein Name Length (aa*) MW * (Da*) pI* GRAVY* AI* II* Charge KSP* Subcellular Localization GhLSD1 97 10608 7.53 -0.161 68.25 61.94 0.7 11 Cyt * , Nuc * GhLSD2 169 17665 9.01 0.192 80.12 35.68 8.3 20 Cyt, Nuc GhLSD3 147 15158 8.21 0.287 74.35 38.66 2.3 12 Cyt, Nuc GhLSD4 170 17929 8.77 0.108 76.24 32.06 6.3 15 Cyt, Nuc GhLSD5 146 15074 8.21 0.359 79.52 39.87 2.3 10 Cyt, Nuc GhLSD6 167 17666 8.62 0.141 75.75 41.21 5.3 15 Cyt, Nuc GhLSD7 97 10618 7.53 -0.169 68.25 64.48 0.7 10 Cyt, Nuc GhLSD8 180 18776 8.86 0.262 82.28 38.09 7.4 20 Cyt, Nuc GhLSD9 147 15095 8.21 0.282 75.71 38.08 2.3 12 Cyt, Nuc GhLSD10 170 178881 8.77 0.116 77.94 31.62 6.3 15 Cyt, Nuc GhLSD11 146 15074 8.21 0.359 79.52 39.87 2.3 10 Cyt, Nuc * Molecular Weight (MW), Dalton (Da), Kinase Specific Phosphorylation (KSP) regions, Isoelectric point (pI), Grand Average f Hydropathy (GRAVY), Aliphatic Index (AI), Instability Index (II) and the protein charge was estimated according to the amino acid composition. Cyt (Cytoplasm), Nuc (Nucleus). Domain and motif analysis Examination of protein domain structures showed that GhLSD proteins contains two or three conserved LSD-type Zinc Finger (ZF) domains which is defined by CxxCxRxxLMYxxGASxVxCxxC. While nine of eleven GhLSD proteins contain three LSD-type ZF domains, only GhLSD1 and GhLSD7 proteins contain two LSD-type ZF domains (Fig. 3 ). The secondary structure predicted for the GhLSD proteins was displayed above the multiple sequence alignments, revealing the presence of one or two α-helices in GhLSD3, GhLSD5, GhLSD8, GhLSD9, and GhLSD11, as well as a variable number of β-strands across all GhLSD proteins. In upland cotton, the conservation analysis of LSD-type ZF domains demonstrated strong sequence conservation across all GhLSD proteins. In particular, cysteine residues at positions 1, 4, 19, and 22 were highly conserved, while leucine (position 8), tyrosine (position 10), and glycine (position 11) were also consistently conserved (Fig. 4 ). To explore the diversity of GhLSD proteins, conserved motif analysis was conducted using the MEME online server. Ten distinct motifs (motif 1–10) were identified. Among them, motif 1 and motif 2, corresponding to LSD-type ZF domains, were present in nearly all GhLSD genes,highlighting their conserved functional significance within the gene family (Fig. 5 ). Cis-Regulatory Element Analysis The promoter regions (2 kb upstream) of GhLSD genes were screened for cis-acting regulatory elements using the PlantCARE database. The results revealed that GhLSD promoters harbor diverse cis-elements associated with plant growth and development, hormone signaling, stress responses, and light regulation at the transcriptional level (Fig. 6 ). Development-related motifs included AC-II (in GhLSD4 and GhLSD10 ), Circadian (in GhLSD8 ), the GCN4 motif (in GhLSD1, GhLSD2, GhLSD3 , and GhLSD8 ), and RY elements (in GhLSD6 and GhLSD7 ). Among hormone-responsive elements, the ERE motif (ethylene-responsive) was notably enriched, appearing four times in the GhLSD4 promoter, whereas the O2-site (involved in zein metabolism) was detected only in GhLSD1 . Numerous stress-responsive elements were identified, including MYB binding sites (present in all GhLSD genes except GhLSD11 ) and MYC binding sites, which were detected in varying copy numbers across all members. W-box motifs, recognized by WRKY transcription factors, were restricted to GhLSD2, GhLSD5, GhLSD8, GhLSD10, and GhLSD11, while the WUN motif (associated with wound responses) was found only in GhLSD1, GhLSD2, and GhLSD8. Light-responsive elements were also abundant, with Box 4 and G-box being the most prominent (Fig. 6 ). Multiple Sequence Alignment and Phylogenetic Analysis To investigate the evolutionary relationships and identify paralogous gene pairs among the eleven Gossypium hirsutum GhLSD genes, multiple sequence alignment of their CDS sequences was performed using CLUSTALW. Pairwise sequence identity analysis of the eleven GhLSD genes revealed five nearly identical paralogous pairs (GhLSD1–GhLSD7, GhLSD2–GhLSD8, GhLSD3–GhLSD9, GhLSD4–GhLSD10, and GhLSD5–GhLSD11) with > 97% identity, indicating recent duplication events. Other gene pairs exhibited moderate (80–85%) or low (37–46%) identity, reflecting more divergent evolutionary relationships within the gene family. Phylogenetic analysis revealed that the eleven LSD transcription factors of upland cotton ( Gossypium hirsutum ) could be classified into three groups (Fig. 7 ). Group I comprised GhLSD1, GhLSD3, GhLSD5, GhLSD7, GhLSD9, and GhLSD11; Group II included GhLSD2 and GhLSD8; while Group III consisted of GhLSD4, GhLSD6, and GhLSD10. To further elucidate the evolutionary history of the LSD gene family in upland cotton, multiple sequence alignments were performed using ClustalW with LSD proteins from G. hirsutum (11 GhLSDs), Arabidopsis thaliana (6 AtLSDs), G. raimondii (8 GrLSDs), and G. arboreum (6 GaLSDs). Phylogenetic analysis of LSD proteins from Gossypium spp and A. thaliana revealed that the LSD gene family clustered into five distinct clades (Clade I–V) (Fig. 7 ). Most clades contained members from at three species, while Clade II contained only LSD proteins of A. thalina . In most clades, GhLSDs were grouped closely with homologs from G. raimondii and G. arboreum , suggesting inheritance from both A- and D-genome progenitors. All GhLSD members of Group I were clustered within Clade V. Within Group III, GhLSD4 and GhLSD10 were clustered in Clade III, while the remaining member GhLSD6 were positioned in Clade I with homologs from G. raimondii and G. Arboreum (Fig. 7 zein metabolism). Protein Structure Analysis AlphaFold3 was used to predict the tertiary structures 11 GhLSD proteins. In all GhLSD proteins, random coil accounted for the largest proportion ranged from 58.50% to 78.35, followed by extended strand (22.65–36.69%). α-Helix structures which are detected on GhLSD3, GhLSD5, GhLSD8, GhLSD9, and GhLSD11 at the low proportines (2.23–6.80%). (Table 3 ). No Beta turn was found in GhLSD proteins. GhLSD proteins have a secondary structure largely composed of random coils and β-strands, with limited α-helices and no β-turns. These properties, along with their flexible, stable structures, suggest that they are proteins that confer stability (Fig. 8 ). Table 3 The secondary structures of GhLSD proteins. Protein α-Helix (%) Extended Strand (%) Beta Turn (%) Random Coil (%) GhLSD1 0 22.68 0 77.32 GhLSD2 0 36.69 0 63.31 GhLSD3 5.44 34.01 0 60.54 GhLSD4 0 31.18 0 68.82 GhLSD5 6.16 33.56 0 60.28 GhLSD6 0 29.94 0 70.06 GhLSD7 0 21.65 0 78.35 GhLSD8 2.23 33.52 0 64.25 GhLSD9 6.80 34.70 0 58.50 GhLSD10 0 31.18 0 68.82 GhLSD11 6.16 33.56 0 60.27 Gene Expression Pattern Analysis Under Abiotic Stress The GhLSD genes in Leaves and roots under drought and salt stresses in the Gossypium hirsutum genotype, the expression patterns of 6 GhLSD genes—including GhLSD1, GhLSD2, GhLSD3, GhLSD4, GhLSD5 and GhLSD6 , were examined using qRT-PCR analysis. Gene Expression Analysis of LSD genes Under Drought Stress The expression profiling of GhLSD genes revealed diverse and tissue-specific regulatory responses to drought stress. The expression levels of GhLSD1 showed significant upregulation, with peak expression at 8 h (2.24-fold in leaf, while the expression was decreased to 4.76-fold in leaf tissues at 2h. GhLSD2 was moderately upregulated in leaves at 8 h (1.48-fold) and 24 h (1.49-fold), but showed only mild induction at 4 h (0.89-fold) and 48 h (1.17-fold). In contrast, it was moderately downregulated in 5leaves at 2 h (1.72-fold). GhLSD3 exhibited strong upregulation (6.30-fold) in leaves at 8 h under drought stress, followed by moderate induction (2.39-fold) at 24 h. GhLSD4 was markedly upregulated (154.04-fold) in leaves at 8 h of drought exposure. GhLSD5 displayed strong upregulation (3.34-fold) in leaves at 8 h, but was slightly downregulated (1.53-fold) at 4 h under drought stress. GhLSD6 was highly upregulated (4.12-fold and 4.88-fold) in cotton leaves at 8 h and 24h following drought exposure, but displayed only a moderate induction (2.75-fold) under drought stress at 48h respectively (Fig. 9 ). The expression levels of GhLSD1 showed significant upregulation, with peak expression at 48 h (40.44-fold) in root, while the expression was decreased to 13.33-fold in root tissues at 2h. GhLSD2 was moderately downregulated in roots at 24 h (1.36-fold). Stronger downregulation was observed in roots at 8 h (2-fold) and 48 h (2.38-fold). In roots, GhLSD3 was strongly downregulated (9.09-fold and 10-fold) at 2 h and 24 h, respectively. GhLSD5 was strongly upregulated (27.5-fold) following 48h of drought exposure in roots. In roots, GhLSD5 showed strong upregulation (27.5-fold) at 48 h, whereas it was highly downregulated (10-fold) at 4 h. GhLSD6 expression in drought-stressed roots peaked at 48 h, reaching a 19-fold upregulation compared to the control. GhLSD6 was moderately downregulated (2.77-fold) in roots at 4h under drought stress (Fig. 9 ). Gene Expression Analysis of LSD genes Under Salt Stress The expression analysis of GhLSD genes under salt stress revealed distinct temporal and tissue-specific patterns. GhLSD1 showed moderate upregulation in leaves at 8 h (1.38-fold), but was moderately downregulated at early time points (2 h, 0.44-fold; 4 h, 0.63-fold). GhLSD2 exhibited strong time-dependent downregulation in leaves (2 h, 0.61-fold; 4 h, 0.51-fold; 24 h, 0.31-fold; 48 h, 0.19-fold). GhLSD3 displayed dynamic regulation, with strong upregulation in leaves at early time points 2h and 8h but marked downregulation at certain time points, reflecting context-dependent regulatory functions. GhLSD4 was early induced in leaves at 2 h (1.61-fold), peaking at 4 h (6.2-fold). GhLSD5 exhibited moderate upregulation in leaves at 2 h (2.42-fold) and 8 h (1.93-fold). Moderate inductions (2.42-fold and 1.93-fold) of GhLSD5 was observed leaves under salt stress at 2h and 8h respectively. GhLSD6 showed slight to moderate downregulation in leaves (2 h, 0.61-fold; 4 h, 0.45-fold; 24 h, 0.85-fold; 48 h, 0.56-fold) (Fig. 10 ). GhLSD1 was moderately downregulated at early time points (2 h, 0.44-fold; 4 h, 0.63-fold), while in roots it peaked at 24 h (5.68-fold) and was strongly downregulated at 2 h (0.17-fold). GhLSD2 was strongly downregulated in roots at 2 h (0.20-fold) and 48 h (0.17-fold). GhLSD3 displayed dynamic regulation, with strong upregulation in roots at later stages 24h and 48h, but marked downregulation at certain time points, reflecting context-dependent regulatory functions. GhLSD3 was strongly downregulated (0.16-fold) (6.25) and (0.31-fold) (3.22) in roots at 2h and 8h under salt stress. GhLSD3 was highly downregulated (0.05-fold) (18.18) in roots at 4h under salt stress. In roots, GhLSD4 showed moderate downregulation at 2 h (0.46-fold) followed by strong upregulation at 4 h (7.39-fold). GhLSD5 exhibited dramatic upregulation in roots at 24 h (131.20-fold) and 48 h (216.88-fold). GhLSD5 was strongly upregulated (131.20-fold and 216.88-fold) in roots at 24h and 48h under salt stress. GhLSD5 showed extreme upregulation in roots at later stages (24–48 h), suggesting a critical role in root-specific salt stress adaptation. GhLSD6 was strongly downregulated at 8 h (0.62-fold), and highly upregulated at 48 h (5.47-fold). GhLSD6 was highly upregulated peak in roots at 48 h, highlighting its late-stage role in root stress response (Fig. 10 ). Overall, these results indicate that the GhLSD gene family operates in a coordinated, tissue- and time-dependent manner under salt stress, with GhLSD1 , GhLSD4 , GhLSD5 , and GhLSD6 emerging as key positive regulators, whereas GhLSD2 may act as a repressor (Fig. 10 ). Discussion Gossypium hirsutum (Upland cotton) is an allotetraploid species (2n = 4x = 52) composed of two subgenomes, A and D, which originated from the hybridization of G. arboreum (A genome) and G. raimondii (D genome) (Chen et al. 2020 ). Its genome size is estimated at 2.25–2.43 Gb and encodes approximately 76,943 genes. The Lesion Simulating Disease (LSD) transcription factor acts as a negative regulator of programmed cell death (PCD) by inhibiting EDS1 and PAD4, and contributes to plant responses to salinity and drought stress (Dietrich et al. 1994 ). LSD proteins contain a conserved zinc-finger (ZF) motif (CxxCRxxLMYxxGASxVxCxxC) (Dietrich et al. 1997 ), enabling DNA/protein interactions and integration of ROS, salicylic acid (SA), and ethylene (ET) signaling to modulate cell death and acclimation. Genome-wide identification and functional characterization of LSD transcription factors have been reported in several model species, including Arabidopsis thaliana , Oryza sativa , and Zea mays (Jiang et al. 2019 ; Sun et al. 2023 ). In this study, we identifed eleven GhLSD genes in the G. hirsutum genome and found to be unevenly distributed across 5 out of the 13 chromosomes (chromosomes 5, 6, 8, 10, and 12) in both the A and D subgenomes. The number of LSD genes vary among species. For example, 6 MsLSD in M. Sativa , 12 AtLSD in A. Thaliana , 20 ZmLSD in Zea mays and 18 GmLSD in Glycine max . Comprehensive characterization of the Gossypium hirsutum LSD (GhLSD) gene family provides important insights into their genomic organization, structural variation, and potential functional roles in cotton. The CDS lengths of GhLSD genes varied from 294 to 513 bp, and exon numbers ranged from four to eight, suggesting moderate structural diversity within the family. The observed correlation between CDS length and exon number indicates evolutionary constraints that maintain functional domains while allowing flexibility in gene structure. Similar structural variation has been reported in Arabidopsis thaliana and soybean (4 to 6 exon and supporting the idea that exon–intron organization contributes to gene diversification and functional specialization of LSD family members (Cabreira et al. 2013 ). On the other hand, most alpha alpha MsLSD genes contain a limited number of exons, one or four. (Sun et al. 2023 ), The chromosomal distribution of GhLSD genes revealed localization on ten of 26 chromosomes, with an almost balanced presence between the A and D subgenomes. The occurrence of duplicated gene pairs on homoeologous chromosomes, such as GhLSD1 on chromosome A5 and its paralog GhLSD7 on chromosome D5, together with the close localization of GhLSD6 and GhLSD7 on chromosome D5, indicates that both segmental and tandem duplication events have played a pivotal role in the expansion of the GhLSD gene family. The allotetraploid nature of upland cotton further explains the presence of multiple paralogous gene pairs with high sequence identity (> 97%), which likely originated from recent duplication events. Comparable expansion patterns were observed in Medicago sativa (6 MsLSD genes) and Glycine max (18 LSD genes) indicating that polyploidy and duplication events are common mechanisms underlying the expansion of LSD transcription factor families in plants (Cabreiara et al. 2013). At the protein level, GhLSD members exhibited lengths of 97–180 amino acids, with predicted molecular weights ranging from 10.6 to 18.7 kDa. Most proteins had basic isoelectric points, consistent with their proposed roles in DNA or protein binding. Variations in hydrophobicity and instability indices suggest that different GhLSD proteins may adopt specialized cellular roles, with GhLSD1, GhLSD6, and GhLSD7 being less stable and potentially subject to post-translational regulation. Similar physicochemical diversity has been reported in LSD proteins of maize ( Zea mays ), where variation in hydrophilicity was suggested to reflect adaptive subfunctionalization (Jiang et al. 2019 ). Subcellular localization predictions identified nuclear and cytoplasmic, implying functional diversity in transcriptional regulation and stress signaling. The nuclear localization is consistent with their role as transcription factors, whereas their cytoplasmic presence suggests potential additional functions, such as acting as scaffold proteins or participating in signaling cascades prior to nuclear translocation. Importantly, consistent with previous findings, (Czarnocka et. al. 2017 ) reported that LSD1 not only functions as a transcriptional regulator but also acts as a scaffold protein, thereby supporting the notion that LSD proteins exhibit dual roles within the cell. Taken together, these results suggest that GhLSD proteins may function as condition-dependent scaffold proteins in the cytoplasm and as transcriptional regulators in the nucleus, reflecting a versatile role in stress response and developmental regulation in cotton. Domain analysis confirmed the presence of highly conserved LSD-type zinc finger (ZF) motifs(CxxCRxxLMYxxGASxVxCxxC), also member of a cysteine-2/cysteine-2-class (C2C2), which were maintained across all members, reinforcing their essential role in programmed cell death (PCD) regulation. Nine GhLSD proteins contained three LSD-type zinc finger (ZF) domains, whereas GhLSD1 and GhLSD7 harbored only two, suggesting potential divergence in their DNA-binding affinity or target specificity. Typically, LSD proteins possess three ZF domains; however, the number of domains can vary among different species. For instance, in maize, ZmLSD proteins have been reported to contain between one and three ZF domains, indicating structural diversification within the family across plant lineages (Jiang et al. 2019 ) Conservation of cysteine residues at positions 1, 4, 19, and 22 across all GhLSD proteins, forming the typical C2C2-type ZF structure, highlights their importance in tertiary structure and structural stability, and protects cell from oxidative damage, redox-mediated regulation as well (Cabreira et al. 2013 ). In conclusion, GhLSD proteins may play a similar role in managing oxidative stress triggered by drought and salinity. Motif and cis-regulatory element analyses further supported functional conservation and specialization. The presence of conserved motifs (motif 1 and 2) across almost all GhLSD proteins reflects their core regulatory role, while additional motifs may contribute to lineage-specific adaptation. Promoter analysis revealed abundant stress- and hormone-responsive cis-elements, including MYB, MYC, ERE, and W-box motifs, which are known to regulate transcriptional reprogramming during abiotic stresses. In particular, the enrichment of ethylene-responsive elements (EREs) in GhLSD4 suggests a strong link between LSD proteins and ethylene-mediated signaling pathways, which play central roles in stress adaptation and PCD regulation. These findings are consistent with earlier reports in Glycine max and Medicago sativa , where LSD genes were shown to integrate reactive oxygen species (ROS), salicylic acid (SA), and ethylene (ET) signaling during stress responses (Cabreira et al. 2013 ; Sun et al. 2023 ). The phylogenetic clustering demonstrates that the LSD gene family is highly conserved across cotton species and Arabidopsis, with distinct subgrouping into five clades. The close association of GhLSDs with both G. arboreum and G. raimondii orthologs indicates that G. hirsutum retained most ancestral copies from its diploid progenitors following polyploidization. This conservation suggests that LSD genes play fundamental roles in stress adaptation and development. The absence of G. hirsutum members in Clade II likely reflects gene loss, pseudogenization, or significant divergence, highlighting evolutionary flexibility within the family. Furthermore, the distribution of GhLSDs across different clades suggests functional diversification, with some members potentially undergoing subfunctionalization or neofunctionalization to meet the adaptive demands of upland cotton. These evolutionary patterns are consistent with previous findings that LSD proteins regulate stress signaling and development, implying that different clades may correspond to distinct functional specializations Protein structural predictions revealed that GhLSD proteins are predominantly composed of random coils and β-strands, with limited α-helices and no β-turns. Such structural flexibility is typical of regulatory proteins that interact with multiple partners within stress signaling networks. The thermostability indicated by aliphatic index values suggests that GhLSD proteins remain stable under variable environmental conditions, a property that has been similarly reported for Alfaalfa and maize LSD proteins (Jiang et al. 2019 ; Sun et al. 2023 ). Taken together, these findings indicate that the LSD transcription factor family in cotton has undergone expansion and diversification through polyploidy and gene duplication while maintaining strong conservation of core domains essential for PCD regulation. The integration of diverse promoter elements, structural variation, and predicted functional divergence highlights the potential of GhLSD genes as key regulators of stress adaptation in cotton. Given the established roles of LSD genes in regulating PCD, ROS signaling, and abiotic stress tolerance in other crops, the GhLSD family represents promising candidates for functional validation studies. Future research focusing on expression profiling under drought and salinity stress, as well as genome-editing approaches such as CRISPR/Cas9, may provide valuable strategies for improving cotton stress tolerance. The expression profiling of GhLSD genes under drought and salt stresses provides novel insights into their temporal and tissue-specific regulatory functions in G. hirsutum . Overall, the results demonstrate that the GhLSD gene family operates in a highly dynamic, coordinated, and context-dependent manner, with distinct members contributing to either activation or repression of stress responses in leaves and roots. The drought-responsive expression profiles revealed a clear division of regulatory roles among GhLSD genes. In leaves, GhLSD1, GhLSD3, GhLSD4, GhLSD5, and GhLSD6 were strongly induced, particularly at 8 h, indicating an early activation of defense and acclimation mechanisms. Notably, GhLSD4 exhibited a dramatic 154-fold upregulation, positioning it as a potential master regulator of drought-induced signaling pathways in cotton. Similar strong induction of LSD genes under drought stress has been observed in Arabidopsis and soybean, where LSD family members were shown to integrate ROS and hormone signals to fine-tune stress responses (Cabreira et al. 2013 ; Szechyn´ska-Hebda et al. 2016) In roots, GhLSD1, GhLSD5 , and GhLSD6 displayed robust late-stage induction (40.44-fold, 27.5-fold, and 19-fold, respectively, at 48 h), suggesting that these genes may contribute to sustained drought adaptation mechanisms, possibly by modulating root growth or enhancing antioxidant defenses. In contrast, GhLSD2 and GhLSD3 were consistently downregulated in roots, implying a negative regulatory role, potentially acting to prevent excessive PCD activation under prolonged water deficit. Such contrasting expression patterns between leaves and roots highlight tissue-specific functional diversification, consistent with earlier reports that LSD homologs act as context-dependent regulators balancing survival and cell death in plants Taken together, GhLSD1, GhLSD4, and GhLSD6 appear to function as positive regulators of drought tolerance, while GhLSD2 may act as a repressor. The strong induction of GhLSD4 in leaves and GhLSD6 in roots emphasizes their importance as candidate genes for improving drought resilience in cotton. Under salt stress, the GhLSD family again exhibited diverse and tissue-dependent regulation. In leaves, GhLSD4 and GhLSD5 showed early induction (up to 6.2-fold at 4 h for GhLSD4, and 2.42-fold for GhLSD5 at 2 h), suggesting their involvement in rapid signaling and protective responses against ionic and osmotic stress. GhLSD3 showed transient upregulation at early time points but was subsequently downregulated, indicating a tightly controlled, context-dependent role. In contrast, GhLSD2 was consistently downregulated, supporting its putative role as a negative regulator during salt stress. In roots, striking differences emerged. GhLSD5 exhibited extremely high induction (131-fold at 24 h and 217-fold at 48 h), suggesting it plays a central role in root-specific salt tolerance, likely through regulation of ion homeostasis, osmotic adjustment, or ROS detoxification. GhLSD6 also showed late-stage induction (5.47-fold at 48 h), pointing to a supportive role in root acclimation. By contrast, GhLSD2 remained strongly repressed across time points, further strengthening its proposed role as a negative regulator. Similar patterns of late-stage activation of LSD homologs have been observed in rice, where LSD family members were implicated in long-term adaptive responses to salinity (Sun et al. 2010 ). Comparing the two stress conditions reveals both overlapping and divergent roles of GhLSD genes. GhLSD1, GhLSD4, GhLSD5, and GhLSD6 consistently emerged as positive regulators under both drought and salt stresses, albeit with tissue- and time-dependent differences. GhLSD4 functioned predominantly in leaves, displaying rapid and strong induction under drought and salt stresses, while GhLSD5 and GhLSD6 were highly responsive in roots, particularly under prolonged stress exposure. GhLSD2 , conversely, was consistently downregulated in both conditions, suggesting it acts as a suppressor of stress responses, possibly to prevent excessive or uncontrolled PCD. The contrasting regulation of GhLSD3 across tissues and stress conditions suggests a dual, context-dependent role in fine-tuning stress signaling. These findings highlight that the GhLSD gene family contributes to cotton stress adaptation through a coordinated network of activators and repressors that function in both early and late phases of stress. The strong upregulation of GhLSD4, GhLSD5, and GhLSD6 suggests that these genes are promising targets for genetic improvement of drought and salinity tolerance in cotton. Their tissue-specific expression further emphasizes the importance of considering organ-level regulation in stress biology. Future functional validation using overexpression and CRISPR/Cas9 knockout strategies will be essential to clarify the precise regulatory roles of GhLSD genes and to exploit them for crop improvement. Given the integration of LSD proteins with ROS, SA, and ET signaling, further investigation into their upstream regulation and downstream target genes will provide critical insights into their role in stress resilience mechanisms in cotton. Conclusion and Recommendation The comprehensive expression profiling of the GhLSD gene family under drought and salinity stress revealed that these transcription factors function in a dynamic, tissue-specific, and time-dependent manner to regulate stress responses in Gossypium hirsutum . Among the six genes analyzed, GhLSD1, GhLSD4, GhLSD5 , and GhLSD6 consistently exhibited strong induction under both drought and salt treatments, highlighting their potential roles as positive regulators of abiotic stress tolerance. In particular, GhLSD4 (in leaves) and GhLSD5 (in roots) demonstrated exceptionally high expression peaks, suggesting specialized roles in tissue-specific stress adaptation. GhLSD6 also showed consistent late-stage upregulation, pointing to its involvement in sustained stress responses. Conversely, GhLSD2 was generally downregulated in both tissues under drought and salinity, suggesting a repressive role in stress signaling pathways. GhLSD3 displayed variable regulation, alternating between induction and suppression depending on tissue type and exposure duration, indicating a context-dependent function. Taken together, these findings emphasize that the GhLSD family contributes to stress acclimation through a finely tuned regulatory network rather than uniform responses. The strong induction of GhLSD4, GhLSD5 , and GhLSD6 highlights them as promising candidate genes for further functional studies and potential genetic improvement strategies to enhance drought and salinity tolerance in cotton. Declarations Funding: This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University (FYL-2024-40510) Conflict of interest/Competing interests: The authors declare no conflict of interest. Ethics approval and consent to participate: Not applicable Consent for publication: Not applicable Data availability: Data are included within the article. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9441383","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":634798573,"identity":"184bc3fe-d9c6-417a-b5df-3b47047713d3","order_by":0,"name":"Ayşe Arucan","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Ayşe","middleName":"","lastName":"Arucan","suffix":""},{"id":634798578,"identity":"9670a9bb-0c57-4906-b6dd-6ce4c8a1833d","order_by":1,"name":"Cüneyt Uçarlı","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIiWNgGAWjYLCCBwYMDPzMYOYBBj4gKUFQSwJQi2QzA2MDSAsbRIsBAS1AbHCAWC0Gxw8/+5BQYJe4+Tj78wc/d9xJbGNgPnibh+FPPk4tZ9KMZyQYJCduO8xj2Nh75hlQC1uyNQ+DgWUDLi03GIyBfjkA0sLYwNt2GKiFx0waqAWnywxusH8Ga9nczP6w8S9YC/83Alp4ILZsYGYwbIbawoZXi+SZnGKglmTjGUC/zJZte2bcxsxmbDnHwBinFr7jxzczfPhjJ9vff/zBx7dtd2T72Zsf3nhTIUcgYlAAOBmQomEUjIJRMApGAQYAAIKgU5U8/sbuAAAAAElFTkSuQmCC","orcid":"","institution":"Istanbul University","correspondingAuthor":true,"prefix":"","firstName":"Cüneyt","middleName":"","lastName":"Uçarlı","suffix":""}],"badges":[],"createdAt":"2026-04-16 18:38:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9441383/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9441383/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108979577,"identity":"944b37a5-eb4e-4b41-9d78-59d2d75c688d","added_by":"auto","created_at":"2026-05-11 11:59:41","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":980956,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distributions of GhLSD genes on sub-genomes A and D of \u003cem\u003eG. hirsutum.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/29222bb93da95f90a9498dc7.jpg"},{"id":108978628,"identity":"a4e0da74-c609-42e5-bd00-2ea4b3285e9a","added_by":"auto","created_at":"2026-05-11 11:47:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":382021,"visible":true,"origin":"","legend":"\u003cp\u003eCDS-intron structure analysis of GhLSD genes. The blue boxes show UTRs, the yellow boxes show CDS and the black lines indicate introns.\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/3eca57cbb6e8106ced80cd20.jpg"},{"id":108978630,"identity":"d412a3de-e157-473c-81c0-5245c47202c3","added_by":"auto","created_at":"2026-05-11 11:47:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6045741,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of the GhLSD proteins. The secondary structural elements, α-helix and β-strand, predicted of GhLSD and LSD-type ZF domains are shown above. Red box, LSD-type ZF domains; blue frame represents similarity across groups. β-strands are shown as black arrows and black frames. α-helices are shown as blue solenoids and blue frames.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/e8af3ad07ec04f34e143a69d.jpg"},{"id":108978639,"identity":"1451d10c-d85e-4a3a-b2f3-caed19cc4d20","added_by":"auto","created_at":"2026-05-11 11:47:12","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2205917,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of LSD-type ZFN domains of GhLSD proteins.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/b4c45167be2faab9c6a3c6aa.jpg"},{"id":108978611,"identity":"094d67bd-9756-45cd-a0c1-19b22ccbb35e","added_by":"auto","created_at":"2026-05-11 11:46:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1292712,"visible":true,"origin":"","legend":"\u003cp\u003eConserved motifs of LSD proteins in \u003cem\u003eGossypium hirsutum\u003c/em\u003e. Motifs 1–10, represented by colored boxes, illustrate the conserved sequence patterns identified within the protein family.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/fe4ddd30805774b7c9de60fa.jpg"},{"id":109081308,"identity":"718bbfa9-7e5f-47ea-a135-793fb68c5a84","added_by":"auto","created_at":"2026-05-12 12:15:05","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2390140,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of cis-acting elements related to stress responses, hormonal regulation, and plant growth in the 2 kb upstream promoter regions of \u003cem\u003eGhLSD\u003c/em\u003e genes.\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/7c7e0a033c87723419c1684d.jpg"},{"id":108978612,"identity":"92388d1d-2e1b-4fbd-9693-39473ca2ed3a","added_by":"auto","created_at":"2026-05-11 11:46:26","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":991204,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of LSD genes among \u003cem\u003eH. hirsutum. Arabiopsis thaliana. G. arboretum, G. raimondii.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/854577e8649456df86412e26.jpg"},{"id":109067795,"identity":"9ea059b4-2188-4776-b5bf-795294c6defe","added_by":"auto","created_at":"2026-05-12 10:01:05","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1651620,"visible":true,"origin":"","legend":"\u003cp\u003eTertiary (3°) structure of eleven GhLSD Proteins.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/bf9a6a92d807b76a6053b9d6.jpg"},{"id":108979599,"identity":"ec2aabf8-99a8-49bf-96b7-fa4e00845aef","added_by":"auto","created_at":"2026-05-11 12:00:10","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2352573,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of GhLSD genes in leaves and roots of \u003cem\u003eGossypium hirsutum\u003c/em\u003e under drought stress at 0, 2, 4, 8, 24 and 48 h. Heat map of \u003cem\u003eGhLSD\u003c/em\u003egene expression patterns in leaves and roots of \u003cem\u003eGossypium hirsutum\u003c/em\u003e under drought stress at 0, 2, 4, 8, 24, and 48 h. Downregulated genes are shown in blue, whereas upregulated genes are shown in red.\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/16351de89d5cabe9ed523ac3.jpg"},{"id":108979601,"identity":"47497114-520b-4083-9e39-dd069a8d4aa0","added_by":"auto","created_at":"2026-05-11 12:00:13","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":2365895,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of GhLSD genes in leaves and roots of \u003cem\u003eGossypium hirsutum\u003c/em\u003e under salt stress at 0, 2, 4, 8, 24 and 48 h. Heat map of \u003cem\u003eGhLSD\u003c/em\u003e gene expression patterns in leaves and roots of \u003cem\u003eGossypium hirsutum\u003c/em\u003e under salt stress at 0, 2, 4, 8, 24, and 48 h. Downregulated genes are shown in blue, whereas upregulated genes are shown in red.\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/a40b990aefb15cc6791a061c.jpg"},{"id":109082873,"identity":"b7c7241c-f9a1-4e4e-bdbd-4ccbff4d3e7c","added_by":"auto","created_at":"2026-05-12 12:45:06","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21220322,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9441383/v1/9a937b02-4436-49d7-b66f-4b83386b466b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Characterization and Expression Profiling of the LSD Gene Family in Cotton (Gossypium hirsutum) Under Drought and Salt Stress","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCotton (\u003cem\u003eGossypium spp\u003c/em\u003e.) is an economically important crop that supplies nearly 35% of the world\u0026rsquo;s natural fiber and also serves as a source of vegetable oil and bioenergy (Abdelraheem et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the 2024\u0026ndash;2025 growing season, global cotton production reached around 121.07\u0026nbsp;million 480-lb bales, with China, India, Brazil, the United States, Australia, Pakistan, and T\u0026uuml;rkiye as the main producers (USDA, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). T\u0026uuml;rkiye ranks seventh globally, contributing about 3% of world production\u0026mdash;mainly from the Southeastern Anatolia and Aegean regions\u0026mdash;with an average yield of 1,799 kg/ha between 2020/2021 and 2024/2025 (Najib et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; USDA, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCotton plays an important role in studying polyploid evolution, fiber development, and stress adaptation (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eGossypium\u003c/em\u003e represents a genus of the Malvaceae family encompesses about 50 species\u0026mdash;45 diploid and 5 allotetraploid \u0026mdash;with \u003cem\u003eG. hirsutum\u003c/em\u003e (AD₁) and \u003cem\u003eG. barbadense\u003c/em\u003e (AD₂) arising from hybridization between \u003cem\u003eG. arboreum\u003c/em\u003e (A₂) and \u003cem\u003eG. raimondii\u003c/em\u003e (D₅) followed by chromosome doubling (Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eG. hirsutum\u003c/em\u003e accounts for nearly 95% of global production, while \u003cem\u003eG. arboreum\u003c/em\u003e and \u003cem\u003eG. raimondii\u003c/em\u003e remain significant genetic resources for plant toleranse in drought and salinity and genomic researches ( Zhu et al. 2013; Yan et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGlobal climate change has intensified abiotic stresses such as drought and salinity, which negatively affect fiber quality, plant development, and overall cotton productivity (Khan et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Although cotton exhibits moderate salt tolerance (\u0026asymp;\u0026thinsp;7.7 dS m⁻\u0026sup1;), severe stress can reduce yield by up to 67%, varying with stress severity and genotype (Maas and Hoffman \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1977\u003c/span\u003e; Abdelraheem et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). These conditions impair photosynthesis, root development, and nutrient uptake, resulting in major agronomic and economic losses (Bano et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalinity is a critical condition that disrupts plant development by inducing osmotic stress, ionic toxicity, and nutrient imbalance (Ahmad et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In cotton, excessive Na⁺ and Cl⁻ accumulation impairs water uptake, damages cellular membranes, and inhibits enzymatic activity, leading to oxidative stress and alleviate fiber characteristics (Lu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Chaudhary et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Plants counteract these effects through ion homeostasis, antioxidant activation, and transcriptional regulation of genes associated with stress tolerance (U\u0026ccedil;arlı \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDrought is a major environmental problem which constrain growth of cotton seeds and fiber quality while promoting soil degradation and erosion (Xiao et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Plants employ avoidance, tolerance, escape, and recovery strategies to withstand drought, involving mechanisms such as leaf rolling, stomatal closure, osmotic setting, and metabolic stabilization (Kneebone et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; U\u0026ccedil;arlı \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Root system architecture is a key determinant of water uptake and signaling under deficit conditions (Zhang et al. 2024).\u003c/p\u003e \u003cp\u003eAt the molecular level, plant adaptation to water scarcity and salinity are governed by complex networks of transcription factors (TFs) that bind cis-regulatory elements to control gene expression (Latchman, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; W\u0026auml;rnmark et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). These TFs contain distinct DNA-binding and activation domains that regulate transcriptional activity and integrate environmental cues into adaptive responses (Nakano et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Hrmova et al. 2021; Dhatterwal et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In adverse condition regulation and secondary metabolism, key TF families are bZIP, MYB, WRKY, NAC, AP2/ERF, and MADS-box (Dubos et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Rushton et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Puranik et al. 2012; Licausi et al. 2013; Yu et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Chowdhary et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Gao \u0026amp; Dubos, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eLesion Simulating Disease\u003c/em\u003e (\u003cem\u003eLSD\u003c/em\u003e) gene encodes a C2C2-type zinc finger protein that functions as a central regulator of programmed cell death (PCD) and stress acclimation (Dietrich et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Takatsuji, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Coll et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eLSD\u003c/em\u003e negatively regulates PCD, with \u003cem\u003elsd1\u003c/em\u003e mutants exhibiting runaway cell death and hypersensitivity to oxidative and pathogen-induced stresses due to disrupted ROS homeostasis (Bernacki et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Functioning within the \u003cem\u003eLSD\u0026ndash;EDS1\u0026ndash;PAD4\u003c/em\u003e regulatory hub, \u003cem\u003eLSD\u003c/em\u003e integrates redox and hormonal signaling to coordinate defense activation and stress adaptation under environmental extremes (M\u0026uuml;hlenbock et al. 2008; Wituszyńska et al. 2013). However, its molecular functions in cotton remain largely unexplored, and elucidating the evolution and expression of \u003cem\u003eLSD1\u003c/em\u003e homologs in \u003cem\u003eGossypium\u003c/em\u003e could advance understanding of stress tolerance and facilitate breeding of resilient cultivars (Dietrich et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Bernacki et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we aimed to characterize and identify the structural features and conserved domains of \u003cem\u003eGhLSDs\u003c/em\u003e through in silico analysis; also analyze the expression profile of the \u003cem\u003eLSD\u003c/em\u003e gene under arid conditions and salinity in upland cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e). Overall, this study provides new insights into the structural and functional attributes of \u003cem\u003eGhLSDs\u003c/em\u003e and its potential role in cotton\u0026rsquo;s adaptive response to environmental stresses including salinity and drought.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eIn Silico Analysis of LSD Genes and Proteins in Cotton (\u003c/b\u003e \u003cb\u003eGossypium hirsutum\u003c/b\u003e \u003cb\u003e)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMembers of the LSD (\u0026ldquo;Lesion Simulating Disease 1\u0026rdquo;) gene family and their corresponding protein sequences were identified in the \u003cem\u003eGossypium hirsutum\u003c/em\u003e genome using the CottonGen database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cottongen.org/\u003c/span\u003e\u003cspan address=\"https://www.cottongen.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the Plant Transcription Factor Database (PlantTFDB v4.0 and v5.0; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://planttfdb.gao-lab.org/\u003c/span\u003e\u003cspan address=\"https://planttfdb.gao-lab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). LSD protein sequences from \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eG. arboreum\u003c/em\u003e, \u003cem\u003eG. raimondii\u003c/em\u003e, and \u003cem\u003eG. hirsutum\u003c/em\u003e were retrieved and confirmed via BLASTP and BLASTN searches (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene characteristics, including nucleotide and protein length, chromosomal location, and exon-intron organization, were obtained from NCBI. The physicochemical properties of GhLSD proteins\u0026mdash;molecular weight, isoelectric point (pI), hydropathicity (GRAVY), and sequence length were computed using ProtParam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), while subcellular localization was estimated through DeepLoc-2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/DeepLoc-2.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/DeepLoc-2.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Conserved domains were validated with CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and secondary structure components (α-helix, β-turn, extended strand, random coil) were analyzed with PSIPRED (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinf.cs.ucl.ac.uk/psipred/\u003c/span\u003e\u003cspan address=\"https://bioinf.cs.ucl.ac.uk/psipred/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Tertiary structures of GhLSD proteins were modeled using AlphaFold3 (Abramson et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The structural organization of the genes was illustrated via GSDS 2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and conserved motifs were identified using MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with maximum of 10 motifs was allowed, with motif lengths ranging from 6 to 50 amino acids. Cis-acting regulatory elements within 2 kb upstream promoter regions were extracted from NCBI and analyzed using the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Multiple sequence alignments were performed with ClustalW, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replicates in MEGA-X (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.megasoftware.net/\u003c/span\u003e\u003cspan address=\"https://www.megasoftware.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), then visualized and annotated with iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials, Growth Conditions, and Treatments\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eCotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e cv. Harem 2) seeds were obtained from the Republic of T\u0026uuml;rkiye Ministry of Agriculture and Forestry, Eastern Mediterranean Agricultural Research Institute. Seeds were surface-sterilized with 70% ethanol for 1 min, followed by 10% H₂O₂ for 2 h, and rinsed three times with sterile distilled water. The seed coats were carefully removed, and embryos were transferred onto Murashige and Skoog (MS) medium (Murashige \u0026amp; Skoog, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1962\u003c/span\u003e). Germination was carried out at 24\u0026deg;C under controlled growth chamber conditions for 3\u0026ndash;4 days.\u003c/p\u003e \u003cp\u003eFor salt stress, 10-day-old seedlings were transferred to MS medium supplemented with 200 mM NaCl and sampled after 0, 2, 4, 8, 24, and 48 h. For drought stress, seedlings were transferred to glass jars containing MS medium infused with 25% polyethylene glycol (PEG6000) following the PEG overlay method described by (Pawar \u0026amp; Veena, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and exposed to stress for the same time intervals.\u003c/p\u003e \u003cp\u003eLeaf and root tissues were collected at each time point, immediately frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until RNA extraction and gene expression analysis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTotal RNA Isolation and cDNA Synthesis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eTotal RNA was isolated from the 10 day-old leaf and root samples using the FastPure Universal Plant Total RNA isolation kit (Vazyme- Nanjing, China), which is suitable for plant species rich in polysaccharides or polyphenols.\u003c/p\u003e \u003cp\u003eThe concentration and purity of total RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). The A260/A280 ratio between 1.8 and 2.1 indicated acceptable purity, while an A260/A230 ratio above 1.8 confirmed the absence of major contaminants. RNA integrity was verified by 1% agarose gel electrophoresis, where distinct 28S and 18S rRNA bands indicated high-quality, intact RNA suitable for downstream applications.\u003c/p\u003e \u003cp\u003eFirst-strand complementary DNA (cDNA) was synthesized from 1 \u0026micro;g of total RNA using the qScript cDNA Synthesis Kit (Quanta Biosciences, Germany) according to the manufacturer\u0026rsquo;s protocol. The synthesized cDNA was diluted 1:10 with nuclease-free water and stored at \u0026minus;\u0026thinsp;20\u0026deg;C until quantitative PCR (qRT-PCR) analysis.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eQuantitative Real-Time PCR (qRT-PCR) Analysis\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eGene-specific primers for six candidate \u003cem\u003eGhLSD\u003c/em\u003e genes (\u003cem\u003eGhLSD1\u0026ndash;GhLSD6\u003c/em\u003e) were designed using the Primer3 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://primer3.ut.ee/\u003c/span\u003e\u003cspan address=\"https://primer3.ut.ee/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The ubq7 gene was used as an internal reference for normalization (Wang et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). qRT-PCR reactions were performed in 20 \u0026micro;L volumes containing 1 \u0026micro;L of cDNA (100 ng), 0.5 \u0026micro;L each of forward and reverse primers \u003cb\u003e(\u003c/b\u003e10 \u0026micro;M), 10 \u0026micro;L of 2\u0026times; SYBR Green Master Mix, and nuclease-free water to volume. Amplification was conducted on an A.B.T. (Atlas Biotechnology) real-time PCR system using the following thermal profile: 95\u0026deg;C for 5 min, followed by 40 cycles of 95\u0026deg;C for 15 s, 57\u0026deg;C for 20 s, and 72\u0026deg;C for 20 \u003cb\u003es\u003c/b\u003e, with a final melting curve analysis to verify amplification specificity. Each reaction included three biological replicates and two technical replicates. Relative gene expression was assessed following the 2⁻\u003csup\u003eΔΔCT\u003c/sup\u003e method (Livak and Schmittgen, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eStatistical Analyses\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe qRT-PCR data were analyzed using one-way ANOVA with the least significant difference Tukey HSD test via the SPSS21 statistical program (IBM). All analyses were performed using three independent biological replicates, each analyzed with two technical replicates.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of GhLSD Family Members\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eEleven LSD genes were identified in the tetraploid cotton species \u003cem\u003eG. hirsutum\u003c/em\u003e genome, and these genes were distributed across the chromosomes of \u003cem\u003eG. hirsutum\u003c/em\u003e (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;52). Eleven \u003cem\u003eGhLSD\u003c/em\u003e genes were located on 5 out of the 13 chromosomes (chromosomes 5, 6, 8, 10, and 12) in both the A and D subgenomes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In \u003cem\u003eGhLSD\u003c/em\u003e genes, the coding sequence (CDS) length varied from 294 bp to 513 bp, while the number of exons ranged from four to eight. Notably, a correlation between exon number and CDS length was observed. Among the \u003cem\u003eGhLSD\u003c/em\u003e genes, five are oriented on the positive strand, whereas six are positioned on the negative strand.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of the \u003cem\u003eGhLSD\u003c/em\u003e genes identified in the cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e L.) genome.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene Name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAccession No\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGene Location (5'-3')\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStrand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCDS\u003c/p\u003e \u003cp\u003e(bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eExon No\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016842666.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053428.1_16065629\u0026ndash;16067143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016896396.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053429.1_ 128108232\u0026ndash;128112426\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e510\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016848346.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053431.1_ 112468123\u0026ndash;112470334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016855484.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053433.1_ 426067\u0026ndash;430393\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_041083378.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053435.1_ 102589789\u0026ndash;102592455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_041093586.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053441.1_ 9470745\u0026ndash;9476686\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016832533.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053441.1_ 14672500\u0026ndash;14674115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e294\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_041096384.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053442.1_ 66551319\u0026ndash;66555576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e543\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016865838\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053444.1_ 56483411\u0026ndash;56485604\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e444\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016862004.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053446.1_ 415235\u0026ndash;419837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePlus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e513\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXM_016881184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eD12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNC_053448.1 _ 56748242\u0026ndash;56750893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMinus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromosomal localization and Gene structure\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eUpland cotton \u003cem\u003eGossypium hirsutum\u003c/em\u003e is allotetraploid (2n\u0026thinsp;=\u0026thinsp;4X\u0026thinsp;=\u0026thinsp;52) with subgenomes A and D. All of the A chromosomes are relatively larger than homoeologous D chromosomes. Eleven \u003cem\u003eGhLSD\u003c/em\u003e genes were found to located on terminal parts of ten chromosomes among 26 chromosomes of \u003cem\u003eG. hirsutum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). While 5 \u003cem\u003eGhLSD\u003c/em\u003e genes were located on chromosomes A (A5, A6, A8, A10, A12), 6 \u003cem\u003eGhLSD\u003c/em\u003e genes were located on D chromosomes (D5, D6, D8, D10, D12) Among these chromosomes, ChrD5 harbored two \u003cem\u003eGhLSD\u003c/em\u003e genes (GhLSD6 and GhLSD7).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eIn order to determine the differences in gene structure, CDS and intron structures of 11 \u003cem\u003eGhLSD\u003c/em\u003e genes were compared and illustrated. The intron count of \u003cem\u003eGhLSD\u003c/em\u003e genes varied from 3 to 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCharacterization of cotton LSD1 proteins\u003c/h3\u003e\n\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe characterization of cotton LSD transcription factors was performed using different bioinformatics tools. The findings obtained are shown in Table\u0026nbsp;3.2. It was found that the length of the proteins ranged from 97 to 180 amino acids. GHLSD1 is the lightest LSD protein with a weight of 10.6 kDa, while the heaviest is GhLSD8 with a weight of 18.7 kDa. It was found that the theoretical isoelectric points (pI) are above 7, and GhLSD proteins have a basic structure. According to GRAVY values, GhLSD1 and GhLSD7 proteins were found to have hydrophilic properties, while other GhLSD proteins were found to have hydrophobic structure (GRAVY\u0026thinsp;\u0026gt;\u0026thinsp;0). The aliphatic index (AI) values of cotton LSD proteins range from 68.25 (GhLS1) to 82.28 (GhLSD8), which indicates that LSD proteins have a thermostable structure. Subcellular localization analysis predicted that GhLSD proteins are localized in both the nucleus and the cytoplasm. According to the Instability Index (II) analysis results, it was determined that LSD proteins were generally stable in structure, while GhLSD1, GhLSD6 and GhLSD7 proteins (II values\u0026thinsp;\u0026gt;\u0026thinsp;40) were determined to be unstable in structure (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCharacterization of Cotton LSD1 proteins\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"10\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003cp\u003eName\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLength\u003c/p\u003e \u003cp\u003e(aa*)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMW \u003csup\u003e*\u003c/sup\u003e(Da*)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epI*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGRAVY*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAI*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eII*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCharge\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eKSP*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eSubcellular Localization\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10608\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.161\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e68.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e61.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt\u003csup\u003e*\u003c/sup\u003e, Nuc\u003csup\u003e*\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17665\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.192\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e80.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e35.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e8.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e74.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e76.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e32.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e79.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e39.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e17666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.141\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e75.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e41.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10618\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e-0.169\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e68.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e64.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e18776\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e82.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e7.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15095\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e75.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e38.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e178881\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.116\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e77.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e31.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e6.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e15074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e79.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e39.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCyt, Nuc\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003csup\u003e*\u003c/sup\u003eMolecular Weight (MW), Dalton (Da), Kinase Specific Phosphorylation (KSP) regions, Isoelectric point (pI), Grand Average f Hydropathy (GRAVY), Aliphatic Index (AI), Instability Index (II) and the protein charge was estimated according to the amino acid composition. Cyt (Cytoplasm), Nuc (Nucleus).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDomain and motif analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eExamination of protein domain structures showed that GhLSD proteins contains two or three conserved LSD-type Zinc Finger (ZF) domains which is defined by CxxCxRxxLMYxxGASxVxCxxC. While nine of eleven GhLSD proteins contain three LSD-type ZF domains, only GhLSD1 and GhLSD7 proteins contain two LSD-type ZF domains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The secondary structure predicted for the GhLSD proteins was displayed above the multiple sequence alignments, revealing the presence of one or two α-helices in GhLSD3, GhLSD5, GhLSD8, GhLSD9, and GhLSD11, as well as a variable number of β-strands across all GhLSD proteins. In upland cotton, the conservation analysis of LSD-type ZF domains demonstrated strong sequence conservation across all GhLSD proteins. In particular, cysteine residues at positions 1, 4, 19, and 22 were highly conserved, while leucine (position 8), tyrosine (position 10), and glycine (position 11) were also consistently conserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore the diversity of GhLSD proteins, conserved motif analysis was conducted using the MEME online server. Ten distinct motifs (motif 1\u0026ndash;10) were identified. Among them, motif 1 and motif 2, corresponding to LSD-type ZF domains, were present in nearly all GhLSD genes,highlighting their conserved functional significance within the gene family (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCis-Regulatory Element Analysis\u003c/h2\u003e \u003cp\u003eThe promoter regions (2 kb upstream) of GhLSD genes were screened for cis-acting regulatory elements using the PlantCARE database. The results revealed that \u003cem\u003eGhLSD\u003c/em\u003e promoters harbor diverse cis-elements associated with plant growth and development, hormone signaling, stress responses, and light regulation at the transcriptional level (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Development-related motifs included AC-II (in \u003cem\u003eGhLSD4\u003c/em\u003e and \u003cem\u003eGhLSD10\u003c/em\u003e), Circadian (in \u003cem\u003eGhLSD8\u003c/em\u003e), the GCN4 motif (in \u003cem\u003eGhLSD1, GhLSD2, GhLSD3\u003c/em\u003e, and \u003cem\u003eGhLSD8\u003c/em\u003e), and RY elements (in \u003cem\u003eGhLSD6\u003c/em\u003e and \u003cem\u003eGhLSD7\u003c/em\u003e). Among hormone-responsive elements, the ERE motif (ethylene-responsive) was notably enriched, appearing four times in the \u003cem\u003eGhLSD4\u003c/em\u003e promoter, whereas the O2-site (involved in zein metabolism) was detected only in \u003cem\u003eGhLSD1\u003c/em\u003e. Numerous stress-responsive elements were identified, including MYB binding sites (present in all \u003cem\u003eGhLSD\u003c/em\u003e genes except \u003cem\u003eGhLSD11\u003c/em\u003e) and MYC binding sites, which were detected in varying copy numbers across all members. W-box motifs, recognized by WRKY transcription factors, were restricted to GhLSD2, GhLSD5, GhLSD8, GhLSD10, and GhLSD11, while the WUN motif (associated with wound responses) was found only in GhLSD1, GhLSD2, and GhLSD8. Light-responsive elements were also abundant, with Box 4 and G-box being the most prominent (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMultiple Sequence Alignment and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eTo investigate the evolutionary relationships and identify paralogous gene pairs among the eleven \u003cem\u003eGossypium hirsutum\u003c/em\u003e GhLSD genes, multiple sequence alignment of their CDS sequences was performed using CLUSTALW. Pairwise sequence identity analysis of the eleven GhLSD genes revealed five nearly identical paralogous pairs (GhLSD1\u0026ndash;GhLSD7, GhLSD2\u0026ndash;GhLSD8, GhLSD3\u0026ndash;GhLSD9, GhLSD4\u0026ndash;GhLSD10, and GhLSD5\u0026ndash;GhLSD11) with \u0026gt;\u0026thinsp;97% identity, indicating recent duplication events. Other gene pairs exhibited moderate (80\u0026ndash;85%) or low (37\u0026ndash;46%) identity, reflecting more divergent evolutionary relationships within the gene family.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis revealed that the eleven LSD transcription factors of upland cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e) could be classified into three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Group I comprised GhLSD1, GhLSD3, GhLSD5, GhLSD7, GhLSD9, and GhLSD11; Group II included GhLSD2 and GhLSD8; while Group III consisted of GhLSD4, GhLSD6, and GhLSD10. To further elucidate the evolutionary history of the LSD gene family in upland cotton, multiple sequence alignments were performed using ClustalW with LSD proteins from \u003cem\u003eG. hirsutum\u003c/em\u003e (11 GhLSDs), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (6 AtLSDs), \u003cem\u003eG. raimondii\u003c/em\u003e (8 GrLSDs), and \u003cem\u003eG. arboreum\u003c/em\u003e (6 GaLSDs). Phylogenetic analysis of LSD proteins from \u003cem\u003eGossypium spp and A. thaliana\u003c/em\u003e revealed that the LSD gene family clustered into five distinct clades (Clade I\u0026ndash;V) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Most clades contained members from at three species, while Clade II contained only LSD proteins of \u003cem\u003eA. thalina\u003c/em\u003e. In most clades, GhLSDs were grouped closely with homologs from \u003cem\u003eG. raimondii\u003c/em\u003e and \u003cem\u003eG. arboreum\u003c/em\u003e, suggesting inheritance from both A- and D-genome progenitors. All GhLSD members of Group I were clustered within Clade V. Within Group III, GhLSD4 and GhLSD10 were clustered in Clade III, while the remaining member GhLSD6 were positioned in Clade I with homologs from \u003cem\u003eG. raimondii\u003c/em\u003e and \u003cem\u003eG. Arboreum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e zein metabolism).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein Structure Analysis\u003c/h2\u003e \u003cp\u003eAlphaFold3 was used to predict the tertiary structures 11 GhLSD proteins. In all GhLSD proteins, random coil accounted for the largest proportion ranged from 58.50% to 78.35, followed by extended strand (22.65\u0026ndash;36.69%). α-Helix structures which are detected on GhLSD3, GhLSD5, GhLSD8, GhLSD9, and GhLSD11 at the low proportines (2.23\u0026ndash;6.80%). (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). No Beta turn was found in GhLSD proteins. GhLSD proteins have a secondary structure largely composed of random coils and β-strands, with limited α-helices and no β-turns. These properties, along with their flexible, stable structures, suggest that they are proteins that confer stability (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe secondary structures of GhLSD proteins.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eα-Helix (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eExtended Strand (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBeta Turn (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRandom Coil (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e77.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e36.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e63.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e29.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e70.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e78.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e64.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e34.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e58.50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e31.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e68.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGhLSD11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e33.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e60.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Pattern Analysis Under Abiotic Stress\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eGhLSD\u003c/em\u003e genes in Leaves and roots under drought and salt stresses in the \u003cem\u003eGossypium hirsutum\u003c/em\u003e genotype, the expression patterns of 6 GhLSD genes\u0026mdash;including \u003cem\u003eGhLSD1, GhLSD2, GhLSD3, GhLSD4, GhLSD5\u003c/em\u003e and \u003cem\u003eGhLSD6\u003c/em\u003e, were examined using qRT-PCR analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Analysis of LSD genes Under Drought Stress\u003c/h2\u003e \u003cp\u003eThe expression profiling of \u003cem\u003eGhLSD\u003c/em\u003e genes revealed diverse and tissue-specific regulatory responses to drought stress. The expression levels of \u003cem\u003eGhLSD1\u003c/em\u003e showed significant upregulation, with peak expression at 8 h (2.24-fold in leaf, while the expression was decreased to 4.76-fold in leaf tissues at 2h. \u003cem\u003eGhLSD2\u003c/em\u003e was moderately upregulated in leaves at 8 h (1.48-fold) and 24 h (1.49-fold), but showed only mild induction at 4 h (0.89-fold) and 48 h (1.17-fold). In contrast, it was moderately downregulated in 5leaves at 2 h (1.72-fold). \u003cem\u003eGhLSD3\u003c/em\u003e exhibited strong upregulation (6.30-fold) in leaves at 8 h under drought stress, followed by moderate induction (2.39-fold) at 24 h. GhLSD4 was markedly upregulated (154.04-fold) in leaves at 8 h of drought exposure. GhLSD5 displayed strong upregulation (3.34-fold) in leaves at 8 h, but was slightly downregulated (1.53-fold) at 4 h under drought stress. GhLSD6 was highly upregulated (4.12-fold and 4.88-fold) in cotton leaves at 8 h and 24h following drought exposure, but displayed only a moderate induction (2.75-fold) under drought stress at 48h respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe expression levels of GhLSD1 showed significant upregulation, with peak expression at 48 h (40.44-fold) in root, while the expression was decreased to 13.33-fold in root tissues at 2h. GhLSD2 was moderately downregulated in roots at 24 h (1.36-fold). Stronger downregulation was observed in roots at 8 h (2-fold) and 48 h (2.38-fold). In roots, GhLSD3 was strongly downregulated (9.09-fold and 10-fold) at 2 h and 24 h, respectively. \u003cem\u003eGhLSD5\u003c/em\u003e was strongly upregulated (27.5-fold) following 48h of drought exposure in roots. In roots, \u003cem\u003eGhLSD5\u003c/em\u003e showed strong upregulation (27.5-fold) at 48 h, whereas it was highly downregulated (10-fold) at 4 h. \u003cem\u003eGhLSD6\u003c/em\u003e expression in drought-stressed roots peaked at 48 h, reaching a 19-fold upregulation compared to the control. \u003cem\u003eGhLSD6\u003c/em\u003e was moderately downregulated (2.77-fold) in roots at 4h under drought stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Analysis of LSD genes Under Salt Stress\u003c/h2\u003e \u003cp\u003eThe expression analysis of \u003cem\u003eGhLSD\u003c/em\u003e genes under salt stress revealed distinct temporal and tissue-specific patterns. \u003cem\u003eGhLSD1\u003c/em\u003e showed moderate upregulation in leaves at 8 h (1.38-fold), but was moderately downregulated at early time points (2 h, 0.44-fold; 4 h, 0.63-fold). \u003cem\u003eGhLSD2\u003c/em\u003e exhibited strong time-dependent downregulation in leaves (2 h, 0.61-fold; 4 h, 0.51-fold; 24 h, 0.31-fold; 48 h, 0.19-fold). \u003cem\u003eGhLSD3\u003c/em\u003e displayed dynamic regulation, with strong upregulation in leaves at early time points 2h and 8h but marked downregulation at certain time points, reflecting context-dependent regulatory functions. \u003cem\u003eGhLSD4\u003c/em\u003e was early induced in leaves at 2 h (1.61-fold), peaking at 4 h (6.2-fold). \u003cem\u003eGhLSD5\u003c/em\u003e exhibited moderate upregulation in leaves at 2 h (2.42-fold) and 8 h (1.93-fold). Moderate inductions (2.42-fold and 1.93-fold) of \u003cem\u003eGhLSD5\u003c/em\u003e was observed leaves under salt stress at 2h and 8h respectively. \u003cem\u003eGhLSD6\u003c/em\u003e showed slight to moderate downregulation in leaves (2 h, 0.61-fold; 4 h, 0.45-fold; 24 h, 0.85-fold; 48 h, 0.56-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eGhLSD1\u003c/em\u003e was moderately downregulated at early time points (2 h, 0.44-fold; 4 h, 0.63-fold), while in roots it peaked at 24 h (5.68-fold) and was strongly downregulated at 2 h (0.17-fold). \u003cem\u003eGhLSD2\u003c/em\u003e was strongly downregulated in roots at 2 h (0.20-fold) and 48 h (0.17-fold). \u003cem\u003eGhLSD3\u003c/em\u003e displayed dynamic regulation, with strong upregulation in roots at later stages 24h and 48h, but marked downregulation at certain time points, reflecting context-dependent regulatory functions. \u003cem\u003eGhLSD3\u003c/em\u003e was strongly downregulated (0.16-fold) (6.25) and (0.31-fold) (3.22) in roots at 2h and 8h under salt stress. \u003cem\u003eGhLSD3\u003c/em\u003e was highly downregulated (0.05-fold) (18.18) in roots at 4h under salt stress. In roots, \u003cem\u003eGhLSD4\u003c/em\u003e showed moderate downregulation at 2 h (0.46-fold) followed by strong upregulation at 4 h (7.39-fold). \u003cem\u003eGhLSD5\u003c/em\u003e exhibited dramatic upregulation in roots at 24 h (131.20-fold) and 48 h (216.88-fold). \u003cem\u003eGhLSD5\u003c/em\u003e was strongly upregulated (131.20-fold and 216.88-fold) in roots at 24h and 48h under salt stress. \u003cem\u003eGhLSD5\u003c/em\u003e showed extreme upregulation in roots at later stages (24\u0026ndash;48 h), suggesting a critical role in root-specific salt stress adaptation. \u003cem\u003eGhLSD6\u003c/em\u003e was strongly downregulated at 8 h (0.62-fold), and highly upregulated at 48 h (5.47-fold). \u003cem\u003eGhLSD6\u003c/em\u003e was highly upregulated peak in roots at 48 h, highlighting its late-stage role in root stress response (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, these results indicate that the \u003cem\u003eGhLSD\u003c/em\u003e gene family operates in a coordinated, tissue- and time-dependent manner under salt stress, with \u003cem\u003eGhLSD1\u003c/em\u003e, \u003cem\u003eGhLSD4\u003c/em\u003e, \u003cem\u003eGhLSD5\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e emerging as key positive regulators, whereas \u003cem\u003eGhLSD2\u003c/em\u003e may act as a repressor (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eGossypium hirsutum\u003c/em\u003e (Upland cotton) is an allotetraploid species (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;52) composed of two subgenomes, A and D, which originated from the hybridization of \u003cem\u003eG. arboreum\u003c/em\u003e (A genome) and \u003cem\u003eG. raimondii\u003c/em\u003e (D genome) (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Its genome size is estimated at 2.25\u0026ndash;2.43 Gb and encodes approximately 76,943 genes. The Lesion Simulating Disease (LSD) transcription factor acts as a negative regulator of programmed cell death (PCD) by inhibiting EDS1 and PAD4, and contributes to plant responses to salinity and drought stress (Dietrich et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). LSD proteins contain a conserved zinc-finger (ZF) motif (CxxCRxxLMYxxGASxVxCxxC) (Dietrich et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), enabling DNA/protein interactions and integration of ROS, salicylic acid (SA), and ethylene (ET) signaling to modulate cell death and acclimation.\u003c/p\u003e \u003cp\u003eGenome-wide identification and functional characterization of LSD transcription factors have been reported in several model species, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, and \u003cem\u003eZea mays\u003c/em\u003e (Jiang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In this study, we identifed eleven \u003cem\u003eGhLSD\u003c/em\u003e genes in the \u003cem\u003eG. hirsutum\u003c/em\u003e genome and found to be unevenly distributed across 5 out of the 13 chromosomes (chromosomes 5, 6, 8, 10, and 12) in both the A and D subgenomes. The number of LSD genes vary among species. For example, 6 MsLSD in \u003cem\u003eM. Sativa\u003c/em\u003e, 12 AtLSD in \u003cem\u003eA. Thaliana\u003c/em\u003e, 20\u003cem\u003eZmLSD\u003c/em\u003e in \u003cem\u003eZea mays\u003c/em\u003e and 18 \u003cem\u003eGmLSD\u003c/em\u003e in \u003cem\u003eGlycine max\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eComprehensive characterization of the \u003cem\u003eGossypium hirsutum\u003c/em\u003e LSD (GhLSD) gene family provides important insights into their genomic organization, structural variation, and potential functional roles in cotton. The CDS lengths of GhLSD genes varied from 294 to 513 bp, and exon numbers ranged from four to eight, suggesting moderate structural diversity within the family. The observed correlation between CDS length and exon number indicates evolutionary constraints that maintain functional domains while allowing flexibility in gene structure. Similar structural variation has been reported in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and soybean (4 to 6 exon and supporting the idea that exon\u0026ndash;intron organization contributes to gene diversification and functional specialization of LSD family members (Cabreira et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). On the other hand, most alpha alpha MsLSD genes contain a limited number of exons, one or four. (Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e),\u003c/p\u003e \u003cp\u003eThe chromosomal distribution of \u003cem\u003eGhLSD\u003c/em\u003e genes revealed localization on ten of 26 chromosomes, with an almost balanced presence between the A and D subgenomes. The occurrence of duplicated gene pairs on homoeologous chromosomes, such as GhLSD1 on chromosome A5 and its paralog GhLSD7 on chromosome D5, together with the close localization of GhLSD6 and GhLSD7 on chromosome D5, indicates that both segmental and tandem duplication events have played a pivotal role in the expansion of the GhLSD gene family. The allotetraploid nature of upland cotton further explains the presence of multiple paralogous gene pairs with high sequence identity (\u0026gt;\u0026thinsp;97%), which likely originated from recent duplication events. Comparable expansion patterns were observed in \u003cem\u003eMedicago sativa\u003c/em\u003e (6 MsLSD genes) and \u003cem\u003eGlycine max\u003c/em\u003e (18 LSD genes) indicating that polyploidy and duplication events are common mechanisms underlying the expansion of LSD transcription factor families in plants (Cabreiara et al. 2013).\u003c/p\u003e \u003cp\u003eAt the protein level, GhLSD members exhibited lengths of 97\u0026ndash;180 amino acids, with predicted molecular weights ranging from 10.6 to 18.7 kDa. Most proteins had basic isoelectric points, consistent with their proposed roles in DNA or protein binding. Variations in hydrophobicity and instability indices suggest that different GhLSD proteins may adopt specialized cellular roles, with GhLSD1, GhLSD6, and GhLSD7 being less stable and potentially subject to post-translational regulation. Similar physicochemical diversity has been reported in LSD proteins of maize (\u003cem\u003eZea mays\u003c/em\u003e), where variation in hydrophilicity was suggested to reflect adaptive subfunctionalization (Jiang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Subcellular localization predictions identified nuclear and cytoplasmic, implying functional diversity in transcriptional regulation and stress signaling. The nuclear localization is consistent with their role as transcription factors, whereas their cytoplasmic presence suggests potential additional functions, such as acting as scaffold proteins or participating in signaling cascades prior to nuclear translocation. Importantly, consistent with previous findings, (Czarnocka et. al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that LSD1 not only functions as a transcriptional regulator but also acts as a scaffold protein, thereby supporting the notion that LSD proteins exhibit dual roles within the cell. Taken together, these results suggest that GhLSD proteins may function as condition-dependent scaffold proteins in the cytoplasm and as transcriptional regulators in the nucleus, reflecting a versatile role in stress response and developmental regulation in cotton.\u003c/p\u003e \u003cp\u003eDomain analysis confirmed the presence of highly conserved LSD-type zinc finger (ZF) motifs(CxxCRxxLMYxxGASxVxCxxC), also member of a cysteine-2/cysteine-2-class (C2C2), which were maintained across all members, reinforcing their essential role in programmed cell death (PCD) regulation. Nine GhLSD proteins contained three LSD-type zinc finger (ZF) domains, whereas GhLSD1 and GhLSD7 harbored only two, suggesting potential divergence in their DNA-binding affinity or target specificity. Typically, LSD proteins possess three ZF domains; however, the number of domains can vary among different species. For instance, in maize, ZmLSD proteins have been reported to contain between one and three ZF domains, indicating structural diversification within the family across plant lineages (Jiang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) Conservation of cysteine residues at positions 1, 4, 19, and 22 across all GhLSD proteins, forming the typical C2C2-type ZF structure, highlights their importance in tertiary structure and structural stability, and protects cell from oxidative damage, redox-mediated regulation as well (Cabreira et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In conclusion, GhLSD proteins may play a similar role in managing oxidative stress triggered by drought and salinity.\u003c/p\u003e \u003cp\u003eMotif and cis-regulatory element analyses further supported functional conservation and specialization. The presence of conserved motifs (motif 1 and 2) across almost all GhLSD proteins reflects their core regulatory role, while additional motifs may contribute to lineage-specific adaptation. Promoter analysis revealed abundant stress- and hormone-responsive cis-elements, including MYB, MYC, ERE, and W-box motifs, which are known to regulate transcriptional reprogramming during abiotic stresses. In particular, the enrichment of ethylene-responsive elements (EREs) in GhLSD4 suggests a strong link between LSD proteins and ethylene-mediated signaling pathways, which play central roles in stress adaptation and PCD regulation. These findings are consistent with earlier reports in \u003cem\u003eGlycine max\u003c/em\u003e and \u003cem\u003eMedicago sativa\u003c/em\u003e, where LSD genes were shown to integrate reactive oxygen species (ROS), salicylic acid (SA), and ethylene (ET) signaling during stress responses (Cabreira et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe phylogenetic clustering demonstrates that the LSD gene family is highly conserved across cotton species and Arabidopsis, with distinct subgrouping into five clades. The close association of GhLSDs with both \u003cem\u003eG. arboreum\u003c/em\u003e and \u003cem\u003eG. raimondii\u003c/em\u003e orthologs indicates that \u003cem\u003eG. hirsutum\u003c/em\u003e retained most ancestral copies from its diploid progenitors following polyploidization. This conservation suggests that LSD genes play fundamental roles in stress adaptation and development. The absence of \u003cem\u003eG. hirsutum\u003c/em\u003e members in Clade II likely reflects gene loss, pseudogenization, or significant divergence, highlighting evolutionary flexibility within the family. Furthermore, the distribution of GhLSDs across different clades suggests functional diversification, with some members potentially undergoing subfunctionalization or neofunctionalization to meet the adaptive demands of upland cotton. These evolutionary patterns are consistent with previous findings that LSD proteins regulate stress signaling and development, implying that different clades may correspond to distinct functional specializations\u003c/p\u003e \u003cp\u003eProtein structural predictions revealed that GhLSD proteins are predominantly composed of random coils and β-strands, with limited α-helices and no β-turns. Such structural flexibility is typical of regulatory proteins that interact with multiple partners within stress signaling networks. The thermostability indicated by aliphatic index values suggests that GhLSD proteins remain stable under variable environmental conditions, a property that has been similarly reported for Alfaalfa and maize LSD proteins (Jiang et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTaken together, these findings indicate that the LSD transcription factor family in cotton has undergone expansion and diversification through polyploidy and gene duplication while maintaining strong conservation of core domains essential for PCD regulation. The integration of diverse promoter elements, structural variation, and predicted functional divergence highlights the potential of GhLSD genes as key regulators of stress adaptation in cotton. Given the established roles of LSD genes in regulating PCD, ROS signaling, and abiotic stress tolerance in other crops, the GhLSD family represents promising candidates for functional validation studies. Future research focusing on expression profiling under drought and salinity stress, as well as genome-editing approaches such as CRISPR/Cas9, may provide valuable strategies for improving cotton stress tolerance.\u003c/p\u003e \u003cp\u003eThe expression profiling of GhLSD genes under drought and salt stresses provides novel insights into their temporal and tissue-specific regulatory functions in \u003cem\u003eG. hirsutum\u003c/em\u003e. Overall, the results demonstrate that the GhLSD gene family operates in a highly dynamic, coordinated, and context-dependent manner, with distinct members contributing to either activation or repression of stress responses in leaves and roots.\u003c/p\u003e \u003cp\u003eThe drought-responsive expression profiles revealed a clear division of regulatory roles among \u003cem\u003eGhLSD\u003c/em\u003e genes. In leaves, \u003cem\u003eGhLSD1, GhLSD3, GhLSD4, GhLSD5, and GhLSD6\u003c/em\u003e were strongly induced, particularly at 8 h, indicating an early activation of defense and acclimation mechanisms. Notably, \u003cem\u003eGhLSD4\u003c/em\u003e exhibited a dramatic 154-fold upregulation, positioning it as a potential master regulator of drought-induced signaling pathways in cotton. Similar strong induction of LSD genes under drought stress has been observed in \u003cem\u003eArabidopsis\u003c/em\u003e and soybean, where LSD family members were shown to integrate ROS and hormone signals to fine-tune stress responses (Cabreira et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Szechyn\u0026acute;ska-Hebda et al. 2016)\u003c/p\u003e \u003cp\u003eIn roots, \u003cem\u003eGhLSD1, GhLSD5\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e displayed robust late-stage induction (40.44-fold, 27.5-fold, and 19-fold, respectively, at 48 h), suggesting that these genes may contribute to sustained drought adaptation mechanisms, possibly by modulating root growth or enhancing antioxidant defenses. In contrast, \u003cem\u003eGhLSD2\u003c/em\u003e and \u003cem\u003eGhLSD3\u003c/em\u003e were consistently downregulated in roots, implying a negative regulatory role, potentially acting to prevent excessive PCD activation under prolonged water deficit. Such contrasting expression patterns between leaves and roots highlight tissue-specific functional diversification, consistent with earlier reports that LSD homologs act as context-dependent regulators balancing survival and cell death in plants\u003c/p\u003e \u003cp\u003eTaken together, \u003cem\u003eGhLSD1, GhLSD4, and GhLSD6\u003c/em\u003e appear to function as positive regulators of drought tolerance, while GhLSD2 may act as a repressor. The strong induction of \u003cem\u003eGhLSD4\u003c/em\u003e in leaves and \u003cem\u003eGhLSD6\u003c/em\u003e in roots emphasizes their importance as candidate genes for improving drought resilience in cotton.\u003c/p\u003e \u003cp\u003eUnder salt stress, the GhLSD family again exhibited diverse and tissue-dependent regulation. In leaves, GhLSD4 and GhLSD5 showed early induction (up to 6.2-fold at 4 h for GhLSD4, and 2.42-fold for GhLSD5 at 2 h), suggesting their involvement in rapid signaling and protective responses against ionic and osmotic stress. \u003cem\u003eGhLSD3\u003c/em\u003e showed transient upregulation at early time points but was subsequently downregulated, indicating a tightly controlled, context-dependent role. In contrast, \u003cem\u003eGhLSD2\u003c/em\u003e was consistently downregulated, supporting its putative role as a negative regulator during salt stress.\u003c/p\u003e \u003cp\u003eIn roots, striking differences emerged. \u003cem\u003eGhLSD5\u003c/em\u003e exhibited extremely high induction (131-fold at 24 h and 217-fold at 48 h), suggesting it plays a central role in root-specific salt tolerance, likely through regulation of ion homeostasis, osmotic adjustment, or ROS detoxification. GhLSD6 also showed late-stage induction (5.47-fold at 48 h), pointing to a supportive role in root acclimation. By contrast, GhLSD2 remained strongly repressed across time points, further strengthening its proposed role as a negative regulator. Similar patterns of late-stage activation of LSD homologs have been observed in rice, where LSD family members were implicated in long-term adaptive responses to salinity (Sun et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eComparing the two stress conditions reveals both overlapping and divergent roles of GhLSD genes. \u003cem\u003eGhLSD1, GhLSD4, GhLSD5, and GhLSD6\u003c/em\u003e consistently emerged as positive regulators under both drought and salt stresses, albeit with tissue- and time-dependent differences. GhLSD4 functioned predominantly in leaves, displaying rapid and strong induction under drought and salt stresses, while \u003cem\u003eGhLSD5\u003c/em\u003e and \u003cem\u003eGhLSD6\u003c/em\u003e were highly responsive in roots, particularly under prolonged stress exposure. \u003cem\u003eGhLSD2\u003c/em\u003e, conversely, was consistently downregulated in both conditions, suggesting it acts as a suppressor of stress responses, possibly to prevent excessive or uncontrolled PCD. The contrasting regulation of \u003cem\u003eGhLSD3\u003c/em\u003e across tissues and stress conditions suggests a dual, context-dependent role in fine-tuning stress signaling.\u003c/p\u003e \u003cp\u003eThese findings highlight that the GhLSD gene family contributes to cotton stress adaptation through a coordinated network of activators and repressors that function in both early and late phases of stress. The strong upregulation of GhLSD4, GhLSD5, and GhLSD6 suggests that these genes are promising targets for genetic improvement of drought and salinity tolerance in cotton. Their tissue-specific expression further emphasizes the importance of considering organ-level regulation in stress biology.\u003c/p\u003e \u003cp\u003eFuture functional validation using overexpression and CRISPR/Cas9 knockout strategies will be essential to clarify the precise regulatory roles of GhLSD genes and to exploit them for crop improvement. Given the integration of LSD proteins with ROS, SA, and ET signaling, further investigation into their upstream regulation and downstream target genes will provide critical insights into their role in stress resilience mechanisms in cotton.\u003c/p\u003e "},{"header":"Conclusion and Recommendation","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003cp\u003eThe comprehensive expression profiling of the \u003cem\u003eGhLSD\u003c/em\u003e gene family under drought and salinity stress revealed that these transcription factors function in a dynamic, tissue-specific, and time-dependent manner to regulate stress responses in \u003cem\u003eGossypium hirsutum\u003c/em\u003e. Among the six genes analyzed, \u003cem\u003eGhLSD1, GhLSD4, GhLSD5\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e consistently exhibited strong induction under both drought and salt treatments, highlighting their potential roles as positive regulators of abiotic stress tolerance. In particular, \u003cem\u003eGhLSD4\u003c/em\u003e (in leaves) and \u003cem\u003eGhLSD5\u003c/em\u003e (in roots) demonstrated exceptionally high expression peaks, suggesting specialized roles in tissue-specific stress adaptation. \u003cem\u003eGhLSD6\u003c/em\u003e also showed consistent late-stage upregulation, pointing to its involvement in sustained stress responses. Conversely, \u003cem\u003eGhLSD2\u003c/em\u003e was generally downregulated in both tissues under drought and salinity, suggesting a repressive role in stress signaling pathways. \u003cem\u003eGhLSD3\u003c/em\u003e displayed variable regulation, alternating between induction and suppression depending on tissue type and exposure duration, indicating a context-dependent function.\u003c/p\u003e \u003cp\u003eTaken together, these findings emphasize that the \u003cem\u003eGhLSD\u003c/em\u003e family contributes to stress acclimation through a finely tuned regulatory network rather than uniform responses. The strong induction of \u003cem\u003eGhLSD4, GhLSD5\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e highlights them as promising candidate genes for further functional studies and potential genetic improvement strategies to enhance drought and salinity tolerance in cotton.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding: This work was supported by the Scientific Research Projects Coordination Unit of Istanbul University (FYL-2024-40510)\u003c/p\u003e\n\u003cp\u003eConflict of interest/Competing interests: The authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate: Not applicable\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable\u003c/p\u003e\n\u003cp\u003eData availability: Data are included within the article.\u003c/p\u003e\n\u003cp\u003eMaterials availability: Materials are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003eCode availability: Not applicable\u003c/p\u003e\n\u003cp\u003eAuthor contribution: C.U. designed the study, analyzed the data and wrote the manuscript. 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J Integr Plant Biol 55:570\u0026ndash;571. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/jipb.12076\u003c/span\u003e\u003cspan address=\"10.1111/jipb.12076\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Lesion Simulating Disease, abiotic stress, salinity, transcription factors, stress tolerance, zinc finger proteins","lastPublishedDoi":"10.21203/rs.3.rs-9441383/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9441383/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCotton (\u003cem\u003eGossypium hirsutum)\u003c/em\u003e is a major natural fiber crop whose productivity is severely constrained by drought and salinity stress. Understanding the molecular mechanisms underlying stress responses is critical for developing stress-resilient cultivars. The Lesion Simulating Disease (LSD) transcription factor family plays a crucial role in regulating programmed cell death and stress signaling pathways in plants. In this study, a genome-wide analysis identified eleven \u003cem\u003eGhLSD\u003c/em\u003e genes \u003cem\u003ein G. hirsutum\u003c/em\u003e, which were unevenly distributed across chromosomes and contained conserved LSD-type zinc finger domains, suggesting their involvement in stress-responsive regulatory networks. Expression profiling of six selected \u003cem\u003eGhLSD\u003c/em\u003e genes under drought and salinity conditions revealed complex, tissue-specific, and time-dependent expression patterns. Under drought stress, \u003cem\u003eGhLSD1, GhLSD4\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e were significantly upregulated, indicating their potential roles as positive regulators of stress tolerance, whereas \u003cem\u003eGhLSD2\u003c/em\u003e was consistently downregulated, suggesting a possible negative regulatory function. Similarly, under salinity stress, \u003cem\u003eGhLSD4, GhLSD5\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e exhibited strong and sustained induction, highlighting their importance in long-term and tissue-specific salt stress responses. In contrast, the persistent downregulation of \u003cem\u003eGhLSD2\u003c/em\u003e further supports its potential role as a negative regulator in salinity stress adaptation. Overall, these findings demonstrate that the \u003cem\u003eGhLSD\u003c/em\u003e gene family functions in a coordinated and dynamic manner in response to abiotic stress, with \u003cem\u003eGhLSD4, GhLSD5\u003c/em\u003e, and \u003cem\u003eGhLSD6\u003c/em\u003e emerging as promising candidate genes for improving drought and salinity tolerance in cotton through molecular breeding approaches.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Characterization and Expression Profiling of the LSD Gene Family in Cotton (Gossypium hirsutum) Under Drought and Salt Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-08 16:34:32","doi":"10.21203/rs.3.rs-9441383/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-11T00:32:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T03:42:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"162505179758033453694923138617285467972","date":"2026-05-05T12:20:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"24085744797103119683384938992195056523","date":"2026-05-02T23:04:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87931886210608153920482984096010852150","date":"2026-05-02T06:08:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"32099792354394948594933181037306017699","date":"2026-05-02T04:30:53+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T11:58:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"294022534972491682519780410031322553136","date":"2026-04-30T11:44:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-30T10:26:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-17T02:57:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-17T02:57:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Functional \u0026 Integrative Genomics","date":"2026-04-16T18:28:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"functional-and-integrative-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fige","sideBox":"Learn more about [Functional \u0026 Integrative Genomics](http://link.springer.com/journal/10142)","snPcode":"10142","submissionUrl":"https://submission.nature.com/new-submission/10142/3","title":"Functional \u0026 Integrative Genomics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0ccfdf62-83e8-4881-9055-554820706053","owner":[],"postedDate":"May 8th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-11T00:32:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-10T03:42:18+00:00","index":29,"fulltext":""},{"type":"reviewerAgreed","content":"162505179758033453694923138617285467972","date":"2026-05-05T12:20:15+00:00","index":28,"fulltext":""},{"type":"reviewerAgreed","content":"24085744797103119683384938992195056523","date":"2026-05-02T23:04:08+00:00","index":27,"fulltext":""},{"type":"reviewerAgreed","content":"87931886210608153920482984096010852150","date":"2026-05-02T06:08:56+00:00","index":25,"fulltext":""},{"type":"reviewerAgreed","content":"32099792354394948594933181037306017699","date":"2026-05-02T04:30:53+00:00","index":24,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-30T11:58:02+00:00","index":22,"fulltext":""},{"type":"reviewerAgreed","content":"294022534972491682519780410031322553136","date":"2026-04-30T11:44:43+00:00","index":20,"fulltext":""},{"type":"reviewersInvited","content":"14","date":"2026-04-30T10:26:38+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-11T00:39:45+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-08 16:34:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9441383","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9441383","identity":"rs-9441383","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}