Overexpression and silencing of the cotton GhABA2 gene reveal its role in salt stress tolerance

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To explore the function of GhABA2 in cotton salt tolerance, we conducted bioinformatic analyses, gene overexpression, and virus-induced gene silencing (VIGS) experiments. Bioinformatic examination revealed that the promoter region of the GhABA2 gene contains multiple cis -acting elements, including those responsive to ABA, light, and various stress signals. Overexpression of GhABA2 in Arabidopsis resulted in significantly increased seed germination rate, root length, leaf relative water content (RWC), catalase (CAT) activity, peroxidase (POD) activity, and proline (Pro) content under salt stress conditions, while malondialdehyde (MDA) content was markedly reduced. Conversely, silencing GhABA2 gene in cotton via virus-induced gene silencing (VIGS) led to significant decreases in ABA content, RWC, superoxide dismutase (SOD) activity, POD activity, and Pro content, along with a significant increase in MDA content. These findings provide important insights into the mechanism of GhABA2 in mediating cotton salt stress response and highlight its potential as a genetic target for breeding of salt-tolerant cotton varieties. Cotton ABA2 Salt stress Short-chain dehydrogenase/reductase (SDR) ABA signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. INTORDUCTION Soil salinization, or salt stress, denotes the detrimental impact of elevated salt concentrations in the growth environment—such as in soil or irrigation water—on plant growth, development, and physiological metabolism. As a major abiotic stress, it significantly constrains agricultural productivity and threatens global food security [ 1 ]. This issue is particularly acute in the arid northwestern regions of China, where low precipitation, high evaporation rates, and saline groundwater contribute to progressive soil salinization. This issue is particularly acute in the arid northwestern regions of China, where low precipitation, high evaporation rates, and saline groundwater contribute to progressive soil salinization [ 2 ]. Cotton, a key economic crop in China, is predominantly cultivated in Xinjiang. In 2024, Xinjiang produced approximately 5, 112 million metric tons of cotton, accounting for 91% of the national total, underscoring its pivotal role in the Chinese cotton industry. However, soil salinization affects nearly 10% of China's arable land, with northwestern regions being especially vulnerable, which poses a serious threat to cotton production[ 3 ]. Notably, saline soils constitute 37.72% of the total cultivated area in Xinjiang's irrigation zones, posing a substantial challenge to local cotton production [ 4 ]. ABA, a critical phytohormone and signaling molecule, plays a central role in mediating plant adaptation to salt stress. It regulates salinity responses by activating ABA-dependent signaling pathways that induce stress-responsive gene expression and through crosstalk with other hormones [ 5 ]. For instance, ABA signals can trigger MAPK cascades via calcium signaling to enhance stress resistance [ 6 ]. In soybean, GmSIN1 (salt-induced NAC1 ) is upregulated by ABA and promotes root growth and salinity tolerance. and promotes root growth and salinity tolerance [ 7 ]. ABA also interacts with auxin (IAA), gibberellin (GA), and cytokinin (CK) to modulate stress responses [ 8 ]. In maize, salt-induced ABA accumulation disrupts polar auxin transport by affecting ZmPIN1 localization, thereby suppressing lateral root formation [ 9 ]. In Arabidopsis , ABI4 overexpression enhances the transcription of NCED6 and GA2ox7 , increasing the ABA/GA ratio and improving salt tolerance [ 10 , 11 ]. Furthermore, ABA suppresses cytokinin biosynthesis via MYB2 , increasing ABA sensitivity and reducing shoot growth as an adaptive mechanism [ 12 ]. Similarly, in tomato, salt stress elev ABA levels while reducing CK content [ 13 ]. These studies collectively underscore the importance of ABA signaling and hormonal interplay in plant salt adaptation. Besides signaling, the ABA biosynthesis pathway is crucial for salt tolerance. The key enzyme encoded by AtABA2 in Arabidopsis —a short-chain dehydrogenase/reductase (SDR1)—catalyzes the conversion of xanthoxin to abscisic aldehyde, a pivotal step in ABA biosynthesis [ 14 , 15 ]. Overexpression of AtABA2 increases endogenous ABA levels and enhances salt tolerance [ 16 ].The AtLEW2 mutant, which exhibits elevated ABA, proline, and soluble sugars, shows upregulated SDR1 expression and improved salt resistance [ 17 ]. Homologs of SDR1 have been identified in halophytes and functionally validated in crops such as sugar beet, where SDR1 overexpression improves salinity tolerance by optimizing physiological responses and suppressing ROS accumulation [ 18 , 19 ]. These findings highlight the conserved role of ABA2 / SDR1 in salt stress adaptation across species. Although the function of the ABA2 gene has been well characterized in model plants and some crops, its role in cotton ( GhABA2 ) remains unexplored. This study aims to bridge this gap through comprehensive bioinformatic analysis of GhABA2 , including protein structure prediction, domain analysis, promoter cis -element profiling, and phylogenetic reconstruction. We further generated GhABA2 -overexpressing Arabidopsis lines and silenced GhABA2 in cotton via VIGS. Comparative phenotypical and physiological assessments under salt stress were conducted to elucidate the contribution of GhABA2 to salt tolerance. Our results provide mechanistic insights into the salt stress response in cotton and identify GhABA2 as a promising genetic target for breeding salt-resistant cotton varieties. 2. METHODS 2.1. Cloning and Bioinformatic Analysis of the GhABA2 Gene The coding sequence (CDS) of GhABA2 ( Ghi_A13G00706 ) was retrieved from the CottonMD database ( https://yanglab.hzau.edu.cn/CottonMD ). Gene-specific primers were designed (Table S1 ) , The target GhABA2 sequence was amplified via polymerase chain reaction (PCR). The PCR product was purified and subsequently sequenced (Shanghai Sangon Biotech) to verify the integrity and accuracy of the amplified fragment. A complete and error-free GhABA2 CDS was thereby obtained. Bioinformatic analyses were performed to characterize the GhABA2 gene and its encoded protein. Properties evaluated included physicochemical parameters, phosphorylation sites, hydrophobicity and hydrophilicity profiles, subcellular localization, secondary and tertiary structures, phylogenetic relationships, and cis-regulatory elements. The tools and online resources used for these analyses are summarized in Table 1 . Table 1 Bioinformatic Analysis Software Software Website Functionality Prot Param http://web.expasy.org/cgi-bin/protparam/protparam Physicochemical property analysis NetPhos 3.1 http://www.cbs.dtu.dk/services/NetPhos Phosphorylation site analysis Prot Scale http://www.expasy.org/tools/protscale.html Hydrophobicity/hydrophilicity analysis PSORT http://www.genscript.com/cgi-bin/tools/psort2.pl Subcellular localization analysis SOPMA https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl Secondary structure analysis SWISS-MODEL http://swissmodel.expasy.org Tertiary structure analysis DNAMAN - Multiple sequence alignment MEGA11.0.13 - Phylogenetic tree construction Plantcare https://bioinformatics.psb.ugent.be Cis-acting element analysis 2.2. Generation of GhABA2 -Overexpressing Transgenic Arabidopsis Lines The amplified GhABA2 fragment was cloned into the restriction-digested pCAMBIA-2300 vector to generate the recombinant plasmid pCAMBIA-2300- GhABA2 . This construct was introduced into Agrobacterium tumefaciens strain GV3101 and subsequently transformed into Arabidopsis thaliana ecotype Col-0 via the floral dip method. Transformants were selected on medium containing 50 µg/mL kanamycin and verified by PCR. The expression level of GhABA2 in transgenic plants was quantified using quantitative real-time PCR (qRT-PCR). Homozygous T 3 lines exhibiting high GhABA2 expression were selected for further functional studies. 2.3. Evaluation of Salt Stress Tolerance, Root Elongation, and Germination Rate in Transgenic Arabidopsis Germination Assay under Salt Stress: Seeds of homozygous T3 transgenic and wild-type (WT) Arabidopsis were surface-sterilized with 75% (v/v) ethanol for 2 min, rinsed three times with sterile water, treated with 5% (v/v) NaClO for 5 min, and then washed five times with sterile water. The sterilized seeds were sown on 1/2 MS solid medium supplemented with 0, 100, 150, or 200 mmol/L NaCl [ 20 ]. After stratification at 4°C for 24 h to break dormancy, the plates were transferred to a growth chamber set at 22 ± 1°C under a 16/8 h light/dark cycle. Germination rates were recorded after 10 days. Root Elongation Assay under Salt Stress: Surface-sterilized T3 transgenic and wild-type seeds were germinated on 1/2 MS solid medium for 4 days. Uniform seedlings were then transferred to vertical plates containing 1/2 MS medium with 0, 100, 150, or 200 mmol/L NaCl. Root length was measured after 4 days of vertical growth [ 20 ]. Phenotypic Evaluation of Salt Stress Tolerance: Fourteen-day-old soil-grown seedlings were subjected to salt stress by irrigation with 300 mM NaCl solution every 3 days for 9 days. Biochemical analyses were conducted after 6 days of treatment, and phenotypic observations were recorded on day 9. 2.4. Cotton Materials and Growth Conditions The seeds of the Gossypium hirsutum variety Zhongmian 113 were provided by Xinjiang Zhongmian Seed Industry Co., Ltd. (Aksu City, Xinjiang, China).Seeds were sterilized by immersion in 75% (v/v) ethanol for 5 min, followed by three rinses with sterile distilled water. Subsequently, seeds were treated with 1% (v/v) NaClO for 10 min and washed thoroughly with sterile distilled water. The sterilized seeds were placed between moist sterile filter papers and incubated at 28°C for 2 days to promote germination. Germinated seedlings were transplanted into pots containing a mixed substrate of nutrient soil, black soil, and vermiculite (3:1:1, v/v/v). Plants were grown in a greenhouse under controlled conditions: 25°C (day)/22°C (night), with a 16 h light/8 h dark photoperiod. 2.5. Generation of GhABA2 -Silenced Cotton Plants via VIGS A 400-bp unique fragment of GhABA2 was selected as the silencing target. A gene-specific primer pair was designed to amplify this fragment from cotton cDNA, and the resulting PCR product was cloned into the TRV2 vector to generate the TRV2: GhABA2 construct. The recombinant TRV2: GhABA2 , along with empty TRV1 and TRV2 vectors, were individually transformed into Agrobacterium tumefaciens strain GV3101. Bacterial cultures containing TRV1 were mixed separately with those harboring TRV2 (empty vector control), TRV2: GhCLA (positive control; CLA silencing induces an albino phenotype), or TRV2: GhABA2 . The mixtures were infiltrated into the cotyledons of cotton seedlings. Plants infiltrated with TRV1 + TRV2 were designated as negative controls (TRV2:00), while those infiltrated with TRV1 + TRV2: GhABA2 were considered GhABA2 -silenced plants (TRV2: GhABA2 ). Silencing efficiency was validated by the emergence of an albino phenotype in TRV2: GhCLA positive control plants. 2.6 RNA Extraction and qRT-PCR Analysis Total RNA was extracted from leaves of TRV2:00 and TRV2: GhABA2 plants using the Plant Polysaccharide & Polyphenol RNA Kit (FOREGENE, Chengdu, China), following the manufacturer’s protocol. RNA quality was verified by agarose gel electrophoresis, and concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized with the FOREGENE Master Premix RT Easy™ II Kit. qRT-PCR was performed using Real Time PCR Easy™-SYBR Green I reagents on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The thermal cycling conditions consisted of an initial denaturation at 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The GhHis3 gene was used as an internal control for normalization. Relative expression levels were calculated using the 2 −ΔΔCT method. Three biological replicates were performed to confirm efficient silencing of GhABA2 . Cotton plants showing significant downregulation of GhABA2 were selected for further experiments. 2.7. Salt Stress Treatment of Cotton Plants Cotton plants at the three-true-leaf stage with confirmed high silencing efficiency were transferred to a hydroponic system and acclimatized for two days. Salt stress was subsequently applied by irrigating the plants with 300 mM NaCl solution. Phenotypic responses were documented after 7 days of treatment. Leaf samples from both TRV2:00 (control) and TRV2: GhABA2 (silenced) plants were collected at the end of the stress period for subsequent analyses. 2.8. Measurement of Physiological and Biochemical Parameters in Transgenic Arabidopsis and VIGS Cotton A series of physiological and biochemical assays were conducted to evaluate oxidative stress and abiotic stress responses in both GhABA2 -overexpressing Arabidopsis and GhABA2 -silenced cotton plants. The following parameters were measured using commercial assay kits according to the manufacturers’ protocols: activities of POD, SOD, and CAT; content of MDA and Pro and endogenous ABA levels. Specifically, kits from Solarbio (Beijing, China) were used for POD, MDA, SOD, Pro, and CAT assays, while ABA content was quantified using a kit from GlidePharma (Wuhan, China). All measurements were performed on plant materials collected both before and after the application of salt stress. 3. RESULTS 3.1. Cloning and Sequence Verification of GhABA2 The coding sequence (CDS) of GhABA2 ( Ghi_A13G00706 ) was retrieved from the CottonMD database. Gene-specific primers were designed to amplify the target sequence by PCR. A clear band of the expected size was observed on an agarose gel. The purified PCR product was sequenced (Shanghai Sangon Biotech), and the resulting sequence was confirmed to be complete and accurate, matching the reference GhABA2 CDS. These results confirm the successful cloning of the full-length GhABA2 CDS for further functional characterization. Bioinformatic analysis indicated that the GhABA2 protein consists of 282 amino acids, with a molecular formula of C₁₃₁₇H₂₀₈₈N₃₇₄O₄₀₅S₁₂ and a molecular weight of approximately 30.03 kDa. The theoretical isoelectric point (pI) was predicted to be 6.1. The protein contains 23 basic (Arg + Lys) and 28 acidic (Asp + Glu) residues, suggesting a capacity for ionic interactions. The instability index was computed as 21.25, classifying the protein as stable. The aliphatic index was 93.62, indicating a high proportion of aliphatic amino acids that may enhance thermal stability. The grand average of hydropathicity (GRAVY) was 0.072. Subcellular localization predictions strongly suggested cytoplasmic localization. Hydrophobicity analysis revealed that the C-terminal region is predominantly hydrophobic, the N-terminal is hydrophilic, and the central region shows alternating hydrophobicity and hydrophilicity. Overall, the protein is hydrophilic, with one prominent hydrophilic peak exceeding a value of 2 (Fig. 1 A). Multiple potential phosphorylation sites were predicted with high confidence (scores ≈ 1) (Fig. 1 B), implying regulatory potential through phosphorylation. Secondary structure prediction indicated that α-helices (45.73%), random coils (28.25%), extended strands (17.07%), and β-turns (8.94%) constitute the protein (Fig. 1 C). The tertiary structure was modeled using 7o6p.1.A as a template (sequence identity: 51%) (Fig. 1 D). Multiple sequence alignment demonstrated that GhABA2 contains regions highly conserved among ABA2 homologs from diverse plant species, including Arabidopsis thaliana , Zea mays , Oryza sativa , Linum usitatissimum , Citrullus lanatus , Corchorus olitorius , Streptocarpus spp ., Sinningia speciosa, Arachis hypogaea, several tropical trees, Solanum tuberosum, and Ricinus communis (Fig. 2 A), underscoring the functional conservation of this gene. Phylogenetic analysis revealed that GhABA2 is most closely related to the ABA2 protein from Citrullus lanatus (Fig. 2 B), suggesting shared functional and regulatory mechanisms. Analysis of the 2,000 bp promoter region upstream of the GhABA2 transcription start site identified canonical core promoter elements (e.g., TATA-box and CAAT-box). In addition, numerous cis-acting elements associated with hormone response and stress signaling were detected, including abscisic acid response elements (ABRE), gibberellin response elements (GARE-motif), jasmonic acid response elements (CGGTA-motif, TGACG-motif), anaerobic response elements (ARE), and light-responsive elements (G-Box, GT1-motif, Box 4) (Table 2 ). The prevalence of these regulatory motifs suggests that GhABA2 expression is likely modulated by multiple hormones (e.g., ABA, GA, and JA) and environmental stimuli (e.g., low oxygen and light), implicating its role in abiotic stress adaptation and hormonal signaling. Table 2 Prediction of cis-acting elements in the GhABA2 promoter Element Number Function TGACG-motif 1 cis-acting regulatory element involved in the MeJA-responsiveness TC-rich repeats 2 cis-acting element involved in defense and stress responsiveness ABRE 1 cis-acting element involved in the abscisic acid responsiveness ARE 1 cis-acting regulatory element essential for the anaerobic induction GA-motif 1 part of a light responsive element GT1-motif 3 light responsive element Box 4 4 part of a conserved DNA module involved in light responsiveness CGTCA-motif 1 cis-acting regulatory element involved in the MeJA-responsiveness G-box 1 cis-acting regulatory element involved in light responsiveness O2-site 1 cis-acting regulatory element involved in zein metabolism regulation 3.2. Expression Analysis of GhABA2 under Salt Stress To examine the response of GhABA2 to salt stress, its transcript abundance was quantified using qRT-PCR in cotton plants grown hydroponically and treated with either distilled water (control) or 300 mM NaCl. Salt stress treatment significantly up-regulated the expression of GhABA2 compared to the control (Fig. 3 A), indicating its involvement in the cotton salt stress response. 3.3. Overexpression of GhABA2 Enhances Salt Tolerance in Arabidopsis To functionally characterize GhABA2 , transgenic Arabidopsis lines overexpressing the gene were generated. Eight independent lines were obtained and verified by PCR and qRT-PCR. Three lines (OE6, OE7, and OE8) with the highest expression levels of GhABA2 (Fig. 3 B) were selected for further phenotypic assays. Under salt stress conditions during seed germination, the germination rate of WT seeds decreased markedly with increasing NaCl concentrations. In contrast, the overexpression (OE) lines maintained significantly higher germination rates (Fig. 4 A and 4 C), indicating reduced sensitivity to salt stress at the germination stage. Consistent with the germination phenotypes, root growth assays revealed that although NaCl suppressed root elongation in all genotypes, the OE lines developed significantly longer roots than the WT under each salinity treatment. Notably, at 200 mM NaCl, root growth was nearly abolished in WT plants, whereas the OE lines retained considerable root elongation capacity (Fig. 4 B and 4 D). To assess salt tolerance at the seedling stage, 14-day-old soil-grown WT and OE plants were treated with 300 mM NaCl. Although all plants showed initial similar appearance, wilting symptoms emerged in WT plants after three days of treatment, whereas OE plants remained less affected. By day 9, WT seedlings exhibited severe wilting and salt damage, while most OE plant leaves remained turgid and viable (Fig. 5 A). To elucidate the physiological mechanisms underlying improved salt tolerance, we measured RWC and key stress-related biochemical parameters. Under salt stress, OE lines had significantly higher RWC, increased activities of POD and CAT, elevated Pro content, and reduced MDA accumulation compared to WT (Fig. 5 B– 5 F). These results suggest that GhABA2 overexpression enhances salt tolerance by alleviating membrane lipid peroxidation (reduced MDA), improving osmotic adjustment (elevated Pro), and boosting antioxidant enzyme activity (increased POD and CAT). 3.4. Silencing of GhABA2 Impairs Salt Tolerance in Cotton To further explore the role of GhABA2 in salt stress response, VIGS was employed to downregulate GhABA2 expression in cotton. Effective silencing was confirmed when positive control plants (TRV2: GhCLA ) displayed the expected albino phenotype (Fig. 6 A). Subsequent qRT-PCR analysis showed that the transcript level of GhABA2 in TRV2: GhABA2 plants was significantly reduced compared to the empty vector control (TRV2:00) (Fig. 6 B). Both TRV2:00 and TRV2: GhABA2 plants at the three-true-leaf stage were treated with 300 mM NaCl. While no visible phenotypic differences were observed at the onset of treatment (0 h), slight wilting appeared in TRV2: GhABA2 plants after 3 hours. After 6 hours of salt stress, wilting became more pronounced in silenced plants compared to controls (Fig. 6 C), indicating compromised salt tolerance upon GhABA2 silencing. To understand the physiological basis of this phenotype, key stress-related parameters were measured. Relative to TRV2:00 plants, TRV2: GhABA2 lines exhibited significantly reduced ABA content and RWC, along with a marked increase in MDA accumulation (Fig. 7 A– 7 C). Additionally, activities of POD and SOD, as well as Pro content, were significantly lower in silenced plants (Fig. 7 D– 7 F). Together, these results demonstrate that silencing GhABA2 diminishes salt tolerance in cotton, likely through impairment of ABA-mediated signaling, reduction in reactive oxygen species (ROS) scavenging capability, and suppression of osmotic adjustment processes such as proline accumulation. 4. DISCUSSION ABA is a pivotal phytohormone that regulates plant growth, development, and adaptation to abiotic stresses [ 21 , 22 ]. The biosynthesis of ABA involves a complex, multi-step pathway finely tuned to ensure plasticity in response to environmental changes [ 23 ]. In plants, the primary route is the C40 indirect pathway, which takes place in plastids. Initiated by β-carotene, this pathway proceeds through epoxidation reactions catalyzed by zeaxanthin epoxidase (ZEP), yielding violaxanthin and neoxanthin. The key cleavage step is mediated by 9-cis-epoxycarotenoid dioxygenase (NCED), which generates the C15 intermediate xanthoxin from 9-cis-violaxanthin or neoxanthin [ 24 ]. Xanthoxin is then translocated to the cytoplasm, where it is converted into ABA aldehyde by the short-chain dehydrogenase/reductase ABA2. Finally, ABA aldehyde is oxidized to ABA by abscisic aldehyde oxidase (AAO3) [ 25 – 27 ]. In addition to this central pathway, two auxiliary routes contribute to ABA biosynthesis. The C15 direct pathway utilizes farnesyl pyrophosphate (FPP) from the mevalonate pathway as a precursor, leading to ABA via a one-step cyclization and oxidation [ 28 ]. A ZEP-independent bypass pathway starts from zeaxanthin and involves a non-ZEP oxidative cleavage directly yielding xanthoxin, which is subsequently channeled into ABA through ABA2 and AAO3 [ 29 ]. Thus, ABA homeostasis is maintained through the coordinated action of three biosynthetic pathways: the canonical C40 indirect route, the C15 direct pathway, and the ZEP-independent bypass. Under salt stress, ABA enhances plant tolerance by promoting stomatal closure to minimize water loss via its signaling cascade [ 30 ]. Accumulating evidence indicates that elevated endogenous ABA levels generally correlate with improved salt resistance [ 31 – 34 ]. ABA2 , which catalyzes a pivotal step in ABA biosynthesis [ 26 , 35 ], plays an essential role in this process. For instance, the AtABA2 mutant in Arabidopsis shows attenuated ABA accumulation under salt stress and reduced tolerance [ 26 , 36 ], which can be partially rescued by exogenous ABA application [ 37 ], underscoring the functional importance of ABA2 . While most studies on ABA2 in salt stress have been conducted in Arabidopsis , little was known about its role in cotton. Our results demonstrate that salt stress up-regulates GhABA2 expression in cotton (Fig. 3 A), concomitant with increased ABA accumulation and enhanced salt tolerance. Furthermore, overexpression of GhABA2 in Arabidopsis improved salt tolerance, whereas silencing GhABA2 in cotton compromised it (Fig. 5 A, 6 C), confirming a strong correlation between ABA2 activity and salt stress resistance. In this study, GhABA2 -overexpressing lines exhibited higher seed germination rates and greater root elongation under salt stress compared to wild-type plants, indicating that GhABA2 alleviates the inhibitory effects of salinity on early growth stages [ 38 ]. Moreover, transgenic lines showed increased activities of CAT and POD, elevated Pro content, higher RWC, and reduced MDA levels (Fig. 5 B– 5 F), suggesting enhanced ROS scavenging capacity, improved osmotic adjustment, and maintained membrane integrity. Conversely, silencing GhABA2 in cotton led to decreased SOD and POD activities, reduced Pro and ABA content, lower RWC, and elevated MDA accumulation (Fig. 7 A– 7 F), resulting in heightened sensitivity to salt stress. Collectively, these physiological data indicate that GhABA2 , as a gene involved in ABA biosynthesis, contributes to salt stress tolerance in cotton by modulating ABA levels, activating antioxidant systems, and maintaining osmotic homeostasis—consistent with the well-established role of ABA in abiotic stress adaptation [ 39 ]。 5. CONCLUSIONS ABA2 is a key enzyme in the ABA biosynthesis pathway and plays an essential role in mediating plant responses to salt stress. In this study, we demonstrated the involvement of the GhABA2 gene in salt tolerance through heterologous overexpression in Arabidopsis and VICS in cotton. However, the precise molecular mechanisms underlying GhABA2 function in cotton's adaptation to salt stress remain to be fully elucidated. This work lays a theoretical foundation for understanding the role of GhABA2 under salt stress and identifies a valuable genetic resource for breeding salt-tolerant cotton varieties. Abbreviations D.S.C., D.L.G., C.Y.G., W.W.F., X.Z., W.H.M., J.C.L. ,H.X.Z.* and H.X.J.* Declarations Compliance with Ethical Standards Clinical trial number: not applicable. Ethics, Consent to Participate, and Consent to Publish declarations: not applicable. Consent for publication* Not applicable. Availability of data and material Data are contained within the article. Conflicts of Interest The authors declare that none of the authors have any competing interests. Funding This research was funded by the National Natural Science Foundation of China (Grant No. 32401847) and Tianchi Yingcai Introduction Program (Young PhDs) under the Talent Development Fund of Xinjiang Uygur Autonomous Region of China. Authors’ contributions D.S.C, D.L.G, H.X.Z and HJ designed the study. D.SC, D.L.G, C.Y.G, W.W.F, X.Z, W.H.M and J.C.L performed the experiments. D.S.C and D.L.G analyzed the data. D.S.C, D.L.G, H.X.Z and H.X.J wrote the manuscript. All authors have read and approved the final manuscript. 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12:45:37","extension":"xml","order_by":39,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126006,"visible":true,"origin":"","legend":"","description":"","filename":"df05a7b31e3f4df186ccda52eba725841structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/acfee6b0cd955491432f33ad.xml"},{"id":95385133,"identity":"4ba597da-372e-4df6-9e1f-fd3a04d9bba1","added_by":"auto","created_at":"2025-11-07 12:45:37","extension":"html","order_by":40,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140689,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/de1ca6500a996afa13a6a1a7.html"},{"id":95385096,"identity":"672eb230-a544-4231-a7ec-bdc5af70d616","added_by":"auto","created_at":"2025-11-07 12:45:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":21785787,"visible":true,"origin":"","legend":"\u003cp\u003eProtein analysis of GhABA2.\u003c/p\u003e\n\u003cp\u003e(A) Hydrophilicity/hydrophobicity analysis; (B) Phosphorylation site prediction; (C) Secondary structure prediction; (D) Tertiary structure prediction\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/f9ff19a31e79cd31a03449e5.png"},{"id":95385123,"identity":"42cf476e-fd88-4861-b111-0db8f070291c","added_by":"auto","created_at":"2025-11-07 12:45:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80127744,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment and phylogenetic analysis of GhABA2 across different species.\u003c/p\u003e\n\u003cp\u003e(A) Multiple sequence alignment among various species; (B) Phylogenetic tree analysis\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/c315c452738c43d690343417.png"},{"id":95385091,"identity":"f06c88ce-5def-4767-b085-e482ac4ca672","added_by":"auto","created_at":"2025-11-07 12:45:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3159240,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of \u003cem\u003eGhABA2\u003c/em\u003e in cotton under salt stress and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Expression levels of \u003cem\u003eGhABA2 \u003c/em\u003ein cotton plants under salt stress and control conditions at different time points; (B) Screening of transgenic \u003cem\u003eArabidopsis \u003c/em\u003elines. Data represent mean±SD of three biological replicates. Significant differences were determined by Student’s t-test: *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/1aa53d2f4cf47bc3dc49e6dc.png"},{"id":95526158,"identity":"6fcd390b-ef68-4833-82aa-1cb0559e6ceb","added_by":"auto","created_at":"2025-11-10 10:06:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54022861,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eGhABA2\u003c/em\u003e enhances salt tolerance during germination in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Representative germination phenotypes; (B) Germination rate; (C) Root growth phenotypes; (D) Root length. Data are presented as mean ± SD of three biological replicates. Significant differences were determined by Student’s t-test: *\u003cem\u003eP \u0026lt; \u003c/em\u003e0.05, **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01, ***\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.001.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/44cb5d12d313e6f6337a68af.png"},{"id":95385108,"identity":"6a07e508-f829-4267-817c-1e706e467349","added_by":"auto","created_at":"2025-11-07 12:45:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63591544,"visible":true,"origin":"","legend":"\u003cp\u003eOverexpression of \u003cem\u003eGhABA2\u003c/em\u003e enhances salt stress tolerance at the seedling stage in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e(A) Phenotypic comparison of WT and transgenic plants under salt stress; (B) RWC; (C) MDA content; (D) Pro content; (E) POD activity; (F) CAT activity. Data represent mean ±SD of three biological replicates. Significant differences were determined by Student’s t-test: *\u003cem\u003eP \u0026lt;\u003c/em\u003e0.05, **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01, ***\u003cem\u003eP \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/15761f38d0373c18fb6204fc.png"},{"id":95385113,"identity":"b2dfdfa4-1a19-4078-b5fa-f9d9357d58c4","added_by":"auto","created_at":"2025-11-07 12:45:37","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69645169,"visible":true,"origin":"","legend":"\u003cp\u003eSilencing of \u003cem\u003eGhABA2 \u003c/em\u003ereduces cotton tolerance to salt stress.\u003c/p\u003e\n\u003cp\u003e(A) Albino marker plants; (B) Relative silencing efficiency in TRV2:00 and TRV2:\u003cem\u003eGhABA2\u003c/em\u003eplants; (C) Phenotypic comparison of TRV2:00 and TRV2:\u003cem\u003eGhABA2\u003c/em\u003e plants under salt treatment.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/99847961e37ec9fe33122198.png"},{"id":95525508,"identity":"46abbe2c-a744-4069-88f7-3c7780205583","added_by":"auto","created_at":"2025-11-10 10:05:10","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9439147,"visible":true,"origin":"","legend":"\u003cp\u003ePhysiological analysis of \u003cem\u003eGhABA2\u003c/em\u003e-silenced cotton plants under salt stress.\u003c/p\u003e\n\u003cp\u003e(A) RWC; (B) ABA content; (C) MDA content; (D) POD activity; (E) Pro content; (F) SOD activity in TRV2:00 (control) and TRV2:\u003cem\u003eGhABA2\u003c/em\u003e plants. Data represent mean ± SD of three biological replicates. Significance was determined by two-tailed Student’s t-test:*\u003cem\u003e P \u0026lt; \u003c/em\u003e0.05, **\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01, ***\u003cem\u003eP \u0026lt; \u003c/em\u003e0.001.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/a00c1ce25b6d00cbbdd73ea1.png"},{"id":95385090,"identity":"91e6a9dc-c05f-4127-b33d-1c001fd1b085","added_by":"auto","created_at":"2025-11-07 12:45:36","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22528,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.doc","url":"https://assets-eu.researchsquare.com/files/rs-7651246/v1/234eaae52c49512790f60a3f.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Overexpression and silencing of the cotton GhABA2 gene reveal its role in salt stress tolerance","fulltext":[{"header":"1. INTORDUCTION","content":"\u003cp\u003eSoil salinization, or salt stress, denotes the detrimental impact of elevated salt concentrations in the growth environment\u0026mdash;such as in soil or irrigation water\u0026mdash;on plant growth, development, and physiological metabolism. As a major abiotic stress, it significantly constrains agricultural productivity and threatens global food security [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This issue is particularly acute in the arid northwestern regions of China, where low precipitation, high evaporation rates, and saline groundwater contribute to progressive soil salinization. This issue is particularly acute in the arid northwestern regions of China, where low precipitation, high evaporation rates, and saline groundwater contribute to progressive soil salinization [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Cotton, a key economic crop in China, is predominantly cultivated in Xinjiang. In 2024, Xinjiang produced approximately 5, 112\u0026nbsp;million metric tons of cotton, accounting for 91% of the national total, underscoring its pivotal role in the Chinese cotton industry. However, soil salinization affects nearly 10% of China's arable land, with northwestern regions being especially vulnerable, which poses a serious threat to cotton production[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, saline soils constitute 37.72% of the total cultivated area in Xinjiang's irrigation zones, posing a substantial challenge to local cotton production [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eABA, a critical phytohormone and signaling molecule, plays a central role in mediating plant adaptation to salt stress. It regulates salinity responses by activating ABA-dependent signaling pathways that induce stress-responsive gene expression and through crosstalk with other hormones [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For instance, ABA signals can trigger MAPK cascades via calcium signaling to enhance stress resistance [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In soybean, \u003cem\u003eGmSIN1\u003c/em\u003e (salt-induced \u003cem\u003eNAC1\u003c/em\u003e) is upregulated by ABA and promotes root growth and salinity tolerance. and promotes root growth and salinity tolerance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. ABA also interacts with auxin (IAA), gibberellin (GA), and cytokinin (CK) to modulate stress responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In maize, salt-induced ABA accumulation disrupts polar auxin transport by affecting \u003cem\u003eZmPIN1\u003c/em\u003e localization, thereby suppressing lateral root formation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eABI4\u003c/em\u003e overexpression enhances the transcription of \u003cem\u003eNCED6\u003c/em\u003e and \u003cem\u003eGA2ox7\u003c/em\u003e, increasing the ABA/GA ratio and improving salt tolerance [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, ABA suppresses cytokinin biosynthesis via \u003cem\u003eMYB2\u003c/em\u003e, increasing ABA sensitivity and reducing shoot growth as an adaptive mechanism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, in tomato, salt stress elev ABA levels while reducing CK content [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. These studies collectively underscore the importance of ABA signaling and hormonal interplay in plant salt adaptation.\u003c/p\u003e\u003cp\u003eBesides signaling, the ABA biosynthesis pathway is crucial for salt tolerance. The key enzyme encoded by \u003cem\u003eAtABA2\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e\u0026mdash;a short-chain dehydrogenase/reductase (SDR1)\u0026mdash;catalyzes the conversion of xanthoxin to abscisic aldehyde, a pivotal step in ABA biosynthesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Overexpression of \u003cem\u003eAtABA2\u003c/em\u003e increases endogenous ABA levels and enhances salt tolerance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].The \u003cem\u003eAtLEW2\u003c/em\u003e mutant, which exhibits elevated ABA, proline, and soluble sugars, shows upregulated \u003cem\u003eSDR1\u003c/em\u003e expression and improved salt resistance [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Homologs of \u003cem\u003eSDR1\u003c/em\u003e have been identified in halophytes and functionally validated in crops such as sugar beet, where \u003cem\u003eSDR1\u003c/em\u003e overexpression improves salinity tolerance by optimizing physiological responses and suppressing ROS accumulation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These findings highlight the conserved role of \u003cem\u003eABA2\u003c/em\u003e/\u003cem\u003eSDR1\u003c/em\u003e in salt stress adaptation across species.\u003c/p\u003e\u003cp\u003eAlthough the function of the \u003cem\u003eABA2\u003c/em\u003e gene has been well characterized in model plants and some crops, its role in cotton (\u003cem\u003eGhABA2\u003c/em\u003e) remains unexplored. This study aims to bridge this gap through comprehensive bioinformatic analysis of \u003cem\u003eGhABA2\u003c/em\u003e, including protein structure prediction, domain analysis, promoter \u003cem\u003ecis\u003c/em\u003e-element profiling, and phylogenetic reconstruction. We further generated \u003cem\u003eGhABA2\u003c/em\u003e-overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e lines and silenced \u003cem\u003eGhABA2\u003c/em\u003e in cotton via VIGS. Comparative phenotypical and physiological assessments under salt stress were conducted to elucidate the contribution of \u003cem\u003eGhABA2\u003c/em\u003e to salt tolerance. Our results provide mechanistic insights into the salt stress response in cotton and identify \u003cem\u003eGhABA2\u003c/em\u003e as a promising genetic target for breeding salt-resistant cotton varieties.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Cloning and Bioinformatic Analysis of the \u003cem\u003eGhABA2\u003c/em\u003e Gene\u003c/h2\u003e\u003cp\u003eThe coding sequence (CDS) of \u003cem\u003eGhABA2\u003c/em\u003e (\u003cem\u003eGhi_A13G00706\u003c/em\u003e) was retrieved from the CottonMD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://yanglab.hzau.edu.cn/CottonMD\u003c/span\u003e\u003cspan address=\"https://yanglab.hzau.edu.cn/CottonMD\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Gene-specific primers were designed (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) ,\u003c/p\u003e\u003cp\u003eThe target \u003cem\u003eGhABA2\u003c/em\u003e sequence was amplified via polymerase chain reaction (PCR). The PCR product was purified and subsequently sequenced (Shanghai Sangon Biotech) to verify the integrity and accuracy of the amplified fragment. A complete and error-free \u003cem\u003eGhABA2\u003c/em\u003e CDS was thereby obtained.\u003c/p\u003e\u003cp\u003eBioinformatic analyses were performed to characterize the \u003cem\u003eGhABA2\u003c/em\u003e gene and its encoded protein. Properties evaluated included physicochemical parameters, phosphorylation sites, hydrophobicity and hydrophilicity profiles, subcellular localization, secondary and tertiary structures, phylogenetic relationships, and cis-regulatory elements. The tools and online resources used for these analyses are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\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\u003eBioinformatic Analysis Software\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSoftware\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWebsite\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFunctionality\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProt Param\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/cgi-bin/protparam/protparam\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/cgi-bin/protparam/protparam\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhysicochemical property analysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eNetPhos 3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/NetPhos\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/NetPhos\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhosphorylation site analysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eProt Scale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.expasy.org/tools/protscale.html\u003c/span\u003e\u003cspan address=\"http://www.expasy.org/tools/protscale.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eHydrophobicity/hydrophilicity analysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePSORT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genscript.com/cgi-bin/tools/psort2.pl\u003c/span\u003e\u003cspan address=\"http://www.genscript.com/cgi-bin/tools/psort2.pl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSubcellular localization analysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSOPMA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eSecondary structure analysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSWISS-MODEL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://swissmodel.expasy.org\u003c/span\u003e\u003cspan address=\"http://swissmodel.expasy.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTertiary structure analysis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDNAMAN\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eMultiple sequence alignment\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMEGA11.0.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePhylogenetic tree construction\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePlantcare\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCis-acting element analysis\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\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Generation of \u003cem\u003eGhABA2\u003c/em\u003e-Overexpressing Transgenic \u003cem\u003eArabidopsis\u003c/em\u003e Lines\u003c/h2\u003e\u003cp\u003eThe amplified \u003cem\u003eGhABA2\u003c/em\u003e fragment was cloned into the restriction-digested pCAMBIA-2300 vector to generate the recombinant plasmid pCAMBIA-2300-\u003cem\u003eGhABA2\u003c/em\u003e. This construct was introduced into Agrobacterium tumefaciens strain GV3101 and subsequently transformed into \u003cem\u003eArabidopsis\u003c/em\u003e thaliana ecotype Col-0 via the floral dip method. Transformants were selected on medium containing 50 \u0026micro;g/mL kanamycin and verified by PCR. The expression level of \u003cem\u003eGhABA2\u003c/em\u003e in transgenic plants was quantified using quantitative real-time PCR (qRT-PCR). Homozygous T\u003csub\u003e3\u003c/sub\u003e lines exhibiting high \u003cem\u003eGhABA2\u003c/em\u003e expression were selected for further functional studies.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Evaluation of Salt Stress Tolerance, Root Elongation, and Germination Rate in Transgenic \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eGermination Assay under Salt Stress: Seeds of homozygous T3 transgenic and wild-type (WT) \u003cem\u003eArabidopsis\u003c/em\u003e were surface-sterilized with 75% (v/v) ethanol for 2 min, rinsed three times with sterile water, treated with 5% (v/v) NaClO for 5 min, and then washed five times with sterile water. The sterilized seeds were sown on 1/2 MS solid medium supplemented with 0, 100, 150, or 200 mmol/L NaCl [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After stratification at 4\u0026deg;C for 24 h to break dormancy, the plates were transferred to a growth chamber set at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a 16/8 h light/dark cycle. Germination rates were recorded after 10 days.\u003c/p\u003e\u003cp\u003eRoot Elongation Assay under Salt Stress: Surface-sterilized T3 transgenic and wild-type seeds were germinated on 1/2 MS solid medium for 4 days. Uniform seedlings were then transferred to vertical plates containing 1/2 MS medium with 0, 100, 150, or 200 mmol/L NaCl. Root length was measured after 4 days of vertical growth [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePhenotypic Evaluation of Salt Stress Tolerance: Fourteen-day-old soil-grown seedlings were subjected to salt stress by irrigation with 300 mM NaCl solution every 3 days for 9 days. Biochemical analyses were conducted after 6 days of treatment, and phenotypic observations were recorded on day 9.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Cotton Materials and Growth Conditions\u003c/h2\u003e\u003cp\u003eThe seeds of the Gossypium hirsutum variety Zhongmian 113 were provided by Xinjiang Zhongmian Seed Industry Co., Ltd. (Aksu City, Xinjiang, China).Seeds were sterilized by immersion in 75% (v/v) ethanol for 5 min, followed by three rinses with sterile distilled water. Subsequently, seeds were treated with 1% (v/v) NaClO for 10 min and washed thoroughly with sterile distilled water. The sterilized seeds were placed between moist sterile filter papers and incubated at 28\u0026deg;C for 2 days to promote germination. Germinated seedlings were transplanted into pots containing a mixed substrate of nutrient soil, black soil, and vermiculite (3:1:1, v/v/v). Plants were grown in a greenhouse under controlled conditions: 25\u0026deg;C (day)/22\u0026deg;C (night), with a 16 h light/8 h dark photoperiod.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Generation of \u003cem\u003eGhABA2\u003c/em\u003e-Silenced Cotton Plants via VIGS\u003c/h2\u003e\u003cp\u003eA 400-bp unique fragment of \u003cem\u003eGhABA2\u003c/em\u003e was selected as the silencing target. A gene-specific primer pair was designed to amplify this fragment from cotton cDNA, and the resulting PCR product was cloned into the TRV2 vector to generate the TRV2:\u003cem\u003eGhABA2\u003c/em\u003e construct. The recombinant TRV2:\u003cem\u003eGhABA2\u003c/em\u003e, along with empty TRV1 and TRV2 vectors, were individually transformed into Agrobacterium tumefaciens strain GV3101. Bacterial cultures containing TRV1 were mixed separately with those harboring TRV2 (empty vector control), TRV2:\u003cem\u003eGhCLA\u003c/em\u003e (positive control; \u003cem\u003eCLA\u003c/em\u003e silencing induces an albino phenotype), or TRV2:\u003cem\u003eGhABA2\u003c/em\u003e. The mixtures were infiltrated into the cotyledons of cotton seedlings. Plants infiltrated with TRV1\u0026thinsp;+\u0026thinsp;TRV2 were designated as negative controls (TRV2:00), while those infiltrated with TRV1\u0026thinsp;+\u0026thinsp;TRV2:\u003cem\u003eGhABA2\u003c/em\u003e were considered \u003cem\u003eGhABA2\u003c/em\u003e-silenced plants (TRV2:\u003cem\u003eGhABA2\u003c/em\u003e). Silencing efficiency was validated by the emergence of an albino phenotype in TRV2:\u003cem\u003eGhCLA\u003c/em\u003e positive control plants.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 RNA Extraction and qRT-PCR Analysis\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from leaves of TRV2:00 and TRV2:\u003cem\u003eGhABA2\u003c/em\u003e plants using the Plant Polysaccharide \u0026amp; Polyphenol RNA Kit (FOREGENE, Chengdu, China), following the manufacturer\u0026rsquo;s protocol. RNA quality was verified by agarose gel electrophoresis, and concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). First-strand cDNA was synthesized with the FOREGENE Master Premix RT Easy\u0026trade; II Kit. qRT-PCR was performed using Real Time PCR Easy\u0026trade;-SYBR Green I reagents on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). The thermal cycling conditions consisted of an initial denaturation at 95\u0026deg;C for 30 seconds, followed by 40 cycles of 95\u0026deg;C for 5 seconds and 60\u0026deg;C for 30 seconds. The \u003cem\u003eGhHis3\u003c/em\u003e gene was used as an internal control for normalization. Relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method. Three biological replicates were performed to confirm efficient silencing of \u003cem\u003eGhABA2\u003c/em\u003e. Cotton plants showing significant downregulation of \u003cem\u003eGhABA2\u003c/em\u003e were selected for further experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Salt Stress Treatment of Cotton Plants\u003c/h2\u003e\u003cp\u003eCotton plants at the three-true-leaf stage with confirmed high silencing efficiency were transferred to a hydroponic system and acclimatized for two days. Salt stress was subsequently applied by irrigating the plants with 300 mM NaCl solution. Phenotypic responses were documented after 7 days of treatment. Leaf samples from both TRV2:00 (control) and TRV2:\u003cem\u003eGhABA2\u003c/em\u003e (silenced) plants were collected at the end of the stress period for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Measurement of Physiological and Biochemical Parameters in Transgenic \u003cem\u003eArabidopsis\u003c/em\u003e and VIGS Cotton\u003c/h2\u003e\u003cp\u003eA series of physiological and biochemical assays were conducted to evaluate oxidative stress and abiotic stress responses in both \u003cem\u003eGhABA2\u003c/em\u003e-overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eGhABA2\u003c/em\u003e-silenced cotton plants. The following parameters were measured using commercial assay kits according to the manufacturers\u0026rsquo; protocols: activities of POD, SOD, and CAT; content of MDA and Pro and endogenous ABA levels. Specifically, kits from Solarbio (Beijing, China) were used for POD, MDA, SOD, Pro, and CAT assays, while ABA content was quantified using a kit from GlidePharma (Wuhan, China). All measurements were performed on plant materials collected both before and after the application of salt stress.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Cloning and Sequence Verification of \u003cem\u003eGhABA2\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe coding sequence (CDS) of \u003cem\u003eGhABA2\u003c/em\u003e (\u003cem\u003eGhi_A13G00706\u003c/em\u003e) was retrieved from the CottonMD database. Gene-specific primers were designed to amplify the target sequence by PCR. A clear band of the expected size was observed on an agarose gel. The purified PCR product was sequenced (Shanghai Sangon Biotech), and the resulting sequence was confirmed to be complete and accurate, matching the reference \u003cem\u003eGhABA2\u003c/em\u003e CDS. These results confirm the successful cloning of the full-length \u003cem\u003eGhABA2\u003c/em\u003e CDS for further functional characterization.\u003c/p\u003e\u003cp\u003eBioinformatic analysis indicated that the GhABA2 protein consists of 282 amino acids, with a molecular formula of C₁₃₁₇H₂₀₈₈N₃₇₄O₄₀₅S₁₂ and a molecular weight of approximately 30.03 kDa. The theoretical isoelectric point (pI) was predicted to be 6.1. The protein contains 23 basic (Arg\u0026thinsp;+\u0026thinsp;Lys) and 28 acidic (Asp\u0026thinsp;+\u0026thinsp;Glu) residues, suggesting a capacity for ionic interactions. The instability index was computed as 21.25, classifying the protein as stable. The aliphatic index was 93.62, indicating a high proportion of aliphatic amino acids that may enhance thermal stability. The grand average of hydropathicity (GRAVY) was 0.072. Subcellular localization predictions strongly suggested cytoplasmic localization. Hydrophobicity analysis revealed that the C-terminal region is predominantly hydrophobic, the N-terminal is hydrophilic, and the central region shows alternating hydrophobicity and hydrophilicity. Overall, the protein is hydrophilic, with one prominent hydrophilic peak exceeding a value of 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Multiple potential phosphorylation sites were predicted with high confidence (scores\u0026thinsp;\u0026asymp;\u0026thinsp;1) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), implying regulatory potential through phosphorylation. Secondary structure prediction indicated that α-helices (45.73%), random coils (28.25%), extended strands (17.07%), and β-turns (8.94%) constitute the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The tertiary structure was modeled using 7o6p.1.A as a template (sequence identity: 51%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eMultiple sequence alignment demonstrated that GhABA2 contains regions highly conserved among ABA2 homologs from diverse plant species, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eLinum usitatissimum\u003c/em\u003e, \u003cem\u003eCitrullus lanatus\u003c/em\u003e, \u003cem\u003eCorchorus olitorius\u003c/em\u003e, \u003cem\u003eStreptocarpus spp\u003c/em\u003e., Sinningia speciosa, Arachis hypogaea, several tropical trees, Solanum tuberosum, and Ricinus communis (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), underscoring the functional conservation of this gene. Phylogenetic analysis revealed that GhABA2 is most closely related to the ABA2 protein from Citrullus lanatus (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), suggesting shared functional and regulatory mechanisms.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAnalysis of the 2,000 bp promoter region upstream of the \u003cem\u003eGhABA2\u003c/em\u003e transcription start site identified canonical core promoter elements (e.g., TATA-box and CAAT-box). In addition, numerous cis-acting elements associated with hormone response and stress signaling were detected, including abscisic acid response elements (ABRE), gibberellin response elements (GARE-motif), jasmonic acid response elements (CGGTA-motif, TGACG-motif), anaerobic response elements (ARE), and light-responsive elements (G-Box, GT1-motif, Box 4) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The prevalence of these regulatory motifs suggests that \u003cem\u003eGhABA2\u003c/em\u003e expression is likely modulated by multiple hormones (e.g., ABA, GA, and JA) and environmental stimuli (e.g., low oxygen and light), implicating its role in abiotic stress adaptation and hormonal signaling.\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\u003ePrediction of cis-acting elements in the \u003cem\u003eGhABA2\u003c/em\u003e promoter\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\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=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eElement\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNumber\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTGACG-motif\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTC-rich repeats\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting element involved in defense and stress responsiveness\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eABRE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting element involved in the abscisic acid responsiveness\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eARE\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting regulatory element essential for the anaerobic induction\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGA-motif\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epart of a light responsive element\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGT1-motif\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003elight responsive element\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBox 4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epart of a conserved DNA module involved in light responsiveness\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCGTCA-motif\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting regulatory element involved in the MeJA-responsiveness\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eG-box\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting regulatory element involved in light responsiveness\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eO2-site\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ecis-acting regulatory element involved in zein metabolism regulation\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\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Expression Analysis of \u003cem\u003eGhABA2\u003c/em\u003e under Salt Stress\u003c/h2\u003e\u003cp\u003eTo examine the response of \u003cem\u003eGhABA2\u003c/em\u003e to salt stress, its transcript abundance was quantified using qRT-PCR in cotton plants grown hydroponically and treated with either distilled water (control) or 300 mM NaCl. Salt stress treatment significantly up-regulated the expression of \u003cem\u003eGhABA2\u003c/em\u003e compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating its involvement in the cotton salt stress response.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3. Overexpression of \u003cem\u003eGhABA2\u003c/em\u003e Enhances Salt Tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo functionally characterize \u003cem\u003eGhABA2\u003c/em\u003e, transgenic \u003cem\u003eArabidopsis\u003c/em\u003e lines overexpressing the gene were generated. Eight independent lines were obtained and verified by PCR and qRT-PCR. Three lines (OE6, OE7, and OE8) with the highest expression levels of \u003cem\u003eGhABA2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) were selected for further phenotypic assays.\u003c/p\u003e\u003cp\u003eUnder salt stress conditions during seed germination, the germination rate of WT seeds decreased markedly with increasing NaCl concentrations. In contrast, the overexpression (OE) lines maintained significantly higher germination rates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), indicating reduced sensitivity to salt stress at the germination stage. Consistent with the germination phenotypes, root growth assays revealed that although NaCl suppressed root elongation in all genotypes, the OE lines developed significantly longer roots than the WT under each salinity treatment. Notably, at 200 mM NaCl, root growth was nearly abolished in WT plants, whereas the OE lines retained considerable root elongation capacity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess salt tolerance at the seedling stage, 14-day-old soil-grown WT and OE plants were treated with 300 mM NaCl. Although all plants showed initial similar appearance, wilting symptoms emerged in WT plants after three days of treatment, whereas OE plants remained less affected. By day 9, WT seedlings exhibited severe wilting and salt damage, while most OE plant leaves remained turgid and viable (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the physiological mechanisms underlying improved salt tolerance, we measured RWC and key stress-related biochemical parameters. Under salt stress, OE lines had significantly higher RWC, increased activities of POD and CAT, elevated Pro content, and reduced MDA accumulation compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). These results suggest that \u003cem\u003eGhABA2\u003c/em\u003e overexpression enhances salt tolerance by alleviating membrane lipid peroxidation (reduced MDA), improving osmotic adjustment (elevated Pro), and boosting antioxidant enzyme activity (increased POD and CAT).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4. Silencing of \u003cem\u003eGhABA2\u003c/em\u003e Impairs Salt Tolerance in Cotton\u003c/h2\u003e\u003cp\u003eTo further explore the role of \u003cem\u003eGhABA2\u003c/em\u003e in salt stress response, VIGS was employed to downregulate \u003cem\u003eGhABA2\u003c/em\u003e expression in cotton. Effective silencing was confirmed when positive control plants (TRV2:\u003cem\u003eGhCLA\u003c/em\u003e) displayed the expected albino phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequent qRT-PCR analysis showed that the transcript level of \u003cem\u003eGhABA2\u003c/em\u003e in TRV2:\u003cem\u003eGhABA2\u003c/em\u003e plants was significantly reduced compared to the empty vector control (TRV2:00) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Both TRV2:00 and TRV2:\u003cem\u003eGhABA2\u003c/em\u003e plants at the three-true-leaf stage were treated with 300 mM NaCl. While no visible phenotypic differences were observed at the onset of treatment (0 h), slight wilting appeared in TRV2:\u003cem\u003eGhABA2\u003c/em\u003e plants after 3 hours. After 6 hours of salt stress, wilting became more pronounced in silenced plants compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), indicating compromised salt tolerance upon \u003cem\u003eGhABA2\u003c/em\u003e silencing.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo understand the physiological basis of this phenotype, key stress-related parameters were measured. Relative to TRV2:00 plants, TRV2:\u003cem\u003eGhABA2\u003c/em\u003e lines exhibited significantly reduced ABA content and RWC, along with a marked increase in MDA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Additionally, activities of POD and SOD, as well as Pro content, were significantly lower in silenced plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Together, these results demonstrate that silencing \u003cem\u003eGhABA2\u003c/em\u003e diminishes salt tolerance in cotton, likely through impairment of ABA-mediated signaling, reduction in reactive oxygen species (ROS) scavenging capability, and suppression of osmotic adjustment processes such as proline accumulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eABA is a pivotal phytohormone that regulates plant growth, development, and adaptation to abiotic stresses [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The biosynthesis of ABA involves a complex, multi-step pathway finely tuned to ensure plasticity in response to environmental changes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In plants, the primary route is the C40 indirect pathway, which takes place in plastids. Initiated by β-carotene, this pathway proceeds through epoxidation reactions catalyzed by zeaxanthin epoxidase (ZEP), yielding violaxanthin and neoxanthin. The key cleavage step is mediated by 9-cis-epoxycarotenoid dioxygenase (NCED), which generates the C15 intermediate xanthoxin from 9-cis-violaxanthin or neoxanthin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Xanthoxin is then translocated to the cytoplasm, where it is converted into ABA aldehyde by the short-chain dehydrogenase/reductase ABA2. Finally, ABA aldehyde is oxidized to ABA by abscisic aldehyde oxidase (AAO3) [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition to this central pathway, two auxiliary routes contribute to ABA biosynthesis. The C15 direct pathway utilizes farnesyl pyrophosphate (FPP) from the mevalonate pathway as a precursor, leading to ABA via a one-step cyclization and oxidation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A ZEP-independent bypass pathway starts from zeaxanthin and involves a non-ZEP oxidative cleavage directly yielding xanthoxin, which is subsequently channeled into ABA through ABA2 and AAO3 [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, ABA homeostasis is maintained through the coordinated action of three biosynthetic pathways: the canonical C40 indirect route, the C15 direct pathway, and the ZEP-independent bypass.\u003c/p\u003e\u003cp\u003eUnder salt stress, ABA enhances plant tolerance by promoting stomatal closure to minimize water loss via its signaling cascade [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Accumulating evidence indicates that elevated endogenous ABA levels generally correlate with improved salt resistance [\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. \u003cem\u003eABA2\u003c/em\u003e, which catalyzes a pivotal step in ABA biosynthesis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], plays an essential role in this process. For instance, the \u003cem\u003eAtABA2\u003c/em\u003e mutant in \u003cem\u003eArabidopsis\u003c/em\u003e shows attenuated ABA accumulation under salt stress and reduced tolerance [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which can be partially rescued by exogenous ABA application [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], underscoring the functional importance of \u003cem\u003eABA2\u003c/em\u003e. While most studies on \u003cem\u003eABA2\u003c/em\u003e in salt stress have been conducted in \u003cem\u003eArabidopsis\u003c/em\u003e, little was known about its role in cotton. Our results demonstrate that salt stress up-regulates \u003cem\u003eGhABA2\u003c/em\u003e expression in cotton (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), concomitant with increased ABA accumulation and enhanced salt tolerance. Furthermore, overexpression of \u003cem\u003eGhABA2\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e improved salt tolerance, whereas silencing \u003cem\u003eGhABA2\u003c/em\u003e in cotton compromised it (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC), confirming a strong correlation between \u003cem\u003eABA2\u003c/em\u003e activity and salt stress resistance.\u003c/p\u003e\u003cp\u003eIn this study, \u003cem\u003eGhABA2\u003c/em\u003e-overexpressing lines exhibited higher seed germination rates and greater root elongation under salt stress compared to wild-type plants, indicating that \u003cem\u003eGhABA2\u003c/em\u003e alleviates the inhibitory effects of salinity on early growth stages [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, transgenic lines showed increased activities of CAT and POD, elevated Pro content, higher RWC, and reduced MDA levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), suggesting enhanced ROS scavenging capacity, improved osmotic adjustment, and maintained membrane integrity. Conversely, silencing \u003cem\u003eGhABA2\u003c/em\u003e in cotton led to decreased SOD and POD activities, reduced Pro and ABA content, lower RWC, and elevated MDA accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF), resulting in heightened sensitivity to salt stress. Collectively, these physiological data indicate that \u003cem\u003eGhABA2\u003c/em\u003e, as a gene involved in ABA biosynthesis, contributes to salt stress tolerance in cotton by modulating ABA levels, activating antioxidant systems, and maintaining osmotic homeostasis\u0026mdash;consistent with the well-established role of ABA in abiotic stress adaptation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]。\u003c/p\u003e"},{"header":"5. CONCLUSIONS","content":"\u003cp\u003eABA2 is a key enzyme in the ABA biosynthesis pathway and plays an essential role in mediating plant responses to salt stress. In this study, we demonstrated the involvement of the \u003cem\u003eGhABA2\u003c/em\u003e gene in salt tolerance through heterologous overexpression in \u003cem\u003eArabidopsis\u003c/em\u003e and VICS in cotton. However, the precise molecular mechanisms underlying \u003cem\u003eGhABA2\u003c/em\u003e function in cotton's adaptation to salt stress remain to be fully elucidated. This work lays a theoretical foundation for understanding the role of \u003cem\u003eGhABA2\u003c/em\u003e under salt stress and identifies a valuable genetic resource for breeding salt-tolerant cotton varieties.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eD.S.C., D.L.G., C.Y.G., W.W.F., X.Z., W.H.M., J.C.L. ,H.X.Z.* and H.X.J.*\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with Ethical Standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial number: not applicable.\u003c/p\u003e\n\u003cp\u003eEthics, Consent to Participate, and Consent to Publish declarations: not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication*\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are contained within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that none of the authors have any competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (Grant No. 32401847) and Tianchi Yingcai Introduction Program (Young PhDs) under the Talent Development Fund of Xinjiang Uygur Autonomous Region of China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.S.C, D.L.G, H.X.Z and HJ designed the study. D.SC, D.L.G, C.Y.G, W.W.F, X.Z, W.H.M and J.C.L performed the experiments. D.S.C and D.L.G analyzed the data. D.S.C, D.L.G, H.X.Z and H.X.J wrote the manuscript. All authors have read and approved the final manuscript. D.S.C and D.L.G contributed equally to the study. These authors \u003cem\u003econtributed\u0026nbsp;\u003c/em\u003eequally \u003cem\u003eto this work.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 32401847) and Tianchi Yingcai Introduction Program (Young PhDs) under the Talent Development Fund of Xinjiang Uygur Autonomous Region of China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePeng Y, Zhu H, Wang Y, Kang J, Hu L, Li L, et al. Revisiting the role of light signaling in plant responses to salt stress. 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Agronomy. 2023;13:2698. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/agronomy13112698\u003c/span\u003e\u003cspan address=\"10.3390/agronomy13112698\" 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":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cotton, ABA2, Salt stress, Short-chain dehydrogenase/reductase (SDR), ABA signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-7651246/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7651246/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eABA2\u003c/em\u003e encodes a short-chain dehydrogenase/reductase involved in the regulation of abscisic acid (ABA) biosynthesis and plays a crucial role in plant responses to salt stress. To explore the function of \u003cem\u003eGhABA2\u003c/em\u003e in cotton salt tolerance, we conducted bioinformatic analyses, gene overexpression, and virus-induced gene silencing (VIGS) experiments. Bioinformatic examination revealed that the promoter region of the \u003cem\u003eGhABA2\u003c/em\u003e gene contains multiple \u003cem\u003ecis\u003c/em\u003e-acting elements, including those responsive to ABA, light, and various stress signals. Overexpression of \u003cem\u003eGhABA2\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e resulted in significantly increased seed germination rate, root length, leaf relative water content (RWC), catalase (CAT) activity, peroxidase (POD) activity, and proline (Pro) content under salt stress conditions, while malondialdehyde (MDA) content was markedly reduced. Conversely, silencing \u003cem\u003eGhABA2\u003c/em\u003e gene in cotton via virus-induced gene silencing (VIGS) led to significant decreases in ABA content, RWC, superoxide dismutase (SOD) activity, POD activity, and Pro content, along with a significant increase in MDA content. These findings provide important insights into the mechanism of \u003cem\u003eGhABA2\u003c/em\u003e in mediating cotton salt stress response and highlight its potential as a genetic target for breeding of salt-tolerant cotton varieties.\u003c/p\u003e","manuscriptTitle":"Overexpression and silencing of the cotton GhABA2 gene reveal its role in salt stress tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 12:45:31","doi":"10.21203/rs.3.rs-7651246/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4476d86b-102c-4b3e-92ae-f565adc206d7","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T10:12:42+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 12:45:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7651246","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7651246","identity":"rs-7651246","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}