AtGASA1 positively regulates Arabidopsis response to salt stress by suppressing accumulation of reactive oxygen species

AtGASA1 positively regulates Arabidopsis response to salt stress by suppressing accumulation of reactive oxygen species | 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 AtGASA1 positively regulates Arabidopsis response to salt stress by suppressing accumulation of reactive oxygen species Jian-Bo Song, Hao-En He, Cai-Feng Wang, Xian-Zhi Zuo, Zi-Xin Zhao, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7978126/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract AtGASA1 is a member of the gibberellin acid-stimulating protein (GASA) family, characterized by a GASA domain containing 12 conserved cysteine-rich peptides (CRP). Its homologous genes are known to play an essential role in plant responses to both biotic and abiotic stresses; however, the function of AtGASA1 remains unclear. In this study, we found that AtGASA1 is involved in regulating plant growth, leaf expansion and flowering time. Moreover, various results showed that gasa1 mutants exhibited sensitivity to salt stress, while overexpression of AtGASA1 conferred increased resistance to salt stress. To explore the role of conserved cysteine residues within the GASA domain, site-directed mutagenesis was performed to substitute Cys-40 and Cys-44 with alanine. Functional assays in a yeast heterologous expression system showed that yeast expressing AtGASA1C40,44A displayed reduced tolerance to salt stress compared with yeast expressing non-mutated AtGASA1, indicating that these residues are critical for salt stress adaptation. Consistently, transgenic Arabidopsis plants overexpressing AtGASA1C40,44A accumulated higher levels of ROS compared with 35S::GASA1 plants. Collectively, our findings demonstrate that AtGASA1 positively regulates salt stress tolerance in Arabidopsis by reducing ROS accumulation. salt stress GA reactive oxygen species (ROS) Arabidopsis AtGASA1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Message AtGASA1 regulates plant growth and development, while conserved Cys-40 and Cys-44 residues in its GASA domain are crucial for controlling ROS accumulation under salt stress. 1. Introduction Reactive oxygen species (ROS) play key roles in plant stress perception, the integration of salt stress-response signaling networks, and the activation of defense mechanisms (Zandalinas and Mittler 2018 ; Medina et al. 2021 ). ROS accumulation in cells can alter expression of resistance genes and influence plant resilience to environmental stresses (Suzuki et al. 2013 ; Zou et al. 2015 ; Nietzel et al. 2020 ). As signal molecules in response to salinity, low-level of ROS participate in the regulation of ABA-dependent stomatal closure, ethylene-dependent leaf abscission and activation of mitogen-activated protein kinase cascades (Zou et al. 2015 ). In contrast, excessive ROS production is harmful to plant growth and development (Tavanti et al. 2021 ). To maintain redox homeostasis, plants have evolved sophisticated systems to balance ROS generation and scavenging (Sachdev et al. 2021 ; Lanza and Reis 2021 ). Overexpression of key antioxidant genes, such as SUPEROXIDE DISMUTASES ( SOD ), CATALASE ( CAT ) and ASCORBATE PEROXIDASE ( APX ), has been demonstrated to improve plant salinity stress tolerance by increasing ROS scavenging capacity in a variety of plant species (Suzuki et al. 2013 , p. 2; Xu et al. 2021 ). Salt stress has emerged as an increasingly prevalent and severe problem worldwide in recent years, leading to reduced crops yield and decreased the quality (Parmar et al. 2017 ; Soltabayeva et al. 2021 ). Hormones, such as gibberellic acid (GA) and abscisic acid (ABA), play pivotal roles in mediating salt stress responses at different developmental stages (Christmann et al. 2006 ; Zhang and Wang 2011 ; Verma et al. 2016 ). ABA inhibit seed germination and seedling growth, promotes stomatal closure to minimize water loss, and regulate the expression of genes associated with salt tolerance (Lata and Prasad 2011 ; Rai et al. 2023 ; Liu et al. 2023 ). Moreover, ABA increases cytosolic pH, thereby activating ion channels and inducing enzymes that stimulate ROS production (Huang et al. 2012 ; Li et al. 2022 ; Liu et al. 2022 ). GA and ABA function as mutually antagonistic hormones in growth regulation. GA accumulation promotes the degradation of DELLA proteins, which act as negative regulators of GA signaling downstream of the GA receptor (Peng et al. 1997 ; Golldack et al. 2013 ; Bouré and Arnaud 2023 ). This degradation process is particularly important under adverse environmental conditions (Jiang and Fu 2007 ). Studies have demonstrated that a quadruple della mutant exhibited reduced inhibition of seed germination under high-salinity condition (Achard et al. 2006 ). Together, these findings underscore the critical roles of GA and ABA in balancing plant growth with stress adaptation. GA regulates the expression of numerous genes in plants, among which the GASAs gene family is particularly notable for its roles in plant growth and development. These genes are present in various species, including tomato GAST (Shi et al. 1992 ), petunia GIP1 / 2 / 3 / 4 (Navarro et al. 2008 ), rice OsGASR1 , OsGASR2 , and OsGSR1 (Furukawa et al. 2006 ; Wang et al. 2009 ), beech ( Fagus sylvatica ) FsGASA4 , and soybean ( Glycine max ) GsGASA1 (Li et al. 2011 ). In Arabidopsis , the GASA family comprises 14 members, each containing three distinct domains: (1) a 18–29 amino acid signal peptide; (2) a variable region that exhibits significant variation in amino acid composition and sequence length among family members; (3) a hydrophilic core and conserved C-terminal region that contain 12 Cys-rich peptides (CRP) termed as “GASA domain” (Wigoda et al. 2006 ). Furthermore, the expression of certain AtGASA genes is affected by phytohormones other than GA. AtGASA4 and AtGASA6 expression is promoted by growth hormones such as auxin, brassinosteroids, cytokinins, and GA, while it is inhibited by stress hormones such as ABA and JA (Qu et al. 2016 ). These findings suggest that GASA genes are integral components of complex hormonal crosstalk, linking growth regulation with stress responses. The 12 Cys residues in GASA proteins have the potential to form up to six disulfide bridges, which are necessary for maintaining protein structure stability, interaction with other proteins and reversible reduction and oxidation, particularly in response to biotic and abiotic stresses (Wigoda et al. 2006 ). Several GASA family genes have been functionally characterized. For instance, FsGASA4 and AtGASA5 regulate the expression of salicylic acid (SA) pathway genes, thereby participating in the response to abiotic stress (Alonso-Ramírez et al. 2009 ; Zhang and Wang 2011 ). AtGASA6 and OsGSR1 are involved in regulating multiple hormones signaling pathways (Wang et al. 2009 ; Zhong et al. 2015 ). Furthermore, AtENO2 is a positive germination regulator under salt stress, and transcriptomic analyses revealed that AtGASA1 expression is markedly reduced in the eno2 mutant (Wu et al. 2022 ). Despite being a homolog of the AtGASA family, the role of AtGASA1 in abiotic stress responses, particularly under salt stress, remains unclear. Given the importance of disulfide bridges in redox regulation and stress adaptation, investigating AtGASA1 under salt stress conditions is critical to elucidate the molecular mechanisms by which the GASA family contributes to salt tolerance in plants. In this study, we found that AtGASA1 may act as a downstream regulator of GA, controlling plant growth, leaf expansion, and flowering time. In addition, the gasa1 mutant was highly sensitive to salt stress, whereas overexpression of AtGASA1 enhanced salt tolerance. Further analyses revealed that the cysteine residues at positions 40 and 44 are critical for reducing ROS accumulation. Collectively, our study provides genetic and molecular evidence that deepens the understanding of AtGASA1-mediated regulation of both plant development and salt stress responses in Arabidopsis . 2. Materials and methods 2.1. Plant materials and growth conditions The wild-type used in our study was Arabidopsis thaliana ecotype Columbia-0 (Col-0). All mutants and transgenic plants used in our work were in the Col-0 background. The homozygous T-DNA knockout (SALK-001187.34.70.x) of AtGASA1 was obtained from the Arabidopsis Biological Resource Center (ABRC). The 35S::GASA1 transgenic plants were generated by transforming pCAMBIA 1302.1 into Col-0 genetic background via A. tumefaciens strain (GV3101) transformation using the Arabidopsis floral-dip method (Bent 2006 ). Seeds were surface sterilized as described previously (Sun et al. 2013 ). Seedlings were grown at 23°C with 60% relative humidity under a 16 h light (photon flux density 70 µmol/m 2 /s)/ 8 h dark cycle. 2.2 Analysis of GUS activity The pGASA1::GUS transgenic lines were obtained by cloning a 1260 bp promoter region of AtGASA1 into the pCAMBIA1305.1 vector. For GUS staining, various tissues of seedlings or adult plants were processed as described previously (Cai et al. 2014 ). Plant material was immersed in GUS staining solution containing 2 mM ferricyanide, and 2 mM ferrocyanide, 20 mM phosphate buffer, 1 mg/ml of X-Gluc, and incubated overnight at 37°C in the dark. Subsequently, the plant materials were washed three times with the buffer solution and decolorized with 70% ethanol to remove chlorophyll. For GA 3 treatment, a working solution of 10 µM GA 3 was prepared by diluting 1 mM GA 3 with water. For each pot, 20 ml of 10 µM GA 3 solution was slowly poured onto the soil. As a control, the control plants were treated with 20 ml of water. 2.3 DAB and NBT staining H 2 O 2 and O 2 − accumulation in seedlings was assessed by DAB and NBT staining, respectively. The two-week-old seedlings grown in petri dishes were transferred to MS 0 medium containing 100mM NaCl and cultured for 3 days. The seedlings were immersed in DAB and NBT staining solution for 5 hours in the dark. Then chlorophyll was eliminated with 95% ethanol. 2.4 H 2 DCF-DA staining H 2 O 2 production in guard cells was detected using the fluorescent probe H 2 DCF-DA. Two-week-old seedlings were treated with NaCl for 3 days. Seedlings were then immersed in Tris-KCl buffer (10 mM Tris, 50 mM KCl, pH 6.5), added with 20 µM H 2 DCF-DA for 30 min at 28°C under dark conditions. Samples were washed five times with fresh Tris-KCl buffer to remove excess dye. Fluorescence was observed using an excitation wavelength of 488 nm, where green fluorescence indicated H₂O₂ accumulation and red fluorescence corresponded to chlorophyll autofluorescence. 2.5 Salt tolerance evaluation For the seed germination assay, seeds were horizontally germinated on 1/2 MS medium with or without 100 mM NaCl, germination (emergence of radicles) and post-germination growth (green cotyledon appearance) were scored at the indicated time points (Zhao et al. 2021 ). Root length was measured on 1/2 MS medium with or without 100 mM NaCl at the 10 day (Chen et al. 2023 ). The relative water content (RWC) was measured as described previously (Zegaoui et al. 2017 ). Arabidopsis seedlings were grown on 1/2 MS medium supplemented with 100 mM NaCl. After 7 days of salt treatment, RWC was determined for each genotype. Chlorophyll content was measured as described previously (He et al. 2021 ; Chen et al. 2023 ). A. thaliana lines were grown in soil for one month and then exposed to 100 mM NaCl for 14 days to evaluate chlorophyll content. 2.6 RNA isolation and quantitative RT-qPCR RNA extraction and cDNA synthesis were performed as described previously(Pu et al. 2020 ). Quantitative reverse transcription PCR (RT-qPCR) was performed using optical 96-well plate in real-time PCR system (Bio-Rad Laboratories; Hercules, CA, USA), with cycling program: denaturation at 95°C for 5 min, 39 cycles of 95°C for 10 s, annealing at corresponding temperature for 30 s. Fold changes of samples were calculated by 2 −ΔΔCt method. The primers are listed in supplementary file 1. 2.7 Functional Identification with a Yeast Expression System A mutated AtGASA1 cDNA construct was generated by substituting specific cysteine residues within the conserved domain with alanine residues. The AtGASA1 ORF was first cloned into the PGAD-T7 vector. Using this construct as a template, primers containing the desired mutations were used for PCR amplification. The resulting PCR products were transformed into E. coli for plasmid amplification. The confirmed plasmids were then introduced into yeast cells using the LiAc/PEG method (Zou et al. 2022 ). For the salt tolerance assay, single yeast colonies were cultured and diluted to the same density. Aliquots (2 µL) of yeast suspensions were spotted onto SD-Leu solid medium containing different concentrations of NaCl. Plates were incubated at 30°C for 2–5 days and then photographed. 2.8 Data analysis Data analysis and graph generation were performed using software program GraphPad Prism V. 9.0. One-way ANOVA was used for comparison of multiple data sets, with p˂ 0.05 as criterion for significant difference. 3. Results 3.1. AtGASA1 expression pattern analysis Previous studies have shown that the expression of certain GASA family genes exhibits tissue-specific and developmental stage-specific patterns (Bouteraa et al. 2023 ). RT-qPCR analysis revealed AtGASA1 expression in multiple tissues, including rosette leaves, cauline leaves, shoots, flowers, and silique (Fig. 1 A). Subsequently, we employed the AtGASA1 promoter to drive β-glucuronidase (GUS) expression. The substrate X-Gluc was hydrolyzed into a blue compound, allowing visualization of GUS activity. In pGASA1::GUS plants, tissue-specific GUS staining patterns were consistent with RT-qPCR results, with GUS activity observed in inflorescences, vascular tissue, shoot apex, cut end of stems (Fig. 1 B). Furthermore, GUS staining was also performed at different developmental stages. At cotyledon stage, GUS expression appeared as blue dots at edges of rosette leaves. As the plants developed, these dots gradually expanded and spread along leaf edges (Fig. 1 C). Since most members of the AtGASA gene family are induced by GA (Bouteraa et al. 2023 ), we investigated the response of AtGASA1 after exogenous application of GA 3 . RT-qPCR analysis showed that AtGASA1 transcript levels increased approximately thirtyfold at 3 h after GA 3 treatment (Fig. 1 D). Consistently, GUS staining of pGASA1::GUS plants treated with 10 µM GA 3 revealed strong GUS signals in rosette leaves at 3 h, which markedly decreased by 24 h (Fig. 1 E). 3.2. Role of AtGASA1 in Arabidopsis Growth Previous research has indicated that most CRP proteins are involved in regulating plant growth and development (Marshall et al. 2011 ). To elucidate the function of the AtGASA1 , we first obtained a T-DNA insertion gasa1 mutant (Fig. 2 A). We then generated a 35S::GASA1 overexpression line and 35S-GASA1::gasa1 line. Compared with Col-0, AtGASA1 expression was significantly reduced in the gasa1 mutant but markedly increased in the overexpression lines (Supplementary file 2). After approximately four weeks of growth, phenotypic analyses revealed that gasa1 mutant exhibited reduced growth, whereas 35S::GASA1 plants showed enhanced growth (Fig. 3 B, C). This difference gradually diminished as plants reached the adult stage. Measurements of leaf area confirmed significant differences among gasa1 , 35S::GASA1 and Col-0 plants (Fig. 3 D). In addition, 35S::GASA1 plants displayed earlier flowering compared with Col-0 (Fig. 3 E). Together, these results indicate that AtGASA1 positively regulates plant growth and development in Arabidopsis . 3.3 AtGASA1 may be involved in the process of salt tolerance Previous studies have shown that several AtGASA genes enhance plant tolerance to abiotic stress by regulating ROS levels (Rubinovich and Weiss 2010 ). To investigate whether AtGASA1 contributes to salt tolerance, we examined its expression under salt stress using RT-qPCR. The results showed that AtGASA1 transcript levels were slightly downregulated at the early stage, but subsequently increased and peaked at 24 h (Fig. 3 A). Consistently, pGASA1::GUS plants displayed stronger GUS staining under salt stress, with larger blue-stained areas observed across developmental stages (Fig. 3 B), indicating that AtGASA1 expression accumulates during the salt stress response. Subsequently, we conducted measurements of germination rate and survival rate for gasa1 mutant, 35S::GASA1 , 35S-GASA1::gasa1 and Col-0. The gasa1 mutant showed a lower germination rate than Col-0 on the fifth day. The 35S::GASA1 line exhibits faster germination, but shows no significant difference in final germination rate compared to Col-0 (Fig. 3 C, 3 D). After 15 days of growth on 100 mM NaCl, the survival rate of the gasa1 mutant was lower, whereas 35S::GASA1 plants displayed higher survival compared with Col-0 (Fig. 3 E, 3 F). These results indicated that overexpression of AtGASA1 in plants enhances their salt tolerance. 3.4 Overexpression of AtGASA1 enhances the tolerance of Arabidopsis to salt stress To further clarify the role of AtGASA1 in salt stress adaptation in Arabidopsis , we measured several physiological indexes in plants of different genotypes. In the root elongation experiment, 35S::GASA1 and 35S-GASA1::gasa1 plants exhibited longer roots than Col-0 (Fig. 4 A). Under the stress of 100 mM NaCl, root lengths of the two transgenic lines hardly changed, while the roots of the gasa1 mutant were significantly shortened (Fig. 4 A, 4 B). Measurements of chlorophyll content revealed that transgenic plants retained more chlorophyll than Col-0 under salt stress (Fig. 4 C). At 100 mM NaCl stress, the relative water content of plants of all genotypes decreased, but the transgenic plants had a significant reduction in water loss (Fig. 4 D). Meanwhile, the transgenic plants had a higher cotyledon greening rate than Col-0 (Fig. 4 E). These findings indicate that overexpression of AtGASA1 enhances salt tolerance in Arabidopsis , thereby improving plant survival under stress conditions. 3.5 AtGASA1 positively modulates salt tolerance in Arabidopsis Salt stress damages the plasma membrane of Arabidopsis cells, leading to electrolyte leakage. The rate of electrolyte leakage in the gasa1 mutant was slightly higher than that of Col-0 after salt stress (Fig. 5 A). Measurement of malondialdehyde (MDA), an indicator of membrane lipid peroxidation, showed that 35S::GASA1 plants accumulated significantly lower MDA levels than Col-0 (Fig. 5 B). Under salt stress, plant cells also accumulate organic substances like proline to alleviate osmotic stress. Consistently, 35S::GASA1 plants had higher proline content than Col-0, whereas the gasa1 mutant contained less (Fig. 5 C). Furthermore, expression analysis of the proline synthetase gene P5CS1 revealed reduced transcript levels in gasa1 compared with Col-0 under salt stress (Fig. 5 D). Together, these results suggest that overexpression of AtGASA1 enhances salt tolerance in Arabidopsis by reducing membrane damage and promoting proline accumulation. 3.6 AtGASA1 modulates the transcription of salt stress-responsive genes The impaired salt tolerance of the gasa1 mutant may be linked to altered expression of stress-responsive genes. To test this, the transcription levels of several selected stress-responsive genes were examined in all lines, including genes known to play a role in ABA signaling or ROS scavenging pathway. Under salt stress, 35S::GASA1 plants exhibited 2–3 fold higher expression of RD20 and NCED3 compared with Col-0, whereas the gasa1 mutant showed reduced expression (Fig. 6 A, 6 B). Similarly, the expression of ROS-scavenging genes CAT2 and APX6 was markedly decreased in gasa1 under salt stress, while expression levels in mock-treated plants were not significantly different among genotypes (Fig. 6 C, 6 D). These findings suggest that defects in H 2 O 2 scavenging in gasa1 lead to excessive ROS accumulation and greater sensitivity to salt stress. 3.7 AtGASA1 improves Arabidopsis salt tolerance by regulating the accumulation of ROS The balance between ROS and antioxidants is important because both extremes of oxidative stress and antioxidant stress are harmful. The content of O 2 − can be visually observed by NBT staining. Compared with Col-0, the accumulation of O 2 − was lower in 35S::GASA1 and higher in gasa1 mutant (Fig. 7 A). The H 2 O 2 accumulation was examined using the DAB staining in leaves. DAB staining result showed there no significant difference in the accumulation of H 2 O 2 in Col-0, gasa1 , 35S::GASA1 and 35S-GASA1::gasa1 plants treated with H 2 O. However, under salt stress treatment, the gasa1 mutant exhibited a darker staining, while the 35S::GASA1 line displayed lighter staining (Fig. 7 B). H 2 O 2 levels in guard cells were further quantified using the fluorescent probe H 2 DCF-DA in 2-week-old seedlings treated with 100 mM NaCl for 3 h. Under salt stress, the fluorescence intensity of Col-0 was significantly higher than that after H 2 O treatment. Moreover, the gasa1 mutant exhibited higher fluorescence, whereas 35S::GASA1 plants showed lower fluorescence, indicating enhanced accumulation of H 2 O 2 in the guard cells of the gasa1 plants and reduced accumulation in the 35S::GASA1 plants (Fig. 7 C). These results indicated that AtGASA1 improves Arabidopsis salt tolerance by suppressing the accumulation of ROS. 3.8 AtGASA1 enhances yeast or plant’s salt tolerance by requiring the presence of 40-CYS and 44-CYS To study the role of the conserved cysteines in AtGASA1 protein, a construct was generated containing AtGASA1 cDNA in which different conserved cysteines were replaced by alanine (Fig. 8 A). The primary functions of AtGASA1 were analyzed with a yeast heterologous expression system. Salt tolerance assays revealed yeast cells expressing AtGASA1 C40,44A was significantly higher sensitive to salt than that only transformed with AtGASA1, but yeast cells expressing AtGASA1 C40,44,48A and AtGASA1 C40,44,48,64A still seem to have some salt tolerance compared to AH109 (Fig. 8 B). The result suggested that disulfide bond containing 40-CYS and 44-CYS might act as key regulators in salt tolerance. We next generated transgenic Arabidopsis plants over-expressing AtGASA1 C40,44A under the CaMV 35S promoter. Compared with the 35S::GASA1 line, the 35S::GASA1 C40,44A -1 and 35S::GASA1 C40,44A -2 lines exhibited reduced growth and lower survival rate under salt stress (Fig. 8 C, 8 D). Besides, H 2 O 2 accumulation was examined using the H 2 DCF-DA. We found that the fluorescence intensity was higher in 35S::GASA1 C40,44A -1 and 35S::GASA1 C40,44A -2 lines compared to 35S::GASA1 line (Fig. 8 E). These results demonstrated that 40-CYS and 44-CYS in AtGASA1 are crucial for inhibiting H 2 O 2 accumulation in Arabidopsis . 4. Discussion Most CRP are involved in regulation of plant growth and development, including the promotion or inhibition of cell elongation and division, as well as the control of flowering time (Shi et al. 1992 ; Ben-Nissan and Weiss 1996 ; de la Fuente et al. 2006 ). AtGASA14 regulates leaf expansion and accelerates flowering by suppressing two DELLA proteins, GAI and RGA (Sun et al. 2013 ). Consistently, we observed that AtGASA1 also regulates flowering time, and may functions downstream of the DELLA protein. In contrast, AtGASA5 suppresses flowering via GA pathway by promoting FLC and downregulating FT and LFY (Zhang et al. 2009 ). Petunia GIP2 and tomato GAST1 promote stem elongation by facilitating cell elongation (Shi et al. 1992 ; Ben-Nissan et al. 2004 ). The function of AtGASA1 is similar to these proteins, as it regulates leaf size and flowering time. Moreover, expression of GASA family genes is associated with young tissues and actively growing organs, indicating their involvement in cellular processes such as cell division or expansion. In the past decade, structures and functions of GASA proteins have become increasingly clear, as numerous GASA -like genes have been identified in a variety of plant species. Silverstein et al. ( 2007 ), in a study of 33 plant species, identified 12824 CRP genes and 445 encoding GASA proteins (Silverstein et al. 2007 ). The CRP within GASA proteins play a crucial role because these cysteine residues have the potential to form up to six disulfide bridges (Wigoda et al. 2006 ). The potato Snakin-1 protein exhibits predicted disulfide bond patterns such as CysI-CysIX, CysII-CysVII, CysIII-CysIV, CysV-CysXI, CysVI-CysXII, and CysVIII-CysX, which are thought to stabilize protein structure and regulate redox-related functions (Porto and Franco 2013 ). In this study, we demonstrated that cysteines 40 and 44 are critical for the function of AtGASA1. The yeast expressing AtGASA1 C40,44A was significantly less sensitive to salt stress than those expressing the non-mutated AtGASA1. Besides, the 35S::GASA1 C40,44A lines exhibited more H 2 O 2 accumulation and lower survival rate compared with 35S::GASA1 lines under salt stress (Fig. 8 ). These disulfide bridges are likely essential for establishing the proper 3D structure of GASA proteins, facilitating their interaction with other proteins and enabling them to undergo reversible reduction and oxidation processes. The disulfide bonds in the GASA protein can act as an electronic donor or acceptor to play catalytic roles in redox reactions (Wigoda et al. 2006 ). Overexpression of AtGASA4 has been shown to enhance antioxidant activity by inhibiting ROS accumulation (Rubinovich and Weiss 2010 ). Interestingly, a partial structural domain of AtGASA4 shares similarity with the cysteine-rich region of the ATP-binding cassette protein ABCE1, and substitution of the cysteine residues with alanine abolished both its ROS-reducing ability and GA responsiveness (Rubinovich and Weiss 2010 ). It shows that GASA domain is the key for AtGASA4. Similarly, our study reveals that AtGASA1 also suppressed H 2 O 2 accumulation to enhance salt stress resistance in Arabidopsis . Under salt stress conditions, the 35S::GASA1 line displays lower levels of H 2 O 2 , while the gasa1 mutant exhibits higher levels of H 2 O 2 compared to Col-0 (Fig. 7 ). AtGASA5 may function as a metalloprotein utilizing iron as a cofactor to exert antioxidant activity (Rubinovich et al. 2014 ). AtGASA14 has also been shown to confer resistance to ABA and salt stress by modulating ROS accumulation (Sun et al. 2013 ). Moreover, overexpression of FsGASA4 enhance the antioxidant capacity of Arabidopsis (Alonso-Ramírez et al. 2009 ). These results indicate that GASA proteins can suppress the levels of ROS to improve the plant tolerance to abiotic stress. Unlike most signaling molecules with defined receptors, ROS signaling primarily occurs through oxidative post-translational modifications (De Smet et al. 2019 ; Huang et al. 2019 ; Nietzel et al. 2020 ). Fluctuations in intracellular ROS levels can alter the structure and function of proteins, thereby modulating multiple signaling pathways (Mittler et al. 2022 ). One of the main physiological targets of H 2 O 2 is the reversible oxidation of cysteine thiolate anions (S⁻) (Rampon et al. 2018 ). H 2 O 2 oxidizes thiolate anion to sulfenic acid (–SOH), an unstable intermediate that can react with neighboring –SH groups to form disulfide bonds. At the same time, H₂O₂ may also be reduced to water, helping to alleviate excess ROS and maintain redox balance (Akter et al. 2015 ). Conversely, under reducing conditions, disulfide bonds can be readily cleaved, restoring the thiol groups. Given that AtGASA1 contains multiple conserved cysteine residues, it is plausible that it contributes to ROS homeostasis through disulfide bond dynamics. In addition, AtGASA1 may act as a signaling mediator through redox-dependent interactions with other proteins, thus fine-tuning ROS balance. Future studies should aim to identify AtGASA1-interacting partners to clarify its broader role in redox signaling and stress adaptation. Most of GASA genes are regulated by various phytohormones. For example, rice OsGSR1 is induced by GA and inhibited by BR, and it directly interacts with the BR synthase DIM/DWF1 to regulate BR biosynthesis (Wang et al. 2009 ). In the OsGSR1 RNAi rice line, the content of GA 4 is increased, but it exhibits a phenotype lacking GA response, suggesting that OsGSR1 is required for GA signal transduction (Wang et al. 2009 ). AtGASA5, as a negative regulatory protein in response to heat stress, by suppressing SA signaling and reduces the antioxidant capacity of Arabidopsis (Zhang and Wang 2011 ). Interestingly, GASA5 functions by reducing the expression level of NPR1 to block the transmission of SA signals, suggesting the GASA family might also be involved in the plant immune response against bacterial pathogens (Rubinovich et al. 2014 ). In FsGASA4 overexpression lines, the levels of ABA and SA increase, whereas in the gasa4 mutant, JA and ABA levels decrease without significantly affecting SA content (Alonso-Ramírez et al. 2009 ). Compared with the Col-0 plants, the transcription levels of RD20 and NCED3 were 2–3 times higher in the salt-treated 35S::GASA1 , indicated that AtGASA1 may also be involved in the ABA signaling pathway to enhance salt tolerance (Fig. 6 ). These studies suggest that the GASA family genes may mediate plant defense responses by participating in the regulation of multiple hormones. Fourteen members of the AtGASA protein family have been identified in Arabidopsis , but their functions and mechanisms are poorly understood at the gene or protein levels. Only four members, AtGASA4, AtGASA5, AtGASA6, and AtGASA14, have been extensively studied and characterized. Therefore, there is still a need to investigate the functions of the remaining other AtGASA proteins. Our current findings suggest that AtGASA1 plays a role in regulating growth and development, responding to salt stress, and participating in hormone crosstalk and redox homeostasis. However, future work is needed to identify and characterize the potential interacting proteins of AtGASA1, which will be essential for elucidating its precise molecular functions and regulatory networks. Overall, these findings provide a valuable foundation for future in-depth investigations into the molecular mechanisms of AtGASA1. Declarations Conflict of interest statement The authors declare that the research described here involved no commercial or financial relationships that could be construed as potential conflicts of interest. Contributions Jian-Bo Song, Hao-En He, Cai-Feng Wang, Xian-Zhi Zuo screened material. Zi-Xin Zhao, Ya-Ru Li designed the study. Jian-Bo Song, Yu-Fan Chen, Shu-Fan Liu analysed data. Jian-Bo Song, Han-Wen Guo, Xuan Huang discussed the results and wrote the paper. Acknowledgements This study was supported by grants from the National Natural Science Foundation of China (31300223), Natural Science Foundation of Shaanxi Province (2025JC-YBMS-241, 2016JM3001), International Science and Technology Cooperation Project (2024GH-YBXM-23), National Training Programs of Innovation and Entrepreneurship for Undergraduate (202210697001). Data Availability Supplementary file 1: Gene-specific primers used in experiments. Supplementary file 2: The relative expression level of AtGASA1 in the different GASA1-genotype plants. Supplementary file 3: Schematic diagram of site-directed mutagenesis of AtGASA1. References Achard P, Cheng H, De Grauwe L et al (2006) Integration of plant responses to environmentally activated phytohormonal signals. Science 311:91–94. https://doi.org/10.1126/science.1118642 Akter S, Huang J, Waszczak C et al (2015) Cysteines under ROS attack in plants: a proteomics view. 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Plant Cell 27:1445–1460. https://doi.org/10.1105/tpc.15.00144 Zou T, Pu L, Lin R et al (2022) Roles of Canavalia rosea metallothioneins in metal tolerance and extreme environmental adaptation to tropical coral reefs. J Plant Physiol 268:153559. https://doi.org/10.1016/j.jplph.2021.153559 Supplementary Files Supplementaryfile1.docx Supplementaryfile2.docx Supplementaryfile3.docx Cite Share Download PDF Status: Published Journal Publication published 17 Apr, 2026 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Minor revisions 10 Feb, 2026 Reviewers agreed at journal 25 Nov, 2025 Reviewers invited by journal 25 Nov, 2025 Editor assigned by journal 07 Nov, 2025 First submitted to journal 03 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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LS: lateral stem; ST: stamen; RL: rosette leaf; SE: sepal; IF: inflorescence; R: root; F: flower; CL: cauline leaf; S: silicle. Values are means ± SD (n = 3). Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P \u0026lt; 0.05) \u003cstrong\u003e(B)\u003c/strong\u003e GUS staining was performed at different tissues. MS: main stem; S: silicle; VT: vascular tissue; SA: shoot apex; F: flower; C: cotyledons; ST: stamen; CL: cauline leaf; RL: rosette leaf; IS: inflorescent stem; R: root; V: leaf vein.\u003cstrong\u003e (C)\u003c/strong\u003e \u003cem\u003eAtGASA1 \u003c/em\u003eexpression at various developmental stages.\u003cstrong\u003e \u003c/strong\u003eTL: two-leaf stage; FL: four-leaf stage; SL: six-leaf stage; EL: eight-leaf stage. \u003cstrong\u003e(D)\u003c/strong\u003e The expression level of \u003cem\u003eAtGASA1\u003c/em\u003e upon exogenous GA\u003csub\u003e3\u003c/sub\u003e treatment was evaluated by RT-qPCR.\u003cstrong\u003e \u003c/strong\u003eValues are means ± SD (n = 3), Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05).\u003cstrong\u003e (E) \u003c/strong\u003e\u003cem\u003eAtGASA1\u003c/em\u003e was induced by GA\u003csub\u003e3\u003c/sub\u003e at 3 h. GA\u003csub\u003e3 \u003c/sub\u003ewas diluted with water to make 10 μM working solution, \u003cem\u003epGASA1\u003c/em\u003e::\u003cem\u003eGUS\u003c/em\u003e plants were soil drenched with 20 mL of each solution.\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/f31ebfeed0e25912f77197ed.png"},{"id":97157402,"identity":"3b5320b2-3789-48b6-a260-7fc77e1a522c","added_by":"auto","created_at":"2025-12-01 11:44:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2377236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRole of AtGASA1 in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eGrowth.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of AtGASA1 gene. Arrows: position and direction of T-DNA insertion in gasa1 mutant. boxes = exons, lines = introns, and gray boxes = 5’ and 3’ UTRs. \u003cstrong\u003e(B)\u003c/strong\u003e Phenotype analysis of the different AtGASA1-genotype plants after 20 days of germination, respectively. \u003cstrong\u003e(C)\u003c/strong\u003e Individual leaves of different AtGASA1-genotype plants after 20 days of germination. \u003cstrong\u003e(D)\u003c/strong\u003eLeaf areas of the different AtGASA1-genotype plants. Values are means ± SD (n=15). Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05). \u003cstrong\u003e(E)\u003c/strong\u003e Early flowering phenotypes of the \u003cem\u003e35S::GASA1\u003c/em\u003eplants.\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/7988358d76878b29ec24dc07.png"},{"id":97248878,"identity":"29cd7e9f-374c-4542-818b-b10ccad4a63e","added_by":"auto","created_at":"2025-12-02 13:07:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4305884,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtGASA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e positively modulates salt tolerance in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Arabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e After 7 days of germination, the seedlings are transferred to MS\u003csub\u003e0\u003c/sub\u003e medium containing 100 mM NaCl. The expression levels of \u003cem\u003eAtGASA1\u003c/em\u003e at different time points were determined using RT-qPCR. \u003cstrong\u003e(B)\u003c/strong\u003e The seedlings are transferred to MS\u003csub\u003e0\u003c/sub\u003e medium containing 100 mM NaCl. The \u003cem\u003epGASA1::GUS\u003c/em\u003e lines were performed GUS staining at 24 h. \u003cstrong\u003e(C)\u003c/strong\u003e Phenotypic comparison of the different plants. \u003cstrong\u003e(D)\u003c/strong\u003e Percentage of germinated seeds of the different lines. \u003cstrong\u003e(E)\u003c/strong\u003e Phenotypic comparison of the different lines under 100 mM NaCl. \u003cstrong\u003e(F)\u003c/strong\u003e Survival rate of germinated seeds of the different lines under 100 mM NaCl. Values are means ± SD, Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/976d85309ede255cdc835d91.png"},{"id":97157404,"identity":"be38c923-2e45-47cd-b05e-298343452616","added_by":"auto","created_at":"2025-12-01 11:44:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1529620,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtGASA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e may be involved in the process of salt tolerance. (A) (B)\u003c/strong\u003eRoot development phenotype and length of different plants under 0 mM or 50 mM NaCl. Values are means ± SD (n = 20). \u003cstrong\u003e(C-E)\u003c/strong\u003e Evaluation of physiological indicators of different plants under salt stress. \u003cstrong\u003e(C)\u003c/strong\u003e Chlorophyll content. \u003cstrong\u003e(D)\u003c/strong\u003e Relative water content. \u003cstrong\u003e(E)\u003c/strong\u003e Cotyledon greening. Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/bd120cf033d4f7bcd6d89118.png"},{"id":97157406,"identity":"ee618dc8-435a-473a-b4ab-5d6f5693fa85","added_by":"auto","created_at":"2025-12-01 11:44:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1614472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAtGASA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eimproves \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e resistance to salt stress. (A)\u003c/strong\u003e The rate of electrolyte leakage in different plants under salt stress. \u003cstrong\u003e(B)\u003c/strong\u003e Measurement of MDA content in different plants under salt stress.\u003cstrong\u003e (C)\u003c/strong\u003eMeasurement of proline content in different plants under salt stress. \u003cstrong\u003e(D)\u003c/strong\u003e Relative transcript abundance of \u003cem\u003eP5CS1\u003c/em\u003ewas determined by RT-qPCR. Values are means ± SD (n = 3), different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/80a423d081038bf8af97a367.png"},{"id":97248641,"identity":"b3c91673-e1b1-4c4e-af8d-7b06ad4ade32","added_by":"auto","created_at":"2025-12-02 13:04:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1235357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtGASA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e modulates the transcription of salt stress-responsive genes in Col-0, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egasa1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e35S::GASA1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e35S-GASA1::gasa1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e line.\u003c/strong\u003e Relative transcript abundance of \u003cem\u003eRD20\u003c/em\u003e, \u003cem\u003eNCED3\u003c/em\u003e, \u003cem\u003eAPX6\u003c/em\u003eand \u003cem\u003eCAT2\u003c/em\u003e was determined by RT-qPCR. The 4-wk-old plants were treated with 100 mM NaCl, and sampled at 0 h, 3 h and 6 h, respectively. Values are means ± SD (n = 3), Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/bba4baa60834916439abd887.png"},{"id":97248448,"identity":"1d924470-daf0-4446-85bb-d103ac608a6d","added_by":"auto","created_at":"2025-12-02 12:58:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":14662755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eAtGASA1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reduces salt-induced ROS production in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. (A)\u003c/strong\u003e NBT staining of Col-0, \u003cem\u003egasa1\u003c/em\u003e mutant, \u003cem\u003e35S::GASA1\u003c/em\u003e and \u003cem\u003e35S-GASA1::gasa1\u003c/em\u003e line leaves were treated with or without 100 mM NaCl. Bar = 1 mm. \u003cstrong\u003e(B)\u003c/strong\u003e DAB staining of the different \u003cem\u003eAtGASA1\u003c/em\u003e-genotype plants was treated with or without 100 mM NaCl. Bar = 1 mm. \u003cstrong\u003e(C)\u003c/strong\u003e H\u003csub\u003e2\u003c/sub\u003eDCF-DA staining in lower epidermal cells of the different \u003cem\u003eAtGASA1\u003c/em\u003e-genotype plants were treated with or without 100 mM NaCl. Bar = 10 μm.\u003c/p\u003e","description":"","filename":"Figure.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/17db03e064418c0d80fdc4f9.png"},{"id":97248667,"identity":"5c94b5bc-1870-4bf8-bc9f-c96ebbf2d898","added_by":"auto","created_at":"2025-12-02 13:05:05","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":16807291,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe presence of CYS-40 and CYS-44 in AtGASA1 is essential for the inhibition of ROS accumulation in plants or yeast. (A)\u003c/strong\u003e Schematic representation of conserved domains in the AtGASA1 protein. The red 'C' and red 'A' represent the mutation of CYS to Ala. The green 'C' represents the non-mutated CYS capable of forming disulfide bonds normally. \u003cstrong\u003e(B) \u003c/strong\u003eThe salt tolerance confirmation of AtGASA1 in yeast by heterologous expression assay.\u003cstrong\u003e (C)\u003c/strong\u003e Phenotypic comparison of the different AtGASA1-genotype plants under 100 mM NaCl. \u003cstrong\u003e(D)\u003c/strong\u003e Survival rate of germinated seeds of the different AtGASA1-genotype plants under 100 mM NaCl. \u003cstrong\u003e(E)\u003c/strong\u003e H\u003csub\u003e2\u003c/sub\u003eDCF-DA staining in lower epidermal cells of the different GASA1-genotype plants were treated with or without 100 mM NaCl. Values are means ± SD (n = 3), Different letters above each bar indicate a significant difference determined by one-way ANOVA Tukey’s multiple range tests with SPSS statistics software (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"Figure.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/fefd79569022c5aa14b49138.png"},{"id":107350779,"identity":"d8592c65-cc2d-4bfe-b248-42425e7de399","added_by":"auto","created_at":"2026-04-20 16:04:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":45128286,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/182ea5af-b15b-4fcf-8979-e56462522197.pdf"},{"id":97248657,"identity":"4ea44130-5140-4be4-a54a-282269bbf6a5","added_by":"auto","created_at":"2025-12-02 13:04:58","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":16752,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/aea0e0f49e0d1e70ba47c7c4.docx"},{"id":97248619,"identity":"939a09e7-9d6e-4a71-9156-50499acde55d","added_by":"auto","created_at":"2025-12-02 13:04:18","extension":"docx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":871729,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/95af1cce3cad51939982e481.docx"},{"id":97157426,"identity":"bbfddc57-cb5d-4511-bca0-f4ab1f8b9c56","added_by":"auto","created_at":"2025-12-01 11:44:17","extension":"docx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":809335,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-7978126/v1/1352917ec5d71c546b07b3c4.docx"}],"financialInterests":"","formattedTitle":"AtGASA1 positively regulates Arabidopsis response to salt stress by suppressing accumulation of reactive oxygen species","fulltext":[{"header":"Key Message","content":"\u003cp\u003eAtGASA1 regulates plant growth and development, while conserved Cys-40 and Cys-44 residues in its GASA domain are crucial for controlling ROS accumulation under salt stress.\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eReactive oxygen species (ROS) play key roles in plant stress perception, the integration of salt stress-response signaling networks, and the activation of defense mechanisms (Zandalinas and Mittler \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Medina et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). ROS accumulation in cells can alter expression of resistance genes and influence plant resilience to environmental stresses (Suzuki et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zou et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nietzel et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As signal molecules in response to salinity, low-level of ROS participate in the regulation of ABA-dependent stomatal closure, ethylene-dependent leaf abscission and activation of mitogen-activated protein kinase cascades (Zou et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In contrast, excessive ROS production is harmful to plant growth and development (Tavanti et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). To maintain redox homeostasis, plants have evolved sophisticated systems to balance ROS generation and scavenging (Sachdev et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lanza and Reis \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Overexpression of key antioxidant genes, such as \u003cem\u003eSUPEROXIDE DISMUTASES\u003c/em\u003e (\u003cem\u003eSOD\u003c/em\u003e), \u003cem\u003eCATALASE\u003c/em\u003e (\u003cem\u003eCAT\u003c/em\u003e) and \u003cem\u003eASCORBATE PEROXIDASE\u003c/em\u003e (\u003cem\u003eAPX\u003c/em\u003e), has been demonstrated to improve plant salinity stress tolerance by increasing ROS scavenging capacity in a variety of plant species (Suzuki et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, p. 2; Xu et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSalt stress has emerged as an increasingly prevalent and severe problem worldwide in recent years, leading to reduced crops yield and decreased the quality (Parmar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Soltabayeva et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Hormones, such as gibberellic acid (GA) and abscisic acid (ABA), play pivotal roles in mediating salt stress responses at different developmental stages (Christmann et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zhang and Wang \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Verma et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). ABA inhibit seed germination and seedling growth, promotes stomatal closure to minimize water loss, and regulate the expression of genes associated with salt tolerance (Lata and Prasad \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Rai et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Moreover, ABA increases cytosolic pH, thereby activating ion channels and inducing enzymes that stimulate ROS production (Huang et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). GA and ABA function as mutually antagonistic hormones in growth regulation. GA accumulation promotes the degradation of DELLA proteins, which act as negative regulators of GA signaling downstream of the GA receptor (Peng et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Golldack et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Bour\u0026eacute; and Arnaud \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This degradation process is particularly important under adverse environmental conditions (Jiang and Fu \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Studies have demonstrated that a quadruple \u003cem\u003edella\u003c/em\u003e mutant exhibited reduced inhibition of seed germination under high-salinity condition (Achard et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Together, these findings underscore the critical roles of GA and ABA in balancing plant growth with stress adaptation.\u003c/p\u003e\u003cp\u003eGA regulates the expression of numerous genes in plants, among which the GASAs gene family is particularly notable for its roles in plant growth and development. These genes are present in various species, including tomato \u003cem\u003eGAST\u003c/em\u003e (Shi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), petunia \u003cem\u003eGIP1\u003c/em\u003e/\u003cem\u003e2\u003c/em\u003e/\u003cem\u003e3\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e (Navarro et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), rice \u003cem\u003eOsGASR1\u003c/em\u003e, \u003cem\u003eOsGASR2\u003c/em\u003e, and \u003cem\u003eOsGSR1\u003c/em\u003e (Furukawa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), beech (\u003cem\u003eFagus sylvatica\u003c/em\u003e) \u003cem\u003eFsGASA4\u003c/em\u003e, and soybean (\u003cem\u003eGlycine max\u003c/em\u003e) \u003cem\u003eGsGASA1\u003c/em\u003e (Li et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In \u003cem\u003eArabidopsis\u003c/em\u003e, the GASA family comprises 14 members, each containing three distinct domains: (1) a 18\u0026ndash;29 amino acid signal peptide; (2) a variable region that exhibits significant variation in amino acid composition and sequence length among family members; (3) a hydrophilic core and conserved C-terminal region that contain 12 Cys-rich peptides (CRP) termed as \u0026ldquo;GASA domain\u0026rdquo; (Wigoda et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, the expression of certain \u003cem\u003eAtGASA\u003c/em\u003e genes is affected by phytohormones other than GA. \u003cem\u003eAtGASA4\u003c/em\u003e and \u003cem\u003eAtGASA6\u003c/em\u003e expression is promoted by growth hormones such as auxin, brassinosteroids, cytokinins, and GA, while it is inhibited by stress hormones such as ABA and JA (Qu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). These findings suggest that GASA genes are integral components of complex hormonal crosstalk, linking growth regulation with stress responses.\u003c/p\u003e\u003cp\u003eThe 12 Cys residues in GASA proteins have the potential to form up to six disulfide bridges, which are necessary for maintaining protein structure stability, interaction with other proteins and reversible reduction and oxidation, particularly in response to biotic and abiotic stresses (Wigoda et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Several \u003cem\u003eGASA\u003c/em\u003e family genes have been functionally characterized. For instance, \u003cem\u003eFsGASA4\u003c/em\u003e and \u003cem\u003eAtGASA5\u003c/em\u003e regulate the expression of salicylic acid (SA) pathway genes, thereby participating in the response to abiotic stress (Alonso-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhang and Wang \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). \u003cem\u003eAtGASA6\u003c/em\u003e and \u003cem\u003eOsGSR1\u003c/em\u003e are involved in regulating multiple hormones signaling pathways (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Zhong et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Furthermore, AtENO2 is a positive germination regulator under salt stress, and transcriptomic analyses revealed that \u003cem\u003eAtGASA1\u003c/em\u003e expression is markedly reduced in the \u003cem\u003eeno2\u003c/em\u003e mutant (Wu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Despite being a homolog of the AtGASA family, the role of AtGASA1 in abiotic stress responses, particularly under salt stress, remains unclear. Given the importance of disulfide bridges in redox regulation and stress adaptation, investigating AtGASA1 under salt stress conditions is critical to elucidate the molecular mechanisms by which the GASA family contributes to salt tolerance in plants.\u003c/p\u003e\u003cp\u003eIn this study, we found that \u003cem\u003eAtGASA1\u003c/em\u003e may act as a downstream regulator of GA, controlling plant growth, leaf expansion, and flowering time. In addition, the \u003cem\u003egasa1\u003c/em\u003e mutant was highly sensitive to salt stress, whereas overexpression of \u003cem\u003eAtGASA1\u003c/em\u003e enhanced salt tolerance. Further analyses revealed that the cysteine residues at positions 40 and 44 are critical for reducing ROS accumulation. Collectively, our study provides genetic and molecular evidence that deepens the understanding of AtGASA1-mediated regulation of both plant development and salt stress responses in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Plant materials and growth conditions\u003c/h2\u003e\u003cp\u003eThe wild-type used in our study was \u003cem\u003eArabidopsis thaliana\u003c/em\u003e ecotype Columbia-0 (Col-0). All mutants and transgenic plants used in our work were in the Col-0 background. The homozygous T-DNA knockout (SALK-001187.34.70.x) of \u003cem\u003eAtGASA1\u003c/em\u003e was obtained from the \u003cem\u003eArabidopsis\u003c/em\u003e Biological Resource Center (ABRC). The \u003cem\u003e35S::GASA1\u003c/em\u003e transgenic plants were generated by transforming pCAMBIA 1302.1 into Col-0 genetic background via \u003cem\u003eA. tumefaciens\u003c/em\u003e strain (GV3101) transformation using the \u003cem\u003eArabidopsis\u003c/em\u003e floral-dip method (Bent \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Seeds were surface sterilized as described previously (Sun et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Seedlings were grown at 23\u0026deg;C with 60% relative humidity under a 16 h light (photon flux density 70 \u0026micro;mol/m\u003csup\u003e2\u003c/sup\u003e/s)/ 8 h dark cycle.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Analysis of GUS activity\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003epGASA1::GUS\u003c/em\u003e transgenic lines were obtained by cloning a 1260 bp promoter region of \u003cem\u003eAtGASA1\u003c/em\u003e into the pCAMBIA1305.1 vector. For GUS staining, various tissues of seedlings or adult plants were processed as described previously (Cai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Plant material was immersed in GUS staining solution containing 2 mM ferricyanide, and 2 mM ferrocyanide, 20 mM phosphate buffer, 1 mg/ml of X-Gluc, and incubated overnight at 37\u0026deg;C in the dark. Subsequently, the plant materials were washed three times with the buffer solution and decolorized with 70% ethanol to remove chlorophyll. For GA\u003csub\u003e3\u003c/sub\u003e treatment, a working solution of 10 \u0026micro;M GA\u003csub\u003e3\u003c/sub\u003e was prepared by diluting 1 mM GA\u003csub\u003e3\u003c/sub\u003e with water. For each pot, 20 ml of 10 \u0026micro;M GA\u003csub\u003e3\u003c/sub\u003e solution was slowly poured onto the soil. As a control, the control plants were treated with 20 ml of water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 DAB and NBT staining\u003c/h2\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e accumulation in seedlings was assessed by DAB and NBT staining, respectively. The two-week-old seedlings grown in petri dishes were transferred to MS\u003csub\u003e0\u003c/sub\u003e medium containing 100mM NaCl and cultured for 3 days. The seedlings were immersed in DAB and NBT staining solution for 5 hours in the dark. Then chlorophyll was eliminated with 95% ethanol.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 H\u003csub\u003e2\u003c/sub\u003eDCF-DA staining\u003c/h2\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production in guard cells was detected using the fluorescent probe H\u003csub\u003e2\u003c/sub\u003eDCF-DA. Two-week-old seedlings were treated with NaCl for 3 days. Seedlings were then immersed in Tris-KCl buffer (10 mM Tris, 50 mM KCl, pH 6.5), added with 20 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eDCF-DA for 30 min at 28\u0026deg;C under dark conditions. Samples were washed five times with fresh Tris-KCl buffer to remove excess dye. Fluorescence was observed using an excitation wavelength of 488 nm, where green fluorescence indicated H₂O₂ accumulation and red fluorescence corresponded to chlorophyll autofluorescence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Salt tolerance evaluation\u003c/h2\u003e\u003cp\u003eFor the seed germination assay, seeds were horizontally germinated on 1/2 MS medium with or without 100 mM NaCl, germination (emergence of radicles) and post-germination growth (green cotyledon appearance) were scored at the indicated time points (Zhao et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Root length was measured on 1/2 MS medium with or without 100 mM NaCl at the 10 day (Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The relative water content (RWC) was measured as described previously (Zegaoui et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). \u003cem\u003eArabidopsis\u003c/em\u003e seedlings were grown on 1/2 MS medium supplemented with 100 mM NaCl. After 7 days of salt treatment, RWC was determined for each genotype. Chlorophyll content was measured as described previously (He et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). \u003cem\u003eA. thaliana\u003c/em\u003e lines were grown in soil for one month and then exposed to 100 mM NaCl for 14 days to evaluate chlorophyll content.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 RNA isolation and quantitative RT-qPCR\u003c/h2\u003e\u003cp\u003eRNA extraction and cDNA synthesis were performed as described previously(Pu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Quantitative reverse transcription PCR (RT-qPCR) was performed using optical 96-well plate in real-time PCR system (Bio-Rad Laboratories; Hercules, CA, USA), with cycling program: denaturation at 95\u0026deg;C for 5 min, 39 cycles of 95\u0026deg;C for 10 s, annealing at corresponding temperature for 30 s. Fold changes of samples were calculated by 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The primers are listed in supplementary file 1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Functional Identification with a Yeast Expression System\u003c/h2\u003e\u003cp\u003eA mutated AtGASA1 cDNA construct was generated by substituting specific cysteine residues within the conserved domain with alanine residues. The AtGASA1 ORF was first cloned into the PGAD-T7 vector. Using this construct as a template, primers containing the desired mutations were used for PCR amplification. The resulting PCR products were transformed into \u003cem\u003eE. coli\u003c/em\u003e for plasmid amplification. The confirmed plasmids were then introduced into yeast cells using the LiAc/PEG method (Zou et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For the salt tolerance assay, single yeast colonies were cultured and diluted to the same density. Aliquots (2 \u0026micro;L) of yeast suspensions were spotted onto SD-Leu solid medium containing different concentrations of NaCl. Plates were incubated at 30\u0026deg;C for 2\u0026ndash;5 days and then photographed.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Data analysis\u003c/h2\u003e\u003cp\u003eData analysis and graph generation were performed using software program GraphPad Prism V. 9.0. One-way ANOVA was used for comparison of multiple data sets, with p˂ 0.05 as criterion for significant difference.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1. \u003cem\u003eAtGASA1\u003c/em\u003e expression pattern analysis\u003c/h2\u003e\u003cp\u003ePrevious studies have shown that the expression of certain \u003cem\u003eGASA\u003c/em\u003e family genes exhibits tissue-specific and developmental stage-specific patterns (Bouteraa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). RT-qPCR analysis revealed \u003cem\u003eAtGASA1\u003c/em\u003e expression in multiple tissues, including rosette leaves, cauline leaves, shoots, flowers, and silique (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Subsequently, we employed the \u003cem\u003eAtGASA1\u003c/em\u003e promoter to drive β-glucuronidase (GUS) expression. The substrate X-Gluc was hydrolyzed into a blue compound, allowing visualization of GUS activity. In \u003cem\u003epGASA1::GUS\u003c/em\u003e plants, tissue-specific GUS staining patterns were consistent with RT-qPCR results, with GUS activity observed in inflorescences, vascular tissue, shoot apex, cut end of stems (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, GUS staining was also performed at different developmental stages. At cotyledon stage, GUS expression appeared as blue dots at edges of rosette leaves. As the plants developed, these dots gradually expanded and spread along leaf edges (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSince most members of the \u003cem\u003eAtGASA\u003c/em\u003e gene family are induced by GA (Bouteraa et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), we investigated the response of \u003cem\u003eAtGASA1\u003c/em\u003e after exogenous application of GA\u003csub\u003e3\u003c/sub\u003e. RT-qPCR analysis showed that AtGASA1 transcript levels increased approximately thirtyfold at 3 h after GA\u003csub\u003e3\u003c/sub\u003e treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). Consistently, GUS staining of \u003cem\u003epGASA1::GUS\u003c/em\u003e plants treated with 10 \u0026micro;M GA\u003csub\u003e3\u003c/sub\u003e revealed strong GUS signals in rosette leaves at 3 h, which markedly decreased by 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2. Role of AtGASA1 in \u003cem\u003eArabidopsis\u003c/em\u003e Growth\u003c/h2\u003e\u003cp\u003ePrevious research has indicated that most CRP proteins are involved in regulating plant growth and development (Marshall et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). To elucidate the function of the \u003cem\u003eAtGASA1\u003c/em\u003e, we first obtained a T-DNA insertion \u003cem\u003egasa1\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). We then generated a \u003cem\u003e35S::GASA1\u003c/em\u003e overexpression line and \u003cem\u003e35S-GASA1::gasa1\u003c/em\u003e line. Compared with Col-0, \u003cem\u003eAtGASA1\u003c/em\u003e expression was significantly reduced in the \u003cem\u003egasa1\u003c/em\u003e mutant but markedly increased in the overexpression lines (Supplementary file 2). After approximately four weeks of growth, phenotypic analyses revealed that \u003cem\u003egasa1\u003c/em\u003e mutant exhibited reduced growth, whereas \u003cem\u003e35S::GASA1\u003c/em\u003e plants showed enhanced growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). This difference gradually diminished as plants reached the adult stage. Measurements of leaf area confirmed significant differences among \u003cem\u003egasa1\u003c/em\u003e, \u003cem\u003e35S::GASA1\u003c/em\u003e and Col-0 plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). In addition, \u003cem\u003e35S::GASA1\u003c/em\u003e plants displayed earlier flowering compared with Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Together, these results indicate that \u003cem\u003eAtGASA1\u003c/em\u003e positively regulates plant growth and development in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 \u003cem\u003eAtGASA1\u003c/em\u003e may be involved in the process of salt tolerance\u003c/h2\u003e\u003cp\u003ePrevious studies have shown that several \u003cem\u003eAtGASA\u003c/em\u003e genes enhance plant tolerance to abiotic stress by regulating ROS levels (Rubinovich and Weiss \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). To investigate whether \u003cem\u003eAtGASA1\u003c/em\u003e contributes to salt tolerance, we examined its expression under salt stress using RT-qPCR. The results showed that \u003cem\u003eAtGASA1\u003c/em\u003e transcript levels were slightly downregulated at the early stage, but subsequently increased and peaked at 24 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Consistently, \u003cem\u003epGASA1::GUS\u003c/em\u003e plants displayed stronger GUS staining under salt stress, with larger blue-stained areas observed across developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), indicating that \u003cem\u003eAtGASA1\u003c/em\u003e expression accumulates during the salt stress response. Subsequently, we conducted measurements of germination rate and survival rate for \u003cem\u003egasa1\u003c/em\u003e mutant, \u003cem\u003e35S::GASA1\u003c/em\u003e, \u003cem\u003e35S-GASA1::gasa1\u003c/em\u003e and Col-0. The \u003cem\u003egasa1\u003c/em\u003e mutant showed a lower germination rate than Col-0 on the fifth day. The \u003cem\u003e35S::GASA1\u003c/em\u003e line exhibits faster germination, but shows no significant difference in final germination rate compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). After 15 days of growth on 100 mM NaCl, the survival rate of the \u003cem\u003egasa1\u003c/em\u003e mutant was lower, whereas \u003cem\u003e35S::GASA1\u003c/em\u003e plants displayed higher survival compared with Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results indicated that overexpression of \u003cem\u003eAtGASA1\u003c/em\u003e in plants enhances their salt tolerance.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Overexpression of \u003cem\u003eAtGASA1\u003c/em\u003e enhances the tolerance of \u003cem\u003eArabidopsis\u003c/em\u003e to salt stress\u003c/h2\u003e\u003cp\u003eTo further clarify the role of \u003cem\u003eAtGASA1\u003c/em\u003e in salt stress adaptation in \u003cem\u003eArabidopsis\u003c/em\u003e, we measured several physiological indexes in plants of different genotypes. In the root elongation experiment, \u003cem\u003e35S::GASA1\u003c/em\u003e and \u003cem\u003e35S-GASA1::gasa1\u003c/em\u003e plants exhibited longer roots than Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Under the stress of 100 mM NaCl, root lengths of the two transgenic lines hardly changed, while the roots of the \u003cem\u003egasa1\u003c/em\u003e mutant were significantly shortened (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Measurements of chlorophyll content revealed that transgenic plants retained more chlorophyll than Col-0 under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). At 100 mM NaCl stress, the relative water content of plants of all genotypes decreased, but the transgenic plants had a significant reduction in water loss (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Meanwhile, the transgenic plants had a higher cotyledon greening rate than Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These findings indicate that overexpression of \u003cem\u003eAtGASA1\u003c/em\u003e enhances salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e, thereby improving plant survival under stress conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5 \u003cem\u003eAtGASA1\u003c/em\u003e positively modulates salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eSalt stress damages the plasma membrane of \u003cem\u003eArabidopsis\u003c/em\u003e cells, leading to electrolyte leakage. The rate of electrolyte leakage in the \u003cem\u003egasa1\u003c/em\u003e mutant was slightly higher than that of Col-0 after salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Measurement of malondialdehyde (MDA), an indicator of membrane lipid peroxidation, showed that \u003cem\u003e35S::GASA1\u003c/em\u003e plants accumulated significantly lower MDA levels than Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Under salt stress, plant cells also accumulate organic substances like proline to alleviate osmotic stress. Consistently, \u003cem\u003e35S::GASA1\u003c/em\u003e plants had higher proline content than Col-0, whereas the \u003cem\u003egasa1\u003c/em\u003e mutant contained less (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Furthermore, expression analysis of the proline synthetase gene \u003cem\u003eP5CS1\u003c/em\u003e revealed reduced transcript levels in \u003cem\u003egasa1\u003c/em\u003e compared with Col-0 under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Together, these results suggest that overexpression of \u003cem\u003eAtGASA1\u003c/em\u003e enhances salt tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e by reducing membrane damage and promoting proline accumulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.6 \u003cem\u003eAtGASA1\u003c/em\u003e modulates the transcription of salt stress-responsive genes\u003c/h2\u003e\u003cp\u003eThe impaired salt tolerance of the \u003cem\u003egasa1\u003c/em\u003e mutant may be linked to altered expression of stress-responsive genes. To test this, the transcription levels of several selected stress-responsive genes were examined in all lines, including genes known to play a role in ABA signaling or ROS scavenging pathway. Under salt stress, \u003cem\u003e35S::GASA1\u003c/em\u003e plants exhibited 2\u0026ndash;3 fold higher expression of \u003cem\u003eRD20\u003c/em\u003e and \u003cem\u003eNCED3\u003c/em\u003e compared with Col-0, whereas the \u003cem\u003egasa1\u003c/em\u003e mutant showed reduced expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Similarly, the expression of ROS-scavenging genes \u003cem\u003eCAT2\u003c/em\u003e and \u003cem\u003eAPX6\u003c/em\u003e was markedly decreased in \u003cem\u003egasa1\u003c/em\u003e under salt stress, while expression levels in mock-treated plants were not significantly different among genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings suggest that defects in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging in \u003cem\u003egasa1\u003c/em\u003e lead to excessive ROS accumulation and greater sensitivity to salt stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.7 \u003cem\u003eAtGASA1\u003c/em\u003e improves \u003cem\u003eArabidopsis\u003c/em\u003e salt tolerance by regulating the accumulation of ROS\u003c/h2\u003e\u003cp\u003eThe balance between ROS and antioxidants is important because both extremes of oxidative stress and antioxidant stress are harmful. The content of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e can be visually observed by NBT staining. Compared with Col-0, the accumulation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e was lower in \u003cem\u003e35S::GASA1\u003c/em\u003e and higher in \u003cem\u003egasa1\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation was examined using the DAB staining in leaves. DAB staining result showed there no significant difference in the accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in Col-0, \u003cem\u003egasa1\u003c/em\u003e, \u003cem\u003e35S::GASA1\u003c/em\u003e and \u003cem\u003e35S-GASA1::gasa1\u003c/em\u003e plants treated with H\u003csub\u003e2\u003c/sub\u003eO. However, under salt stress treatment, the \u003cem\u003egasa1\u003c/em\u003e mutant exhibited a darker staining, while the \u003cem\u003e35S::GASA1\u003c/em\u003e line displayed lighter staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels in guard cells were further quantified using the fluorescent probe H\u003csub\u003e2\u003c/sub\u003eDCF-DA in 2-week-old seedlings treated with 100 mM NaCl for 3 h. Under salt stress, the fluorescence intensity of Col-0 was significantly higher than that after H\u003csub\u003e2\u003c/sub\u003eO treatment. Moreover, the \u003cem\u003egasa1\u003c/em\u003e mutant exhibited higher fluorescence, whereas \u003cem\u003e35S::GASA1\u003c/em\u003e plants showed lower fluorescence, indicating enhanced accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the guard cells of the \u003cem\u003egasa1\u003c/em\u003e plants and reduced accumulation in the \u003cem\u003e35S::GASA1\u003c/em\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These results indicated that \u003cem\u003eAtGASA1\u003c/em\u003e improves \u003cem\u003eArabidopsis\u003c/em\u003e salt tolerance by suppressing the accumulation of ROS.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.8 AtGASA1 enhances yeast or plant\u0026rsquo;s salt tolerance by requiring the presence of 40-CYS and 44-CYS\u003c/h2\u003e\u003cp\u003eTo study the role of the conserved cysteines in AtGASA1 protein, a construct was generated containing AtGASA1 cDNA in which different conserved cysteines were replaced by alanine (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). The primary functions of AtGASA1 were analyzed with a yeast heterologous expression system. Salt tolerance assays revealed yeast cells expressing AtGASA1\u003csup\u003eC40,44A\u003c/sup\u003e was significantly higher sensitive to salt than that only transformed with AtGASA1, but yeast cells expressing AtGASA1 \u003csup\u003eC40,44,48A\u003c/sup\u003e and AtGASA1 \u003csup\u003eC40,44,48,64A\u003c/sup\u003e still seem to have some salt tolerance compared to AH109 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). The result suggested that disulfide bond containing 40-CYS and 44-CYS might act as key regulators in salt tolerance. We next generated transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants over-expressing \u003cem\u003eAtGASA1\u003c/em\u003e \u003csup\u003e\u003cem\u003eC40,44A\u003c/em\u003e\u003c/sup\u003e under the CaMV 35S promoter. Compared with the \u003cem\u003e35S::GASA1\u003c/em\u003e line, the \u003cem\u003e35S::GASA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eC40,44A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-1\u003c/em\u003e and \u003cem\u003e35S::GASA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eC40,44A\u003c/em\u003e\u003c/sup\u003e \u003cem\u003e-2\u003c/em\u003e lines exhibited reduced growth and lower survival rate under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Besides, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation was examined using the H\u003csub\u003e2\u003c/sub\u003eDCF-DA. We found that the fluorescence intensity was higher in \u003cem\u003e35S::GASA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eC40,44A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-1\u003c/em\u003e and \u003cem\u003e35S::GASA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eC40,44A\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e-2\u003c/em\u003e lines compared to \u003cem\u003e35S::GASA1\u003c/em\u003e line (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). These results demonstrated that 40-CYS and 44-CYS in AtGASA1 are crucial for inhibiting H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation in \u003cem\u003eArabidopsis\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMost CRP are involved in regulation of plant growth and development, including the promotion or inhibition of cell elongation and division, as well as the control of flowering time (Shi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Ben-Nissan and Weiss \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; de la Fuente et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). AtGASA14 regulates leaf expansion and accelerates flowering by suppressing two DELLA proteins, GAI and RGA (Sun et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Consistently, we observed that AtGASA1 also regulates flowering time, and may functions downstream of the DELLA protein. In contrast, \u003cem\u003eAtGASA5\u003c/em\u003e suppresses flowering via GA pathway by promoting \u003cem\u003eFLC\u003c/em\u003e and downregulating \u003cem\u003eFT\u003c/em\u003e and \u003cem\u003eLFY\u003c/em\u003e (Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Petunia \u003cem\u003eGIP2\u003c/em\u003e and tomato \u003cem\u003eGAST1\u003c/em\u003e promote stem elongation by facilitating cell elongation (Shi et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Ben-Nissan et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). The function of AtGASA1 is similar to these proteins, as it regulates leaf size and flowering time. Moreover, expression of \u003cem\u003eGASA\u003c/em\u003e family genes is associated with young tissues and actively growing organs, indicating their involvement in cellular processes such as cell division or expansion.\u003c/p\u003e\u003cp\u003eIn the past decade, structures and functions of GASA proteins have become increasingly clear, as numerous \u003cem\u003eGASA\u003c/em\u003e-like genes have been identified in a variety of plant species. Silverstein et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), in a study of 33 plant species, identified 12824 \u003cem\u003eCRP\u003c/em\u003e genes and 445 encoding GASA proteins (Silverstein et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The CRP within GASA proteins play a crucial role because these cysteine residues have the potential to form up to six disulfide bridges (Wigoda et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The potato Snakin-1 protein exhibits predicted disulfide bond patterns such as CysI-CysIX, CysII-CysVII, CysIII-CysIV, CysV-CysXI, CysVI-CysXII, and CysVIII-CysX, which are thought to stabilize protein structure and regulate redox-related functions (Porto and Franco \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). In this study, we demonstrated that cysteines 40 and 44 are critical for the function of AtGASA1. The yeast expressing AtGASA1\u003csup\u003eC40,44A\u003c/sup\u003e was significantly less sensitive to salt stress than those expressing the non-mutated AtGASA1. Besides, the \u003cem\u003e35S::GASA1\u003c/em\u003e\u003csup\u003e\u003cem\u003eC40,44A\u003c/em\u003e\u003c/sup\u003e lines exhibited more H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation and lower survival rate compared with \u003cem\u003e35S::GASA1\u003c/em\u003e lines under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These disulfide bridges are likely essential for establishing the proper 3D structure of GASA proteins, facilitating their interaction with other proteins and enabling them to undergo reversible reduction and oxidation processes.\u003c/p\u003e\u003cp\u003eThe disulfide bonds in the GASA protein can act as an electronic donor or acceptor to play catalytic roles in redox reactions (Wigoda et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Overexpression of \u003cem\u003eAtGASA4\u003c/em\u003e has been shown to enhance antioxidant activity by inhibiting ROS accumulation (Rubinovich and Weiss \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Interestingly, a partial structural domain of AtGASA4 shares similarity with the cysteine-rich region of the ATP-binding cassette protein ABCE1, and substitution of the cysteine residues with alanine abolished both its ROS-reducing ability and GA responsiveness (Rubinovich and Weiss \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It shows that GASA domain is the key for AtGASA4. Similarly, our study reveals that AtGASA1 also suppressed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation to enhance salt stress resistance in \u003cem\u003eArabidopsis\u003c/em\u003e. Under salt stress conditions, the \u003cem\u003e35S::GASA1\u003c/em\u003e line displays lower levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, while the \u003cem\u003egasa1\u003c/em\u003e mutant exhibits higher levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e compared to Col-0 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). AtGASA5 may function as a metalloprotein utilizing iron as a cofactor to exert antioxidant activity (Rubinovich et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). AtGASA14 has also been shown to confer resistance to ABA and salt stress by modulating ROS accumulation (Sun et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Moreover, overexpression of \u003cem\u003eFsGASA4\u003c/em\u003e enhance the antioxidant capacity of \u003cem\u003eArabidopsis\u003c/em\u003e (Alonso-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). These results indicate that GASA proteins can suppress the levels of ROS to improve the plant tolerance to abiotic stress.\u003c/p\u003e\u003cp\u003eUnlike most signaling molecules with defined receptors, ROS signaling primarily occurs through oxidative post-translational modifications (De Smet et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Nietzel et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Fluctuations in intracellular ROS levels can alter the structure and function of proteins, thereby modulating multiple signaling pathways (Mittler et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). One of the main physiological targets of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is the reversible oxidation of cysteine thiolate anions (S⁻) (Rampon et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidizes thiolate anion to sulfenic acid (\u0026ndash;SOH), an unstable intermediate that can react with neighboring \u0026ndash;SH groups to form disulfide bonds. At the same time, H₂O₂ may also be reduced to water, helping to alleviate excess ROS and maintain redox balance (Akter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Conversely, under reducing conditions, disulfide bonds can be readily cleaved, restoring the thiol groups. Given that AtGASA1 contains multiple conserved cysteine residues, it is plausible that it contributes to ROS homeostasis through disulfide bond dynamics. In addition, AtGASA1 may act as a signaling mediator through redox-dependent interactions with other proteins, thus fine-tuning ROS balance. Future studies should aim to identify AtGASA1-interacting partners to clarify its broader role in redox signaling and stress adaptation.\u003c/p\u003e\u003cp\u003eMost of \u003cem\u003eGASA\u003c/em\u003e genes are regulated by various phytohormones. For example, rice OsGSR1 is induced by GA and inhibited by BR, and it directly interacts with the BR synthase DIM/DWF1 to regulate BR biosynthesis (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In the OsGSR1 RNAi rice line, the content of GA\u003csub\u003e4\u003c/sub\u003e is increased, but it exhibits a phenotype lacking GA response, suggesting that OsGSR1 is required for GA signal transduction (Wang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). AtGASA5, as a negative regulatory protein in response to heat stress, by suppressing SA signaling and reduces the antioxidant capacity of \u003cem\u003eArabidopsis\u003c/em\u003e (Zhang and Wang \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Interestingly, GASA5 functions by reducing the expression level of \u003cem\u003eNPR1\u003c/em\u003e to block the transmission of SA signals, suggesting the GASA family might also be involved in the plant immune response against bacterial pathogens (Rubinovich et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In \u003cem\u003eFsGASA4\u003c/em\u003e overexpression lines, the levels of ABA and SA increase, whereas in the \u003cem\u003egasa4\u003c/em\u003e mutant, JA and ABA levels decrease without significantly affecting SA content (Alonso-Ram\u0026iacute;rez et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Compared with the Col-0 plants, the transcription levels of \u003cem\u003eRD20\u003c/em\u003e and \u003cem\u003eNCED3\u003c/em\u003e were 2\u0026ndash;3 times higher in the salt-treated \u003cem\u003e35S::GASA1\u003c/em\u003e, indicated that \u003cem\u003eAtGASA1\u003c/em\u003e may also be involved in the ABA signaling pathway to enhance salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These studies suggest that the GASA family genes may mediate plant defense responses by participating in the regulation of multiple hormones.\u003c/p\u003e\u003cp\u003eFourteen members of the AtGASA protein family have been identified in \u003cem\u003eArabidopsis\u003c/em\u003e, but their functions and mechanisms are poorly understood at the gene or protein levels. Only four members, AtGASA4, AtGASA5, AtGASA6, and AtGASA14, have been extensively studied and characterized. Therefore, there is still a need to investigate the functions of the remaining other AtGASA proteins. Our current findings suggest that AtGASA1 plays a role in regulating growth and development, responding to salt stress, and participating in hormone crosstalk and redox homeostasis. However, future work is needed to identify and characterize the potential interacting proteins of AtGASA1, which will be essential for elucidating its precise molecular functions and regulatory networks. Overall, these findings provide a valuable foundation for future in-depth investigations into the molecular mechanisms of AtGASA1.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest statement\u003c/h2\u003e\u003cp\u003eThe authors declare that the research described here involved no commercial or financial relationships that could be construed as potential conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eContributions\u003c/h2\u003e\u003cp\u003eJian-Bo Song, Hao-En He, Cai-Feng Wang, Xian-Zhi Zuo screened material. Zi-Xin Zhao, Ya-Ru Li designed the study. Jian-Bo Song, Yu-Fan Chen, Shu-Fan Liu analysed data. Jian-Bo Song, Han-Wen Guo, Xuan Huang discussed the results and wrote the paper.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThis study was supported by grants from the National Natural Science Foundation of China (31300223), Natural Science Foundation of Shaanxi Province (2025JC-YBMS-241, 2016JM3001), International Science and Technology Cooperation Project (2024GH-YBXM-23), National Training Programs of Innovation and Entrepreneurship for Undergraduate (202210697001).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eSupplementary file 1: Gene-specific primers used in experiments.\u003c/p\u003e\u003cp\u003eSupplementary file 2: The relative expression level of \u003cem\u003eAtGASA1\u003c/em\u003e in the different GASA1-genotype plants.\u003c/p\u003e\u003cp\u003eSupplementary file 3: Schematic diagram of site-directed mutagenesis of AtGASA1.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAchard P, Cheng H, De Grauwe L et al (2006) Integration of plant responses to environmentally activated phytohormonal signals. 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J Plant Physiol 268:153559. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jplph.2021.153559\u003c/span\u003e\u003cspan address=\"10.1016/j.jplph.2021.153559\" 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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"salt stress, GA, reactive oxygen species (ROS), Arabidopsis, AtGASA1","lastPublishedDoi":"10.21203/rs.3.rs-7978126/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7978126/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"AtGASA1 is a member of the gibberellin acid-stimulating protein (GASA) family, characterized by a GASA domain containing 12 conserved cysteine-rich peptides (CRP). Its homologous genes are known to play an essential role in plant responses to both biotic and abiotic stresses; however, the function of AtGASA1 remains unclear. In this study, we found that AtGASA1 is involved in regulating plant growth, leaf expansion and flowering time. Moreover, various results showed that gasa1 mutants exhibited sensitivity to salt stress, while overexpression of AtGASA1 conferred increased resistance to salt stress. To explore the role of conserved cysteine residues within the GASA domain, site-directed mutagenesis was performed to substitute Cys-40 and Cys-44 with alanine. Functional assays in a yeast heterologous expression system showed that yeast expressing AtGASA1C40,44A displayed reduced tolerance to salt stress compared with yeast expressing non-mutated AtGASA1, indicating that these residues are critical for salt stress adaptation. Consistently, transgenic Arabidopsis plants overexpressing AtGASA1C40,44A accumulated higher levels of ROS compared with 35S::GASA1 plants. Collectively, our findings demonstrate that AtGASA1 positively regulates salt stress tolerance in Arabidopsis by reducing ROS accumulation.","manuscriptTitle":"AtGASA1 positively regulates Arabidopsis response to salt stress by suppressing accumulation of reactive oxygen species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 11:44:12","doi":"10.21203/rs.3.rs-7978126/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Minor revisions","date":"2026-02-11T04:18:16+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-11-25T17:22:21+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-25T13:16:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-07T16:26:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2025-11-03T21:56:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2e255324-b0c5-4367-882d-ae9df9197b02","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:01:32+00:00","versionOfRecord":{"articleIdentity":"rs-7978126","link":"https://doi.org/10.1007/s00299-026-03818-5","journal":{"identity":"plant-cell-reports","isVorOnly":false,"title":"Plant Cell Reports"},"publishedOn":"2026-04-17 15:57:11","publishedOnDateReadable":"April 17th, 2026"},"versionCreatedAt":"2025-12-01 11:44:12","video":"","vorDoi":"10.1007/s00299-026-03818-5","vorDoiUrl":"https://doi.org/10.1007/s00299-026-03818-5","workflowStages":[]},"version":"v1","identity":"rs-7978126","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7978126","identity":"rs-7978126","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}