ThASR4 enhances salt tolerances in transgenic Tamarix and Arabidopsis by Scavenging Reactive Oxygen Species

ThASR4 enhances salt tolerances in transgenic Tamarix and Arabidopsis by Scavenging 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 ThASR4 enhances salt tolerances in transgenic Tamarix and Arabidopsis by Scavenging Reactive Oxygen Species Zhenyu Zhu, Jie Wang, Hanyang Liu, Yu Zhang, Xin Zhao, Chao Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8909679/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract ASR genes are a class of genes that are expressed in plants in response to abscisic acid, abiotic stress, and fruit ripening. While they have been cloned and functionally characterized in various plant species, their study in Tamarix hispida has been limited. In this study, six ThASR genes were cloned from Tamarix hispida . Expression analysis revealed that ThASR genes respond to salt stress. Specifically, ThASR4 was found to be highly induced in both shoots and root of Tamarix hispida under salt stress, thus prompting its selection for further functional characterization in salt stress responses. ThASR4 is targeted to the nucleus and possesses transcriptional activity. Under salt stress conditions, compared with the wild-type (WT) lines, ThASR4 -overexpressing Arabidopsis thaliana exhibited enhanced germination rate, longer primary roots, and higher fresh weight, indicating a marked improvement in salt tolerance. Correspondingly, both ThASR4 -overexpressing T. hispida and A. thaliana plants exhibited significantly diminished levels of reactive oxygen species (ROS), malondialdehyde (MDA), and electrolyte leakage, along with elevated activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as increased proline (Pro) content, compared to Control plants. In contrast, ThASR4 RNA interference (RNAi) transgenic T. hispida displayed the opposite phenotypic and physiological outcomes. Furthermore, ThASR4 was found to upregulate the expression of ROS scavenger-associated genes ( ThPOD1, ThSOD1 and ThCAT3 ) and salt Stress-Responsive genes ( ThSOS3 , ThPIP2;5 , and ThDREB ). Collectively, our results indicate that ThASR4 enhances salt tolerance in T. hispida and A. thaliana by improving ROS scavenging capacity. The present study provides a theoretical foundation for further investigation into the regulatory mechanism of ThASR4 in the salt stress adaptation of T. hispida . ThASR4 Tamarix hispida Salt Stress ROS Scavenging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Highlights ThASR genes respond to salt stress Overexpression of ThASR4 enhances salt tolerance in transgenic Tamarix hispida and Arabidopsis thaliana ThASR4 reduces reactive oxygen species (ROS) accumulation and increases antioxidant enzyme activities ThASR4 upregulates the expression of the ThPOD1 , ThSOD1 , ThCAT3 , ThSOS3 , ThPIP2;5 and ThDREB gene. Introduction Abscisic acid-, stress-, and ripening-induced (ASR) genes are a class of plant-specific transcription factors (TFs), which play pivotal roles in plant growth, development, and responses to abiotic stress. The identification of ASR genes first occurred in tomato in 1993 (Iusem et al. 1993 ). Subsequent detection of these genes has been documented in maize (Liang et al. 2019 ), rice(Li et al. 2018 ), foxtail millet (Feng et al. 2016 ), banana (Miao et al. 2014), and the halophyte plant Suaeda liaotungensis (Hu et al. 2014 ). All ASR genes identified to date possess a simple structure, with two exons separated by a single intron. Correspondingly, ASR proteins contain two highly conserved domains: an N-terminal zinc-binding domain and a C-terminal ABA/WDS (abscisic acid/water deficit stress) domain that harbors a putative nuclear localization signal (NLS). This feature that may be critical for their transcription factor function. ASR plays a key role in promoting plant growth and fruit ripening by regulating the biosynthesis and metabolism of various compounds, including glucose, cell wall components, amino acids, ABA, and carotenoids (Zhang et al. 2024 ). Research on ASR in promoting fruit ripening has primarily focused on Rosaceae, Solanaceae, and cereal crops. Overexpression of ASR genes in tomatoes and strawberries has been shown to promote fruit ripening, resulting in a softer fruit texture and increased coloration, thereby further regulating fruit flavor and quality (Jia et al. 2016 ). The ASR1 gene specifically influences glucose accumulation in potato tubers (Frankel et al. 2007 ). In tobacco cells, the SlASR1 gene (from Solanum lycopersicum ) has been shown to regulate glucose metabolism by influencing the uptake of hexoses in heterotrophic organs, thereby contributing to carbon fixation in leaves (Dominguez et al. 2013 ). A lily ASR is preferentially expressed in the vegetative cell for pollen maturation. while the banana ASR ( MaASR ) can delay flowering time in Arabidopsis thaliana (Sun et al. 2016 ). Furthermore, ASR genes have been shown to enhance tolerance to multiple abiotic stresses, including drought, salinity, cold, and heavy metal toxicity. Overexpression of the BdASR1 gene has been shown to substantially increase drought tolerance in transgenic tobacco plants (Wang et al. 2016 ). OsASR1 plays a pivotal role in ABA-mediated regulation of stomatal closure, which helps conserve water during salt- and drought-stress conditions (Li et al. 2017 ). Moreover, OsASR1 overexpression can enhance salinity and drought tolerance, thereby resulting in improved crop yields (Park et al. 2020 ). Ectopic expression of OsASR1 has also been shown to improve cold tolerance in plants (Kim et al. 2009 ). Additionally, Maize ASR1 conferred Cd-tolerance in tobacco (Zhang et al. 2019 ). Both OsASR1 and OsASR5 are involved in regulating aluminum (Al)-responsive genes, which help reduce Al toxicity in rice (Arenhart et al. 2016 ). Salt stress has been demonstrated to induce an imbalance in ion homeostasis within plants. Specifically, an imbalance between Na + and K + ions has been shown to result in alterations to the intracellular osmotic pressure (Blumwald 2000 ). Furthermore, the presence of high concentrations of Na + ions within cells induces the production of large amounts of reactive oxygen species (ROS), including superoxide anions, hydroxyl radicals, hydrogen peroxide, and singlet oxygen (Qi et al. 2018 ). Excessive concentrations of reactive oxygen species can cause oxidative damage to cell membranes, proteins, RNA, and DNA molecules (Mittler 2002 ). It is therefore vital that antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are present in order to eliminate the toxicity of reactive oxygen species to cells (Petrov et al. 2012). In order to cope with salt stress, plants adopt a series of physiological, morphological, and molecular strategies, such as reducing ion absorption or accumulating ions in vacuoles to avoid the accumulation of toxic ions within cells. In addition, stress response factors activate protective enzyme activity to reduce ROS damage to cells(Choudhury 2017). Previous studies have demonstrated the involvement of ASR genes in plant salt stress tolerance. Research has shown that the OsASR1 gene from Oryza sativa affects net Na + uptake in plants. Under salt stress, the over-expression OsASR1 lines exhibited a reduced accumulation of Na + in comparison to the wild type (Park et al. 2020 ). Similarly, the IpASR -overexpressing transgenic Arabidopsis showed enhanced tolerance to salt and drought stress in comparison to the wild type, accompanied by a diminished accumulation of hydrogen peroxide (H 2 O 2 ) and superoxide (O2· − ) and an augmented activity of antioxidant enzymes in vivo. Kim et al. discovered that OsASR1 could function as an effective reactive oxygen species scavenger (Kim et al. 2012 ). Furthermore, ASR genes have been identified and functionally characterized in halophytes, includ ing Salicornia brachiata and Suaeda liaotungensis K (Hu et al. 2012). Halophytes are able to survive in the extreme environmental conditions due to their unique adaptation of tissue tolerance mechanisms. Tamarix hispida is a woody halophyte with strong salt tolerance, which is an excellent material for cloning salt-responsive genes and investigating salt tolerance mechanisms. However, the role of the ASR gene family in regulating salt tolerance in Tamarix hispida remains to be systematically elucidated. In this study, we identified six T. hispida ASR genes and analyzed their expression profiles in response to salt stress. Functional analysis demonstrated that ThASR4 overexpression enhanced the salt stress tolerance of transgenic T. hispida and A. thaliana. Under salt stress conditions, compared with wild-type (WT) plants, ThASR4 -overexpressing T. hispida exhibited significantly lower levels of H 2 O 2 and O2· − . Meanwhile, transgenic A. thaliana showed a higher seed germination rate, together with increased root length and fresh weight. Notably, the extent of cellular damage in transgenic lines was lower than that in WT plants. Furthermore, we verified that ThASR4 could enhance ROS scavenging capacity by upregulating the expression of antioxidant enzyme genes and the activities of corresponding enzymes. Under salt stress, the activities of POD and SOD were increased in transgenic plants. Consistently, the transcript levels of ROS scavenger-associated genes ( ThPOD1, ThSOD1 and ThCAT3 ) and salt stress-responsive genes ( ThSOS3 , ThPIP2;5 , and ThDREB ) were significantly elevated in transgenic lines compared with Control. This finding indicates that ThASR4 enhances the ROS scavenging capacity by upregulating the expression of ROS-related genes, thereby improving plant tolerance to salt stress. Experimental procedures 1.Plant material and treatment The Tamarix hispida were obtained from the State Key Laboratory of Tree Genetics and Breeding ( Northeast Forestry University). These plants were cultivated in vitro under a photoperiod of 16 hours of light and 8 hours of darkness on 1/2 MS medium supplemented with 0.6% powdered activated carbon. The greenhouse environment was maintained at a temperature of 24°C and a relative humidity level of 70%. Three-month-old Tamarix seedlings were irrigated with 200 mM NaCl for 1, 2, 6, 12, 24 and 48 hours. For each time point, the seedlings that were irrigated with tap water were used as the control. The roots and shoots of at least 10 seedlings were immediately harvested, pooled and frozen in liquid nitrogen and then stored at -80°C until required. Arabidopsis seeds were surface-sterilized with 75% (v/v) ethanol for three minutes and 25% (v/v) sodium hypochlorite for one minute, after which they were washed six times in sterile water. The sterilized seeds were germinated on MS solid medium. After seven days, the seedlings were transferred to a mixed soil substrate (black soil: vermiculite: perlite = 3:1:1) for cultivation. The seedlings were subsequently grown and cultured in growth chambers (16 h light/8 h dark cycle with a light intensity of 200 mmol m −2 s − 1 at 25°C). For the selection of transgenic Arabidopsis thaliana lines, the seedlings were placed on MS solid medium with 50 mg/ml kanamycin, with all other growth conditions kept consistent. The transgenic plants were likewise cultivated under these standardized conditions. 2.Cloning and bioinformatics analysis of 2.Cloning and bioinformatics analysis of ThASRs Encoding ASR family genes in Tamarix hispida were got with the gene-specific primers based on the transcriptome database (the primers used are shown in Supporting Information Table S1). PCR was performed using cDNA extracted as template, and the PCR products were linked to the PMD18-T vector (TaKaRa, Beijing, China) for sequencing. The potential ThASR proteins were analyzed using the PFAM database to confirm the presence of an ABA/WDS domain ( http://pfam.sanger.ac.uk/ ). Structural analysis of conserved regions of each non-redundant sequence was executed by SMART ( http://smart.embl-heidelberg.de/ ), Pfam( http://pfam.sanger.ac.uk/ ) and conserved domain database (CDD) searchesMultiple alignments of ASR protein sequences were carried out with ClustalX2.0. Phylogenetic trees were constructed by (iTOL( https://itol.embl.de/tree/ ). 3.Quantitative Real-time polymerase chain reaction Total RNA was extracted from T. hispida plants with a Plant RNeasy Extraction Kit (BioTeke, China). First-strand cDNA was synthesized from 1 mg of purified RNA using a PrimeScriptTM RT Reagent Kit (TaKaRa, Beijing, China). qRT-PCR was performed on a qTOWER3 G (Analytik Jene AG, Germany) with the β-actin (FJ618517) and α-tubulin (FJ618518) genes as internal controls. Each 20 mL reaction mixture contained 10 mL of SYBR-Green Real-time PCR Master Mix (Toyobo, Shanghai, China), specific primers (0.5 mM each), and 2 mL of cDNA template. The amplification was performed using the following cycling parameters: 94℃ for 30 s; 45 cycles of 94℃ for 12 s, 58℃ for 30 s, and 72℃ for 45 s; and then 82℃ for 1 s for plate reading. Three replicates were included for each sample (The primers used for qRT-PCR are detailed in Supporting Information Table S2). The Actin gene of T. hispida was used as the internal reference, and all experiments were performed with three biological replicates and three technical replicates; statistical significance was determined by one-way analysis of variance (ANOVA) (P < 0.05). 4.Subcellular localization of ThASR4 protein The complete coding sequence of ThASR4 was amplified by PCR using primers containing NcoI/SpeI restriction sites. The PCR product was digested with NcoI/SpeI (LABLEAD, Beijing, China), and cloned into the pBI121 vector so that the gene is under control of Cauliflower mosaic virus 35S (CaMV 35S) promoter to create a fusion construct (the primers used are shown in Table S1). Recombinant plasmids pBI121-ThASR4-GFP and pBI121-GFP (used as a control) were separately transferred into onion epidermal cells using particle bombardment. The transformed cells were then examined by laser-scanning confocal microscopy (LSM800: Karl Zeiss, Jena, Germany) after incubation at 25°C for 24 h on MS medium. 5.Transcriptional activation analysis in yeast The transcriptional activation activity of ThASR4 was determined in yeast cells. The complete coding sequence of ThASR4 , N-terminal ( ThASR4 -N) and C-terminal ( ThASR4 -C) fragments were amplified by PCR using primers, respectively. The PCR products were then cloned into EcoRI/BamHI sites in the pGBKT7 vector, resulting in the constructs pGBKT7- ThASR4 , pGBKT7- ThASR4 -N, and pGBKT7- ThASR4 -C (the primers used are shown in Table S1). The three constructs, along with the negative control pGBKT7 plasmid, were used to transform the Y2H yeast strain. The transformants were streaked on SD/Trp- and SD/Trp-/His-/X- \(\:{\alpha\:}\) -gol and incubated at 28°C for 3 days, after which the growth of the transformants was assessed. 6.Plant transformation and generation of transgenic plants The full-length coding sequence of ThASR4 was cloned into the NcoI/XbaI sites of the pROKII vector to create the overexpression vector pROKII-ThASR4. To silence ThASR4, a 300 bp inverted repeat truncated cDNA fragment of ThASR4 was inserted flanking the CHSA intron of the RNAi vector pFGC5941. The recombinant plasmids pROKII- ThASR4 and pFGC5941- ThASR4 under the control of the CaMV 35S promoter were introduced into Agrobacterium tumefaciens strain EHA105, respectively. The transient transformation of T. hispida was performed using an Agrobacterium-mediated transformation. As a result, transient overexpressing T. hispida (OE) and transient RNAi silencing T. hispida (RNAi) for ThASR4 were obtained. Transgenic Arabidopsis plants were generated using the Agrobacterium-mediated floral dip method. Seeds from these transgenic plants were selected on MS medium containing 50 mg/L of kanamycin. The T1 to T3 transgenic lines were confirmed by PCR using ThASR4 primers. Two independent T3 transgenic line seedlings, OX2 and OX5, which exhibited higher ThASR4 transcript levels, were selected for the stress tolerance assay. The primers employed for these assays are shown in Table S1. 7.Stress tolerance assay for transgenic plants For salt stress tolerance assay, Transient T. hispida OE, RNAi and Control were cultured on one-half MS medium with 200 mM NaCl respectively. We took samples at stress times of 0, 6 and 12 hours, snap-frozen them in liquid nitrogen and stored them in a -80 degree refrigerator. MDA and PRO content was determined using the MDA and PRO Detection Kit (A003 and A107; Jiancheng, Nanjing, China). while the electrolyte leakage measurement was conducted as described by Liang et al. ( 2019 ). For transgenic Arabidopsis seed germination and seedling growth under the salt stress assay, we put the sterilized transgenic Arabidopsis seeds OX2, OX5 and WT in the one-half MS medium with or without 200 mM NaCl respectively. After 7 days, we count the germination of seeds and the length of roots and fresh weight. 8.Physiological and histochemical staining analysis of transgenic plants After culturing for 48 h on 1/2 MS agar medium, the seedlings from transient transgenic T. hispida (OE, RNAi, Control) and transgenic Arabidopsis (OX2, OX5, WT) were transferred to 1/2 MS with 200 mM NaCl for 0, 6 and 12 h. Subsequently,, seedlings were harvested. For physiological analyses. SOD and POD activities were measured using the SOD and POD Detection Kit (A001 and A084; Jiancheng, Nanjing, China) according to the manufacturer’s instructions. The content of H 2 O 2 and O2· − was measured following the protocol of Liang et al. ( 2019 ). At least eight seedlings were used for the physiological index analysis per sample, and three independent biological repetitions were used to ensure the accuracy of the analysis. For histochemical staining analysis, nitroblue tetrazolium (NBT) (LABLEAD, Beijing, China), diaminobenzidine (DAB) (LABLEAD, Beijing, China), and Evans Blue (Beyotime, Shanghai, China) staining assays were performed according to the protocol of Liang et al. ( 2019 ). For each independent biological replicate, at least nine seedlings were collected for histochemical staining analysis. Representative images were captured from two randomly selected seedlings per sample. 9.ThASR4-Regulated Gene Expression Analysis Total RNA from T. hispida and ThASR4 -overexpressing T. hispida was used to analyze the expression levels of ThPOD1, ThSOD1 , ThCAT3, ThSOS3 , ThPIP2;5 , and ThDREB through qRT-PCR. The primers employed for these assays are shown in Table S2. RESULTS 1. Identification and Phylogenetic analysis of ThASR genes Six ASR genes were identified and isolated from Tamarix hispida . These ThASR genes encoded proteins with lengths ranging from 102 to 228 amino acids, a theoretical isoelectric point (pI) of 5.33 to 10.09. The stability indices of the proteins ranged from 26.08 to 46.26, indicating stability for all six proteins. The GRAVY values of the proteins fell between − 1.4 and − 1.0, confirming them as hydrophobic proteins (Table 1 ). Amino acid sequence alignment revealed the presence of two highly conserved regions in ThASRs (Fig. 1a). The first is located in the N-terminal region and consists of a consensus sequence of 10 amino acids, which includes a stretch of six histidine (His) residues, characteristic of zinc-binding domains. The second region is found in the C-terminal region and contains an ABA/WDS domain made up of 80 amino acids, which features two potential nuclear localization signals (NLS). The tertiary structure analysis of Tamarix hispida ASR genes shows that all six genes encode proteins containing a similar helix domain in structure (Fig. 1b). However, ThASR4 and ThASR6 only have an independent helix domain, whilst the remaining four proteins exhibit a loop-helix-loop domain. To gain an insight into the evolutionary relationships of the ASR proteins, a phylogenetic tree was constructed using thirty-nine ASR proteins from five different plant species, including tomato (Solanum lycopersicum), Populus trichocarpa, Oryza sativa, Sorghum bicolor and Zea mays. The phylogenetic tree revealed that ThASRs consisted of three major groups (Fig. 1c). The results demonstrate that ThASR1, ThASR2, ThASR3 and ThASR5 belong to the same branch and are closely related to OsASR1, OsASR2, and OsASR3. Indeed, OsASR1 and OsASR3 are upregulated by Fe and Al stresses. OsASR1 acts as a transcription factor in regulating the aluminum stress response in rice. Additionally, Rafael Augusto also found that OsASR5 regulates a large number of Al-responsive genes, suggesting that ThASR1, ThASR2, ThASR3 and ThASR5 may play a role in the response to metal stress. Furthermore, ThASR4 and ThASR6 are located in the same branch and exhibit strong similarity with OsASR4 and ZmASR4. ZmASR4 is thought to be associated with heavy metal transport and tolerance. The expression of OsASR4 was significantly increased in rice in response to abiotic stress. Therefore, ThASR4 and ThASR6 may also contribute to abiotic stress response. Table 1 Physicochemical properties and subcellular localization of ThASR transcription factor family proteins Gene name Number of amino acids Molecular weight (KD) Theoretical pI Instability index Aliphatic index Grand average of hydropathicity (GRAVY) Subcellular Localization Prediction ThASR1 112 12.78 10.09 26.92 75.09 -1.032 nucl,cyto ThASR2 106 12.12 9.7 36.35 57.17 -1.304 nucl ThASR3 108 12.37 6.65 45.1 53.61 -1.414 mito,nucl ThASR4 118 13.78 6.25 40.09 36.61 -1.622 cyto,nucl ThASR5 102 11.43 9.1 26.08 50 -1.226 mito ThASR6 228 24.44 5.33 48.26 22.81 -1.441 nucl Figure 1. Multiple sequence alignment and phylogenetic analysis of ThASRs A: Alignment of predicted amino acid sequences of ASR genes. B: Tertiary structure analysis of ThASR Family genes proteins. C: Phylogenetic relationships of ASR protein 2. Expression pattern of the in response to salt stress 2. Expression pattern of the ThASRs in response to salt stress To clarify the transcriptional response of ThASR genes to salt stress, the expression levels of six ThASR genes in T. hispida were analyzed at various time points (1 h, 2h, 6 h, 12 h, 24 h, 48 h) under NaCl treatment using quantitative real-time PCR (qRT-PCR). qRT-PCR results showed that all six ThASR genes were transcriptionally regulated by NaCl stress, and their expression was detected in both leaves and roots (Fig. 2 ). Notably, the ThASR gene family showed a uniform up-regulation pattern in roots, contrasting with a pattern of partial down-regulation and low abundance in shoots. These findings indicate that ThASRs likely exert a more pivotal function in mediating the salt stress response within the roots of T. hispida. Moreover, the expression of six ThASRs genes expression was detected significant differences under salt stress (Fig. 2 ). In roots, their expression levels exhibited a trend of increasing, then decreasing, and increasing again over time. Specifically, transcript levels rose to a peak at 2 or 6 hours, subsequently declined, and peaked again at 48 hours. Among these, ThASR1 reached its maximum (7.9-fold) at 6 hours, while ThASR 4 peaked at 2 hours (9.7-fold). Furthermore, In the leaves, the expression of ThASR2 , ThASR3 , ThASR5 , and ThASR6 was either downregulated or remained unchanged. In contrast, ThASR1 and ThASR4 were upregulated at all time points, generally following a trend of decreasing, then increasing, and decreasing again. Notably, ThASR4 showed the highest expression level among all genes tested. Among six ASR genes, ThASR4 showed the most significant up-regulation after salt treatment, indicated that ThASR4 expression was obviously induced in shoots and roots, so ThASR4 was selected for further investigation. 3. Subcellular Localization and Transcription Activation Assay of ThASR4 protein The subcellular localization of the ThASR4 protein was investigated in a transient expression assay with 35S:: ThASR4 -GFP (pBI121- ThASR4 -GFP) translational fusion in onion epidermal cells using particle bombardment. Fluorescence of the 35S:: ThASR4 -GFP chimera was observed in both the cellular nucleus and cytoplasm of onion epidermal cells. In contrast, the cells transformed with the control 35S::GFP (pBI121-GFP) exhibited fluorescence distributed throughout the entire cell, including the nucleus and plasma membrane (Fig. 3 a). This indicates that the ThASR4 protein is localized in both the nucleus and the cytoplasm. To ascertain the transcriptional activation potential of the ThASR4 protein and the location of its transcription activation domain, three recombinant vectors were constructed and transformed into Y2H Gold yeast cells. The vectors were pGBKT7- ThASR4 -FL (full-length ThASR4 ), pGBKT7- ThASR4 -N ( ThASR4 N-terminal), and pGBKT7- ThASR4 -C ( ThASR4 C-terminal) (Fig. 3 b). Following transformation, the transformants were cultured on SD/-Trp medium (to verify successful transformation) and SD/-Trp/-His/X-α-gal medium (to detect transcriptional activation activity). After three days, all yeast transformants exhibited normal growth on SD/-Trp medium, indicating that the vectors were successfully introduced into yeast cells. On SD/-Trp/-His/X-α-gal medium, yeast cells transformed with pGBKT7- ThASR4 -FL or pGBKT7- ThASR4 -C grew well and formed blue colonies. In contrast, yeast cells transformed with pGBKT7- ThASR4 -N exhibited no observable growth (Fig. 3 c). The results indicate that ThASR4 has transcriptional activation activity, and its transcriptional activation domain is localized in the C-terminal. In summary, ThASR4 protein was a nuclear and cytoplasm-localized protein with transcriptional activation activity. These findings suggest that ThASR4 may function as a transcription factor to participate in the transcriptional regulation of salt stress response-related genes in T. hispida . a: Subcellular localization of ThASR4 protein. b: Diagram of the pGBKT7- ThASR4 construct. c: Transactivation activity of the ThASR4 protein in yeast. 4. Overexpression of enhances salt tolerance of 4. Overexpression of ThASR4 enhances salt tolerance of Arabidopsis To assess the function of ThASR4 in stress tolerance, transgenic Arabidopsis plants overexpressing ThASR4 under the control of the CaMV35S promoter were generated. In eight independent homozygous ThASR4 lines, OX2 and OX5, with high expression levels were selected for further functional analysis (Fig. S1a,b). In normal conditions (MS medium), there were no significant differences in the rate of seed germination or the length of seedling roots between the transgenic and wild type (WT) Arabidopsis (Fig. 4 a,d). However, the OX2 and OX5 lines exhibited superior growth in comparison to the WT plants when cultivated on MS medium supplemented with 200 mM NaCl (Fig. 4 b). The transgenic lines displayed larger cotyledons compared to the WT plants, and their seed germination rates and root lengths were significantly greater (Fig. 4 b,e). Almost 83.7% of OX seeds were able to germinate, while WT seeds hardly germinated (Fig. 4 c). In addition, the root length of OX seedlings was longer than that of wild-type under salt treatment (Fig. 4 g). Furthermore, following a two-week growth period in soil irrigated with a 200 mM NaCl solution, the fresh weight of transgenic plants was significantly higher than that of wild-type plants (Fig. 4 f,h). These results indicate that ThASR4 plays a positive regulatory role in plant salt tolerance. a: Germination of WT and ThASR4 transgenic seeds on MS medium. b: Germination of WT and ThASR4 transgenic seeds on MS medium with 200mM NaCl. c: Statistical analysis of the seed germination rates. d: Root length of WT and ThASR4 transgenic seeds on MS medium. e: Root length of WT and ThASR4 transgenic seeds on MS medium with 200mM NaCl. f: The photographs of adult WT, OX2, OX5 seedlings after 200mM NaCl treatment for 5 days. g: Statistical analysis of the seed Root length. h: Statistical analysis of the fresh weights. 5. Generation of transiently transformed Tamarix hispida plants with transient overexpression or RNAi silencing of ThASR4 To further elucidate the role of ThASR4 in T. hispida salt tolerance, transgenic T. hispida plants exhibiting transient overexpression and RNAi silencing of ThASR4 were generated using transient transformation technology. Control plants were obtained by transforming T. hispida with the empty pROKII vector. Following the transient transformation method previously established for T. hispida , we successfully generated ThASR4 -overexpressing lines (OE), ThASR4 - silenced lines (RNAi) and Control. qRT-PCR results showed that the relative transcript level of ThASR4 in OE plants was significantly higher than that in the control plants, while the level in RNAi plants was significantly lower (Fig. 5 ). These results indicate that transient OE, RNAi and Control transgenic T. hispida plants were successfully obtained, and subsequently used for further investigation. qRT-PCR analysis of ThASR4 genes in different kinds of transiently transformed T. hispida , four-week-old T. hispida plants were transiently transformed with empty pROKII, 35S: ThASR4 or pFGC5941: ThASR4 , The expression of ThASR4 in whole OE, RNAi and Control plants was measured. The error bars are standard deviations, which were calculated from multiple replicates of qRT-PCR. OE: transgenic T.hispida overexpressing ThASR4 ; RNAi: ThASR4 RNAi-silenced T. hispida plants; Control: pROKII vector-transformed T. hispida plants. 6. improves ROS-scavenging capability and antioxidant enzyme activity Reactive oxygen species (ROS), such as superoxide (O2· − ) and hydrogen peroxide (H 2 O 2 ), are unavoidable byproducts of aerobic metabolism. The accumulation of ROS under salt stress can lead to significant oxidative damage in plants. Superoxide dismutase (SOD) and peroxidase (POD) are core antioxidant enzymes that scavenge ROS. To further confirm the function of the ThASR4 gene in scavenging ROS, we explored the accumulation of ROS and ROS-scavenging capability in ThASR4 transgenic plants. This included ThASR4 -overexpressing (OE) plants, ThASR4 -RNAi silencing (RNAi) plants, and control plants with empty pROKII vector, all transiently transformed in Tamarix hispida . Additionally, we analyzed ROS accumulation in ThASR4 -overexpressing Arabidopsis lines (OX2 and OX5). DAB and NBT staining results demonstrated that under normal growth conditions, there was no significant difference between the OE, RNAi, and Control plants. However, after 6 hours of 200 mM NaCl treatment, the OE plants showed the lightest brown (DAB staining) and blue (NBT staining) colors compared to the control plants, while the RNAi plants showed the darkest colors. Furthermore, with longer treatment, the staining deepened in all plants and the relative difference was maintained, with OE consistently showing the lightest colors and RNAi the darkest colors (Fig. 6 a, b). This indicates that following salt stress treatment, H 2 O 2 and O2· − levels decreased in the OE plants, whereas they significantly increased in the RNAi plants compared to the control plants (Fig. 6 c, d). Additionally, under salt stress conditions, SOD and POD activities were higher in OE plants than in control plants, whereas they were lower in RNAi plants. As the duration of stress increased, the differences in SOD and POD activities between the transgenic and control Tamarix plants became significantly greater (Fig. 6 e, f). Simultaneously, similar results were obtained in transgenic Arabidopsis plants. Under salt stress, H 2 O 2 and O2· − levels in the OX2 and OX5 lines were significantly lower than in the WT plants (Fig. 7 a-d), while SOD and POD activities in the OX2 and OX5 lines were markedly higher (Fig. 7 e, f). These findings suggest that ThASR4 overexpression enhances SOD and POD activity under salt stress, thereby promoting the scavenging of reactive oxygen species. A: DAB staining. B: NBT staining. C: NaCl induced H 2 O 2 content in transgenic Tamarix hispida plants. D: NaCl induced O2· − content in transgenic Tamarix hispida plants. E:NaCl induced SOD activity in transgenic Tamarix hispida plants. F:NaCl induced POD activity in transgenic Tamarix hispida plants. A: DAB staining. B: NBT staining. C: A.NaCl induced H 2 O 2 content in transgenic Arabidopsis plants. D: NaCl induced O2· − content in transgenic Arabidopsis plants. E:NaCl induced SOD activity in transgenic Arabidopsis plants. F:NaCl induced POD activity in transgenic Arabidopsis plants. 7. decreases cell membrane damage We further examined the cell membrane integrity of transgenic plants including Tamarix hispida transient transformants (OE, RNAi, Control) and Arabidopsis thaliana stable transformants (OX2, OX5) under salt stress. Evans Blue staining results showed that under normal conditions, all lines had no obvious blue staining. After salt stress, OE leaves showed faint blue color, while RNAi leaves were deeply stained (darker than Control), With prolonged salt stress (6 h, 12 h), the staining intensity of all lines gradually deepened, but the increase rate of OE was significantly lower than that of Control and RNAi (Fig. 8 a). Moreover, electrolyte leakage also showed consistent results, After salt stress, the electrolyte leakage rate of OE significantly lower than that of Control. For RNAi, the leakage rate increased by 4.5-fold, significantly higher than that of Control (Fig. 8 b). These result indicating that ThASR4 overexpression alleviated cell membrane damage, whereas ThASR4 silencing exacerbated it. Confirming that ThASR4 delays salt-induced cell membrane damage. Proline acts as an osmotic protectant, while malondialdehyde (MDA) is a key marker of lipid peroxidation reflecting oxidative damage. We determined proline accumulating in ThASR4 -overexpressing transgenic plants after salt stress. We find proline accumulate in OE was slightly higher than in Control, and RNAi contained significantly lower proline content than Control (Fig. 8 d).The MDA content was increased abruptly in the Control plants upon stress treatment relative to the OE. It increased by three and two-fold in salinity stress relative to OE, respectively. The MDA content in the RNAi also increased slightly, but the increase was not comparable to the Control plants (Fig. 8 c). Inaddion, as treatment time increased, the difference in proline content and MDA content between transgenic Tamarix hispida and Control became more significant. To verify the conservation of ThASR4 function, the same staining experiments were performed on Arabidopsis. Evans Blue staining showed that under salt stress, OX2/OX5 leaves had weaker blue color than WT, the leakage rate of OX2/OX5 lower than that of WT, proline accumulate higher, MDA content was lower (Fig. 9 ). These results are consistent in both T. hispida (native species) and Arabidopsis (model plant), confirming that ThASR4 plays a conserved role in protecting cell membrane integrity under salt stress. a: Evans blue staining. b: electrolyte leakage. c: NaCl induced proline accumulation in transgenic Tamarix hispida plants. d: NaCl induced MDA activity in transgenic Tamarix hispida plants. A: Evans blue staining. B: electrolyte leakage. C: NaCl induced proline accumulation in transgenic Arabidopsis plants. D: NaCl induced MDA activity in transgenic Arabidopsis plants. 8. ASR4 Regulates ROS Scavenger-Associated and Salt Stress-Responsive Gene Expression It has been reported that TaASR1 and SbASR1 induce the transcription of ROS scavenger-associated genes and reduce the accumulation of ROS. To analyze the expression of ROS scavenger-associated genes in ThASR4 -overexpressing Tamarix hispida , the ThPOD1, ThSOD1 and ThCAT3 genes, which are involved in ROS detoxification, were selected. Under non-stressed conditions, the ranscript levels of ThPOD1, ThSOD1 and ThCAT3 were increased significantly in OE compared to the Control. After salt treatment 12 hours, the expression of these genes were upregulated in both OE and Control plants, but the expression of these genes was increased markedly in OE (Fig. 10 a-c). A similar regulatory pattern was observed for salt Stress-Responsive genes ThSOS3 , ThPIP2;5 , and ThDREB . Under non-stress conditions, ThSOS3 , ThPIP2;5 , and ThDREB genes were expressed in both OE and Control. However, with salt treatment, their expression levels in OE increased significantly (Fig. 10 d-f). Collectively, these results indicate that in ThASR4 -overexpressing lines, ROS scavenger-associated genes and salt stress-related genes are highly expressed and are strongly induced by salt stress. This suggests that the ThASR4 gene directly regulates the expression of these genes to mediate the salt stress response. a: Comparison of the expression of ROS scavenger-associated genes ThPOD1 between transgenic T. hispida. b: Comparison of the expression of ROS scavenger-associated genes ThSOD1 between transgenic T. hispida. c: Comparison of the expression of ROS scavenger-associated genes ThCAT3 between transgenic T. hispida. d:Comparison of the expression of salt stress-responsive genes ThSOS3 between transgenic T. hispida. e:Comparison of the expression of salt stress-responsive genes ThPIP2;5 between transgenic T. hispida. f:Comparison of the expression of salt stress-responsive genes ThDREB between transgenic T. hispida. transgenic Tamarix hispida including ThASR4 overexpression OE and Control, treated with water or 200 mM NaCl for 12 h, Each experiment was repeated independently for at least three times. Discussion Since the first discovery of tomato ASR1 , more than 20 years have passed. An increasing number of ASR families have been characterized in various plants. The functions of these proteins are involved in the responses to ABA and abiotic stress, as well as, the process of fruit ripening. ASR gene families are widely distributed in monocots , dicots , herbs , and xylophyta , but no ASR ortholog has been identified in Arabidopsis . However, Tamarix hispida , which is an important woody halophyte with strong salt tolerance in China, has been less well-studied than other plants, and the ASR regulatory pathway in this species has been unclear. In this study, six ThASR genes were identified in T. hispida named ThASR1 to ThASR6 . All of the ThASRs had two highly conserved regions including N-terminal zinc-binding regions and C-terminal an ABA/WDS domain, for T. hispida All of the ThASRs had two potential NLS in there ABA/WDS domain (Fig. 1a). The structure was similar to ASR genes from banana and Suaeda liaotungensis , and therefore was characterized as a potential ASR family member. Tertiary structures of ThASR4 and ThASR6 have only one independent helical domains, however other proteins have loop-helix-loop domains (Fig. 1b). This structural difference may influence their functions. Previous studies have identified ASR family members in many plants, including Populus trichocarpa , Oryza sativa , Sorghum bicolor and Zea mays . Then multiple alignments and phylogenetic tree analysis revealed that ThASRs are more similar to OsASR1 , OsASR2 , OsASR3 and ZmASR3, ZmASR5, ZmASR9 (Fig. 1c). ZmASRs are involved in abiotic stress tolerance (Hou et al. 2021 ). OsASR1 and OsASR3 are up regulated by Fe and Al stresses, and OsASR1 is involved in regulating the Al stress response in rice, acting as a transcription factor (Foy 1988 ). OsASR2 is a DNA-binding protein that directly regulates a number of Al-responsive genes and associated with responses to abiotic stresses such as drought, salt, cold,and submergence (Iusem et al. 1993 ). Thus we speculated that ThASR genes have a similar function to OsASRs and ZmASRs. In our study, qRT-PCR showed that all six ThASR genes are transcriptionally regulated by salt stress and are expressed in both roots and shoots of T. hispida . Among them, ThASR4 exhibited the most significant upregulation under salt stress (Fig. 2 ), indicating its high sensitivity to salt stress and making it a key candidate gene for further functional verification. Different ASR proteins' subcellular distribution patterns were observed in tomato , litchi , wheat , and lily . The ASR1 from tomato was first reported as a nuclear protein (Iusem et al. 1993 ). Most ASR proteins such as lily (Wang et al. 2005 ), l itchi (Liu et al. 2013 ) and wheat (Hu et al. 2013 ) were found in the nucleus. However, Kalifa et al. reported that tomato ASR1 was localized in both the cytosol and the nucleus (Kalifa et al. 2004 ). In banana, MaASR1 protein was localized in the nucleus and plasma membrane (Sun et al. 2016 ). In this study, Subcellular localization indicated that the ThASR4 -GFP fusion protein was located in the nucleus and cytomembrane (Fig. 3 a). In addition, ThASR4 could activate transcription in yeast cells and the activation domain located in the N-terminal region (Fig. 3 c), which is consistent with the recent study on the tomato ASR gene. These results indicate that ThASR4 functions as a transcription factor, which provides a molecular basis for its regulatory role in downstream gene expression. To further clarify the function of ThASR4 ,we obtained ThASR4 -overexpressing transiently transformed T. hispida (OE) and ThASR4 RNAi silencing Transient T. hispida (RNAi) (Fig. 5 , OE and RNAi), and stable overexpression of ThASR4 in Arabidopsis thaliana (Fig. S1, OX2 and OX5 ) were used here to study the role of ThASR4 . ThASR4 -overexpressing transgenic Arabidopsis resulted in better germination and growth under saline treated conditions. Longer root length and higher fresh weight to dry weight ratio than the WT demonstrating that the ThASR4 gene has the function of improving salt stress tolerence. High cellular concentrations of NaCl causes increased formation of reactive oxygen species (ROS), ROS is an important substance in the cell,when the level of ROS is low in cells, they act as signal molecules (Choudhury et al. 2017 ). However, under severe biotic and abiotic stress, ROS can be overproduced and cause damage to cell growth by oxidizing proteins, lipids and DNA. Therefore, plants have developed complete systems to protect themselves against oxidative stress caused by abiotic stresses through adjusting ROS homeostasis (Choudhury et al. 2012). Some reports have indicated that plant ASR genes have important functions in response to salt stress in many plant species. MaASR1 confers salt stress tolerance by regulating the expression of ABA/stress-responsive genes, and SbASR1 enhances the salinity and drought stress tolerance by functioning as an LEA protein and transcription factor (Tiwari et al. 2015 ). SlASR protein may act as a protective molecule to help plants adapt to abiotic stresses (Hu et al. 2014 ). Overexpression of MaASR1 , Banana ( Musa acuminataL .) ASR gene, in Arabidopsis confers salt stress tolerance by reducing the expression of ABA/stress-responsive genes SiASR4 functions in the adaption to drought and salt stress and is regulated by SiARDP via an ABA-dependent pathway (Li et al. 2017 ). Our study indicate that overexpression of ThASR4 increased proline accumulation and reduced malondialdehyde (MDA) content in both Tamarix hispida and Arabidopsis under salt stress, thereby enhancing osmotic adjustment capacity and alleviating membrane damage. ThASR4 overexpression significantly reduced the accumulation of reactive oxygen species (ROS, including H 2 O 2 and O2· − ) under salt stress, as evidenced by DAB and NBT staining. Correspondingly, the activities of antioxidant enzymes (SOD and POD) were significantly higher in overexpression plants than in Control, which enhanced ROS detoxification efficiency and protected cells from oxidative damage. ThASR4 overexpression upregulated the transcription of ROS-scavenging-related genes ( ThPOD1, ThSOD1, ThCAT3 ) and salt stress-responsive genes ( ThSOS3 , ThPIP2;5 , ThDREB ) in both with or without salt stress. These suggested that ThASR4 gene enhanced the activation of antioxidant defence system, which in turn protected T. hispida against ROS-mediated injury under salt stress. In summary, this study demonstrates that ThASR4 from T. hispida enhances plant salt tolerance through a comprehensive mechanism: as a transcription factor, ThASR4 activates the expression of ROS-scavenging and salt stress-responsive genes, thereby promoting antioxidant enzyme activity, reducing ROS accumulation, enhancing osmotic adjustment, and protecting cell membrane integrity. These findings not only enrich our understanding of the functional diversity of ASR family genes in woody halophytes but also provide a potential candidate gene for genetic improvement of plant salt tolerance. Conclusions In the present study, six ThASR genes were identified and obtained from wild-type Tamarix hispida plants. Among these genes, ThASR4 was selected as the focus for in-depth investigation into its function and regulatory mechanism in salt stress tolerance, based on its significant upregulation under NaCl treatment. Our results systematically demonstrate that ThASR4 plays a positive regulatory role in plant salt stress tolerance. Mechanistically, ThASR4 enhances salt tolerance primarily through two interconnected pathways: first, as a nuclear-localized transcription factor, it directly or indirectly upregulates the expression of a suite of salt stress-responsive genes ( ThSOS3 , ThPIP2;5 , ThDREB ); second, it strengthens the antioxidant system by promoting the activity of key antioxidant enzymes ( ThPOD1, ThSOD1, ThCAT3 ) and the accumulation of osmotic protectants (proline). These combined effects effectively reduce the overaccumulation of reactive oxygen species (ROS) under salt stress, alleviate ROS-mediated oxidative damage, and maintain cell membrane integrity, thereby improving plant adaptation to high-salt environments. To fully elucidate the regulatory network of ThASR4 , future studies should focus on identifying its direct downstream target genes—specifically, the cis-acting elements in target gene promoters that ThASR4 binds to. Such investigations will not only refine our understanding of the molecular mechanism underlying ThASR4 -mediated salt tolerance via ROS scavenging but also provide a more solid theoretical basis for leveraging ThASR4 as a candidate gene in genetic engineering for improving salt tolerance in woody plants. Declarations Ethics, Consent to Participate, and Consent to Publish declarations not applicable. Funding This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572023CT03) and the Innovation Project of the State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (No. 2014B03). Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author’s Contributions WC designed the research. ZY and ZX per formed most experiments and analyzed experimental data. WJ and LHY conducted a part of experiments. ZZY wrote the manuscript, and WC revised the paper. All authors approved the final manuscript. Data Availability The data used to support the findings of this study are available from the corresponding author upon request. Acknowledgements This work was supported by the Fundamental Research Funds for the Central Universities (No. 2572023CT03) and the Innovation Project of the State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (No. 2014B03). 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8909679","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":597656206,"identity":"edd21bb4-9e49-45d1-a595-2a9fb7bc4db5","order_by":0,"name":"Zhenyu Zhu","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Zhenyu","middleName":"","lastName":"Zhu","suffix":""},{"id":597656207,"identity":"9fdd95ed-742c-4d79-b984-04d98240f902","order_by":1,"name":"Jie Wang","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Wang","suffix":""},{"id":597656208,"identity":"3ad4328d-123f-4ad7-a23e-66e7ae75cd56","order_by":2,"name":"Hanyang Liu","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Hanyang","middleName":"","lastName":"Liu","suffix":""},{"id":597656209,"identity":"3b4df57c-37b0-446a-b2a6-bab78a19f69e","order_by":3,"name":"Yu Zhang","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Zhang","suffix":""},{"id":597656210,"identity":"65211923-f988-48f0-8255-61232f684b91","order_by":4,"name":"Xin Zhao","email":"","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zhao","suffix":""},{"id":597656211,"identity":"0ca976b5-3ff0-4690-8958-0d7caaaa77b5","order_by":5,"name":"Chao Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYDACCSjND8QfgJixgWgtkg0MjDNI02JwgFgt/LObHz7mqbljt/l288FmHgYb2Q0HmJ89wGvJnWPGxjzHniVvu3MsEaglzXjDATZzA3xaDCQSzKRz2A4nm93IMX/Mw3A4ccMBHjYJ/FrSv0nn/DucbDwj/yPQlv/EaMkxk85tO2wHZDACtRwgrEXiRk6x8d++wwkSN9IMG+cYJBvPPMxmhlcL/4z0jQ9nfDtszz8j+WHDmwo72b7jzc/waoGBxAaIO4GYmRj1QGBPpLpRMApGwSgYiQAALbpLPfZdhA0AAAAASUVORK5CYII=","orcid":"","institution":"Northeast Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Chao","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2026-02-18 13:09:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8909679/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8909679/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103755593,"identity":"da10b036-9bef-4721-b6a6-4e52ee5096af","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":209154,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple sequence alignment and phylogenetic analysis of ThASRs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: Alignment of predicted amino acid sequences of ASR genes. B: Tertiary structure analysis of ThASRFamily genes proteins. C: Phylogenetic relationships of ASR protein\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/10f5b30098bbc130098c7ce5.jpg"},{"id":104400211,"identity":"6ee8c294-0c16-44a0-800d-f8a280b2daed","added_by":"auto","created_at":"2026-03-11 12:09:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49094,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression levels of ThASR family genes after NaCl treatment\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/044dae89e7843777f5de38db.jpg"},{"id":104779185,"identity":"3260333c-1632-4569-843b-007a456e7c53","added_by":"auto","created_at":"2026-03-17 07:36:12","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":84842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization and transcription activation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea: Subcellular localization of ThASR4 protein . b: Diagram of the pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e construct. c: Transactivation activity of the ThASR4 protein in yeast.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/c584a78995452dd5857db8f9.jpg"},{"id":103755594,"identity":"67cc1c6d-2b62-4cd4-8442-296fb9dc6a2f","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":479116,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSalt stress tolerance associated with \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4-\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eoverexpression\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Arabidopsis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Germination of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium. b: Germination of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium with 200mM NaCl. c：Statistical analysis of the seed germination rates. d: Root length of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium. e: Root length of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium with 200mM NaCl. f: The photographs of adult WT, OX2, OX5 seedlings after 200mM NaCl treatment for 5 days. g: Statistical analysis of the seed Root length. h: Statistical analysis of the fresh weights.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/25423dc4bde806c95e84decf.jpg"},{"id":104400092,"identity":"bb7f5ae7-9b0f-4574-8deb-d305e6d05aa9","added_by":"auto","created_at":"2026-03-11 12:08:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":28827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRelative transcript level of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e in transiently transformed \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTamarix hispida\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eqRT-PCR analysis of \u003cem\u003eThASR4\u003c/em\u003e genes in different kinds of transiently transformed \u003cem\u003eT. hispida, \u003c/em\u003efour-week-old\u003cem\u003e T. hispida \u003c/em\u003eplants were transiently transformed with empty pROKII, 35S:\u003cem\u003eThASR4\u003c/em\u003e or pFGC5941:\u003cem\u003eThASR4\u003c/em\u003e , The expression of \u003cem\u003eThASR4\u003c/em\u003e in whole OE, RNAi and Control plants was measured. The error bars are standard deviations, which were calculated from multiple replicates of qRT-PCR. OE: transgenic \u003cem\u003eT.hispida\u003c/em\u003e overexpressing \u003cem\u003eThASR4\u003c/em\u003e; RNAi: \u003cem\u003eThASR4\u003c/em\u003e RNAi-silenced \u003cem\u003eT. hispida \u003c/em\u003eplants; Control: pROKII vector-transformed \u003cem\u003eT. hispida\u003c/em\u003e plants.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/799728681d5d9ef2df9e32f6.jpg"},{"id":103755598,"identity":"0e18da18-e5fe-4305-94f1-7fcb78d0db5b","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":92067,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROS-scavenging capability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing transgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTamarix hispida\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA: DAB staining. B: NBT staining. C: NaCl induced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. D: NaCl induced O2·\u003csup\u003e−\u003c/sup\u003e content in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. E:NaCl induced SOD activity in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. F:NaCl induced POD activity in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/66958e89dafd4236db2fb5f4.jpg"},{"id":103755599,"identity":"71630ebc-2169-4c11-8779-72879ae2c1c3","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":78853,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eROS-scavenging capability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing transgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA: DAB staining. B: NBT staining. C: A.NaCl induced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. D: NaCl induced O2·\u003csup\u003e−\u003c/sup\u003e content in transgenic \u003cem\u003eArabidopsis \u003c/em\u003eplants. E:NaCl induced SOD activity in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. F:NaCl induced POD activity in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/fc4162e68e0e6ad624072b11.jpg"},{"id":103755597,"identity":"1077c1dd-883f-4255-be1f-82635466695b","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":101259,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edecreases cell membrane damage in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing transgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTamarix hispida\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Evans blue staining. b: electrolyte leakage. c: NaCl induced proline accumulation in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. d: NaCl induced MDA activity in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/1eb747a1d8febf1bae8c4625.jpg"},{"id":103755600,"identity":"61f69a66-558f-4709-931d-8fba626d7633","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":79779,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edecreases cell membrane damage in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-overexpressing transgenic \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eArabidopsis\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA: Evans blue staining. B: electrolyte leakage. C: NaCl induced proline accumulation in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. D: NaCl induced MDA activity in transgenic \u003cem\u003eArabidopsis \u003c/em\u003eplants.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/2221aca8dbc7fa695dc0ad3c.jpg"},{"id":103755601,"identity":"726bbe14-b456-4881-8a98-7db3eb419db7","added_by":"auto","created_at":"2026-03-02 13:57:38","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":93040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eThASR4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e regulates ROS scavenger-associated and Salt stress-responsive gene expression levels in transgenic\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eTamarix hispida\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ea: Comparison of the expression of ROS scavenger-associated genes \u003cem\u003eThPOD1\u003c/em\u003ebetween transgenic \u003cem\u003eT. hispida.\u003c/em\u003e b: Comparison of the expression of ROS scavenger-associated genes \u003cem\u003eThSOD1\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003ec: Comparison of the expression of ROS scavenger-associated genes \u003cem\u003eThCAT3 \u003c/em\u003ebetween transgenic \u003cem\u003eT. hispida. \u003c/em\u003ed:Comparison of the expression of salt stress-responsive genes \u003cem\u003eThSOS3\u003c/em\u003e between transgenic \u003cem\u003eT. hispida. \u003c/em\u003ee:Comparison of the expression of salt stress-responsive genes \u003cem\u003eThPIP2;5\u003c/em\u003e between transgenic \u003cem\u003eT. hispida. \u003c/em\u003ef:Comparison of the expression of salt stress-responsive genes \u003cem\u003eThDREB\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003etransgenic \u003cem\u003eTamarix hispida\u003c/em\u003e including \u003cem\u003eThASR4 \u003c/em\u003eoverexpression OE and Control, treated with water or 200 mM NaCl for 12 h, Each experiment was repeated independently for at least three times.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/a98c89c0499c4178c8b780c0.jpg"},{"id":104783692,"identity":"ece57e0a-8809-4777-a4e3-f28d792f4811","added_by":"auto","created_at":"2026-03-17 08:03:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3011680,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8909679/v1/2dc87027-4c0a-4b37-9ef8-91fc14835ae2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"ThASR4 enhances salt tolerances in transgenic Tamarix and Arabidopsis by Scavenging Reactive Oxygen Species","fulltext":[{"header":"Highlights","content":"\u003col\u003e\n \u003cli\u003eThASR genes respond to salt stress\u003c/li\u003e\n \u003cli\u003eOverexpression of \u003cem\u003eThASR4\u003c/em\u003e enhances salt tolerance in transgenic \u003cem\u003eTamarix hispida\u0026nbsp;\u003c/em\u003eand \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eThASR4\u003c/em\u003e reduces reactive oxygen species (ROS) accumulation and increases antioxidant enzyme activities\u003c/li\u003e\n \u003cli\u003e\u003cem\u003eThASR4\u003c/em\u003e upregulates the expression of the \u003cem\u003eThPOD1\u003c/em\u003e, \u003cem\u003eThSOD1\u003c/em\u003e , \u003cem\u003eThCAT3\u003c/em\u003e, \u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e and \u003cem\u003eThDREB\u003c/em\u003e gene.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Introduction","content":"\u003cp\u003eAbscisic acid-, stress-, and ripening-induced (ASR) genes are a class of plant-specific transcription factors (TFs), which play pivotal roles in plant growth, development, and responses to abiotic stress. The identification of ASR genes first occurred in tomato in 1993 (Iusem et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Subsequent detection of these genes has been documented in maize (Liang et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), rice(Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), foxtail millet (Feng et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), banana (Miao et al. 2014), and the halophyte plant \u003cem\u003eSuaeda liaotungensis\u003c/em\u003e (Hu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). All ASR genes identified to date possess a simple structure, with two exons separated by a single intron. Correspondingly, ASR proteins contain two highly conserved domains: an N-terminal zinc-binding domain and a C-terminal ABA/WDS (abscisic acid/water deficit stress) domain that harbors a putative nuclear localization signal (NLS). This feature that may be critical for their transcription factor function.\u003c/p\u003e \u003cp\u003eASR plays a key role in promoting plant growth and fruit ripening by regulating the biosynthesis and metabolism of various compounds, including glucose, cell wall components, amino acids, ABA, and carotenoids (Zhang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Research on ASR in promoting fruit ripening has primarily focused on Rosaceae, Solanaceae, and cereal crops. Overexpression of ASR genes in tomatoes and strawberries has been shown to promote fruit ripening, resulting in a softer fruit texture and increased coloration, thereby further regulating fruit flavor and quality (Jia et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The ASR1 gene specifically influences glucose accumulation in potato tubers (Frankel et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In tobacco cells, the \u003cem\u003eSlASR1\u003c/em\u003e gene (from \u003cem\u003eSolanum lycopersicum\u003c/em\u003e) has been shown to regulate glucose metabolism by influencing the uptake of hexoses in heterotrophic organs, thereby contributing to carbon fixation in leaves (Dominguez et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A lily ASR is preferentially expressed in the vegetative cell for pollen maturation. while the banana ASR (\u003cem\u003eMaASR\u003c/em\u003e) can delay flowering time in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Sun et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Furthermore, ASR genes have been shown to enhance tolerance to multiple abiotic stresses, including drought, salinity, cold, and heavy metal toxicity. Overexpression of the \u003cem\u003eBdASR1\u003c/em\u003e gene has been shown to substantially increase drought tolerance in transgenic tobacco plants (Wang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). \u003cem\u003eOsASR1\u003c/em\u003e plays a pivotal role in ABA-mediated regulation of stomatal closure, which helps conserve water during salt- and drought-stress conditions (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Moreover, \u003cem\u003eOsASR1\u003c/em\u003e overexpression can enhance salinity and drought tolerance, thereby resulting in improved crop yields (Park et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ectopic expression of \u003cem\u003eOsASR1\u003c/em\u003e has also been shown to improve cold tolerance in plants (Kim et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Additionally, Maize ASR1 conferred Cd-tolerance in tobacco (Zhang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Both \u003cem\u003eOsASR1\u003c/em\u003e and \u003cem\u003eOsASR5\u003c/em\u003e are involved in regulating aluminum (Al)-responsive genes, which help reduce Al toxicity in rice (Arenhart et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSalt stress has been demonstrated to induce an imbalance in ion homeostasis within plants. Specifically, an imbalance between Na\u003csup\u003e+\u003c/sup\u003e\u0026thinsp;and K\u003csup\u003e+\u003c/sup\u003e ions has been shown to result in alterations to the intracellular osmotic pressure (Blumwald \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Furthermore, the presence of high concentrations of Na\u003csup\u003e+\u003c/sup\u003e ions within cells induces the production of large amounts of reactive oxygen species (ROS), including superoxide anions, hydroxyl radicals, hydrogen peroxide, and singlet oxygen (Qi et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Excessive concentrations of reactive oxygen species can cause oxidative damage to cell membranes, proteins, RNA, and DNA molecules (Mittler \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). It is therefore vital that antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) are present in order to eliminate the toxicity of reactive oxygen species to cells (Petrov et al. 2012). In order to cope with salt stress, plants adopt a series of physiological, morphological, and molecular strategies, such as reducing ion absorption or accumulating ions in vacuoles to avoid the accumulation of toxic ions within cells. In addition, stress response factors activate protective enzyme activity to reduce ROS damage to cells(Choudhury 2017). Previous studies have demonstrated the involvement of ASR genes in plant salt stress tolerance. Research has shown that the \u003cem\u003eOsASR1\u003c/em\u003e gene from \u003cem\u003eOryza sativa\u003c/em\u003e affects net Na\u003csup\u003e+\u003c/sup\u003e uptake in plants. Under salt stress, the over-expression \u003cem\u003eOsASR1\u003c/em\u003e lines exhibited a reduced accumulation of Na\u003csup\u003e+\u003c/sup\u003e in comparison to the wild type (Park et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, the \u003cem\u003eIpASR\u003c/em\u003e-overexpressing transgenic \u003cem\u003eArabidopsis\u003c/em\u003e showed enhanced tolerance to salt and drought stress in comparison to the wild type, accompanied by a diminished accumulation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and superoxide (O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e) and an augmented activity of antioxidant enzymes in vivo. Kim \u003cem\u003eet al.\u003c/em\u003e discovered that \u003cem\u003eOsASR1\u003c/em\u003e could function as an effective reactive oxygen species scavenger (Kim et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, ASR genes have been identified and functionally characterized in halophytes, includ\u003cem\u003eing Salicornia brachiata\u003c/em\u003e and \u003cem\u003eSuaeda liaotungensis\u003c/em\u003e K (Hu et al. 2012).\u003c/p\u003e \u003cp\u003eHalophytes are able to survive in the extreme environmental conditions due to their unique adaptation of tissue tolerance mechanisms. \u003cem\u003eTamarix hispida\u003c/em\u003e is a woody halophyte with strong salt tolerance, which is an excellent material for cloning salt-responsive genes and investigating salt tolerance mechanisms. However, the role of the ASR gene family in regulating salt tolerance in \u003cem\u003eTamarix hispida\u003c/em\u003e remains to be systematically elucidated. In this study, we identified six \u003cem\u003eT. hispida\u003c/em\u003e ASR genes and analyzed their expression profiles in response to salt stress.\u003c/p\u003e \u003cp\u003eFunctional analysis demonstrated that \u003cem\u003eThASR4\u003c/em\u003e overexpression enhanced the salt stress tolerance of transgenic \u003cem\u003eT. hispida\u003c/em\u003e and \u003cem\u003eA. thaliana.\u003c/em\u003e Under salt stress conditions, compared with wild-type (WT) plants, \u003cem\u003eThASR4\u003c/em\u003e-overexpressing \u003cem\u003eT. hispida\u003c/em\u003e exhibited significantly lower levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e. Meanwhile, transgenic \u003cem\u003eA. thaliana\u003c/em\u003e showed a higher seed germination rate, together with increased root length and fresh weight. Notably, the extent of cellular damage in transgenic lines was lower than that in WT plants. Furthermore, we verified that \u003cem\u003eThASR4\u003c/em\u003e could enhance ROS scavenging capacity by upregulating the expression of antioxidant enzyme genes and the activities of corresponding enzymes. Under salt stress, the activities of POD and SOD were increased in transgenic plants. Consistently, the transcript levels of ROS scavenger-associated genes (\u003cem\u003eThPOD1, ThSOD1\u003c/em\u003e and \u003cem\u003eThCAT3\u003c/em\u003e) and salt stress-responsive genes (\u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, and \u003cem\u003eThDREB\u003c/em\u003e) were significantly elevated in transgenic lines compared with Control.\u003c/p\u003e \u003cp\u003eThis finding indicates that \u003cem\u003eThASR4\u003c/em\u003e enhances the ROS scavenging capacity by upregulating the expression of ROS-related genes, thereby improving plant tolerance to salt stress.\u003c/p\u003e"},{"header":"Experimental procedures","content":"\n\u003ch3\u003e1.Plant material and treatment\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eTamarix hispida\u003c/em\u003e were obtained from the State Key Laboratory of Tree Genetics and Breeding ( Northeast Forestry University). These plants were cultivated in vitro under a photoperiod of 16 hours of light and 8 hours of darkness on 1/2 MS medium supplemented with 0.6% powdered activated carbon. The greenhouse environment was maintained at a temperature of 24\u0026deg;C and a relative humidity level of 70%. Three-month-old \u003cem\u003eTamarix\u003c/em\u003e seedlings were irrigated with 200 mM NaCl for 1, 2, 6, 12, 24 and 48 hours. For each time point, the seedlings that were irrigated with tap water were used as the control. The roots and shoots of at least 10 seedlings were immediately harvested, pooled and frozen in liquid nitrogen and then stored at -80\u0026deg;C until required.\u003c/p\u003e \u003cp\u003eArabidopsis seeds were surface-sterilized with 75% (v/v) ethanol for three minutes and 25% (v/v) sodium hypochlorite for one minute, after which they were washed six times in sterile water. The sterilized seeds were germinated on MS solid medium. After seven days, the seedlings were transferred to a mixed soil substrate (black soil: vermiculite: perlite\u0026thinsp;=\u0026thinsp;3:1:1) for cultivation. The seedlings were subsequently grown and cultured in growth chambers (16 h light/8 h dark cycle with a light intensity of 200 mmol m\u003csup\u003e\u0026minus;2\u003c/sup\u003es\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 25\u0026deg;C). For the selection of transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e lines, the seedlings were placed on MS solid medium with 50 mg/ml kanamycin, with all other growth conditions kept consistent. The transgenic plants were likewise cultivated under these standardized conditions.\u003c/p\u003e\n\u003ch3\u003e2.Cloning and bioinformatics analysis of \u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e2.Cloning and bioinformatics analysis of \u003cem\u003eThASRs\u003c/em\u003e\u003c/div\u003e \u003cp\u003eEncoding ASR family genes in \u003cem\u003eTamarix hispida\u003c/em\u003e were got with the gene-specific primers based on the transcriptome database (the primers used are shown in Supporting Information Table S1). PCR was performed using cDNA extracted as template, and the PCR products were linked to the PMD18-T vector (TaKaRa, Beijing, China) for sequencing. The potential ThASR proteins were analyzed using the PFAM database to confirm the presence of an ABA/WDS domain (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.sanger.ac.uk/\u003c/span\u003e\u003cspan address=\"http://pfam.sanger.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Structural analysis of conserved regions of each non-redundant sequence was executed by SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), Pfam(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.sanger.ac.uk/\u003c/span\u003e\u003cspan address=\"http://pfam.sanger.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and conserved domain database (CDD) searchesMultiple alignments of ASR protein sequences were carried out with ClustalX2.0. Phylogenetic trees were constructed by (iTOL(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/tree/\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/tree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003e3.Quantitative Real-time polymerase chain reaction\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from \u003cem\u003eT. hispida\u003c/em\u003e plants with a Plant RNeasy Extraction Kit (BioTeke, China). First-strand cDNA was synthesized from 1 mg of purified RNA using a PrimeScriptTM RT Reagent Kit (TaKaRa, Beijing, China). qRT-PCR was performed on a qTOWER3 G (Analytik Jene AG, Germany) with the β-actin (FJ618517) and α-tubulin (FJ618518) genes as internal controls. Each 20 mL reaction mixture contained 10 mL of SYBR-Green Real-time PCR Master Mix (Toyobo, Shanghai, China), specific primers (0.5 mM each), and 2 mL of cDNA template. The amplification was performed using the following cycling parameters: 94℃ for 30 s; 45 cycles of 94℃ for 12 s, 58℃ for 30 s, and 72℃ for 45 s; and then 82℃ for 1 s for plate reading. Three replicates were included for each sample (The primers used for qRT-PCR are detailed in Supporting Information Table S2). The Actin gene of \u003cem\u003eT. hispida\u003c/em\u003e was used as the internal reference, and all experiments were performed with three biological replicates and three technical replicates; statistical significance was determined by one-way analysis of variance (ANOVA) (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\n\u003ch3\u003e4.Subcellular localization of ThASR4 protein\u003c/h3\u003e\n\u003cp\u003eThe complete coding sequence of \u003cem\u003eThASR4\u003c/em\u003e was amplified by PCR using primers containing NcoI/SpeI restriction sites. The PCR product was digested with NcoI/SpeI (LABLEAD, Beijing, China), and cloned into the pBI121 vector so that the gene is under control of Cauliflower mosaic virus 35S (CaMV 35S) promoter to create a fusion construct (the primers used are shown in Table S1). Recombinant plasmids pBI121-ThASR4-GFP and pBI121-GFP (used as a control) were separately transferred into onion epidermal cells using particle bombardment. The transformed cells were then examined by laser-scanning confocal microscopy (LSM800: Karl Zeiss, Jena, Germany) after incubation at 25\u0026deg;C for 24 h on MS medium.\u003c/p\u003e\n\u003ch3\u003e5.Transcriptional activation analysis in yeast\u003c/h3\u003e\n\u003cp\u003eThe transcriptional activation activity of \u003cem\u003eThASR4\u003c/em\u003e was determined in yeast cells. The complete coding sequence of \u003cem\u003eThASR4\u003c/em\u003e, N-terminal (\u003cem\u003eThASR4\u003c/em\u003e-N) and C-terminal (\u003cem\u003eThASR4\u003c/em\u003e-C) fragments were amplified by PCR using primers, respectively. The PCR products were then cloned into EcoRI/BamHI sites in the pGBKT7 vector, resulting in the constructs pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e, pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-N, and pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-C (the primers used are shown in Table S1). The three constructs, along with the negative control pGBKT7 plasmid, were used to transform the Y2H yeast strain. The transformants were streaked on SD/Trp- and SD/Trp-/His-/X-\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\alpha\\:}\\)\u003c/span\u003e\u003c/span\u003e-gol and incubated at 28\u0026deg;C for 3 days, after which the growth of the transformants was assessed.\u003c/p\u003e\n\u003ch3\u003e6.Plant transformation and generation of transgenic plants\u003c/h3\u003e\n\u003cp\u003eThe full-length coding sequence of \u003cem\u003eThASR4\u003c/em\u003e was cloned into the NcoI/XbaI sites of the pROKII vector to create the overexpression vector pROKII-ThASR4. To silence ThASR4, a 300 bp inverted repeat truncated cDNA fragment of ThASR4 was inserted flanking the CHSA intron of the RNAi vector pFGC5941.\u003c/p\u003e \u003cp\u003eThe recombinant plasmids pROKII-\u003cem\u003eThASR4\u003c/em\u003e and pFGC5941-\u003cem\u003eThASR4\u003c/em\u003e under the control of the CaMV 35S promoter were introduced into Agrobacterium tumefaciens strain EHA105, respectively. The transient transformation of \u003cem\u003eT. hispida\u003c/em\u003e was performed using an Agrobacterium-mediated transformation. As a result, transient overexpressing \u003cem\u003eT. hispida\u003c/em\u003e (OE) and transient RNAi silencing \u003cem\u003eT. hispida\u003c/em\u003e (RNAi) for \u003cem\u003eThASR4\u003c/em\u003e were obtained. Transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants were generated using the Agrobacterium-mediated floral dip method. Seeds from these transgenic plants were selected on MS medium containing 50 mg/L of kanamycin. The T1 to T3 transgenic lines were confirmed by PCR using \u003cem\u003eThASR4\u003c/em\u003e primers. Two independent T3 transgenic line seedlings, OX2 and OX5, which exhibited higher \u003cem\u003eThASR4\u003c/em\u003e transcript levels, were selected for the stress tolerance assay. The primers employed for these assays are shown in Table S1.\u003c/p\u003e\n\u003ch3\u003e7.Stress tolerance assay for transgenic plants\u003c/h3\u003e\n\u003cp\u003eFor salt stress tolerance assay, Transient \u003cem\u003eT. hispida\u003c/em\u003e OE, RNAi and Control were cultured on one-half MS medium with 200 mM NaCl respectively. We took samples at stress times of 0, 6 and 12 hours, snap-frozen them in liquid nitrogen and stored them in a -80 degree refrigerator. MDA and PRO content was determined using the MDA and PRO Detection Kit (A003 and A107; Jiancheng, Nanjing, China). while the electrolyte leakage measurement was conducted as described by Liang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFor transgenic \u003cem\u003eArabidopsis\u003c/em\u003e seed germination and seedling growth under the salt stress assay, we put the sterilized transgenic \u003cem\u003eArabidopsis\u003c/em\u003e seeds OX2, OX5 and WT in the one-half MS medium with or without 200 mM NaCl respectively. After 7 days, we count the germination of seeds and the length of roots and fresh weight.\u003c/p\u003e\n\u003ch3\u003e8.Physiological and histochemical staining analysis of transgenic plants\u003c/h3\u003e\n\u003cp\u003eAfter culturing for 48 h on 1/2 MS agar medium, the seedlings from transient transgenic \u003cem\u003eT. hispida\u003c/em\u003e (OE, RNAi, Control) and transgenic \u003cem\u003eArabidopsis\u003c/em\u003e (OX2, OX5, WT) were transferred to 1/2 MS with 200 mM NaCl for 0, 6 and 12 h. Subsequently,, seedlings were harvested.\u003c/p\u003e \u003cp\u003eFor physiological analyses. SOD and POD activities were measured using the SOD and POD Detection Kit (A001 and A084; Jiancheng, Nanjing, China) according to the manufacturer\u0026rsquo;s instructions. The content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e was measured following the protocol of Liang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). At least eight seedlings were used for the physiological index analysis per sample, and three independent biological repetitions were used to ensure the accuracy of the analysis.\u003c/p\u003e \u003cp\u003eFor histochemical staining analysis, nitroblue tetrazolium (NBT) (LABLEAD, Beijing, China), diaminobenzidine (DAB) (LABLEAD, Beijing, China), and Evans Blue (Beyotime, Shanghai, China) staining assays were performed according to the protocol of Liang et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For each independent biological replicate, at least nine seedlings were collected for histochemical staining analysis. Representative images were captured from two randomly selected seedlings per sample.\u003c/p\u003e\n\u003ch3\u003e9.ThASR4-Regulated Gene Expression Analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA from \u003cem\u003eT. hispida\u003c/em\u003e and \u003cem\u003eThASR4\u003c/em\u003e-overexpressing \u003cem\u003eT. hispida\u003c/em\u003e was used to analyze the expression levels of \u003cem\u003eThPOD1, ThSOD1\u003c/em\u003e, \u003cem\u003eThCAT3, ThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, and \u003cem\u003eThDREB\u003c/em\u003e through qRT-PCR. The primers employed for these assays are shown in Table S2.\u003c/p\u003e"},{"header":"RESULTS","content":"\n\u003ch3\u003e1. Identification and Phylogenetic analysis of ThASR genes\u003c/h3\u003e\n\u003cp\u003eSix ASR genes were identified and isolated from \u003cem\u003eTamarix hispida\u003c/em\u003e. These ThASR genes encoded proteins with lengths ranging from 102 to 228 amino acids, a theoretical isoelectric point (pI) of 5.33 to 10.09. The stability indices of the proteins ranged from 26.08 to 46.26, indicating stability for all six proteins. The GRAVY values of the proteins fell between \u0026minus;\u0026thinsp;1.4 and \u0026minus;\u0026thinsp;1.0, confirming them as hydrophobic proteins (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmino acid sequence alignment revealed the presence of two highly conserved regions in ThASRs (Fig.\u0026nbsp;1a). The first is located in the N-terminal region and consists of a consensus sequence of 10 amino acids, which includes a stretch of six histidine (His) residues, characteristic of zinc-binding domains. The second region is found in the C-terminal region and contains an ABA/WDS domain made up of 80 amino acids, which features two potential nuclear localization signals (NLS). The tertiary structure analysis of \u003cem\u003eTamarix hispida\u003c/em\u003e ASR genes shows that all six genes encode proteins containing a similar helix domain in structure (Fig.\u0026nbsp;1b). However, ThASR4 and ThASR6 only have an independent helix domain, whilst the remaining four proteins exhibit a loop-helix-loop domain.\u003c/p\u003e \u003cp\u003eTo gain an insight into the evolutionary relationships of the ASR proteins, a phylogenetic tree was constructed using thirty-nine ASR proteins from five different plant species, including tomato (Solanum lycopersicum), Populus trichocarpa, Oryza sativa, Sorghum bicolor and Zea mays. The phylogenetic tree revealed that ThASRs consisted of three major groups (Fig.\u0026nbsp;1c). The results demonstrate that ThASR1, ThASR2, ThASR3 and ThASR5 belong to the same branch and are closely related to OsASR1, OsASR2, and OsASR3. Indeed, OsASR1 and OsASR3 are upregulated by Fe and Al stresses. OsASR1 acts as a transcription factor in regulating the aluminum stress response in rice. Additionally, Rafael Augusto also found that OsASR5 regulates a large number of Al-responsive genes, suggesting that ThASR1, ThASR2, ThASR3 and ThASR5 may play a role in the response to metal stress. Furthermore, ThASR4 and ThASR6 are located in the same branch and exhibit strong similarity with OsASR4 and ZmASR4. ZmASR4 is thought to be associated with heavy metal transport and tolerance. The expression of OsASR4 was significantly increased in rice in response to abiotic stress. Therefore, ThASR4 and ThASR6 may also contribute to abiotic stress response.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical properties and subcellular localization of ThASR transcription factor family proteins\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003cp\u003ename\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNumber of amino acids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMolecular weight\u003c/p\u003e \u003cp\u003e(KD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTheoretical pI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eInstability index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAliphatic index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGrand average of hydropathicity\u003c/p\u003e \u003cp\u003e(GRAVY)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSubcellular Localization Prediction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThASR1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e112\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.032\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enucl,cyto\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThASR2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e106\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e57.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThASR3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e108\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e53.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.414\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003emito,nucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThASR4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e118\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e6.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e40.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e36.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003ecyto,nucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThASR5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003emito\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eThASR6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-1.441\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003enucl\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure 1. Multiple sequence alignment and phylogenetic analysis of ThASRs\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA: Alignment of predicted amino acid sequences of ASR genes. B: Tertiary structure analysis of ThASR Family genes proteins. C: Phylogenetic relationships of ASR protein\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e2. Expression pattern of the in response to salt stress\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e2. Expression pattern of the \u003cem\u003eThASRs\u003c/em\u003e in response to salt stress\u003c/div\u003e \u003cp\u003eTo clarify the transcriptional response of ThASR genes to salt stress, the expression levels of six ThASR genes in \u003cem\u003eT. hispida\u003c/em\u003e were analyzed at various time points (1 h, 2h, 6 h, 12 h, 24 h, 48 h) under NaCl treatment using quantitative real-time PCR (qRT-PCR).\u003c/p\u003e \u003cp\u003eqRT-PCR results showed that all six ThASR genes were transcriptionally regulated by NaCl stress, and their expression was detected in both leaves and roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Notably, the \u003cem\u003eThASR\u003c/em\u003e gene family showed a uniform up-regulation pattern in roots, contrasting with a pattern of partial down-regulation and low abundance in shoots. These findings indicate that ThASRs likely exert a more pivotal function in mediating the salt stress response within the roots of \u003cem\u003eT. hispida.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eMoreover, the expression of six \u003cem\u003eThASRs\u003c/em\u003e genes expression was detected significant differences under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In roots, their expression levels exhibited a trend of increasing, then decreasing, and increasing again over time. Specifically, transcript levels rose to a peak at 2 or 6 hours, subsequently declined, and peaked again at 48 hours. Among these, \u003cem\u003eThASR1\u003c/em\u003e reached its maximum (7.9-fold) at 6 hours, while \u003cem\u003eThASR 4\u003c/em\u003e peaked at 2 hours (9.7-fold). Furthermore, In the leaves, the expression of \u003cem\u003eThASR2\u003c/em\u003e, \u003cem\u003eThASR3\u003c/em\u003e, \u003cem\u003eThASR5\u003c/em\u003e, and \u003cem\u003eThASR6\u003c/em\u003e was either downregulated or remained unchanged. In contrast, \u003cem\u003eThASR1\u003c/em\u003e and \u003cem\u003eThASR4\u003c/em\u003e were upregulated at all time points, generally following a trend of decreasing, then increasing, and decreasing again. Notably, \u003cem\u003eThASR4\u003c/em\u003e showed the highest expression level among all genes tested.\u003c/p\u003e \u003cp\u003eAmong six ASR genes, \u003cem\u003eThASR4\u003c/em\u003e showed the most significant up-regulation after salt treatment, indicated that \u003cem\u003eThASR4\u003c/em\u003e expression was obviously induced in shoots and roots, so \u003cem\u003eThASR4\u003c/em\u003e was selected for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003e3. Subcellular Localization and Transcription Activation Assay of ThASR4 protein\u003c/h3\u003e\n\u003cp\u003eThe subcellular localization of the ThASR4 protein was investigated in a transient expression assay with 35S::\u003cem\u003eThASR4\u003c/em\u003e-GFP (pBI121-\u003cem\u003eThASR4\u003c/em\u003e-GFP) translational fusion in onion epidermal cells using particle bombardment. Fluorescence of the 35S::\u003cem\u003eThASR4\u003c/em\u003e-GFP chimera was observed in both the cellular nucleus and cytoplasm of onion epidermal cells. In contrast, the cells transformed with the control 35S::GFP (pBI121-GFP) exhibited fluorescence distributed throughout the entire cell, including the nucleus and plasma membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This indicates that the ThASR4 protein is localized in both the nucleus and the cytoplasm.\u003c/p\u003e \u003cp\u003eTo ascertain the transcriptional activation potential of the ThASR4 protein and the location of its transcription activation domain, three recombinant vectors were constructed and transformed into Y2H Gold yeast cells. The vectors were pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-FL (full-length \u003cem\u003eThASR4\u003c/em\u003e), pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-N (\u003cem\u003eThASR4\u003c/em\u003e N-terminal), and pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-C (\u003cem\u003eThASR4\u003c/em\u003e C-terminal) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Following transformation, the transformants were cultured on SD/-Trp medium (to verify successful transformation) and SD/-Trp/-His/X-α-gal medium (to detect transcriptional activation activity). After three days, all yeast transformants exhibited normal growth on SD/-Trp medium, indicating that the vectors were successfully introduced into yeast cells. On SD/-Trp/-His/X-α-gal medium, yeast cells transformed with pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-FL or pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-C grew well and formed blue colonies. In contrast, yeast cells transformed with pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e-N exhibited no observable growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The results indicate that ThASR4 has transcriptional activation activity, and its transcriptional activation domain is localized in the C-terminal.\u003c/p\u003e \u003cp\u003eIn summary, ThASR4 protein was a nuclear and cytoplasm-localized protein with transcriptional activation activity. These findings suggest that ThASR4 may function as a transcription factor to participate in the transcriptional regulation of salt stress response-related genes in \u003cem\u003eT. hispida\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ea: Subcellular localization of ThASR4 protein. b: Diagram of the pGBKT7-\u003cem\u003eThASR4\u003c/em\u003e construct. c: Transactivation activity of the ThASR4 protein in yeast.\u003c/p\u003e\n\u003ch3\u003e4. Overexpression of enhances salt tolerance of \u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003e4. Overexpression of \u003cem\u003eThASR4\u003c/em\u003e enhances salt tolerance of \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/div\u003e \u003cp\u003eTo assess the function of \u003cem\u003eThASR4\u003c/em\u003e in stress tolerance, transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants overexpressing \u003cem\u003eThASR4\u003c/em\u003e under the control of the CaMV35S promoter were generated. In eight independent homozygous \u003cem\u003eThASR4\u003c/em\u003e lines, OX2 and OX5, with high expression levels were selected for further functional analysis (Fig. S1a,b).\u003c/p\u003e \u003cp\u003eIn normal conditions (MS medium), there were no significant differences in the rate of seed germination or the length of seedling roots between the transgenic and wild type (WT) Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,d). However, the OX2 and OX5 lines exhibited superior growth in comparison to the WT plants when cultivated on MS medium supplemented with 200 mM NaCl (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The transgenic lines displayed larger cotyledons compared to the WT plants, and their seed germination rates and root lengths were significantly greater (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eb,e). Almost 83.7% of OX seeds were able to germinate, while WT seeds hardly germinated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). In addition, the root length of OX seedlings was longer than that of wild-type under salt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eg). Furthermore, following a two-week growth period in soil irrigated with a 200 mM NaCl solution, the fresh weight of transgenic plants was significantly higher than that of wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003ef,h). These results indicate that ThASR4 plays a positive regulatory role in plant salt tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ea: Germination of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium. b: Germination of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium with 200mM NaCl. c: Statistical analysis of the seed germination rates. d: Root length of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium. e: Root length of WT and \u003cem\u003eThASR4\u003c/em\u003e transgenic seeds on MS medium with 200mM NaCl. f: The photographs of adult WT, OX2, OX5 seedlings after 200mM NaCl treatment for 5 days. g: Statistical analysis of the seed Root length. h: Statistical analysis of the fresh weights.\u003c/p\u003e\n\u003cdiv class=\"Heading\"\u003e5. \u003cb\u003eGeneration of transiently transformed\u003c/b\u003e \u003cb\u003eTamarix hispida\u003c/b\u003e \u003cb\u003eplants with transient overexpression or RNAi silencing of\u003c/b\u003e \u003cb\u003eThASR4\u003c/b\u003e\u003c/div\u003e \u003cp\u003eTo further elucidate the role of \u003cem\u003eThASR4\u003c/em\u003e in \u003cem\u003eT. hispida\u003c/em\u003e salt tolerance, transgenic \u003cem\u003eT. hispida\u003c/em\u003e plants exhibiting transient overexpression and RNAi silencing of \u003cem\u003eThASR4\u003c/em\u003e were generated using transient transformation technology. Control plants were obtained by transforming \u003cem\u003eT. hispida\u003c/em\u003e with the empty pROKII vector.\u003c/p\u003e \u003cp\u003eFollowing the transient transformation method previously established for \u003cem\u003eT. hispida\u003c/em\u003e, we successfully generated \u003cem\u003eThASR4\u003c/em\u003e-overexpressing lines (OE), \u003cem\u003eThASR4\u003c/em\u003e- silenced lines (RNAi) and Control. qRT-PCR results showed that the relative transcript level of \u003cem\u003eThASR4\u003c/em\u003e in OE plants was significantly higher than that in the control plants, while the level in RNAi plants was significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results indicate that transient OE, RNAi and Control transgenic \u003cem\u003eT. hispida\u003c/em\u003e plants were successfully obtained, and subsequently used for further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eqRT-PCR analysis of \u003cem\u003eThASR4\u003c/em\u003e genes in different kinds of transiently transformed \u003cem\u003eT. hispida\u003c/em\u003e, four-week-old \u003cem\u003eT. hispida\u003c/em\u003e plants were transiently transformed with empty pROKII, 35S:\u003cem\u003eThASR4\u003c/em\u003e or pFGC5941:\u003cem\u003eThASR4\u003c/em\u003e, The expression of \u003cem\u003eThASR4\u003c/em\u003e in whole OE, RNAi and Control plants was measured. The error bars are standard deviations, which were calculated from multiple replicates of qRT-PCR. OE: transgenic \u003cem\u003eT.hispida\u003c/em\u003e overexpressing \u003cem\u003eThASR4\u003c/em\u003e; RNAi: \u003cem\u003eThASR4\u003c/em\u003e RNAi-silenced \u003cem\u003eT. hispida\u003c/em\u003e plants; Control: pROKII vector-transformed \u003cem\u003eT. hispida\u003c/em\u003e plants.\u003c/p\u003e\n\u003ch3\u003e6. improves ROS-scavenging capability and antioxidant enzyme activity\u003c/h3\u003e\n \u003cp\u003eReactive oxygen species (ROS), such as superoxide (O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e), are unavoidable byproducts of aerobic metabolism. The accumulation of ROS under salt stress can lead to significant oxidative damage in plants. Superoxide dismutase (SOD) and peroxidase (POD) are core antioxidant enzymes that scavenge ROS.\u003c/p\u003e \u003cp\u003eTo further confirm the function of the \u003cem\u003eThASR4\u003c/em\u003e gene in scavenging ROS, we explored the accumulation of ROS and ROS-scavenging capability in \u003cem\u003eThASR4\u003c/em\u003e transgenic plants. This included \u003cem\u003eThASR4\u003c/em\u003e-overexpressing (OE) plants, \u003cem\u003eThASR4\u003c/em\u003e-RNAi silencing (RNAi) plants, and control plants with empty pROKII vector, all transiently transformed in \u003cem\u003eTamarix hispida\u003c/em\u003e. Additionally, we analyzed ROS accumulation in \u003cem\u003eThASR4\u003c/em\u003e-overexpressing \u003cem\u003eArabidopsis\u003c/em\u003e lines (OX2 and OX5).\u003c/p\u003e \u003cp\u003eDAB and NBT staining results demonstrated that under normal growth conditions, there was no significant difference between the OE, RNAi, and Control plants. However, after 6 hours of 200 mM NaCl treatment, the OE plants showed the lightest brown (DAB staining) and blue (NBT staining) colors compared to the control plants, while the RNAi plants showed the darkest colors. Furthermore, with longer treatment, the staining deepened in all plants and the relative difference was maintained, with OE consistently showing the lightest colors and RNAi the darkest colors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b). This indicates that following salt stress treatment, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e levels decreased in the OE plants, whereas they significantly increased in the RNAi plants compared to the control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ec, d). Additionally, under salt stress conditions, SOD and POD activities were higher in OE plants than in control plants, whereas they were lower in RNAi plants. As the duration of stress increased, the differences in SOD and POD activities between the transgenic and control \u003cem\u003eTamarix\u003c/em\u003e plants became significantly greater (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee, f).\u003c/p\u003e \u003cp\u003eSimultaneously, similar results were obtained in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. Under salt stress, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e levels in the OX2 and OX5 lines were significantly lower than in the WT plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ea-d), while SOD and POD activities in the OX2 and OX5 lines were markedly higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e7\u003c/span\u003ee, f). These findings suggest that \u003cem\u003eThASR4\u003c/em\u003e overexpression enhances SOD and POD activity under salt stress, thereby promoting the scavenging of reactive oxygen species.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA: DAB staining. B: NBT staining. C: NaCl induced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. D: NaCl induced O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e content in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. E:NaCl induced SOD activity in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. F:NaCl induced POD activity in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA: DAB staining. B: NBT staining. C: A.NaCl induced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. D: NaCl induced O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e content in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. E:NaCl induced SOD activity in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. F:NaCl induced POD activity in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants.\u003c/p\u003e\n\u003ch3\u003e7. decreases cell membrane damage\u003c/h3\u003e\n\u003cp\u003eWe further examined the cell membrane integrity of transgenic plants including \u003cem\u003eTamarix hispida\u003c/em\u003e transient transformants (OE, RNAi, Control) and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e stable transformants (OX2, OX5) under salt stress.\u003c/p\u003e \u003cp\u003eEvans Blue staining results showed that under normal conditions, all lines had no obvious blue staining. After salt stress, OE leaves showed faint blue color, while RNAi leaves were deeply stained (darker than Control), With prolonged salt stress (6 h, 12 h), the staining intensity of all lines gradually deepened, but the increase rate of OE was significantly lower than that of Control and RNAi (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Moreover, electrolyte leakage also showed consistent results, After salt stress, the electrolyte leakage rate of OE significantly lower than that of Control. For RNAi, the leakage rate increased by 4.5-fold, significantly higher than that of Control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). These result indicating that \u003cem\u003eThASR4\u003c/em\u003e overexpression alleviated cell membrane damage, whereas \u003cem\u003eThASR4\u003c/em\u003e silencing exacerbated it. Confirming that \u003cem\u003eThASR4\u003c/em\u003e delays salt-induced cell membrane damage.\u003c/p\u003e \u003cp\u003eProline acts as an osmotic protectant, while malondialdehyde (MDA) is a key marker of lipid peroxidation reflecting oxidative damage. We determined proline accumulating in \u003cem\u003eThASR4\u003c/em\u003e-overexpressing transgenic plants after salt stress. We find proline accumulate in OE was slightly higher than in Control, and RNAi contained significantly lower proline content than Control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).The MDA content was increased abruptly in the Control plants upon stress treatment relative to the OE. It increased by three and two-fold in salinity stress relative to OE, respectively. The MDA content in the RNAi also increased slightly, but the increase was not comparable to the Control plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Inaddion, as treatment time increased, the difference in proline content and MDA content between transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e and Control became more significant.\u003c/p\u003e \u003cp\u003eTo verify the conservation of ThASR4 function, the same staining experiments were performed on Arabidopsis. Evans Blue staining showed that under salt stress, OX2/OX5 leaves had weaker blue color than WT, the leakage rate of OX2/OX5 lower than that of WT, proline accumulate higher, MDA content was lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese results are consistent in both \u003cem\u003eT. hispida\u003c/em\u003e (native species) and \u003cem\u003eArabidopsis\u003c/em\u003e (model plant), confirming that \u003cem\u003eThASR4\u003c/em\u003e plays a conserved role in protecting cell membrane integrity under salt stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ea: Evans blue staining. b: electrolyte leakage. c: NaCl induced proline accumulation in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants. d: NaCl induced MDA activity in transgenic \u003cem\u003eTamarix hispida\u003c/em\u003e plants.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA: Evans blue staining. B: electrolyte leakage. C: NaCl induced proline accumulation in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants. D: NaCl induced MDA activity in transgenic \u003cem\u003eArabidopsis\u003c/em\u003e plants.\u003c/p\u003e\n\u003ch3\u003e8. ASR4 Regulates ROS Scavenger-Associated and Salt Stress-Responsive Gene Expression\u003c/h3\u003e\n\u003cp\u003eIt has been reported that \u003cem\u003eTaASR1\u003c/em\u003e and \u003cem\u003eSbASR1\u003c/em\u003e induce the transcription of ROS scavenger-associated genes and reduce the accumulation of ROS. To analyze the expression of ROS scavenger-associated genes in \u003cem\u003eThASR4\u003c/em\u003e-overexpressing \u003cem\u003eTamarix hispida\u003c/em\u003e, the \u003cem\u003eThPOD1, ThSOD1\u003c/em\u003e and \u003cem\u003eThCAT3\u003c/em\u003e genes, which are involved in ROS detoxification, were selected. Under non-stressed conditions, the ranscript levels of \u003cem\u003eThPOD1, ThSOD1\u003c/em\u003e and \u003cem\u003eThCAT3\u003c/em\u003e were increased significantly in OE compared to the Control. After salt treatment 12 hours, the expression of these genes were upregulated in both OE and Control plants, but the expression of these genes was increased markedly in OE (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003ea-c). A similar regulatory pattern was observed for salt Stress-Responsive genes \u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, and \u003cem\u003eThDREB\u003c/em\u003e. Under non-stress conditions, \u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, and \u003cem\u003eThDREB\u003c/em\u003e genes were expressed in both OE and Control. However, with salt treatment, their expression levels in OE increased significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e10\u003c/span\u003ed-f).\u003c/p\u003e \u003cp\u003eCollectively, these results indicate that in \u003cem\u003eThASR4\u003c/em\u003e-overexpressing lines, ROS scavenger-associated genes and salt stress-related genes are highly expressed and are strongly induced by salt stress. This suggests that the ThASR4 gene directly regulates the expression of these genes to mediate the salt stress response.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ea: Comparison of the expression of ROS scavenger-associated genes \u003cem\u003eThPOD1\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e b: Comparison of the expression of ROS scavenger-associated genes \u003cem\u003eThSOD1\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e c: Comparison of the expression of ROS scavenger-associated genes \u003cem\u003eThCAT3\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e d:Comparison of the expression of salt stress-responsive genes \u003cem\u003eThSOS3\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e e:Comparison of the expression of salt stress-responsive genes \u003cem\u003eThPIP2;5\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e f:Comparison of the expression of salt stress-responsive genes \u003cem\u003eThDREB\u003c/em\u003e between transgenic \u003cem\u003eT. hispida.\u003c/em\u003e\u003c/p\u003e \u003cp\u003etransgenic \u003cem\u003eTamarix hispida\u003c/em\u003e including \u003cem\u003eThASR4\u003c/em\u003e overexpression OE and Control, treated with water or 200 mM NaCl for 12 h, Each experiment was repeated independently for at least three times.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSince the first discovery of tomato \u003cem\u003eASR1\u003c/em\u003e, more than 20 years have passed. An increasing number of ASR families have been characterized in various plants. The functions of these proteins are involved in the responses to ABA and abiotic stress, as well as, the process of fruit ripening. \u003cem\u003eASR\u003c/em\u003e gene families are widely distributed in \u003cem\u003emonocots\u003c/em\u003e, \u003cem\u003edicots\u003c/em\u003e, \u003cem\u003eherbs\u003c/em\u003e, and \u003cem\u003exylophyta\u003c/em\u003e, but no \u003cem\u003eASR\u003c/em\u003e ortholog has been identified in \u003cem\u003eArabidopsis\u003c/em\u003e. However, \u003cem\u003eTamarix hispida\u003c/em\u003e, which is an important woody halophyte with strong salt tolerance in China, has been less well-studied than other plants, and the ASR regulatory pathway in this species has been unclear.\u003c/p\u003e \u003cp\u003eIn this study, six \u003cem\u003eThASR\u003c/em\u003e genes were identified in \u003cem\u003eT. hispida\u003c/em\u003e named \u003cem\u003eThASR1\u003c/em\u003e to \u003cem\u003eThASR6\u003c/em\u003e. All of the ThASRs had two highly conserved regions including N-terminal zinc-binding regions and C-terminal an ABA/WDS domain, for \u003cem\u003eT. hispida\u003c/em\u003e All of the ThASRs had two potential NLS in there ABA/WDS domain (Fig.\u0026nbsp;1a). The structure was similar to \u003cem\u003eASR\u003c/em\u003e genes from banana and \u003cem\u003eSuaeda liaotungensis\u003c/em\u003e, and therefore was characterized as a potential ASR family member. Tertiary structures of ThASR4 and ThASR6 have only one independent helical domains, however other proteins have loop-helix-loop domains (Fig.\u0026nbsp;1b). This structural difference may influence their functions.\u003c/p\u003e \u003cp\u003ePrevious studies have identified ASR family members in many plants, including \u003cem\u003ePopulus trichocarpa\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eSorghum bicolor\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e. Then multiple alignments and phylogenetic tree analysis revealed that ThASRs are more similar to \u003cem\u003eOsASR1\u003c/em\u003e, \u003cem\u003eOsASR2\u003c/em\u003e, \u003cem\u003eOsASR3\u003c/em\u003e and \u003cem\u003eZmASR3, ZmASR5, ZmASR9\u003c/em\u003e (Fig.\u0026nbsp;1c). \u003cem\u003eZmASRs\u003c/em\u003e are involved in abiotic stress tolerance (Hou et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). \u003cem\u003eOsASR1\u003c/em\u003e and \u003cem\u003eOsASR3\u003c/em\u003e are up regulated by Fe and Al stresses, and \u003cem\u003eOsASR1\u003c/em\u003e is involved in regulating the Al stress response in rice, acting as a transcription factor (Foy \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). \u003cem\u003eOsASR2\u003c/em\u003e is a DNA-binding protein that directly regulates a number of Al-responsive genes and associated with responses to abiotic stresses such as drought, salt, cold,and submergence (Iusem et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Thus we speculated that \u003cem\u003eThASR\u003c/em\u003e genes have a similar function to\u003cem\u003eOsASRs\u003c/em\u003e and \u003cem\u003eZmASRs.\u003c/em\u003e In our study, qRT-PCR showed that all six \u003cem\u003eThASR\u003c/em\u003e genes are transcriptionally regulated by salt stress and are expressed in both roots and shoots of \u003cem\u003eT. hispida\u003c/em\u003e. Among them, \u003cem\u003eThASR4\u003c/em\u003e exhibited the most significant upregulation under salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating its high sensitivity to salt stress and making it a key candidate gene for further functional verification.\u003c/p\u003e \u003cp\u003eDifferent ASR proteins' subcellular distribution patterns were observed in \u003cem\u003etomato\u003c/em\u003e, \u003cem\u003elitchi\u003c/em\u003e, \u003cem\u003ewheat\u003c/em\u003e, and \u003cem\u003elily\u003c/em\u003e. The ASR1 from tomato was first reported as a nuclear protein (Iusem et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Most ASR proteins such as \u003cem\u003elily\u003c/em\u003e (Wang et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), l\u003cem\u003eitchi\u003c/em\u003e (Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and \u003cem\u003ewheat\u003c/em\u003e (Hu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) were found in the nucleus. However, Kalifa et al. reported that tomato ASR1 was localized in both the cytosol and the nucleus (Kalifa et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In banana, MaASR1 protein was localized in the nucleus and plasma membrane (Sun et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In this study, Subcellular localization indicated that the \u003cem\u003eThASR4\u003c/em\u003e-GFP fusion protein was located in the nucleus and cytomembrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In addition, ThASR4 could activate transcription in yeast cells and the activation domain located in the N-terminal region (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), which is consistent with the recent study on the tomato ASR gene. These results indicate that ThASR4 functions as a transcription factor, which provides a molecular basis for its regulatory role in downstream gene expression.\u003c/p\u003e \u003cp\u003eTo further clarify the function of \u003cem\u003eThASR4\u003c/em\u003e,we obtained \u003cem\u003eThASR4\u003c/em\u003e-overexpressing transiently transformed \u003cem\u003eT. hispida\u003c/em\u003e (OE) and \u003cem\u003eThASR4\u003c/em\u003e RNAi silencing Transient \u003cem\u003eT. hispida\u003c/em\u003e (RNAi) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e, OE and RNAi), and stable overexpression of \u003cem\u003eThASR4\u003c/em\u003e in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Fig. S1, OX2 and OX5 ) were used here to study the role of \u003cem\u003eThASR4\u003c/em\u003e. \u003cem\u003eThASR4\u003c/em\u003e-overexpressing transgenic \u003cem\u003eArabidopsis\u003c/em\u003e resulted in better germination and growth under saline treated conditions. Longer root length and higher fresh weight to dry weight ratio than the WT demonstrating that the \u003cem\u003eThASR4\u003c/em\u003e gene has the function of improving salt stress tolerence.\u003c/p\u003e \u003cp\u003eHigh cellular concentrations of NaCl causes increased formation of reactive oxygen species (ROS), ROS is an important substance in the cell,when the level of ROS is low in cells, they act as signal molecules (Choudhury et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, under severe biotic and abiotic stress, ROS can be overproduced and cause damage to cell growth by oxidizing proteins, lipids and DNA. Therefore, plants have developed complete systems to protect themselves against oxidative stress caused by abiotic stresses through adjusting ROS homeostasis (Choudhury et al. 2012). Some reports have indicated that plant ASR genes have important functions in response to salt stress in many plant species. \u003cem\u003eMaASR1\u003c/em\u003e confers salt stress tolerance by regulating the expression of ABA/stress-responsive genes, and \u003cem\u003eSbASR1\u003c/em\u003e enhances the salinity and drought stress tolerance by functioning as an LEA protein and transcription factor (Tiwari et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eSlASR\u003c/em\u003e protein may act as a protective molecule to help plants adapt to abiotic stresses (Hu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Overexpression of \u003cem\u003eMaASR1\u003c/em\u003e, Banana (\u003cem\u003eMusa acuminataL\u003c/em\u003e.) ASR gene, in \u003cem\u003eArabidopsis\u003c/em\u003e confers salt stress tolerance by reducing the expression of ABA/stress-responsive genes \u003cem\u003eSiASR4\u003c/em\u003e functions in the adaption to drought and salt stress and is regulated by \u003cem\u003eSiARDP\u003c/em\u003e via an ABA-dependent pathway (Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur study indicate that overexpression of \u003cem\u003eThASR4\u003c/em\u003e increased proline accumulation and reduced malondialdehyde (MDA) content in both \u003cem\u003eTamarix hispida\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e under salt stress, thereby enhancing osmotic adjustment capacity and alleviating membrane damage. \u003cem\u003eThASR4\u003c/em\u003e overexpression significantly reduced the accumulation of reactive oxygen species (ROS, including H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O2\u0026middot;\u003csup\u003e\u0026minus;\u003c/sup\u003e) under salt stress, as evidenced by DAB and NBT staining. Correspondingly, the activities of antioxidant enzymes (SOD and POD) were significantly higher in overexpression plants than in Control, which enhanced ROS detoxification efficiency and protected cells from oxidative damage. \u003cem\u003eThASR4\u003c/em\u003e overexpression upregulated the transcription of ROS-scavenging-related genes (\u003cem\u003eThPOD1, ThSOD1, ThCAT3\u003c/em\u003e) and salt stress-responsive genes (\u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, \u003cem\u003eThDREB\u003c/em\u003e) in both with or without salt stress. These suggested that \u003cem\u003eThASR4\u003c/em\u003e gene enhanced the activation of antioxidant defence system, which in turn protected \u003cem\u003eT. hispida\u003c/em\u003e against ROS-mediated injury under salt stress.\u003c/p\u003e \u003cp\u003eIn summary, this study demonstrates that \u003cem\u003eThASR4\u003c/em\u003e from \u003cem\u003eT. hispida\u003c/em\u003e enhances plant salt tolerance through a comprehensive mechanism: as a transcription factor, \u003cem\u003eThASR4\u003c/em\u003e activates the expression of ROS-scavenging and salt stress-responsive genes, thereby promoting antioxidant enzyme activity, reducing ROS accumulation, enhancing osmotic adjustment, and protecting cell membrane integrity. These findings not only enrich our understanding of the functional diversity of ASR family genes in woody halophytes but also provide a potential candidate gene for genetic improvement of plant salt tolerance.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn the present study, six \u003cem\u003eThASR\u003c/em\u003e genes were identified and obtained from wild-type \u003cem\u003eTamarix hispida\u003c/em\u003e plants. Among these genes, \u003cem\u003eThASR4\u003c/em\u003e was selected as the focus for in-depth investigation into its function and regulatory mechanism in salt stress tolerance, based on its significant upregulation under NaCl treatment.\u003c/p\u003e \u003cp\u003eOur results systematically demonstrate that \u003cem\u003eThASR4\u003c/em\u003e plays a positive regulatory role in plant salt stress tolerance. Mechanistically, \u003cem\u003eThASR4\u003c/em\u003e enhances salt tolerance primarily through two interconnected pathways: first, as a nuclear-localized transcription factor, it directly or indirectly upregulates the expression of a suite of salt stress-responsive genes (\u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, \u003cem\u003eThDREB\u003c/em\u003e); second, it strengthens the antioxidant system by promoting the activity of key antioxidant enzymes (\u003cem\u003eThPOD1, ThSOD1, ThCAT3\u003c/em\u003e) and the accumulation of osmotic protectants (proline). These combined effects effectively reduce the overaccumulation of reactive oxygen species (ROS) under salt stress, alleviate ROS-mediated oxidative damage, and maintain cell membrane integrity, thereby improving plant adaptation to high-salt environments.\u003c/p\u003e \u003cp\u003eTo fully elucidate the regulatory network of \u003cem\u003eThASR4\u003c/em\u003e, future studies should focus on identifying its direct downstream target genes\u0026mdash;specifically, the cis-acting elements in target gene promoters that \u003cem\u003eThASR4\u003c/em\u003e binds to. Such investigations will not only refine our understanding of the molecular mechanism underlying \u003cem\u003eThASR4\u003c/em\u003e-mediated salt tolerance via ROS scavenging but also provide a more solid theoretical basis for leveraging \u003cem\u003eThASR4\u003c/em\u003e as a candidate gene in genetic engineering for improving salt tolerance in woody plants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics, Consent to Participate, and Consent to Publish declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Fundamental Research Funds for the Central Universities (No. 2572023CT03) and the Innovation Project of the State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (No. 2014B03).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWC designed the research. ZY and ZX per formed most experiments and analyzed experimental data. WJ and LHY conducted a part of experiments. ZZY wrote the manuscript, and WC revised the paper. All authors approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used to support the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Fundamental Research Funds for the Central Universities (No. 2572023CT03) and the Innovation Project of the State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (No. 2014B03).\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e\u003cstrong\u003eArenhart\u003c/strong\u003e RA, Schunemann M, Bucker Neto L, Margis R, Wang ZY, Margis-Pinheiro M (2016) Rice ASR1 and ASR5 are complementary transcription factors regulating aluminium responsive genes. 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Int J Mol Sci. 25(19): 10283. https://doi.org/10.3390/ijms251910283\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ThASR4, Tamarix hispida, Salt Stress, ROS Scavenging","lastPublishedDoi":"10.21203/rs.3.rs-8909679/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8909679/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eASR genes are a class of genes that are expressed in plants in response to abscisic acid, abiotic stress, and fruit ripening. While they have been cloned and functionally characterized in various plant species, their study in \u003cem\u003eTamarix hispida\u003c/em\u003e has been limited. In this study, six \u003cem\u003eThASR\u003c/em\u003e genes were cloned from \u003cem\u003eTamarix hispida\u003c/em\u003e. Expression analysis revealed that \u003cem\u003eThASR\u003c/em\u003e genes respond to salt stress. Specifically, \u003cem\u003eThASR4\u003c/em\u003e was found to be highly induced in both shoots and root of \u003cem\u003eTamarix hispida\u003c/em\u003e under salt stress, thus prompting its selection for further functional characterization in salt stress responses. \u003cem\u003eThASR4\u003c/em\u003e is targeted to the nucleus and possesses transcriptional activity. Under salt stress conditions, compared with the wild-type (WT) lines, \u003cem\u003eThASR4\u003c/em\u003e-overexpressing \u003cem\u003eArabidopsis thaliana\u003c/em\u003e exhibited enhanced germination rate, longer primary roots, and higher fresh weight, indicating a marked improvement in salt tolerance. Correspondingly, both \u003cem\u003eThASR4\u003c/em\u003e-overexpressing \u003cem\u003eT. hispida\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e plants exhibited significantly diminished levels of reactive oxygen species (ROS), malondialdehyde (MDA), and electrolyte leakage, along with elevated activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as increased proline (Pro) content, compared to Control plants. In contrast, \u003cem\u003eThASR4\u003c/em\u003e RNA interference (RNAi) transgenic \u003cem\u003eT. hispida\u003c/em\u003e displayed the opposite phenotypic and physiological outcomes. Furthermore, \u003cem\u003eThASR4\u003c/em\u003e was found to upregulate the expression of ROS scavenger-associated genes (\u003cem\u003eThPOD1, ThSOD1\u003c/em\u003e and \u003cem\u003eThCAT3\u003c/em\u003e) and salt Stress-Responsive genes (\u003cem\u003eThSOS3\u003c/em\u003e, \u003cem\u003eThPIP2;5\u003c/em\u003e, and \u003cem\u003eThDREB\u003c/em\u003e). Collectively, our results indicate that \u003cem\u003eThASR4\u003c/em\u003e enhances salt tolerance in \u003cem\u003eT. hispida\u003c/em\u003e and \u003cem\u003eA. thaliana\u003c/em\u003e by improving ROS scavenging capacity. The present study provides a theoretical foundation for further investigation into the regulatory mechanism of \u003cem\u003eThASR4\u003c/em\u003e in the salt stress adaptation of \u003cem\u003eT. hispida\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"ThASR4 enhances salt tolerances in transgenic Tamarix and Arabidopsis by Scavenging Reactive Oxygen Species","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-02 13:57:33","doi":"10.21203/rs.3.rs-8909679/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-28T14:22:30+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-26T01:29:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"292602778680455350671912234716672538970","date":"2026-03-20T13:26:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101710045434261437945301898381706447678","date":"2026-03-18T09:05:18+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-12T16:44:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"120840765643132798427396509584302794966","date":"2026-03-06T04:34:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-25T16:59:17+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-02-23T13:12:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-20T13:28:55+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-20T13:26:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-02-18T13:00:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6be112f4-2946-466b-89c7-5bedc296d4e6","owner":[],"postedDate":"March 2nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-19T06:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-02 13:57:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8909679","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8909679","identity":"rs-8909679","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}