De novo characterization of AcABI5 transcription factor and physiological responses to salt stress in Alhagi camelorum callus

De novo characterization of AcABI5 transcription factor and physiological responses to salt stress in Alhagi camelorum callus | 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 De novo characterization of AcABI5 transcription factor and physiological responses to salt stress in Alhagi camelorum callus Zhengtao Yan, Xiangyi Li, Bo Zhang, Gangliang Tang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8449616/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Alhagi camelorum , a dominant leguminous shrub in the saline–hyperarid Taklimakan Desert, can complete its life cycle in salty soils, yet the molecular basis of seedling-stage salt tolerance remains unknown. Here, we developed an in-vitro callus system to dissect this trait without soil heterogeneity. Among 36 hormone regimes, 1.5 mg/L 2,4-D plus 0.5 mg/L 6-BA produced 100 % induction. Exposure of 28-d-old calli to 200 mM NaCl for 48 h caused transient swelling of cortical cells and a 5.3-fold rise in malondialdehyde (MDA). Antioxidant enzymes responded sequentially: superoxide dismutase (SOD) peaked at 6 h to scavenge superoxide, peroxidase (POD) maintained high activity throughout the first 24 h, and catalase (CAT) stabilized after 48 h, jointly keeping H₂O₂ below toxic levels. RNA-seq has identified an up-regulated transcription factor (log₂FC = 7) which was a basic leucine-zipper (bZIP) homologue of abscisic acid-insensitive protein 5 (ABI5). Quantitative RT-PCR confirmed 17-fold induction by NaCl and rapid decay after stress removal. Sub-cellular localization of a 35S::GFP-AcABI5 fusion in Nicotiana benthamiana epidermis showed exclusive nuclear fluorescence, consistent with a transcriptional regulator. Therefore, our study provides both optimized callus protocols and a candidate gene for engineering salt tolerance in A. camelorum and related desert legumes. Alhagi camelorum Callus induction Salt stress Antioxidant enzymes AcABI5 bZIP transcription factor Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Soil salinization, exacerbated by climate extremes and land misuse, imposes ionic toxicity and osmotic stress that globally curtail root elongation, biomass allocation, and crop yield potential (van Zelm et al. 2020 ). Shrinking freshwater reserves now make the reclamation of saline–alkali land a critical imperative for global food security. Alhagi camelorum Fisch. (Fig. 1 ), a deep-rooted perennial legume of Central Asian deserts, forms 40–130 cm thickets of thorny, grey-green shoots that carry obovate leaves and short axillary racemes whose red-purple petals give way to spiralled loment pods (Dong and Zhang 2000 ; Muhammad et al. 2015 ; Ullah et al. 2022 ). A. camelorum spans the arid corridor from the Taklimakan to the Iranian Plateau, occurring in northwestern China, Kazakhstan, Afghanistan, Iran, Pakistan, Iraq, Mongolia and India; across these regions it routinely colonizes saline soils with > 200 mM NaCl, making it one of the most salt-tolerant wild legumes (Manafu et al. 2024 ; Pirasteh-Anosheh et al. 2022 ; Srivastava et al. 2014 ). On the southern rim of the Taklamakan, A. camelorum carpets the shifting ecotone between oasis cropland and drifting dunes, forming the keystone thicket that stabilizes soils and defines the desert margin (Tang et al. 2013 ). A single stand delivers a triple dividend: its thorny lattice brakes the wind and anchors shifting sand, its shoots furnish protein-rich fodder equal to alfalfa, and its resin-rich exudates remain a staple of traditional pharmacopoeias (Kazemi and Ghasemi Bezdi 2021 ; Muhammad et al. 2014 ; Zeng et al. 2002 ), and is also an excellent candidate plant for saline-alkali land remediation (Zheng et al. 2023 ). Nevertheless, the molecular determinants enabling A. camelorum to maintain growth under saline conditions remain largely uncharacterized. Systematic discovery and validation of the underlying salt tolerance genes will provide strategic targets for molecular breeding programs aimed at enhancing crop resilience to soil salinity. The basic leucine zipper transcription factors (bZIP, TFs) form a highly conserved and functionally diverse transcriptional regulatory family in plants. They are characterized by a conserved bZIP core domain that encompasses a DNA-binding region and a leucine zipper dimerization domain. Through post-translational modifications, these factors participate in the assembly of multiprotein complexes to regulate downstream gene networks. This family plays a central role in the growth, development, and stress adaptation of leguminous plants (Yue et al. 2023 ). In stress responses, bZIP TFs exhibit distinct functional specificity and diversity. GmbZIP60 in soybean ( Glycine max ) can be induced by salt and drought stresses, overexpression of GmbZIP60 enhances soybean tolerance to salt and drought by directly binding to the promoters of stress-responsive genes (Chai et al. 2025 ). GmbZIP19 is a positive regulator of pathogen resistance and a negative regulator to modulate stomatal closure, thereby reducing plant tolerance to salt and drought stresses (He et al. 2020 ). The nuclear protein GmbZIP110 significantly improves the salt stress adaptability of soybean by binding to the ACGT motif to regulate proline accumulation and Na⁺/K⁺ homeostasis (Xu et al. 2016 ). As a dominant sand-fixing plant in arid and semi-arid saline-alkali regions, A. camelorum exerts a central role in improving regional soil structure and maintaining ecosystem stability by virtue of its excellent salt and drought tolerance. Therefore, as an important class of transcription factors, it is necessary to investigate the mechanism by which bZIPs regulate salt tolerance-related genes in A. camelorum. In this work, aseptic seedlings of A. camelorum served as the experimental material, and young stem explants were subjected to a hormone matrix screen that yielded a single optimized medium supporting rapid, highly synchronous callus proliferation. Exposure of these calli to MS medium supplemented with 200 mM NaCl revealed a temporally orchestrated antioxidant response: early superoxide dismutase (SOD) peak, sustained peroxidase (POD) activity, late catalase (CAT) stabilization and corresponding ion-leakage dynamics, validating the in vitro system as a physiologically relevant salt-stress proxy. Transcriptomic profiling of challenged seedling identified a bZIP TF (Tang et al. 2024 ), ABI5-homologous, as the most strongly up-regulated regulatory gene; its full-length CDS (1080 bp; 359 amino acids) was cloned and designated AcABI5 . In-silico analysis predicted a canonical basic leucine-zipper domain and a bipartite nuclear-localization signal (NLS); transient expression of a 35S::GFP-AcABI5 fusion in Nicotiana benthamiana confirmed exclusive nuclear accumulation. Quantitative RT-PCR (qRT-PCR) time courses demonstrated a 17-fold induction within 12 h of salt treatment, followed by rapid decay upon stress relief, consistent with a primary transcriptional regulator. These results provide the first functional annotation of a salt-responsive transcription factor in A. camelorum , establish a reproducible callus-based transformation platform, and identify AcABI5 as a candidate gene for molecular breeding of salt-tolerant crops. Materials and Methods Material treatment and callus induction A. camelorum seeds used in this study were collected in July 2023 from the Cele Desert Grassland Ecosystem National Field Scientific Observation and Research Station, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. Mature and plump seeds were vernalized for 48 h, soaked in 75% ethanol for 30 s, and rinsed 3 times with sterile distilled water, followed by immersion in 5% NaOCl for 5 min and washed 5 times with sterile distilled water. The seeds were inoculated on MS medium (Murashige and Skoog 1962 ) supplemented with 3% (w/v) sucrose and 0.3% (w/v) phytagel (pH = 5.8-6.0), and grown under conditions of 25 ± 1°C, 3000 lx, 16 h light and 8 h dark. Select aseptic seedlings that are in good growth condition and have a seedling age of approximately 28 days for the callus culture of A. camelorum .Young stem segments of approximately 0.5 cm were cut as explants and transferred to culture media containing different concentration combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA) for callus induction. The culture conditions were the same as those described previously. To evaluate the growth dynamics of calli, the callus induction rate and relative growth of A. camelorum were recorded after 28 days of culture, and the fresh weight of calli was measured using the difference method.The callus induction rate (%), callus growth (g) and relative growth rate (%) were calculated as below: Callus induction rate (%) = (Number of explants producing calli / Total number of inoculated explants) × 100% Callus growth (g) = Weight of calli after culture − Weight of calli before culture Relative growth rate (%) = (Callus growth / Initial fresh weight of calli) × 100% Determination methods of growth indicators physiological and biochemical indexes To analyze the physiological status of calli under long-term salt stress, the salt stress treatment group was cultured on callus induction medium (MS medium supplemented with 1.5 mg/L 2,4-D and 0.5 mg/L 6-BA) supplemented with an additional 200 mM NaCl, whereas the control group was maintained on basic callus induction medium without NaCl supplementation.The calli with robust growth and uniform size were transferred to the respective media, with 12 calli per medium and 3 replicates set for each treatment. The culture conditions were as follows: temperature of 25 ± 1°C, 3000 lx, 16 h light and 8 h dark. The fresh weight of calli in both the control and treatment groups was measured, histochemical staining was performed, and the phenotypes of calli were recorded by photography on day 0, day 7, and day 14. DAB staining and NBT staining: A. camelorum calli with uniform size after stress treatment were placed in 50 mL centrifuge tubes. Prepared DAB staining solution and NBT staining solution were added respectively to submerge the calli. Vacuum was slowly applied to 0.8 MPa and maintained for 5 min, then slowly restored to normal pressure. Staining was performed at room temperature in the dark for 12 h. After discarding the staining solution, the calli were rinsed repeatedly with sterile distilled water to remove residual staining solution. Decolorizing solution was added, and the samples were incubated in a 95°C water bath for 10–30 min until chlorophyll was completely removed. Evans blue staining: A. camelorum calli with uniform size after stress treatment were placed in 50 mL centrifuge tubes. Prepared Evans blue staining solution was added to submerge the calli. Vacuum was slowly applied to 0.8 MPa and maintained for 5 min, then slowly restored to normal pressure. Staining was performed at room temperature in the dark for 6 h. After discarding the staining solution, the calli were rinsed repeatedly with sterile distilled water to remove residual staining solution. Decolorizing solution was added, and the samples were incubated in a boiling water bath for 10–30 min until chlorophyll was completely removed. DAB solution (1 mg/mL): Weigh out 100 mg of DAB powder and dissolve it in 100 ml of 0.1 mol/L phosphate buffer solution (pH = 7.4) to prepare a 1 mg/mL aqueous solution. Adjust the pH to 3.8 with hydrochloric acid, and store the solution at 4°C in the dark. NBT solution (0.5 mg/mL): Weigh out 50 mg of NBT powder and dissolve it in 100 mL of 0.1 mol/L phosphate buffer solution (pH = 7.4). Store the solution at 4°C in the dark. Evans blue solution (0.5 mg/mL): Weigh out 50 mg of Evans blue powder and dissolve it in 100 mL of double-distilled water. After dissolving thoroughly, store the solution at room temperature in the dark. Decolorizing solution: 95% ethanol, acetic acid, and glycerol were mixed at a volume ratio of 3:1:1 and stored at room temperature in the dark. To determine the physiological and biochemical indices of A. camelorum calli under salt stress, the calli with robust growth and uniform size cultured on the same medium were selected and separately transferred to the media of the salt stress treatment group and the control group. After treatment for 0 h, 6 h, 12 h and 48 h, the calli were snap-frozen in liquid nitrogen and subsequently ground into powder. Crude enzyme extract was prepared to determine the malondialdehyde (MDA) content and antioxidant enzyme activities. Samples were also stored at -80°C for subsequent RNA extraction. Determination of MDA content and antioxidant enzyme activities: 0.1 g samples were taken from both the control and salt stress treatment groups, with 3 replicates per treatment. Kits purchased from Shanghai Fenxi Biotechnology Co., Ltd. were used to determine the contents of MDA, SOD, POD, and CAT. Detailed instructions are available at http://www.shfxsw.cn . POD activity unit: One unit (U) of POD activity was defined as a 0.005 change in A 470 per minute per gram of tissue in each milliliter of reaction system. SOD activity unit: In the xanthine oxidase-coupled reaction system of the kit, one unit (U) of SOD activity was defined as the amount of enzyme required to achieve a 50% inhibition rate. CAT activity unit: One unit (U) of CAT activity was defined as the amount of enzyme that catalyzes the degradation of 1 µmol H₂O₂ per minute per gram of tissue. Total RNA extraction and first-strand cDNA synthesis Total RNA was extracted from the samples treated in Section 1.5 according to the instructions of the TIANGEN Total RNA Extraction Kit. The concentration and purity of RNA were determined using a Nanodrop, and the integrity of RNA was detected by agarose gel electrophoresis.RNA was reverse-transcribed into cDNA as a template using the TIANGEN FastKing One-Step Genomic DNA Removal and cDNA Synthesis SuperMix according to the instructions. Screening of candidate genes and analysis of gene expression levels Based on the research findings of (Tang et al. 2024 ), we identified 10 candidate genes highly responsive to osmotic stress, namely Asp01G030840, Asp01G035580, Asp02G012850, Asp02G017010, Asp02G034200, Asp04G012530, Asp04G017080, Asp07G002390, Asp07G015930, and Asp08G003230. Using A. camelorum EF-1α (Asp06G015730) as the internal reference gene, specific primers were designed using Primer Premier 6.0 (Table S2). cDNA from the control and treatment groups at 6 h as mentioned above was used as the template. qRT-PCR was performed according to the instructions of Vazyme SYBR qRT-PCR Master Mix. The relative expression levels of each gene were calculated using the 2 −ΔΔ Ct method to screen the gene with the highest expression level under salt stress for subsequent experiments. The gene with the highest expression level was selected for qRT-PCR using cDNA from the control and treatment groups at 0 h, 6 h, 18 h, and 36 h as mentioned above as templates. A. camelorum EF-1α and ACT (Asp07G013640) were used as internal reference genes. qRT‒PCR was performed using Applied Biosystems QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific, USA), the 2 −ΔΔ Ct method was used to calculate the expression levels of this gene in A. camelorum calli at different stress durations (Livak and Schmittgen 2001 ). Bioinformatics analysis The Expasy-ProtParam tool ( https://web.expasy.org/protparam/ ) was used to analyze the physicochemical properties of the predicted Asp07G015930 protein, including molecular weight, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydropathicity. SignalP 6.0 ( https://services.healthtech.dtu.dk/services/SignalP-6.0/ ) was used to predict the signal peptide of the full-length sequence to determine its subcellular localization characteristics. Homologous sequences of Asp07G015930 were obtained using NCBI Blast ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) and Uniprot ( https://www.uniprot.org/ ), and 38 homologous sequences were screened for subsequent analysis. A phylogenetic tree was constructed using MEGA software, with the Neighbor-Joining method for tree reconstruction, and 1000 bootstrap replicates to evaluate branch support. After construction, the generated phylogenetic tree was optimized and visualized using the online tool ITOL ( http://itol.embl.de/ ). The protein sequences was submitted to the NCBI-CDD database ( https://www.ncbi.nlm.nih.gov/cdd/ ) to predict conserved domains and identify the bZIP superfamily domain and other related functional domains. The online tool MEME ( https://meme-suite.org/meme/tools/meme ) was used for conserved motif analysis, with 10 motifs set and a motif length range of 6–50 amino acids. Cis-acting regulatory elements of the promoter were predicted using PlantCARE ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).TBtool s software was used to visualize conserved motifs and generate domain and motif band diagrams(Chen et al. 2023 ). The online tool SOPMA program ( https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html ) was used to predict the secondary structure of the protein. The online tool SWISS MODEL ( https://swissmodel.expasy.org/ ) was used to predict the tertiary structure of the protein, generate a 3D structure model based on homology modeling, and evaluate the structure quality. Uniprot and WoLF PSORT ( https://wolfpsort.hgc.jp/ ) were used for subcellular localization prediction. Subcellular localization Using SnapGene 6.0.2, specific primers for the AcABI5 gene were designed based on its CDS sequence, incorporating attB-specific recombination sites of the Invitrogen Gateway® donor vector pDONR-Zeo (Table S3). The gene was cloned using cDNA as mentioned above as the template according to the instructions of Vazyme 2×Phanta Flash Master Mix. The amplified product was detected by 1% agarose gel electrophoresis, and the target fragment was purified by gel extraction and sent for sequencing. Using pDONR-Zeo as the donor vector, the entry vector was obtained through BP reaction according to the instructions of Invitrogen Gateway™ BP Clonase™ Ⅱ Enzyme Mix and transformed into Escherichia coli DH5α competent cells. The cells were cultured on LB solid medium (containing 50 µg/mL Zeocin) at 37°C for 12 h. Single colonies were picked for bacterial liquid PCR identification and sent for sequencing. Plasmids were extracted from correctly sequenced clones for LR reaction. Using pK7WGF2, a recipient vector containing the enhanced green fluorescent protein (eGFP) reporter gene as the recipient vector, the expression vector was obtained through LR reaction according to the instructions of Invitrogen Gateway® LR Clonase™ Ⅱ Enzyme Mix and transformed into E. coli DH5α competent cells. The cells were cultured on LB solid medium (containing 50 µg/mL Spec) at 37°C for 12 h. Single colonies were picked for bacterial liquid PCR identification and sent for sequencing. Plasmids were extracted from correctly sequenced clones for subsequent experiments. The constructed pK7WGF2-35S::GFP-AcABI5 vector plasmid was transformed into Agrobacterium GV3101 and cultured on YEB solid medium (containing 50 µg/mL Spec and 25 µg/mL Rif) at 28°C for 24 h. Correctly sequenced single-colony Agrobacterium was expanded and resuspended in an infection solution containing 100 µM Acetosyringone, then incubated statically for 3 h. The empty GFP vector was used as a control and mCherry (NLS) was used as a marker for co-injection into N. benthamiana leaves. Observation was performed using a laser confocal microscope after 48 h. Fluorescence was detected with a Zeiss LSM 800 (Carl Zeiss AG, Germany) confocal microscope. Statistical analysis IBM SPSS Statistics software (Version 27.0, IBM Corp., Armonk, NY, USA) was used for one-way ANOVA, Duncan’s multiple range tests, and Student's t -test. Error bars on the graphs indicate the standard deviation of the mean (± SD), calculated from three independent biological replicates ( n = 3) for each treatment. Data were analyzed, and means with identical letters are considered not significantly different at P < 0.05. Asterisks are used to indicate significant differences in Student's t -test ( P < 0.05). GraphPad Prism software was used for plotting graphs (Version 10.1.2, GraphPad Software Inc., San Diego, CA, USA). Results Effects of different hormone concentrations on callus induction of A. camelorum A broad spectrum of auxin–cytokinin regimes (Table S1) evoked callus formation from A. camelorum stem explants, yet macroscopic texture, pigmentation and growth kinetics varied markedly (Fig. 2 ). Sub-threshold concentrations (< 0.2 mg /L 2,4-D; 2,4-D; groups 4–6, 11–12) canalized tissues toward shoot organogenesis. A positive dose–response relationship between 2,4-D and callus induction rate was evident up to 1.5 mg/L, peaking at a relative growth increment of 110.93 ± 12.52% (group 27). Supra-optimal auxin levels reversed this trend, yielding friable, necrotic tissues. Within the window 1.0–2.0 mg/L 2,4-D and 0.5–1.0 mg/L 6-BA, auxin:cytokinin ratios between 3:1 and 2:1 consistently generated compact, cream-coloured, rapidly proliferating calli. Consequently, the formulation 1.5 mg/L 2,4-D plus 0.5 mg/L 6-BA (group 27) was adopted as the benchmark for all subsequent experimentation. Determination of physiological and biochemical indexes of A. camelorum calli under salt stress Exposure to 200 mM NaCl for 14 days imposed a sustained growth arrest on A. camelorum calli (Fig. 3 A-B). Control and salt-treated tissues exhibited equivalent fresh mass at t₀; thereafter, control calli expanded linearly, retaining a bright-green, nodular morphology, whereas treated calli remained quiescent through day 7 and developed a yellow-green hue. By day 14, mean fresh mass of the stressed population had declined by 15.8% relative to t₀, accompanied by marginal necrosis (Fig. 3 A-B), indicating that chronic osmotic stress not only blocked biomass accretion but also drove gradual water loss and cellular decompartmentalization. Plants rapidly accumulate large amounts of reactive oxygen species (ROS) under stress conditions. The main ROS include hydrogen peroxide (H₂O₂), hydroxyl radicals (∙OH), and superoxide anion radicals (O₂ ⁻ ∙) (Juan et al. 2021 ). Excessive ROS act on unsaturated fatty acids in lipids to form peroxides (such as MDA), causing damage to membrane structure and function, oxidizing DNA bases to induce gene mutations or cell apoptosis (Hossain et al. 2015 ). POD decomposes H₂O₂ to release oxygen ions, which oxidize DAB to form brown water-insoluble precipitates. The darker the precipitate color, the higher the H₂O₂ content in calli and the more severe the damage. As shown in Fig. 3 C, the treatment group showed slight damage on day 7, and H₂O₂ accumulated gradually with time, resulting in more severe damage on day 14. O₂ ⁻ ∙, one of the ROS, can reduce NBT to form blue deposits, thus enabling localization of O₂ ⁻ ∙ production sites in calli. The darker the color, the higher the ROS content and the more severe the cell damage. Comparison showed that O₂ ⁻ ∙ in the control group mainly accumulated on the side of calli in contact with the medium, and no obvious O₂ ⁻ ∙ was observed on the top of calli far from the medium. In the treatment group, obvious O₂ ⁻ ∙ accumulation was observed on the top of calli on day 7, and O₂ ⁻ ∙ was distributed throughout the calli on day 14, indicating severe ROS damage to the entire calli. Evans blue is a cell viability dye that stains dead cells light blue but cannot enter living cells. Thus, the darker the staining, the higher the damage degree of calli from the outside to the inside. As shown in Fig. 3 C, the control group showed no obvious change after 14 days of treatment, while the cell death degree of the treatment group gradually increased with the extension of stress time. Combined with the results of the three histochemical stainings, we concluded that the regulatory mechanism in A. camelorum calli could maintain basic cell survival under moderate to high salt stress for 7 days, but the calli suffered obvious damage after 14 days of continuous salt stress. Temporal Response Characteristics of the Antioxidant Enzyme System in A. camelorum Calli Under Salt Stress The enzyme activities and MDA content of A. camelorum calli treated with 200 mM NaCl for 0 h, 6 h, 12 h, and 48 h are shown in Fig. 4 . As shown in Fig. 4 A, compared with the control group, SOD activity began to increase at 6 h after treatment and reached a peak at 12 h, indicating that SOD was activated immediately in the early stage of stress to dismutate O₂ ⁻ ∙ into oxygen and H₂O₂, which reflects the ability of plants to scavenge O₂ ⁻ ∙ and also leads to H₂O₂ accumulation. SOD activity then decreased to the initial stress level at 48 h. The activity of POD was similar to that of SOD: it was activated immediately in the early stage of stress and reached a peak at 12 h. Although it decreased at 48 h, there was no significant difference compared with 12 h, and high activity was still maintained (Fig. 4 B). The activity of CAT increased significantly at 12 h, while there was no significant difference at other time points compared with the control group, but a downward trend was observed (Fig. 4 C). MDA is a marker product of membrane lipid peroxidation. As shown in Fig. 4 D, MDA began to accumulate in calli in the first 12 h of stress but showed no significant increase, indicating that the antioxidant enzyme system in A. camelorum calli effectively scavenged ROS and avoided cell damage. With the extension of stress time, a large amount of MDA accumulated in calli after 48 h of long-term stress, indicating that the scavenging capacity of the antioxidant system in the later stage was insufficient to offset ROS accumulation, leading to aggravated damage to the membrane system of calli. Screening of candidate genes and determination of their expression levels in calli under salt stress Specific primers were designed based on the CDS sequences of 10 candidate genes, and qRT-PCR analysis was performed. After salt stress treatment of A. camelorum calli, the expression levels of six genes were significantly upregulated, while the expression of two genes (Asp08G003230 and Asp02G034200) exhibited a downregulated trend (Fig. 5 A). This may be due to differences in expression regulatory mechanisms between calli (as dedifferentiated non-organs) and organs. Among them, Asp07G015930 showed the highest upregulation (8-fold) and was highly responsive to salt stress. Thus, this gene was selected as the target for subsequent studies. After 48 h of salt stress treatment, compared with the control group, the average relative expression level of AcABI5 (Asp07G015930) was upregulated 7-fold at 6 h, reached a maximum of 17-fold at 12 h, and was upregulated 12.9-fold at 48 h (Fig. 5 B). This indicates that AcABI5 is highly responsive to salt stress in A. camelorum calli, with its expression level significantly increasing as the stress intensifies. Although there was a slight decrease at 48 h, there was no significant difference compared with 12 h. This suggests that AcABI5 can rapidly respond to salt stress in A. camelorum calli and alleviate the damage caused by salt stress by continuously and highly expressing to regulate downstream genes. Bioinformatics analysis of AcABI5 Based on transcriptome data (Tang et al. 2024 ), the CDS length of Asp07G015930 is 1080 bp, encoding 359 amino acids. BLAST analysis using NCBI and UniProtKB databases showed that the protein encoded by this gene has high homology with ABI5 in various plants and has conserved functional domains typical of the bZIP family. Based on its function and sequence characteristics, this gene was named AcABI5 . Phylogenetic tree reconstruction was performed using 38 homologous sequences from plants such as Medicago truncatula and Cicer arietinum obtained by homologous protein alignment of AcABI5. AcABI5 is localized in the Group B branch of the phylogenetic tree and is closely clustered with homologous sequences from species such as Astragalus alpinus and C. arietinum , forming a legume-specific subclade with high support (Fig. 6 A). This branch forms a clear differentiation boundary with multiple G. max sequences in Groups C and D, indicating obvious species differentiation of AcABI5 within legumes. According to the protein domain analysis, the AcABI5 protein possesses a typical bZIP TF domain located in the central region of the protein, consisting of a basic region with the conserved motif N-X7-R/K and a leucine zipper region with the conserved motif L-X6-L (Figs. 6 B and S1 ). This domain serves as the core functional region determining DNA binding and dimerization.A small number of COG2433 and Men1-related motifs are embedded at both ends, which are highly consistent with other legume homologous proteins. The presence of the bZIP domain indicates that this protein may bind to cis-acting elements to regulate the transcription of downstream genes and play a key role in plant stress responses (such as drought and salinity). Compared with sequences in Group D that contain additional extended domains such as cc_RasGRP1_C or bZIP_plant_BZIP46, AcABI5 maintains a relatively streamlined domain architecture, suggesting that its function is biased towards basic transcriptional regulation rather than multi-motif signal integration. The order of conserved motifs is basically consistent with that of homologous sequences, with no significant deletions or insertions, indicating that the DNA binding and dimerization mechanisms of this protein are highly conserved. In addition, PlantCARE prediction results showed that the promoter of AcABI5 contains the abscisic acid-responsive element (ABRE, i.e., the cis-acting element involved in abscisic acid responsiveness), MYB binding sites involved in drought inducibility, as well as hormone-responsive elements and transcription factor binding sites, among other functional elements (Fig. S2). These elements suggest that the expression of AcABI5 may be regulated by abiotic stresses such as salt stress, thereby providing a basis for its involvement in the transcriptional regulation of salt stress responses. Secondary structure prediction showed that AcABI5 is mainly composed of random coils (60.45%), with α-helix accounting for 37.88%, β-sheet and β-turn accounting for only 1.11% and 0.56%, respectively, which is consistent with the typical long helix-coil-helix structure of bZIP TFs (Fig. S3B). The linear structure diagram reveals alternating helix/random coil segments at the N-terminus and C-terminus, with a continuous α-helix in the middle, which is a potential DNA binding and dimerization region (Fig. S3B). The tertiary structure model further shows an extended helix spanning the main body of the protein, accompanied by several flexible loops and secondary helix bundles, indicating that the protein may form a stable dimer through helix bundles during DNA binding (Fig. S3C). The synchronous fluctuation of helix and coil probabilities in the curve prediction diagram also supports its ability to undergo regulated conformational changes. ProtParam analysis showed (Table S4) that AcABI5 is a basic protein consisting of 359 amino acids, with a molecular weight of approximately 40.21 kDa and a theoretical isoelectric point of 9.51. An instability index of 56.46 indicates certain instability in vitro, and an aliphatic index of 66.57 indicates moderate thermal stability at higher temperatures. A grand average of hydropathicity (GRAVY) of − 0.845 indicates that the protein has significant hydrophilicity and is more likely to be located in the nucleus or cytoplasm rather than membranous compartments. SignalP 6.0 predicted no signal peptide in the full-length sequence and classified it as "OTHER", supporting the speculation that it is a nuclear-localized TF, which is consistent with the predictions of Uniprot and WoLF PSORT (Fig. S3A). Results of bioinformatics analysis indicated that AcABI5 is a member of the bZIP superfamily with typical bZIP transcription factor characteristics: hydrophilic, no signal peptide, and mainly composed of helix structures. It maintains a close evolutionary relationship with multiple forage and legume homologous proteins in legumes and has a highly consistent domain composition with multi-copy sequences. These results suggest that ABI5 in A. camelorum may undertake a conserved transcriptional regulatory function and be closely related to the regulatory network of legumes adapting to drought, salinity, and other stresses. Subcellular localization PCR amplified the AcABI5 full-length CDS (1138 bp) from A. camelorum cDNA; electrophoresis and sequencing confirmed its validity (Fig. S4A). Purified products underwent BP recombination with pDONR-Zeo and E. coli transformation, and bacterial PCR/sequencing verified entry vector construction for plasmid extraction (Fig. S4C). Subsequent LR recombination between AcABI5 -pDONR and pK7WGF2, plus E. coli transformation, was validated by colony PCR/sequencing, with positive plasmids extracted and preserved (Fig. S4B).The constructed vector was transiently expressed in the leaves of N. benthamiana . As shown in Fig. 7 , green fluorescence of 35S::GFP was detected throughout the cells, while green fluorescence was only detected in the nucleus of cells transfected with the fusion vector 35S::GFP-AcABI5. Moreover, the green fluorescence overlapped with the red fluorescence of 35S::mCherry, indicating that the AcABI5 protein is localized in the nucleus and is a typical transcription factor. Discussion 2,4-D and 6-BA are two commonly used plant growth regulators. 2,4-D is an auxin-like regulator that promotes callus growth at low concentrations and inhibits differentiation at high concentrations. 6-BA is a cytokinin-like regulator that can induce callus differentiation into buds. This experiment comprehensively analyzed the effects of different concentrations of the two hormones on A. camelorum callus formation through a full-factorial experiment with concentrations ranging from 0 to 2 mg/L. The results showed that the combination of 1.5 mg/L 2,4-D and 0.5 mg/L 6-BA was the optimal hormone ratio for A. camelorum callus proliferation. Bu et al. ( 2000 ) reported that MS medium containing 1.5–2.0 mg/L 2,4-D and 0.5–1.0 mg/L 6-BA was the optimal medium for inducing callus formation and subculture of A. camelorum hypocotyl segments. When 2,4-D and 6-BA reach a specific balance, calli of Alhagi graecorum form somatic embryos through somatic embryogenesis, which further develop into regenerated plants. When cytokinin is dominant (e.g., high concentration of 6-BA), explants dedifferentiate to form calli and then differentiate into adventitious buds through organogenesis (Hassanein 2004 ). These results are consistent with those of this experiment. In evaluating the callus induction effect of A. camelorum , compared with the traditional qualitative judgment of callus induction efficiency using callus induction rate, callus status, and growth rate (Bu et al. 2000 ), this experiment also quantitatively compared the induction effects of different groups by determining the relative growth rate of calli, providing a new reference index. Changes in fresh weight, phenotype, antioxidant enzyme activity, ROS accumulation, MDA accumulation, and cell mortality of calli are comprehensive and intuitive manifestations of callus response to salt stress. This study investigated the responses of the above physiological and biochemical indexes of A. camelorum calli at different time points under high salt stress. Song ( 2019 ) reported that salt stress treatment of Zoysia matrella calli showed that callus growth was inhibited when NaCl concentration was ≥ 0.6% (approximately 100 mM). Jing et al. Jing et al. ( 2025 ) pointed out that under 150 mM NaCl treatment, the growth of wild-type apple calli was significantly inhibited and the fresh weight was significantly reduced compared with MdSPL13B-OE apple calli. The results of this experiment showed that under 200 mM NaCl stress, the fresh weight of A. camelorum calli decreased by 15.8% after 14 days and showed slight browning, which is consistent with previous research results. Previous studies believed that ROS are harmful to plant growth and development, so plants have evolved various strategies to alleviate oxidative stress (Ding et al. 2015 ), such as the antioxidant enzyme system that can scavenge or reduce ROS produced by abiotic stress and reduce cell damage. However, with in-depth research on ROS, it has been found that ROS actually play a complex dual role. Plants can use potentially toxic ROS molecules as beneficial signals for development to precisely and temporally inhibit premature maturation of the shoot apical meristem and ensure orderly flowering (Huang et al. 2021 ). ROS signals produced in callus cells also play an important regulatory role in the embryogenic capacity of calli (Zhou et al. 2016 ). Histochemical staining showed that in the absence of stress, ROS produced by A. camelorum calli mainly accumulated on the side in contact with the medium, with a small amount on the upper surface. This may be because changes in cell volume and number of calli are easily restricted by the cell wall, and the production of ROS signals can break down the cell wall, promote cell wall loosening, and thereby regulate cell growth (Zhang et al. 2024 ). However, under long-term high-concentration salt stress, calli are in a state of oxidative stress for a long time, the scavenging efficiency of the antioxidant enzyme system decreases, and high ROS levels cause damage to calli, which is not conducive to embryonic development (Ren et al. 2022 ), leading to browning and death. Under NaCl stress, the activities of SOD, POD, CAT, as well as the content of MDA, all increased significantly. In the early stage of stress, the antioxidant enzyme system of A. camelorum calli was activated immediately. Compared with POD and CAT, SOD was activated more rapidly and catalyzed the dismutation of O 2 − ∙ into O₂ and H₂O₂ through redox reactions. CAT has high specificity and fast turnover rate for H₂O₂ (Zhang et al. 2016 ), while POD uses H₂O₂ as an oxidant to catalyze various substrates such as phenols and lipid peroxides, and the two synergistically scavenge the produced H₂O₂. In the late stage of stress, MDA content continued to increase, SOD activity decreased to the initial stress level, CAT activity decreased significantly, while POD always maintained high activity. This may be because long-term stress provided more substrates such as phenols for POD, so POD played a major role in the late stage of stress and undertook important antioxidant or secondary metabolic regulatory functions. The decrease in SOD and CAT activities may be related to the consumption of the enzyme system caused by continuous stress. H₂O₂, as a signaling molecule, can induce CAT to initiate defense responses, while excessive ROS can cause oxidative damage to calli, resulting in fluctuating CAT activity. Plants can maintain the active function of SOD and POD for a longer period than CAT when facing stress, and SOD and POD show better synergy (Zhang et al. 2006 ). Long-term stress leads to the decline of antioxidant system function, and the activities of SOD and POD show a trend of first increasing and then decreasing (Zhang et al. 2025 ), which is similar to the results of this experiment. In conclusion, the three key protective enzymes regulated by the antioxidant enzyme system of A. camelorum calli maintain the normal level of ROS in the body through coordinated interaction, reducing ROS damage to calli. Based on the research results of (Tang et al. 2024 ), we screened Asp07G015930, which showed the highest upregulation of gene expression in A. camelorum calli under salt stress, from 10 candidate genes. The CDS length of this gene is 1080 bp, encoding 359 amino acids. Bioinformatics analysis showed that the protein encoded by this gene is hydrophilic, has no signal peptide, is mainly composed of helix structures, has high homology with ABI5 proteins in various plants, and has conserved functional domains typical of the bZIP family. Abscisic acid (ABA) is involved in regulating signal transduction in various defense processes to resist abiotic and biotic stresses and promote plant development (Sah et al. 2016 ). The bZIP family is divided into 13 major groups, among which Group A mainly consists of ABA-responsive element-binding protein (AREB), ABA-responsive element-binding factor (ABF), and ABI5 subfamily members (Collin et al. 2021 ). As a core protein in the ABA signal transduction pathway, ABI5 plays an important regulatory role in seed germination, early seedling development, and stress adaptation. Research findings indicate that numerous ABA-insensitive ( ABI ) genes exist in Arabidopsis thaliana , including ABI1 , ABI2 , ABI3 , ABI4 , and ABI5 . Among them, ABI1 and ABI2 are two highly homologous protein kinases that act as negative regulators in the ABA signaling pathway (Merlot et al. 2001 ); ABI3 belongs to the B3 protein family (Xu et al. 2022 ) and plays a key role in regulating plant flowering; ABI4 belongs to the CBF/DREB subfamily, is a member of the AP2/ERF family, contains an AP2 domain, and is involved in ABA signal transduction (Yang 2025 ); ABI5 belongs to the bZIP transcription factor and is a core transcription factor in the ABA signal transduction pathway (Du et al. 2025 ; Du et al. 2024 ). Based on its function and sequence characteristics, the gene was named AcABI5 . Zinsmeister et al. confirmed the role of ABI5 in regulating ROS accumulation and seed maturation in Pisum sativum, a leguminous plant ; Finkelstein et al. found that ABI5 regulates the expression of downstream genes such as late embryogenesis abundant (LEA) proteins, and is involved in processes including seed germination and dehydration tolerance (Zinsmeister et al. 2016 ); Albertos et al. revealed that nitric oxide can counteract ABA during plant development and regulate the molecular interaction mechanism of ABI5 through post-translational modification (Albertos et al. 2015 ); Basso et al. ( 2025 ) comprehensively analyzed the AREB/ABF/ABI5 genes belonging to the subfamily A of plant bZIP TFs in C. arietinum and Lens culinaris , which are up-regulated under different abiotic and biotic stress conditions, exhibit dynamic expression in different tissues, and show higher expression levels in drought-tolerant varieties. We speculate that AcABI5 has similar functions to these homologous proteins, possibly binding to cis-acting elements to regulate the transcriptional expression of downstream genes, and playing a key role in plant responses to abiotic stress and seed germination. Most studies have shown that typical TFs are usually localized in the nucleus of plant cells (Wang et al. 2021 ). By constructing the pK7WGF2-35S::GFP-AcABI5 fusion expression vector and using Agrobacterium to infect N. benthamiana leaves for transient expression, the results showed that the fusion protein emitted fluorescence only in the nucleus, indicating that AcABI5, like most TFs, is a nuclear-localized protein. Through salt stress treatment of A. camelorum calli, we found that AcABI5 can respond rapidly to salt stress and maintain high expression during the stress process; in the absence of abiotic stress, ABI5 undergoes dephosphorylation, and the dephosphorylated ABI5 is neither active nor stable and will be degraded through the 26S proteasome pathway (Lopez-Molina et al. 2001 ), which is consistent with the low expression level of AcABI5 observed in our control group. In future research, we will further establish an AcABI5 overexpression system in A. camelorum calli, identify the cis-acting elements bound by AcABI5, analyze the downstream target genes regulated by AcABI5, and investigate whether AcABI5 regulates the expression of salt resistance-related genes by binding to these cis-acting elements. Conclusions This study determined that the optimal proliferation medium for A. camelorum calli is 1.5 mg/L 2,4-D combined with 0.5 mg/L 6-BA. In response to salt stress, SOD in A. camelorum calli is activated first to scavenge intracellular O 2 − ∙; POD and CAT synergistically scavenge ROS in the early stage of stress, and POD also exhibits high activity in the late stage of stress. A salt-tolerant gene AcABI5 of A. camelorum was screened out by qRT-PCR, with a full-length CDS sequence of 1080 bp encoding 359 amino acids. Bioinformatics analysis revealed that AcABI5 from A. camelorum is a nuclear-localized bZIP family TF with typical conserved domains; its promoter region contains stress-responsive cis-acting elements. This TF harbors conserved transcriptional regulatory functions, and the gene is inducible in A. camelorum calli under salt stress, suggesting that AcABI5 may be involved in the regulatory network of A. camelorum adapting to abiotic stresses including drought and salt stress. This study provides a research basis for further investigating the molecular mechanism of AcABI5 regulating A. camelorum 's response to salt stress, and offers theoretical support for the establishment of a genetic transformation system for A. camelorum . Declarations Data Availability Data of this study will be made available on reasonable request. Funding This research was funded by the Key Laboratory of Ecological Safety and Sustainable Development in Arid Lands Young Scientists Interdisciplinary Team Project (grant No.E552020301), the National Key Research and Development Program of China (grant No.2022YFF1302505-03), and the National Young Talent Program (grant No.2022000007). Competing Interests The authors have no competing interests to declare that are relevant to the content of this article. Ethics Approval Not applicable. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by, Xiangyi Li, Pingyin Guan, Bo Zhang and Gangliang Tang. The first draft of the manuscript was written by Zhengtao Yan and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Albertos P, Romero-Puertas MC, Tatematsu K, Mateos I, Sánchez-Vicente I, Nambara E, Lorenzo O (2015) S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth. Nat Commun 6:8669. https://doi.org/10.1038/ncomms9669 Basso MF, Iovieno P, Capuana M, Contaldi F, Ieri F, Menicucci F, Celso FL, Barone G, Martinelli F (2025) Identification and expression of the AREB/ABF/ABI5 subfamily genes in chickpea and lentil reveal major players involved in ABA-mediated defense response to drought stress. 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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-8449616","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":578645295,"identity":"cd515a3a-e981-4454-8bfa-5d67ae9b6e06","order_by":0,"name":"Zhengtao Yan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACNvaGhAMJBv/k+JmZDxCnhY/nwMMHHyoOGEu2syUQp0VOIvGx4YwzBxINzvMYEOkwieQ0ad62OwkMh3k+3njDYCen20BIC88zkJZneYzNvJst5zAkG5sdIKSFPQekhbmYmZl3mzQPw4HEbQS1MOR/A2lJbGPmeUakFo6EZKD3Dyf2MPOwEamF50AiMJDTjCWY2Ywt5xgQ4Rf5dnBU2sjZnz/88MabCjs5glpQgASxUYOshVQdo2AUjIJRMCIAAD4MQgNS6OBhAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0009-0003-1090-2219","institution":"Xinjiang Institute of Ecology and Geography","correspondingAuthor":true,"prefix":"","firstName":"Zhengtao","middleName":"","lastName":"Yan","suffix":""},{"id":578645299,"identity":"6c9cfbdd-e833-4f65-99d5-3084f088daf1","order_by":1,"name":"Xiangyi Li","email":"","orcid":"","institution":"Xinjiang Institute of Ecology and Geography","correspondingAuthor":false,"prefix":"","firstName":"Xiangyi","middleName":"","lastName":"Li","suffix":""},{"id":578645302,"identity":"15825e37-5113-49a6-933c-e02aabf2ecfd","order_by":2,"name":"Bo Zhang","email":"","orcid":"","institution":"Xinjiang Institute of Ecology and Geography","correspondingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"Zhang","suffix":""},{"id":578645308,"identity":"4a9fcaf4-5584-44e6-b222-df0597e1d092","order_by":3,"name":"Gangliang Tang","email":"","orcid":"https://orcid.org/0009-0002-1322-7047","institution":"Xinjiang Institute of Ecology and Geography","correspondingAuthor":false,"prefix":"","firstName":"Gangliang","middleName":"","lastName":"Tang","suffix":""}],"badges":[],"createdAt":"2025-12-25 14:24:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8449616/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8449616/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101249479,"identity":"f9bf15dc-6fd6-46ce-b018-ba4e3ea2cd70","added_by":"auto","created_at":"2026-01-27 17:20:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":759909,"visible":true,"origin":"","legend":"\u003cp\u003e(A) \u003cem\u003eA. camelorum\u003c/em\u003ethrives in arid and saline-alkali habitat. (B) The fruit of \u003cem\u003eA. camelorum.\u003c/em\u003e(C) Leaves and thorns of \u003cem\u003eA. camelorum\u003c/em\u003e. The images were taken in the southern margin of the Taklamakan Desert\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/45cdab9d72af1512918642ae.png"},{"id":101249481,"identity":"fceabdff-d4bc-46cd-aa66-830b6da83780","added_by":"auto","created_at":"2026-01-27 17:20:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":234407,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of different hormone concentrations on callus induction.The abscissa represents the concentration of 2,4-D (0–2 mg/L), the ordinate represents the concentration of 6-BA (0–2 mg/L), and each grid corresponds to one type of hormone combination. Bar = 1 cm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/d6915cf1aea3ef2696541cc6.png"},{"id":101249482,"identity":"6a0bfab4-23e3-4eca-81e5-6ff45f11845b","added_by":"auto","created_at":"2026-01-27 17:20:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":456943,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Phenotypic changes (Bar = 1 cm) and (B) fresh weight changes of calli under salt stress. (C) DAB, NBT and Evans Blue Staining of \u003cem\u003eA. camelorum\u003c/em\u003e calli (Bar = 2 mm). Different lowercase letters indicate significant differences according to the Duncan’s test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Data are presented as the mean ± SD (\u003cem\u003en\u003c/em\u003e = 3 independent biological replicates)\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/ad92e24de8adef35ac007c32.png"},{"id":101249483,"identity":"21e0c6e3-368f-4bf7-9d75-738532d4f2cc","added_by":"auto","created_at":"2026-01-27 17:20:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":70230,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of (A) SOD,(B) POD and (C) CAT \u0026nbsp;activities as well as (D) MDA content. Different lowercase letters indicate significant differences according to the Duncan’s test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Data are presented as the mean ± SD (\u003cem\u003en\u003c/em\u003e = 3 independent biological replicates)\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/1222a27269654eaf0de337e3.png"},{"id":101297693,"identity":"ee18e90f-75a2-4f2b-a983-1844e6c24d25","added_by":"auto","created_at":"2026-01-28 09:28:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":85986,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Relative expression levels of different genes in callus. Each gene was individually analyzed using Student's t-test, asterisks indicate significant differences compared to the control group (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) (B) Relative expression level of \u003cem\u003eAcABI5\u003c/em\u003e in callus at different time points. Different lowercase letters indicate significant differences according to the Duncan’s test (\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05). Data are presented as the mean ± SD (\u003cem\u003en\u003c/em\u003e = 3 independent biological replicates)\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/36333ca6220d96b76b853b6f.png"},{"id":101249477,"identity":"8bd6025e-699c-4f1e-8628-09de7ba43262","added_by":"auto","created_at":"2026-01-27 17:20:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":204223,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Phylogenetic tree constructed based on ABI5 homologous proteins identified from \u003cem\u003eA. camelorum\u003c/em\u003e, \u003cem\u003eM. truncatula\u003c/em\u003e , \u003cem\u003eC. arietinum\u003c/em\u003e and other plants. (B) Conserved domain and motif analysis was performed on ABI5 homologous proteins identified from \u003cem\u003eA. camelorum\u003c/em\u003e,\u003cem\u003eM. truncatula\u003c/em\u003e,\u003cem\u003e C. arietinum\u003c/em\u003e and other plants\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/9ddb0cbacc526bf06ffede2c.png"},{"id":101297448,"identity":"3863eee6-0982-4cff-a3f1-8fdd4052c0c3","added_by":"auto","created_at":"2026-01-28 09:27:15","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":335837,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular localization of AcABI5 in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. The signals from GFP fluorescence, mCherry, merged, and bright field images are shown.EGFP: excitation at 488 nm, emission at 507 nm; mCherry: excitation at 587 nm, emission at 610 nm (Bar = 50 μm)\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/7a1ee2415ccce125bd46c2df.png"},{"id":104399329,"identity":"8c7e268f-1598-4af1-9463-ab1537a8fcac","added_by":"auto","created_at":"2026-03-11 12:05:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3140506,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8449616/v1/2cf8d0f5-f3e2-4de6-a207-40892dfaf4f5.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDe novo characterization of AcABI5 transcription factor and physiological responses to salt stress in Alhagi camelorum callus\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil salinization, exacerbated by climate extremes and land misuse, imposes ionic toxicity and osmotic stress that globally curtail root elongation, biomass allocation, and crop yield potential (van Zelm et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Shrinking freshwater reserves now make the reclamation of saline\u0026ndash;alkali land a critical imperative for global food security. \u003cem\u003eAlhagi camelorum\u003c/em\u003e Fisch. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), a deep-rooted perennial legume of Central Asian deserts, forms 40\u0026ndash;130 cm thickets of thorny, grey-green shoots that carry obovate leaves and short axillary racemes whose red-purple petals give way to spiralled loment pods (Dong and Zhang \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Muhammad et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Ullah et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eA. camelorum\u003c/em\u003e spans the arid corridor from the Taklimakan to the Iranian Plateau, occurring in northwestern China, Kazakhstan, Afghanistan, Iran, Pakistan, Iraq, Mongolia and India; across these regions it routinely colonizes saline soils with \u0026gt;\u0026thinsp;200 mM NaCl, making it one of the most salt-tolerant wild legumes (Manafu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Pirasteh-Anosheh et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Srivastava et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the southern rim of the Taklamakan, \u003cem\u003eA. camelorum\u003c/em\u003e carpets the shifting ecotone between oasis cropland and drifting dunes, forming the keystone thicket that stabilizes soils and defines the desert margin (Tang et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). A single stand delivers a triple dividend: its thorny lattice brakes the wind and anchors shifting sand, its shoots furnish protein-rich fodder equal to alfalfa, and its resin-rich exudates remain a staple of traditional pharmacopoeias (Kazemi and Ghasemi Bezdi \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Muhammad et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zeng et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), and is also an excellent candidate plant for saline-alkali land remediation (Zheng et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Nevertheless, the molecular determinants enabling \u003cem\u003eA. camelorum\u003c/em\u003e to maintain growth under saline conditions remain largely uncharacterized. Systematic discovery and validation of the underlying salt tolerance genes will provide strategic targets for molecular breeding programs aimed at enhancing crop resilience to soil salinity.\u003c/p\u003e \u003cp\u003eThe basic leucine zipper transcription factors (bZIP, TFs) form a highly conserved and functionally diverse transcriptional regulatory family in plants. They are characterized by a conserved bZIP core domain that encompasses a DNA-binding region and a leucine zipper dimerization domain.\u003c/p\u003e \u003cp\u003eThrough post-translational modifications, these factors participate in the assembly of multiprotein complexes to regulate downstream gene networks. This family plays a central role in the growth, development, and stress adaptation of leguminous plants (Yue et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In stress responses, bZIP TFs exhibit distinct functional specificity and diversity. \u003cem\u003eGmbZIP60\u003c/em\u003e in soybean (\u003cem\u003eGlycine max\u003c/em\u003e) can be induced by salt and drought stresses, overexpression of \u003cem\u003eGmbZIP60\u003c/em\u003e enhances soybean tolerance to salt and drought by directly binding to the promoters of stress-responsive genes (Chai et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). \u003cem\u003eGmbZIP19\u003c/em\u003e is a positive regulator of pathogen resistance and a negative regulator to modulate stomatal closure, thereby reducing plant tolerance to salt and drought stresses (He et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The nuclear protein GmbZIP110 significantly improves the salt stress adaptability of soybean by binding to the ACGT motif to regulate proline accumulation and Na⁺/K⁺ homeostasis (Xu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As a dominant sand-fixing plant in arid and semi-arid saline-alkali regions, \u003cem\u003eA. camelorum\u003c/em\u003e exerts a central role in improving regional soil structure and maintaining ecosystem stability by virtue of its excellent salt and drought tolerance. Therefore, as an important class of transcription factors, it is necessary to investigate the mechanism by which bZIPs regulate salt tolerance-related genes in \u003cem\u003eA. camelorum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eIn this work, aseptic seedlings of \u003cem\u003eA. camelorum\u003c/em\u003e served as the experimental material, and young stem explants were subjected to a hormone matrix screen that yielded a single optimized medium supporting rapid, highly synchronous callus proliferation. Exposure of these calli to MS medium supplemented with 200 mM NaCl revealed a temporally orchestrated antioxidant response: early superoxide dismutase (SOD) peak, sustained peroxidase (POD) activity, late catalase (CAT) stabilization and corresponding ion-leakage dynamics, validating the in vitro system as a physiologically relevant salt-stress proxy. Transcriptomic profiling of challenged seedling identified a bZIP TF (Tang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), ABI5-homologous, as the most strongly up-regulated regulatory gene; its full-length CDS (1080 bp; 359 amino acids) was cloned and designated \u003cem\u003eAcABI5\u003c/em\u003e. In-silico analysis predicted a canonical basic leucine-zipper domain and a bipartite nuclear-localization signal (NLS); transient expression of a 35S::GFP-AcABI5 fusion in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e confirmed exclusive nuclear accumulation. Quantitative RT-PCR (qRT-PCR) time courses demonstrated a 17-fold induction within 12 h of salt treatment, followed by rapid decay upon stress relief, consistent with a primary transcriptional regulator. These results provide the first functional annotation of a salt-responsive transcription factor in \u003cem\u003eA. camelorum\u003c/em\u003e, establish a reproducible callus-based transformation platform, and identify \u003cem\u003eAcABI5\u003c/em\u003e as a candidate gene for molecular breeding of salt-tolerant crops.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eMaterial treatment and callus induction\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eA. camelorum\u003c/em\u003e seeds used in this study were collected in July 2023 from the Cele Desert Grassland Ecosystem National Field Scientific Observation and Research Station, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences. Mature and plump seeds were vernalized for 48 h, soaked in 75% ethanol for 30 s, and rinsed 3 times with sterile distilled water, followed by immersion in 5% NaOCl for 5 min and washed 5 times with sterile distilled water. The seeds were inoculated on MS medium (Murashige and Skoog \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) supplemented with 3% (w/v) sucrose and 0.3% (w/v) phytagel (pH\u0026thinsp;=\u0026thinsp;5.8-6.0), and grown under conditions of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 3000 lx, 16 h light and 8 h dark.\u003c/p\u003e \u003cp\u003eSelect aseptic seedlings that are in good growth condition and have a seedling age of approximately 28 days for the callus culture of \u003cem\u003eA. camelorum\u003c/em\u003e.Young stem segments of approximately 0.5 cm were cut as explants and transferred to culture media containing different concentration combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (6-BA) for callus induction. The culture conditions were the same as those described previously. To evaluate the growth dynamics of calli, the callus induction rate and relative growth of \u003cem\u003eA. camelorum\u003c/em\u003e were recorded after 28 days of culture, and the fresh weight of calli was measured using the difference method.The callus induction rate (%), callus growth (g) and relative growth rate (%) were calculated as below:\u003c/p\u003e \u003cp\u003eCallus induction rate (%) = (Number of explants producing calli / Total number of inoculated explants) \u0026times; 100%\u003c/p\u003e \u003cp\u003eCallus growth (g)\u0026thinsp;=\u0026thinsp;Weight of calli after culture\u0026thinsp;\u0026minus;\u0026thinsp;Weight of calli before culture\u003c/p\u003e \u003cp\u003eRelative growth rate (%) = (Callus growth / Initial fresh weight of calli) \u0026times; 100%\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eDetermination methods of growth indicators physiological and biochemical indexes\u003c/h2\u003e \u003cp\u003eTo analyze the physiological status of calli under long-term salt stress, the salt stress treatment group was cultured on callus induction medium (MS medium supplemented with 1.5 mg/L 2,4-D and 0.5 mg/L 6-BA) supplemented with an additional 200 mM NaCl, whereas the control group was maintained on basic callus induction medium without NaCl supplementation.The calli with robust growth and uniform size were transferred to the respective media, with 12 calli per medium and 3 replicates set for each treatment. The culture conditions were as follows: temperature of 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 3000 lx, 16 h light and 8 h dark. The fresh weight of calli in both the control and treatment groups was measured, histochemical staining was performed, and the phenotypes of calli were recorded by photography on day 0, day 7, and day 14.\u003c/p\u003e \u003cp\u003eDAB staining and NBT staining: \u003cem\u003eA. camelorum\u003c/em\u003e calli with uniform size after stress treatment were placed in 50 mL centrifuge tubes. Prepared DAB staining solution and NBT staining solution were added respectively to submerge the calli. Vacuum was slowly applied to 0.8 MPa and maintained for 5 min, then slowly restored to normal pressure. Staining was performed at room temperature in the dark for 12 h. After discarding the staining solution, the calli were rinsed repeatedly with sterile distilled water to remove residual staining solution. Decolorizing solution was added, and the samples were incubated in a 95\u0026deg;C water bath for 10\u0026ndash;30 min until chlorophyll was completely removed.\u003c/p\u003e \u003cp\u003eEvans blue staining: \u003cem\u003eA. camelorum\u003c/em\u003e calli with uniform size after stress treatment were placed in 50 mL centrifuge tubes. Prepared Evans blue staining solution was added to submerge the calli. Vacuum was slowly applied to 0.8 MPa and maintained for 5 min, then slowly restored to normal pressure. Staining was performed at room temperature in the dark for 6 h. After discarding the staining solution, the calli were rinsed repeatedly with sterile distilled water to remove residual staining solution. Decolorizing solution was added, and the samples were incubated in a boiling water bath for 10\u0026ndash;30 min until chlorophyll was completely removed.\u003c/p\u003e \u003cp\u003eDAB solution (1 mg/mL): Weigh out 100 mg of DAB powder and dissolve it in 100 ml of 0.1 mol/L phosphate buffer solution (pH\u0026thinsp;=\u0026thinsp;7.4) to prepare a 1 mg/mL aqueous solution. Adjust the pH to 3.8 with hydrochloric acid, and store the solution at 4\u0026deg;C in the dark.\u003c/p\u003e \u003cp\u003eNBT solution (0.5 mg/mL): Weigh out 50 mg of NBT powder and dissolve it in 100 mL of 0.1 mol/L phosphate buffer solution (pH\u0026thinsp;=\u0026thinsp;7.4). Store the solution at 4\u0026deg;C in the dark.\u003c/p\u003e \u003cp\u003eEvans blue solution (0.5 mg/mL): Weigh out 50 mg of Evans blue powder and dissolve it in 100 mL of double-distilled water. After dissolving thoroughly, store the solution at room temperature in the dark.\u003c/p\u003e \u003cp\u003eDecolorizing solution: 95% ethanol, acetic acid, and glycerol were mixed at a volume ratio of 3:1:1 and stored at room temperature in the dark.\u003c/p\u003e \u003cp\u003eTo determine the physiological and biochemical indices of \u003cem\u003eA. camelorum\u003c/em\u003e calli under salt stress, the calli with robust growth and uniform size cultured on the same medium were selected and separately transferred to the media of the salt stress treatment group and the control group. After treatment for 0 h, 6 h, 12 h and 48 h, the calli were snap-frozen in liquid nitrogen and subsequently ground into powder. Crude enzyme extract was prepared to determine the malondialdehyde (MDA) content and antioxidant enzyme activities. Samples were also stored at -80\u0026deg;C for subsequent RNA extraction.\u003c/p\u003e \u003cp\u003eDetermination of MDA content and antioxidant enzyme activities: 0.1 g samples were taken from both the control and salt stress treatment groups, with 3 replicates per treatment. Kits purchased from Shanghai Fenxi Biotechnology Co., Ltd. were used to determine the contents of MDA, SOD, POD, and CAT. Detailed instructions are available at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.shfxsw.cn\u003c/span\u003e\u003cspan address=\"http://www.shfxsw.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003ePOD activity unit: One unit (U) of POD activity was defined as a 0.005 change in \u003cem\u003eA\u003c/em\u003e\u003csub\u003e470\u003c/sub\u003e per minute per gram of tissue in each milliliter of reaction system.\u003c/p\u003e \u003cp\u003eSOD activity unit: In the xanthine oxidase-coupled reaction system of the kit, one unit (U) of SOD activity was defined as the amount of enzyme required to achieve a 50% inhibition rate.\u003c/p\u003e \u003cp\u003eCAT activity unit: One unit (U) of CAT activity was defined as the amount of enzyme that catalyzes the degradation of 1 \u0026micro;mol H₂O₂ per minute per gram of tissue.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eTotal RNA extraction and first-strand cDNA synthesis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from the samples treated in Section 1.5 according to the instructions of the TIANGEN Total RNA Extraction Kit. The concentration and purity of RNA were determined using a Nanodrop, and the integrity of RNA was detected by agarose gel electrophoresis.RNA was reverse-transcribed into cDNA as a template using the TIANGEN FastKing One-Step Genomic DNA Removal and cDNA Synthesis SuperMix according to the instructions.\u003c/p\u003e\n\u003ch3\u003eScreening of candidate genes and analysis of gene expression levels\u003c/h3\u003e\n\u003cp\u003eBased on the research findings of (Tang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we identified 10 candidate genes highly responsive to osmotic stress, namely Asp01G030840, Asp01G035580, Asp02G012850, Asp02G017010, Asp02G034200, Asp04G012530, Asp04G017080, Asp07G002390, Asp07G015930, and Asp08G003230. Using \u003cem\u003eA. camelorum EF-1α\u003c/em\u003e (Asp06G015730) as the internal reference gene, specific primers were designed using Primer Premier 6.0 (Table S2). cDNA from the control and treatment groups at 6 h as mentioned above was used as the template. qRT-PCR was performed according to the instructions of Vazyme SYBR qRT-PCR Master Mix. The relative expression levels of each gene were calculated using the 2\u003csup\u003e\u0026minus;ΔΔ\u003cem\u003eCt\u003c/em\u003e\u003c/sup\u003e method to screen the gene with the highest expression level under salt stress for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe gene with the highest expression level was selected for qRT-PCR using cDNA from the control and treatment groups at 0 h, 6 h, 18 h, and 36 h as mentioned above as templates. \u003cem\u003eA. camelorum EF-1α\u003c/em\u003e and \u003cem\u003eACT\u003c/em\u003e (Asp07G013640) were used as internal reference genes. qRT‒PCR was performed using Applied Biosystems QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific, USA), the 2\u003csup\u003e\u0026minus;ΔΔ\u003cem\u003eCt\u003c/em\u003e\u003c/sup\u003e method was used to calculate the expression levels of this gene in \u003cem\u003eA. camelorum\u003c/em\u003e calli at different stress durations (Livak and Schmittgen \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBioinformatics analysis\u003c/h3\u003e\n\u003cp\u003eThe Expasy-ProtParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to analyze the physicochemical properties of the predicted Asp07G015930 protein, including molecular weight, theoretical isoelectric point, instability index, aliphatic index, and grand average of hydropathicity. SignalP 6.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://services.healthtech.dtu.dk/services/SignalP-6.0/\u003c/span\u003e\u003cspan address=\"https://services.healthtech.dtu.dk/services/SignalP-6.0/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the signal peptide of the full-length sequence to determine its subcellular localization characteristics.\u003c/p\u003e \u003cp\u003eHomologous sequences of Asp07G015930 were obtained using NCBI Blast (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and Uniprot (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and 38 homologous sequences were screened for subsequent analysis. A phylogenetic tree was constructed using MEGA software, with the Neighbor-Joining method for tree reconstruction, and 1000 bootstrap replicates to evaluate branch support. After construction, the generated phylogenetic tree was optimized and visualized using the online tool ITOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itol.embl.de/\u003c/span\u003e\u003cspan address=\"http://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe protein sequences was submitted to the NCBI-CDD database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to predict conserved domains and identify the bZIP superfamily domain and other related functional domains. The online tool MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used for conserved motif analysis, with 10 motifs set and a motif length range of 6\u0026ndash;50 amino acids. Cis-acting regulatory elements of the promoter were predicted using PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/).TBtool\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/).TBtool\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003es software was used to visualize conserved motifs and generate domain and motif band diagrams(Chen et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe online tool SOPMA program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html\u003c/span\u003e\u003cspan address=\"https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_server.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the secondary structure of the protein. The online tool SWISS MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the tertiary structure of the protein, generate a 3D structure model based on homology modeling, and evaluate the structure quality. Uniprot and WoLF PSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp/\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used for subcellular localization prediction.\u003c/p\u003e\n\u003ch3\u003eSubcellular localization\u003c/h3\u003e\n\u003cp\u003eUsing SnapGene 6.0.2, specific primers for the \u003cem\u003eAcABI5\u003c/em\u003e gene were designed based on its CDS sequence, incorporating attB-specific recombination sites of the Invitrogen Gateway\u0026reg; donor vector pDONR-Zeo (Table S3). The gene was cloned using cDNA as mentioned above as the template according to the instructions of Vazyme 2\u0026times;Phanta Flash Master Mix. The amplified product was detected by 1% agarose gel electrophoresis, and the target fragment was purified by gel extraction and sent for sequencing.\u003c/p\u003e \u003cp\u003eUsing pDONR-Zeo as the donor vector, the entry vector was obtained through BP reaction according to the instructions of Invitrogen Gateway\u0026trade; BP Clonase\u0026trade; Ⅱ Enzyme Mix and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e DH5α competent cells. The cells were cultured on LB solid medium (containing 50 \u0026micro;g/mL Zeocin) at 37\u0026deg;C for 12 h. Single colonies were picked for bacterial liquid PCR identification and sent for sequencing. Plasmids were extracted from correctly sequenced clones for LR reaction.\u003c/p\u003e \u003cp\u003eUsing pK7WGF2, a recipient vector containing the enhanced green fluorescent protein (eGFP) reporter gene as the recipient vector, the expression vector was obtained through LR reaction according to the instructions of Invitrogen Gateway\u0026reg; LR Clonase\u0026trade; Ⅱ Enzyme Mix and transformed into \u003cem\u003eE. coli\u003c/em\u003e DH5α competent cells. The cells were cultured on LB solid medium (containing 50 \u0026micro;g/mL Spec) at 37\u0026deg;C for 12 h. Single colonies were picked for bacterial liquid PCR identification and sent for sequencing. Plasmids were extracted from correctly sequenced clones for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe constructed pK7WGF2-35S::GFP-AcABI5 vector plasmid was transformed into \u003cem\u003eAgrobacterium\u003c/em\u003e GV3101 and cultured on YEB solid medium (containing 50 \u0026micro;g/mL Spec and 25 \u0026micro;g/mL Rif) at 28\u0026deg;C for 24 h. Correctly sequenced single-colony \u003cem\u003eAgrobacterium\u003c/em\u003e was expanded and resuspended in an infection solution containing 100 \u0026micro;M Acetosyringone, then incubated statically for 3 h. The empty GFP vector was used as a control and mCherry (NLS) was used as a marker for co-injection into \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Observation was performed using a laser confocal microscope after 48 h. Fluorescence was detected with a Zeiss LSM 800 (Carl Zeiss AG, Germany) confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eIBM SPSS Statistics software (Version 27.0, IBM Corp., Armonk, NY, USA) was used for one-way ANOVA, Duncan\u0026rsquo;s multiple range tests, and Student's \u003cem\u003et\u003c/em\u003e-test. Error bars on the graphs indicate the standard deviation of the mean (\u0026plusmn;\u0026thinsp;SD), calculated from three independent biological replicates (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3) for each treatment. Data were analyzed, and means with identical letters are considered not significantly different at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Asterisks are used to indicate significant differences in Student's \u003cem\u003et\u003c/em\u003e-test (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). GraphPad Prism software was used for plotting graphs (Version 10.1.2, GraphPad Software Inc., San Diego, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of different hormone concentrations on callus induction of\u003c/b\u003e \u003cb\u003eA. camelorum\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA broad spectrum of auxin\u0026ndash;cytokinin regimes (Table S1) evoked callus formation from \u003cem\u003eA. camelorum\u003c/em\u003e stem explants, yet macroscopic texture, pigmentation and growth kinetics varied markedly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Sub-threshold concentrations (\u0026lt;\u0026thinsp;0.2 mg /L 2,4-D; \u0026lt; 0.5 mg /L 6-BA) failed to trigger dedifferentiation, whereas cytokinin dominance (6-BA\u0026gt;2,4-D; groups 4\u0026ndash;6, 11\u0026ndash;12) canalized tissues toward shoot organogenesis. A positive dose\u0026ndash;response relationship between 2,4-D and callus induction rate was evident up to 1.5 mg/L, peaking at a relative growth increment of 110.93\u0026thinsp;\u0026plusmn;\u0026thinsp;12.52% (group 27). Supra-optimal auxin levels reversed this trend, yielding friable, necrotic tissues. Within the window 1.0\u0026ndash;2.0 mg/L 2,4-D and 0.5\u0026ndash;1.0 mg/L 6-BA, auxin:cytokinin ratios between 3:1 and 2:1 consistently generated compact, cream-coloured, rapidly proliferating calli. Consequently, the formulation 1.5 mg/L 2,4-D plus 0.5 mg/L 6-BA (group 27) was adopted as the benchmark for all subsequent experimentation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of physiological and biochemical indexes of\u003c/b\u003e \u003cb\u003eA. camelorum\u003c/b\u003e \u003cb\u003ecalli under salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExposure to 200 mM NaCl for 14 days imposed a sustained growth arrest on \u003cem\u003eA. camelorum\u003c/em\u003e calli (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B). Control and salt-treated tissues exhibited equivalent fresh mass at t₀; thereafter, control calli expanded linearly, retaining a bright-green, nodular morphology, whereas treated calli remained quiescent through day 7 and developed a yellow-green hue. By day 14, mean fresh mass of the stressed population had declined by 15.8% relative to t₀, accompanied by marginal necrosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-B), indicating that chronic osmotic stress not only blocked biomass accretion but also drove gradual water loss and cellular decompartmentalization.\u003c/p\u003e \u003cp\u003ePlants rapidly accumulate large amounts of reactive oxygen species (ROS) under stress conditions. The main ROS include hydrogen peroxide (H₂O₂), hydroxyl radicals (∙OH), and superoxide anion radicals (O₂\u003csup\u003e⁻\u003c/sup\u003e∙) (Juan et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Excessive ROS act on unsaturated fatty acids in lipids to form peroxides (such as MDA), causing damage to membrane structure and function, oxidizing DNA bases to induce gene mutations or cell apoptosis (Hossain et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). POD decomposes H₂O₂ to release oxygen ions, which oxidize DAB to form brown water-insoluble precipitates. The darker the precipitate color, the higher the H₂O₂ content in calli and the more severe the damage. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the treatment group showed slight damage on day 7, and H₂O₂ accumulated gradually with time, resulting in more severe damage on day 14. O₂\u003csup\u003e⁻\u003c/sup\u003e∙, one of the ROS, can reduce NBT to form blue deposits, thus enabling localization of O₂\u003csup\u003e⁻\u003c/sup\u003e∙ production sites in calli. The darker the color, the higher the ROS content and the more severe the cell damage. Comparison showed that O₂\u003csup\u003e⁻\u003c/sup\u003e∙ in the control group mainly accumulated on the side of calli in contact with the medium, and no obvious O₂\u003csup\u003e⁻\u003c/sup\u003e∙ was observed on the top of calli far from the medium. In the treatment group, obvious O₂\u003csup\u003e⁻\u003c/sup\u003e∙ accumulation was observed on the top of calli on day 7, and O₂\u003csup\u003e⁻\u003c/sup\u003e∙ was distributed throughout the calli on day 14, indicating severe ROS damage to the entire calli.\u003c/p\u003e \u003cp\u003eEvans blue is a cell viability dye that stains dead cells light blue but cannot enter living cells. Thus, the darker the staining, the higher the damage degree of calli from the outside to the inside. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, the control group showed no obvious change after 14 days of treatment, while the cell death degree of the treatment group gradually increased with the extension of stress time. Combined with the results of the three histochemical stainings, we concluded that the regulatory mechanism in \u003cem\u003eA. camelorum\u003c/em\u003e calli could maintain basic cell survival under moderate to high salt stress for 7 days, but the calli suffered obvious damage after 14 days of continuous salt stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTemporal Response Characteristics of the Antioxidant Enzyme System in\u003c/b\u003e \u003cb\u003eA. camelorum\u003c/b\u003e \u003cb\u003eCalli Under Salt Stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe enzyme activities and MDA content of \u003cem\u003eA. camelorum\u003c/em\u003e calli treated with 200 mM NaCl for 0 h, 6 h, 12 h, and 48 h are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, compared with the control group, SOD activity began to increase at 6 h after treatment and reached a peak at 12 h, indicating that SOD was activated immediately in the early stage of stress to dismutate O₂\u003csup\u003e⁻\u003c/sup\u003e∙ into oxygen and H₂O₂, which reflects the ability of plants to scavenge O₂\u003csup\u003e⁻\u003c/sup\u003e∙ and also leads to H₂O₂ accumulation. SOD activity then decreased to the initial stress level at 48 h.\u003c/p\u003e \u003cp\u003eThe activity of POD was similar to that of SOD: it was activated immediately in the early stage of stress and reached a peak at 12 h. Although it decreased at 48 h, there was no significant difference compared with 12 h, and high activity was still maintained (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). The activity of CAT increased significantly at 12 h, while there was no significant difference at other time points compared with the control group, but a downward trend was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eMDA is a marker product of membrane lipid peroxidation. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, MDA began to accumulate in calli in the first 12 h of stress but showed no significant increase, indicating that the antioxidant enzyme system in \u003cem\u003eA. camelorum\u003c/em\u003e calli effectively scavenged ROS and avoided cell damage. With the extension of stress time, a large amount of MDA accumulated in calli after 48 h of long-term stress, indicating that the scavenging capacity of the antioxidant system in the later stage was insufficient to offset ROS accumulation, leading to aggravated damage to the membrane system of calli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eScreening of candidate genes and determination of their expression levels in calli under salt stress\u003c/h3\u003e\n\u003cp\u003eSpecific primers were designed based on the CDS sequences of 10 candidate genes, and qRT-PCR analysis was performed. After salt stress treatment of \u003cem\u003eA. camelorum\u003c/em\u003e calli, the expression levels of six genes were significantly upregulated, while the expression of two genes (Asp08G003230 and Asp02G034200) exhibited a downregulated trend (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). This may be due to differences in expression regulatory mechanisms between calli (as dedifferentiated non-organs) and organs. Among them, Asp07G015930 showed the highest upregulation (8-fold) and was highly responsive to salt stress. Thus, this gene was selected as the target for subsequent studies.\u003c/p\u003e \u003cp\u003eAfter 48 h of salt stress treatment, compared with the control group, the average relative expression level of \u003cem\u003eAcABI5\u003c/em\u003e (Asp07G015930) was upregulated 7-fold at 6 h, reached a maximum of 17-fold at 12 h, and was upregulated 12.9-fold at 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This indicates that \u003cem\u003eAcABI5\u003c/em\u003e is highly responsive to salt stress in \u003cem\u003eA. camelorum\u003c/em\u003e calli, with its expression level significantly increasing as the stress intensifies. Although there was a slight decrease at 48 h, there was no significant difference compared with 12 h. This suggests that \u003cem\u003eAcABI5\u003c/em\u003e can rapidly respond to salt stress in \u003cem\u003eA. camelorum\u003c/em\u003e calli and alleviate the damage caused by salt stress by continuously and highly expressing to regulate downstream genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatics analysis of AcABI5\u003c/h2\u003e \u003cp\u003eBased on transcriptome data (Tang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the CDS length of Asp07G015930 is 1080 bp, encoding 359 amino acids. BLAST analysis using NCBI and UniProtKB databases showed that the protein encoded by this gene has high homology with ABI5 in various plants and has conserved functional domains typical of the bZIP family. Based on its function and sequence characteristics, this gene was named \u003cem\u003eAcABI5\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePhylogenetic tree reconstruction was performed using 38 homologous sequences from plants such as \u003cem\u003eMedicago truncatula\u003c/em\u003e and \u003cem\u003eCicer arietinum\u003c/em\u003e obtained by homologous protein alignment of AcABI5. AcABI5 is localized in the Group B branch of the phylogenetic tree and is closely clustered with homologous sequences from species such as \u003cem\u003eAstragalus alpinus\u003c/em\u003e and \u003cem\u003eC. arietinum\u003c/em\u003e, forming a legume-specific subclade with high support (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This branch forms a clear differentiation boundary with multiple \u003cem\u003eG. max\u003c/em\u003e sequences in Groups C and D, indicating obvious species differentiation of AcABI5 within legumes.\u003c/p\u003e \u003cp\u003eAccording to the protein domain analysis, the AcABI5 protein possesses a typical bZIP TF domain located in the central region of the protein, consisting of a basic region with the conserved motif N-X7-R/K and a leucine zipper region with the conserved motif L-X6-L (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). This domain serves as the core functional region determining DNA binding and dimerization.A small number of COG2433 and Men1-related motifs are embedded at both ends, which are highly consistent with other legume homologous proteins. The presence of the bZIP domain indicates that this protein may bind to cis-acting elements to regulate the transcription of downstream genes and play a key role in plant stress responses (such as drought and salinity). Compared with sequences in Group D that contain additional extended domains such as cc_RasGRP1_C or bZIP_plant_BZIP46, AcABI5 maintains a relatively streamlined domain architecture, suggesting that its function is biased towards basic transcriptional regulation rather than multi-motif signal integration. The order of conserved motifs is basically consistent with that of homologous sequences, with no significant deletions or insertions, indicating that the DNA binding and dimerization mechanisms of this protein are highly conserved. In addition, PlantCARE prediction results showed that the promoter of AcABI5 contains the abscisic acid-responsive element (ABRE, i.e., the cis-acting element involved in abscisic acid responsiveness), MYB binding sites involved in drought inducibility, as well as hormone-responsive elements and transcription factor binding sites, among other functional elements (Fig. S2). These elements suggest that the expression of AcABI5 may be regulated by abiotic stresses such as salt stress, thereby providing a basis for its involvement in the transcriptional regulation of salt stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSecondary structure prediction showed that AcABI5 is mainly composed of random coils (60.45%), with α-helix accounting for 37.88%, β-sheet and β-turn accounting for only 1.11% and 0.56%, respectively, which is consistent with the typical long helix-coil-helix structure of bZIP TFs (Fig. S3B). The linear structure diagram reveals alternating helix/random coil segments at the N-terminus and C-terminus, with a continuous α-helix in the middle, which is a potential DNA binding and dimerization region (Fig. S3B). The tertiary structure model further shows an extended helix spanning the main body of the protein, accompanied by several flexible loops and secondary helix bundles, indicating that the protein may form a stable dimer through helix bundles during DNA binding (Fig. S3C). The synchronous fluctuation of helix and coil probabilities in the curve prediction diagram also supports its ability to undergo regulated conformational changes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eProtParam analysis showed (Table S4) that AcABI5 is a basic protein consisting of 359 amino acids, with a molecular weight of approximately 40.21 kDa and a theoretical isoelectric point of 9.51. An instability index of 56.46 indicates certain instability in vitro, and an aliphatic index of 66.57 indicates moderate thermal stability at higher temperatures. A grand average of hydropathicity (GRAVY) of \u0026minus;\u0026thinsp;0.845 indicates that the protein has significant hydrophilicity and is more likely to be located in the nucleus or cytoplasm rather than membranous compartments. SignalP 6.0 predicted no signal peptide in the full-length sequence and classified it as \"OTHER\", supporting the speculation that it is a nuclear-localized TF, which is consistent with the predictions of Uniprot and WoLF PSORT (Fig. S3A).\u003c/p\u003e \u003cp\u003eResults of bioinformatics analysis indicated that AcABI5 is a member of the bZIP superfamily with typical bZIP transcription factor characteristics: hydrophilic, no signal peptide, and mainly composed of helix structures. It maintains a close evolutionary relationship with multiple forage and legume homologous proteins in legumes and has a highly consistent domain composition with multi-copy sequences. These results suggest that ABI5 in \u003cem\u003eA. camelorum\u003c/em\u003e may undertake a conserved transcriptional regulatory function and be closely related to the regulatory network of legumes adapting to drought, salinity, and other stresses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization\u003c/h2\u003e \u003cp\u003ePCR amplified the \u003cem\u003eAcABI5\u003c/em\u003e full-length CDS (1138 bp) from \u003cem\u003eA. camelorum\u003c/em\u003e cDNA; electrophoresis and sequencing confirmed its validity (Fig. S4A). Purified products underwent BP recombination with pDONR-Zeo and \u003cem\u003eE. coli\u003c/em\u003e transformation, and bacterial PCR/sequencing verified entry vector construction for plasmid extraction (Fig. S4C). Subsequent LR recombination between \u003cem\u003eAcABI5\u003c/em\u003e-pDONR and pK7WGF2, plus \u003cem\u003eE. coli\u003c/em\u003e transformation, was validated by colony PCR/sequencing, with positive plasmids extracted and preserved (Fig. S4B).The constructed vector was transiently expressed in the leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e7\u003c/span\u003e, green fluorescence of 35S::GFP was detected throughout the cells, while green fluorescence was only detected in the nucleus of cells transfected with the fusion vector 35S::GFP-AcABI5. Moreover, the green fluorescence overlapped with the red fluorescence of 35S::mCherry, indicating that the AcABI5 protein is localized in the nucleus and is a typical transcription factor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e2,4-D and 6-BA are two commonly used plant growth regulators. 2,4-D is an auxin-like regulator that promotes callus growth at low concentrations and inhibits differentiation at high concentrations. 6-BA is a cytokinin-like regulator that can induce callus differentiation into buds. This experiment comprehensively analyzed the effects of different concentrations of the two hormones on \u003cem\u003eA. camelorum\u003c/em\u003e callus formation through a full-factorial experiment with concentrations ranging from 0 to 2 mg/L. The results showed that the combination of 1.5 mg/L 2,4-D and 0.5 mg/L 6-BA was the optimal hormone ratio for \u003cem\u003eA. camelorum\u003c/em\u003e callus proliferation.\u003c/p\u003e \u003cp\u003eBu et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) reported that MS medium containing 1.5\u0026ndash;2.0 mg/L 2,4-D and 0.5\u0026ndash;1.0 mg/L 6-BA was the optimal medium for inducing callus formation and subculture of \u003cem\u003eA. camelorum\u003c/em\u003e hypocotyl segments. When 2,4-D and 6-BA reach a specific balance, calli of \u003cem\u003eAlhagi graecorum\u003c/em\u003e form somatic embryos through somatic embryogenesis, which further develop into regenerated plants. When cytokinin is dominant (e.g., high concentration of 6-BA), explants dedifferentiate to form calli and then differentiate into adventitious buds through organogenesis (Hassanein \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). These results are consistent with those of this experiment. In evaluating the callus induction effect of \u003cem\u003eA. camelorum\u003c/em\u003e, compared with the traditional qualitative judgment of callus induction efficiency using callus induction rate, callus status, and growth rate (Bu et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), this experiment also quantitatively compared the induction effects of different groups by determining the relative growth rate of calli, providing a new reference index.\u003c/p\u003e \u003cp\u003eChanges in fresh weight, phenotype, antioxidant enzyme activity, ROS accumulation, MDA accumulation, and cell mortality of calli are comprehensive and intuitive manifestations of callus response to salt stress. This study investigated the responses of the above physiological and biochemical indexes of \u003cem\u003eA. camelorum\u003c/em\u003e calli at different time points under high salt stress.\u003c/p\u003e \u003cp\u003eSong (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) reported that salt stress treatment of \u003cem\u003eZoysia matrella\u003c/em\u003e calli showed that callus growth was inhibited when NaCl concentration was \u0026ge;\u0026thinsp;0.6% (approximately 100 mM). Jing et al. Jing et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) pointed out that under 150 mM NaCl treatment, the growth of wild-type apple calli was significantly inhibited and the fresh weight was significantly reduced compared with \u003cem\u003eMdSPL13B-OE\u003c/em\u003e apple calli. The results of this experiment showed that under 200 mM NaCl stress, the fresh weight of \u003cem\u003eA. camelorum\u003c/em\u003e calli decreased by 15.8% after 14 days and showed slight browning, which is consistent with previous research results.\u003c/p\u003e \u003cp\u003ePrevious studies believed that ROS are harmful to plant growth and development, so plants have evolved various strategies to alleviate oxidative stress (Ding et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), such as the antioxidant enzyme system that can scavenge or reduce ROS produced by abiotic stress and reduce cell damage. However, with in-depth research on ROS, it has been found that ROS actually play a complex dual role. Plants can use potentially toxic ROS molecules as beneficial signals for development to precisely and temporally inhibit premature maturation of the shoot apical meristem and ensure orderly flowering (Huang et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). ROS signals produced in callus cells also play an important regulatory role in the embryogenic capacity of calli (Zhou et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Histochemical staining showed that in the absence of stress, ROS produced by \u003cem\u003eA. camelorum\u003c/em\u003e calli mainly accumulated on the side in contact with the medium, with a small amount on the upper surface. This may be because changes in cell volume and number of calli are easily restricted by the cell wall, and the production of ROS signals can break down the cell wall, promote cell wall loosening, and thereby regulate cell growth (Zhang et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, under long-term high-concentration salt stress, calli are in a state of oxidative stress for a long time, the scavenging efficiency of the antioxidant enzyme system decreases, and high ROS levels cause damage to calli, which is not conducive to embryonic development (Ren et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), leading to browning and death.\u003c/p\u003e \u003cp\u003eUnder NaCl stress, the activities of SOD, POD, CAT, as well as the content of MDA, all increased significantly. In the early stage of stress, the antioxidant enzyme system of \u003cem\u003eA. camelorum\u003c/em\u003e calli was activated immediately. Compared with POD and CAT, SOD was activated more rapidly and catalyzed the dismutation of O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e∙ into O₂ and H₂O₂ through redox reactions. CAT has high specificity and fast turnover rate for H₂O₂ (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), while POD uses H₂O₂ as an oxidant to catalyze various substrates such as phenols and lipid peroxides, and the two synergistically scavenge the produced H₂O₂.\u003c/p\u003e \u003cp\u003eIn the late stage of stress, MDA content continued to increase, SOD activity decreased to the initial stress level, CAT activity decreased significantly, while POD always maintained high activity. This may be because long-term stress provided more substrates such as phenols for POD, so POD played a major role in the late stage of stress and undertook important antioxidant or secondary metabolic regulatory functions. The decrease in SOD and CAT activities may be related to the consumption of the enzyme system caused by continuous stress. H₂O₂, as a signaling molecule, can induce CAT to initiate defense responses, while excessive ROS can cause oxidative damage to calli, resulting in fluctuating CAT activity.\u003c/p\u003e \u003cp\u003ePlants can maintain the active function of SOD and POD for a longer period than CAT when facing stress, and SOD and POD show better synergy (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Long-term stress leads to the decline of antioxidant system function, and the activities of SOD and POD show a trend of first increasing and then decreasing (Zhang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), which is similar to the results of this experiment. In conclusion, the three key protective enzymes regulated by the antioxidant enzyme system of \u003cem\u003eA. camelorum\u003c/em\u003e calli maintain the normal level of ROS in the body through coordinated interaction, reducing ROS damage to calli.\u003c/p\u003e \u003cp\u003eBased on the research results of (Tang et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we screened Asp07G015930, which showed the highest upregulation of gene expression in \u003cem\u003eA. camelorum\u003c/em\u003e calli under salt stress, from 10 candidate genes. The CDS length of this gene is 1080 bp, encoding 359 amino acids. Bioinformatics analysis showed that the protein encoded by this gene is hydrophilic, has no signal peptide, is mainly composed of helix structures, has high homology with ABI5 proteins in various plants, and has conserved functional domains typical of the bZIP family.\u003c/p\u003e \u003cp\u003eAbscisic acid (ABA) is involved in regulating signal transduction in various defense processes to resist abiotic and biotic stresses and promote plant development (Sah et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The bZIP family is divided into 13 major groups, among which Group A mainly consists of ABA-responsive element-binding protein (AREB), ABA-responsive element-binding factor (ABF), and ABI5 subfamily members (Collin et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). As a core protein in the ABA signal transduction pathway, ABI5 plays an important regulatory role in seed germination, early seedling development, and stress adaptation. Research findings indicate that numerous ABA-insensitive (\u003cem\u003eABI\u003c/em\u003e) genes exist in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, including \u003cem\u003eABI1\u003c/em\u003e, \u003cem\u003eABI2\u003c/em\u003e, \u003cem\u003eABI3\u003c/em\u003e, \u003cem\u003eABI4\u003c/em\u003e, and \u003cem\u003eABI5\u003c/em\u003e. Among them, ABI1 and ABI2 are two highly homologous protein kinases that act as negative regulators in the ABA signaling pathway (Merlot et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e); ABI3 belongs to the B3 protein family (Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and plays a key role in regulating plant flowering; ABI4 belongs to the CBF/DREB subfamily, is a member of the AP2/ERF family, contains an AP2 domain, and is involved in ABA signal transduction (Yang \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2025\u003c/span\u003e); ABI5 belongs to the bZIP transcription factor and is a core transcription factor in the ABA signal transduction pathway (Du et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Du et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Based on its function and sequence characteristics, the gene was named \u003cem\u003eAcABI5\u003c/em\u003e. Zinsmeister et al. confirmed the role of ABI5 in regulating ROS accumulation and seed maturation in Pisum sativum, a leguminous plant ; Finkelstein et al. found that ABI5 regulates the expression of downstream genes such as late embryogenesis abundant (LEA) proteins, and is involved in processes including seed germination and dehydration tolerance (Zinsmeister et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); Albertos et al. revealed that nitric oxide can counteract ABA during plant development and regulate the molecular interaction mechanism of ABI5 through post-translational modification (Albertos et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e); Basso et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) comprehensively analyzed the \u003cem\u003eAREB/ABF/ABI5\u003c/em\u003e genes belonging to the subfamily A of plant bZIP TFs in \u003cem\u003eC. arietinum\u003c/em\u003e and \u003cem\u003eLens culinaris\u003c/em\u003e, which are up-regulated under different abiotic and biotic stress conditions, exhibit dynamic expression in different tissues, and show higher expression levels in drought-tolerant varieties. We speculate that AcABI5 has similar functions to these homologous proteins, possibly binding to cis-acting elements to regulate the transcriptional expression of downstream genes, and playing a key role in plant responses to abiotic stress and seed germination. Most studies have shown that typical TFs are usually localized in the nucleus of plant cells (Wang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). By constructing the pK7WGF2-35S::GFP-AcABI5 fusion expression vector and using \u003cem\u003eAgrobacterium\u003c/em\u003e to infect \u003cem\u003eN. benthamiana\u003c/em\u003e leaves for transient expression, the results showed that the fusion protein emitted fluorescence only in the nucleus, indicating that AcABI5, like most TFs, is a nuclear-localized protein. Through salt stress treatment of \u003cem\u003eA. camelorum\u003c/em\u003e calli, we found that \u003cem\u003eAcABI5\u003c/em\u003e can respond rapidly to salt stress and maintain high expression during the stress process; in the absence of abiotic stress, ABI5 undergoes dephosphorylation, and the dephosphorylated ABI5 is neither active nor stable and will be degraded through the 26S proteasome pathway (Lopez-Molina et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), which is consistent with the low expression level of \u003cem\u003eAcABI5\u003c/em\u003e observed in our control group. In future research, we will further establish an \u003cem\u003eAcABI5\u003c/em\u003e overexpression system in \u003cem\u003eA. camelorum\u003c/em\u003e calli, identify the cis-acting elements bound by AcABI5, analyze the downstream target genes regulated by AcABI5, and investigate whether AcABI5 regulates the expression of salt resistance-related genes by binding to these cis-acting elements.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study determined that the optimal proliferation medium for \u003cem\u003eA. camelorum\u003c/em\u003e calli is 1.5 mg/L 2,4-D combined with 0.5 mg/L 6-BA. In response to salt stress, SOD in \u003cem\u003eA. camelorum\u003c/em\u003e calli is activated first to scavenge intracellular O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e∙; POD and CAT synergistically scavenge ROS in the early stage of stress, and POD also exhibits high activity in the late stage of stress. A salt-tolerant gene \u003cem\u003eAcABI5\u003c/em\u003e of \u003cem\u003eA. camelorum\u003c/em\u003e was screened out by qRT-PCR, with a full-length CDS sequence of 1080 bp encoding 359 amino acids. Bioinformatics analysis revealed that AcABI5 from \u003cem\u003eA. camelorum\u003c/em\u003e is a nuclear-localized bZIP family TF with typical conserved domains; its promoter region contains stress-responsive cis-acting elements. This TF harbors conserved transcriptional regulatory functions, and the gene is inducible in \u003cem\u003eA. camelorum\u003c/em\u003e calli under salt stress, suggesting that AcABI5 may be involved in the regulatory network of \u003cem\u003eA. camelorum\u003c/em\u003e adapting to abiotic stresses including drought and salt stress. This study provides a research basis for further investigating the molecular mechanism of \u003cem\u003eAcABI5\u003c/em\u003e regulating \u003cem\u003eA. camelorum\u003c/em\u003e's response to salt stress, and offers theoretical support for the establishment of a genetic transformation system for \u003cem\u003eA. camelorum\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData of this study will be made available on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the Key Laboratory of Ecological Safety and Sustainable Development in Arid Lands Young Scientists Interdisciplinary Team Project (grant No.E552020301), the National Key Research and Development Program of China (grant No.2022YFF1302505-03), and the National Young Talent Program (grant No.2022000007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no competing interests to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by, Xiangyi Li, Pingyin Guan, Bo Zhang and Gangliang Tang. The first draft of the manuscript was written by Zhengtao Yan and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlbertos P, Romero-Puertas MC, Tatematsu K, Mateos I, S\u0026aacute;nchez-Vicente I, Nambara E, Lorenzo O (2015) S-nitrosylation triggers ABI5 degradation to promote seed germination and seedling growth. Nat Commun 6:8669. https://doi.org/10.1038/ncomms9669\u003c/li\u003e\n\u003cli\u003eBasso MF, Iovieno P, Capuana M, Contaldi F, Ieri F, Menicucci F, Celso FL, Barone G, Martinelli F (2025) Identification and expression of the AREB/ABF/ABI5 subfamily genes in chickpea and lentil reveal major players involved in ABA-mediated defense response to drought stress. 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Plant Cell 28(11):2735-2754. https://doi.org/10.1105/tpc.16.00470\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alhagi camelorum, Callus induction, Salt stress, Antioxidant enzymes, AcABI5, bZIP transcription factor","lastPublishedDoi":"10.21203/rs.3.rs-8449616/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8449616/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAlhagi camelorum\u003c/em\u003e, a dominant leguminous shrub in the saline–hyperarid Taklimakan Desert, can complete its life cycle in salty soils, yet the molecular basis of seedling-stage salt tolerance remains unknown. Here, we developed an in-vitro callus system to dissect this trait without soil heterogeneity. Among 36 hormone regimes, 1.5 mg/L 2,4-D plus 0.5 mg/L 6-BA produced 100 % induction. Exposure of 28-d-old calli to 200 mM NaCl for 48 h caused transient swelling of cortical cells and a 5.3-fold rise in malondialdehyde (MDA). Antioxidant enzymes responded sequentially: superoxide dismutase (SOD) peaked at 6 h to scavenge superoxide, peroxidase (POD) maintained high activity throughout the first 24 h, and catalase (CAT) stabilized after 48 h, jointly keeping H₂O₂ below toxic levels. RNA-seq has identified an up-regulated transcription factor (log₂FC = 7) which was a basic leucine-zipper (bZIP) homologue of abscisic acid-insensitive protein 5 (ABI5). Quantitative RT-PCR confirmed 17-fold induction by NaCl and rapid decay after stress removal. Sub-cellular localization of a 35S::GFP-AcABI5 fusion in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e epidermis showed exclusive nuclear fluorescence, consistent with a transcriptional regulator. Therefore, our study provides both optimized callus protocols and a candidate gene for engineering salt tolerance in \u003cem\u003eA. camelorum\u003c/em\u003e and related desert legumes.\u003c/p\u003e","manuscriptTitle":"De novo characterization of AcABI5 transcription factor and physiological responses to salt stress in Alhagi camelorum callus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 17:20:41","doi":"10.21203/rs.3.rs-8449616/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"841b8d67-cd6f-4288-8dfd-ccac95dfb851","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-27T14:44:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 17:20:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8449616","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8449616","identity":"rs-8449616","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}