Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance

Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance | 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 Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance Yi Zhou, Pengpeng Fang, Jia Zeng, Xinpeng Li, Qiang He, Wuhan Zhang, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8764411/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract Background Salt stress is a key abiotic stress factor limiting rice growth and development. Previous studies have shown that OsWRKY53 , OsARF18 , and OsRR22 not only serve as important negative regulators of salt tolerance in rice but are also crucial for growth and development. However, the relative strengths of salt tolerance among these three genes and their combined effects within the same rice variety have not yet been reported. Results In this study, we employed CRISPR/Cas9-mediated genome editing to simultaneously disrupt OsWRKY53 , OsARF18 , and OsRR22 in the rice cultivar Shuanghui 459. Salt tolerance increases sequentially in single-gene, double-gene, and triple-gene mutants. Under 1.0% NaCl stress, the triple mutant exhibited approximately 80% survival versus complete lethality in the wild type (WT). Physiologically, ROS accumulation progressively declined, while key antioxidant enzyme activities (CAT, SOD, POD) significantly increased. More importantly, molecular analyses revealed that the OsWRKY53 WRKY domain binds W-box elements in the OsRR22 and OsARF18 promoters, repressing their transcription. Loss function of OsWRKY53 depresses both genes. Reciprocally, OsARF18 and OsRR22 knockout downregulated OsWRKY53 . Furthermore, OsWRKY53 and OsRR22 directly interact at the protein level. Conclusions Taken together, our results reveal that OsWRKY53 , OsARF18 , and OsRR22 constitute a reciprocal negative-feedback loop, wherein these three transcriptional regulators mutually antagonize each other. Disruption of this antagonistic network likely represents a core mechanism responsible for the robust salt tolerance observed in the triple mutant. Importantly, the polygenic aggregation of OsWRKY53 - OsARF18 - OsRR22 not only significantly enhances rice salt tolerance but also does not affect normal plant growth and development. These findings provide new strategies for polygenic aggregation-based genetic improvement of salt-tolerant rice varieties. Salt stress tolerance OsWRKY53 OsARF18 OsRR22 Genome editing Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Soil salinity stress constitutes a primary global abiotic constraint that severely limits crop growth, development, and yield [ 1 , 2 ] . Among cereal crops, rice (Oryza sativa L.) is particularly sensitive to salinity stress [ 3 ] , yet progress in developing salt‑tolerant rice cultivars has been slow. This is primarily because salt tolerance in rice entails the coordinated action of numerous physiological and biochemical processes, constituting a polygenic quantitative trait with a complex genetic architecture that is strongly influenced by environmental factors [ 4 ] . Previous attempts at single‑gene‑based improvement have yielded modest outcomes, and the repertoire of validated genes available for genetic enhancement remains constrained [ 5 – 8 ] , therefore, innovation and breakthroughs in genetic improvement methods are needed. Previous studies have identified OsRR22 [ 9 ] , OsWRKY53 [ 10 ] and OsARF18 [ 11 ] as key negative regulators of salt tolerance in rice. OsRR22 encodes a B‑type response regulator of 696 amino acids [ 12 ] , predominantly expressed in the root cortex and vascular tissues, where it directly upregulates OsHKT2;1 expression. OsSLR1 functions as a transcriptional co‑activator in this process. Loss‑of‑function mutations in either OsSLR1 or OsRR22 disrupt OsHKT2;1 activation, leading to its downregulation and reduced Na⁺ accumulation in roots [ 13 ] . Yu et al. [ 10 ] identified OsWRKY53 as a pivotal negative regulator of salt tolerance in rice via genome‑wide association analysis. Their study revealed that OsWRKY53 specifically binds to W‑box cis‑elements in the promoters of OsMKK10.2 and OsHKT1;5 , leading to transcriptional repression of both genes. Loss‑of‑function of OsWRKY53 relieved this repression, resulting in upregulated expression of OsMKK10.2 and OsHKT1;5 . This regulatory change promotes Na⁺ efflux from xylem and phloem parenchyma cells, thereby enhancing salinity tolerance. Furthermore, in the rice cultivar Zhonghua 11, mutation of OsWRKY53 upregulates OsMYB63 expression, which contributes to stronger resistance against bacterial leaf blight compared with the WT during the booting stage [ 14 ] . Tang et al. [ 15 ] exposed the rice variety LJ11 to low‑temperature (15°C) treatment during the booting stage. They reported that loss‑of‑function of OsWRKY53 upregulated gibberellin biosynthesis genes, leading to increased accumulation of bioactive gibberellins in anthers. This enhancement in cold tolerance at booting occurred without compromising grain yield. In a separate study, Deng et al. [ 11 ] identified RST1 as OsARF18 . Analysis of an OsARF18 loss‑of‑function mutant derived from a Nipponbare population demonstrated that the mutation promotes asparagine synthesis, reduces excessive NH₄⁺ accumulation in shoots, and maintains elevated K⁺/Na⁺ ratios in aerial tissues, collectively contributing to enhanced salt tolerance. Furthermore, in the Zhonghua 11 background, OsARF18 loss‑of‑function elevates transcript levels of OsGS1;1 and OsGS1;2 , increases glutamine synthetase (GS) activity, and upregulates detoxification‑related genes, conferring stronger glufosinate resistance compared to the WT [ 16 ] . CRISPR/Cas9 is the third-generation gene editing technology following zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) [ 17 – 19 ] . In contrast to conventional breeding methods, which are often time‑consuming and labor‑intensive, CRISPR/Cas9‑mediated editing enables precise, simultaneous modification of multiple target genes within a relatively short timeframe, thereby facilitating targeted trait enhancement. This technology significantly accelerates the breeding process and shortens variety development cycles [ 20 – 22 ] . To date, the CRISPR/Cas9 system has been successfully implemented for genome editing in a broad range of organisms, including rice [ 17 ] . For instance, Zhang et al. [ 23 ] substantially improved salt tolerance in rice by targeting OsRR22 using CRISPR/Cas9. In another study, Zhou et al. [ 19 ] applied CRISPR/Cas9 to the widely used two‑line male‑sterile line Longke 638S, simultaneously editing three known broad‑spectrum blast resistance genes— Bsr‑d1 , Pi21 , and ERF922 —to generate triple‑gene and corresponding single‑gene mutants. Phenotypic assessment revealed that the triple-gene mutant and the ERF922 single-gene mutant exhibited the most pronounced disease resistance among all mutant lines and the WT control. OsWRKY53 , OsARF18 , and OsRR22 serve as key regulators not only in the salt‑stress response but also in the growth and development of rice [ 10 , 11 , 13 , 15 , 16 , 24 – 27 ] . To date, the relative contributions of these three genes to salt tolerance and whether their aggregation within the same rice variety exhibits an additive effect remain unknown. In this study, CRISPR/Cas9-mediated genome editing was employed to simultaneously disrupt these three salt-tolerance repressors in the rice cultivar Shuanghui 459, successfully generating single-gene, double-gene (excluding the OsWRKY53 – OsRR22 combination), and triple-gene mutant lines. The results showed that after treatment with 1.0% NaCl, the survival rates of single-gene, double-gene, and triple-gene mutants increased sequentially, with the survival rate of the triple-gene mutant reaching about 80%. More importantly, under non‑stress conditions, loss of OsWRKY53 function increased the transcript levels of OsARF18 and OsRR22 compared with the WT. Conversely, loss of function of OsARF18 and OsRR22 decreased OsWRKY53 expression. These results indicate that OsWRKY53 forms a mutually antagonistic negative‑feedback loop with OsARF18 and OsRR22 . The improved salt tolerance of the double and triple mutants is likely attributable to an additive effect resulting from the disruption of this antagonistic interaction. These findings provide new strategies for improving salt tolerance in rice through multi‑gene pyramiding. Materials and methods Generation of transgenic plants Based on the principle of CRISPR/Cas9-mediated genome editing, a specific protospacer adjacent motif (PAM) sequence was selected within the first exon of each target gene ( OsWRKY53 , OsARF18 , and OsRR22 ). The corresponding single-guide RNA (sgRNA) was assembled and cloned into the binary vector pEGCas9Pubi-H. The resulting construct was introduced into rice cultivar Shuanghui 459 via Agrobacterium tumefaciens (strain EHA105)-mediated transformation. Mutations at the target loci in transgenic plants were validated by Sanger sequencing. The following mutant lines were successfully generated: single-gene knockout mutants of OsWRKY53 ( oswrky53-1 , oswrky53-2 ), OsARF18 ( osarf18-1 , osarf18-2 ), and OsRR22 ( osrr22-1 , osrr22-2 ), double-gene knockout mutants of OsWRKY53 - OsRR22 ( oswrky53 - osrr22-1 , oswrky53 - osrr22-2 ) and OsARF18 - OsRR22 ( osarf18 - osrr22-1 , osarf18 - osrr22 - 2 ), and the triple-gene knockout mutant of OsWRKY53 - OsARF18 - OsRR22 ( oswrky53 - osarf18 - osrr22-1 , oswrky53 - osarf18 - osrr22-2 ). Salt tolerance treatment Seeds of rice cultivar Shuanghui 459 and transgenic materials were incubated at 37°C for 48 h to break dormancy. They were then surface-sterilized with 15% sodium hypochlorite for 15 min, rinsed thoroughly five times with distilled water, and soaked in distilled water at room temperature for 48 h. Subsequently, seeds were returned to 37°C for an additional 48 h to promote germination. Germinated seeds were transferred to a perforated cultivation plate and grown in a controlled-environment chamber under a 14-h-light/10-h-dark photoperiod. Day/night temperatures were maintained at 28°C/26°C, with a light intensity of 30,000 lx and 70% relative humidity. Two-week-old seedlings were subjected to salt stress by treatment with 1.0% or 1.2% NaCl solution for 7 d, followed by a 7‑d recovery period in Yoshida nutrient solution without NaCl. Each treatment consisted of three biological replicates, with 48 seedlings per replicate. Salt tolerance was quantified by comparing the survival rates of salt-treated seedlings to those of the untreated control group. After 7 days of treatment with 1.0% NaCl and a subsequent 7-day recovery period, whole seedlings from both control and treated groups were harvested for fresh weight measurement. Following this, shoot and root lengths were determined using five randomly selected seedlings per biological replicate, and mean values were calculated. DAB and NBT staining Leaves were harvested from 14‑day‑old seedlings that had been treated with 1.0% NaCl for 0 or 24 h. The experiment consisted of three biological replicates, each comprising a pool of leaves from five seedlings. Histochemical staining to visualize H₂O₂ and O₂⁻ accumulation was performed using 3,3’‑diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) kits, respectively, according to the manufacturer’s instructions (Leagene Biotechnology, China). Briefly, leaf samples were vacuum‑infiltrated with the corresponding staining working solution in the dark for 30 min. After staining, samples were rinsed 3–5 times with distilled water to remove excess dye. Chlorophyll was then removed by incubating the samples in 95% ethanol in an 80°C water bath; the ethanol was replaced every 10 min until the leaf tissue became completely clear. Quantification of Na⁺ Roots, stems, and leaves were harvested from seedlings treated with 1.0% NaCl for 0 h or 24 h. Each treatment consisted of three biological replicates, each comprising tissues pooled from 5–7 seedlings. Samples were transferred to pre‑cleaned polytetrafluoroethylene (PTFE) microwave digestion vessels. Then, 10 mL of high‑purity concentrated HNO₃ was added to each vessel, followed by vortex mixing for 1 min. An additional 5 mL of ultra‑pure concentrated HNO₃ was added before sealing. Digestion was performed in a closed‑vessel microwave system using a stepped temperature program: 120°C (5 min), 150°C (5 min), and 180°C (20 min). After digestion, the vessels were transferred to an acid evaporation cabinet and held at 180°C for 30 min to reduce the residual volume to approximately 1 mL. The digestates were subsequently diluted threefold with ultra‑pure water and finally adjusted to a final volume of 50 mL in centrifuge tubes. After thorough mixing (1 min), samples were filtered through a 0.22‑µm membrane. The filtrates were appropriately diluted with 2% HNO₃ as required and analyzed for Na⁺ content using inductively coupled plasma mass spectrometry (ICP‑MS). Quantification of endogenous compounds Leaves were harvested from 14-day-old seedlings exposed to 1.0% NaCl for 0 h or 24 h. The experiment included three biological replicates, with each replicate consisting of leaves pooled from 5 to 7 seedlings. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the malondialdehyde (MDA) content, were quantified in leaf tissues. All measurements were conducted using fluorescence spectrophotometry following the protocols provided with the respective commercial assay kits (BOXBIO, Beijing, China). Reverse transcription quantitative PCR Total RNA was isolated from the roots of mutant plants treated with 1.0% NaCl for 0 or 24 h, using the Eastep Super Total RNA Extraction Kit (Promega, China). Subsequently, cDNA was synthesized from equal amounts of total RNA using the HiScript IV All-in-One Ultra RT SuperMix for qPCR (Novizan, China). QPCR was performed on a Quant Studio 3 Real-Time PCR System. Each reaction contained TB Green Premix Ex Taq II (Tli RNaseH Plus) and gene-specific primers. The actin gene was used as an internal control for normalization. Relative gene expression levels were calculated using the 2 −ΔΔCt method, with the WT sample at 0 h serving as the calibrator. Yeast one-hybrid assay Analysis of the OsRR22 and OsARF18 promoter regions identified two and one W‑box elements within the 1500 bp and 1000 bp upstream sequences of their respective ATG start codons. The corresponding promoter fragments were PCR‑amplified and cloned into the pHis2 reporter vector. Separately, the full‑length coding sequence (CDS) of OsWRKY53 was inserted into the pGADT7 vector to generate the OsWRKY53 -CDS effector construct. After sequence verification, the reporter and effector plasmids were co‑transformed into the Saccharomyces cerevisiae strain Y187. Transformants were first selected on synthetic dropout (SD) medium lacking leucine and tryptophan (SD/–Leu/–Trp). Positive clones were then screened on SD/–Leu/–Trp/–His plates supplemented with 3‑amino‑1, 2, 4‑triazole (3‑AT). Plates were incubated at 30°C for 4 days to assess the protein‑DNA interactions. Dual-luciferase reporter in vivo imaging To analyze the transcriptional activity of OsWRKY53 on the promoters of OsRR22 and OsARF18 , the effector and reporter vectors were constructed. The effector vector 62-SK- OsWRKY53 expressed the full-length OsWRKY53 protein, while the reporter vectors pro OsRR22 -0800-Luc and pro OsARF18 -0800-Luc contained the respective promoter fragments driving the firefly luciferase gene. Each construct was introduced into Agrobacterium tumefaciens strain GV3101 (pSoup-p19). Bacterial cultures carrying the effector and a reporter were mixed and co-infiltrated into the leaves of 4-week-old tobacco plants for transient expression. Luminescence imaging was performed 72 h post-infiltration using an LB985 Night SHADE Plant Molecular Imaging System. Yeast two-hybrid assay The full-length coding sequences (CDS) of OsWRKY53 were cloned into the pGADT7 vector (activation domain), while the CDS of OsRR22 and OsARF18 were cloned into the pGBKT7 vector (DNA-binding domain). After sequence verification, the resulting constructs were pairwise co-transformed (pGADT7- OsWRKY53 with pGBKT7- OsRR22 , and pGADT7- OsWRKY53 with pGBKT7- OsARF18 ) into the Saccharomyces cerevisiae strain Y2HGold. Transformants were first selected on synthetic dropout (SD) medium lacking leucine and tryptophan (SD/–Leu/–Trp). Positive colonies were then screened on SD medium additionally lacking adenine and histidine (SD/–Ade/–Leu/–Trp/–His), supplemented with 3-amino-1,2,4-triazole (3-AT). Plates were incubated at 30°C for 4 days to assess protein-protein interactions. Bifc assay To test for protein-protein interaction in vivo, the full-length coding sequences (CDSs) of OsWRKY53 and OsRR22 were cloned into the pUC‑SPYNE and pUC‑SPYCE vectors, respectively, to generate OsWRKY53 ‑nYFP and OsRR22 ‑cYFP fusion constructs. The known interacting pair BZIP63 ‑nYFP/ BZIP63 ‑cYFP served as a positive control, while empty nYFP and cYFP vectors were used as negative controls. Different plasmid combinations were co‑transfected into rice protoplasts. Fluorescence signals were captured 16–24 h after transfection using a Leica SP8 confocal laser‑scanning microscope equipped with appropriate filter sets (excitation: 488 nm and 587 nm). RNA-seq analysis Root tissues were harvested from WT and mutant plants treated with 1.0% NaCl for 0 or 30 h, with each treatment performed in biological triplicate. Total RNA was extracted using the MagPure Universal RNA LQ Kit 2 (Magen, China). Sequencing libraries were constructed with the VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme, China) and sequenced on an Illumina NovaSeq platform to generate paired-end reads. Differentially expressed genes (DEGs) were identified using the DESeq2 package with an adjusted P-value (padj) cutoff of ≤ 0.05, using the WT samples as the reference group. Gene Ontology (GO) enrichment analysis of the DEGs was subsequently performed using the GOSeq software (v1.34.1) with default parameters. Statistical analysis Data are presented as mean ± standard deviation (SD). Differences among groups were assessed by one-way analysis of variance (ANOVA) followed by post-hoc comparisons, using SPSS 27.0. Significance levels are denoted as follows: * P < 0.05, ** P < 0.01, *** P < 0.001. All oligonucleotide primers used in this study for vector construction, yeast one-hybrid (Y1H), yeast two-hybrid (Y2H), dual-luciferase reporter (LUC), bimolecular fluorescence complementation (BiFC), and quantitative real-time PCR (qPCR) assays are listed in Supplementary Table 1. Results Loss of function of OsWRKY53 - OsARF18 - OsRR22 significantly enhances rice salt tolerance To investigate whether OsWRKY53 , OsARF18 , and OsRR22 have additive effects on salt tolerance in rice, we generated single, double, and triple mutants using CRISPR/Cas9. Each mutant line comprised two independent alleles (Fig. S1 ), all of which carried frameshift mutations leading to disruption of the functional protein domains (Fig. S2 –4). Subsequently, plants were subjected to salt stress. After a 7‑day treatment with 1.0% NaCl followed by 7‑day recovery, all WT plants died. In contrast, the OsWRKY53 - OsARF18 - OsRR22 triple mutant exhibited the strongest tolerance, with survival rates of 79.86% and 83.33%, significantly exceeding all other genotypes (Fig. 1A, B). The OsWRKY53 - OsRR22 and OsARF18 - OsRR22 double mutants showed comparable survival, both significantly higher than the three single mutants (Fig. 1A, B). Among single mutants, a survival gradient was observed: OsARF18 > OsWRKY53 > OsRR22 (Fig. 1A, B). Similarly, under 1.2% NaCl treatment, Consistent with the trend observed at 1.0% NaCl, the OsWRKY53 - OsARF18 - OsRR22 triple mutant remained the most tolerant under 1.2% NaCl stress, with survival rates of 20.83% and 21.53% (Fig. 1C, D), and the OsWRKY53 - OsRR22 double mutant outperformed all other mutant combinations (Fig. 1C, D). Following treatment with 1.0% NaCl, root length, shoot length, and fresh weight were measured for all plant materials. With the exception of the OsRR22 single mutant, which did not differ significantly from the WT in shoot or root length, all other mutants showed significant increases in all three traits relative to the WT. Among them, the triple mutant ( OsWRKY53 - OsARF18 - OsRR22 ) had the longest roots, whereas the OsWRKY53 - OsRR22 double mutant and the OsARF18 single mutant exhibited the greatest shoot length and the highest fresh weight, respectively (Fig. S5A-C). Under normal field conditions, no significant differences were observed between the mutants and the WT in key agronomic traits such as plant height, tiller number, and grain-setting rate (Fig. S6). Collectively, these results demonstrate that the combined loss of function of OsWRKY53 , OsARF18 , and OsRR22 significantly enhances salt tolerance in Shuanghui 459 without compromising normal growth and development. The collective action of OsWRKY53 , OsRR22 , and OsARF18 enhances rice salt tolerance by elevating antioxidant enzyme activity Under salt stress, excessive uptake and accumulation of Na⁺ in plant tissues leads to ionic imbalance and cytotoxicity [ 28 ] . Quantitative analysis of Na⁺ in roots, stems, and leaves showed that, under non‑saline conditions, all mutants maintained comparable, low Na⁺ levels to the WT (Fig. S7A). Across all genotypes, Na⁺ content was significantly higher in roots than in stems or leaves, with no significant difference between the latter two tissues (Fig. S7B). However, following 24 h of treatment with 1.0% NaCl, distinct accumulation patterns emerged. The OsWRKY53 single mutant, the OsARF18 - OsRR22 double mutant, and the triple mutant all accumulated significantly more Na⁺ in roots, stems, and leaves than the WT. In a tissue‑specific manner, the OsRR22 single mutant showed higher Na⁺ content specifically in roots, while the OsARF18 single mutant and the OsWRKY53 - OsRR22 double mutant accumulated more Na⁺ predominantly in leaves (Fig. 2A-C). Compared to pre-salt treatment, significant differences in Na⁺ content were observed in stems and leaves across all treated plants. Na⁺ content in roots, stems, and leaves of WT, single-gene, and OsWRKY53 - OsRR22 double-gene and triple-gene mutants decreased significantly in that order, with the triple-gene mutant exhibiting the most pronounced trend of change. Na⁺ content in leaves of the OsARF18 - OsRR22 double mutant was significantly higher than in other mutants. Furthermore, the OsARF18 - OsRR22 double mutant exhibited uniquely high Na⁺ content in leaves compared to other mutants. The OsARF18 - OsRR22 double mutant exhibited higher Na⁺ content in leaves compared with the other mutants, and in the osarf18 - osrr22 - 2 mutant, leaf Na⁺ levels were even significantly higher than those in stems. (Fig. S7C). Collectively, these results suggest that these mutants alter Na⁺ partitioning, likely to enhance its retention in roots and stems to limit flux into young leaves, while promoting vacuolar sequestration to mitigate cellular toxicity. Salt stress triggers an overproduction of reactive oxygen species (ROS), resulting in oxidative stress that disrupts cellular metabolism and can lead to cytotoxic effects [ 29 ] . DAB and NBT staining revealed that WT leaves exhibited significantly higher localized production of H₂O₂ and O₂⁻ compared to mutants. The accumulation of H₂O₂ and O₂⁻ progressively decreased in monogenic, bigene, and trigenic mutants, respectively (Fig. 2D). Simultaneously, the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) increased sequentially and were significantly higher than those in WT (Fig. 2E-G), while malondialdehyde (MDA) levels decreased sequentially and were significantly lower than those in WT (Fig. 2H). This result indicates that the enhanced antioxidant enzyme activity in the mutant significantly improves salt tolerance. OsWRKY53 antagonizes OsARF18 and OsRR22 in response to salt stress WRKY transcription factors recognize and bind to the W-box cis-element in promoter regions via their conserved WRKY domain to regulate gene expression [ 30 ] , we identified W-box motifs within the promoters of both OsRR22 and OsARF18 (Fig. 3 A), suggesting they are potential direct targets of OsWRKY53 . Yeast one-hybrid analysis confirmed the direct binding of OsWRKY53 to these promoters, supporting its role as a transcriptional regulator. Dual‑luciferase reporter assays further showed that OsWRKY53 represses the transcriptional activity of the OsRR22 and OsARF18 promoters under non‑stress conditions (Fig. 3 B-E). Consistent with this, RT‑qPCR analysis revealed that loss of OsWRKY53 function relieved its repression, leading to significantly elevated transcript levels of both OsRR22 and OsARF18 compared with the WT (Fig. 4 A, C). Moreover, in the OsWRKY53 - OsRR22 double mutant, OsARF18 expression was also markedly upregulated (Fig. 4 C). Compared with the pre‑treatment levels, OsRR22 expression was upregulated in both the WT and OsARF18 single‑gene mutant after salt treatment, although not statistically significant, whereas it was significantly downregulated in the OsWRKY53 single‑gene mutant (Fig. 4 D). OsWRKY53 expression was significantly downregulated in the WT and OsARF18 single‑gene mutant, significantly upregulated in the OsRR22 single‑gene mutant, and showed a non‑significant increasing trend in the OsARF18 – OsRR22 double‑gene mutant (Fig. 4 E). OsARF18 was significantly upregulated in the WT and OsRR22 single‑gene mutant, and significantly downregulated in the OsWRKY53 single‑gene mutant and the OsWRKY53 – OsRR22 double‑gene mutant (Fig. 4 F).Considering the salt tolerance phenotypes (Fig. 1), OsWRKY53 , OsARF18 , and OSRR22 may not exhibit a linear regulatory relationship but rather involve complex feedback and antagonistic interactions. Therefore, functional redundancy may exist among these three genes under salt stress. OsWRKY53 physically interacts with OsRR22 To further elucidate the molecular mechanism underlying the salt stress response mediated by the OsWRKY53 – OsARF18 – OsRR22 module, protein-protein interaction visualization analysis revealed a potential binding site between OsWRKY53 and amino acids 28–409 of the OsRR22 protein (Fig. 5A). This suggests that OsWRKY53 and OsRR22 may physically interact. Yeast two‑hybrid assays demonstrated that co‑transformation with OsWRKY53 ‑pGADT7 and OsRR22 ‑pGBKT7 supported normal growth and produced blue colonies on SD-ALWH/–Ade/–Leu/–Trp/–His medium supplemented with 10 mM 3‑AT and X‑α‑Gal. In contrast, the negative control (pGADT7 + OsRR22 ‑pGBKT7) showed effective suppression of self‑activation on the same selective medium, confirming a specific interaction between OsWRKY53 and OsRR22 (Fig. 5B). Bimolecular fluorescence complementation (BiFC) assays showed that co‑expression of OsWRKY53 ‑nYFP and OsRR22 ‑cYFP in rice protoplasts produced a reconstituted YFP signal localized in the nucleus, while control combinations yielded no detectable fluorescence (Fig. 5C). These results further confirm that OsWRKY53 and OsRR22 interact specifically within the nuclear compartment. Thus, OsWRKY53 and OsRR22 likely form a protein complex that functions as a transcriptional repressor of downstream salt‑stress‑responsive target genes. Loss‑of‑function mutations in either OsWRKY53 or OsRR22 , or their combined knockout, relieves this repression, resulting in the upregulation of these target genes and thereby enhancing salt tolerance. RNA-seq Comparative Analysis To comprehensively dissect the downstream signaling pathway of the OsWRKY53 – OsARF18 – OsRR22 module in response to salt stress, transcriptome sequencing was performed on roots of two‑week‑old WT and mutant plants treated with 1.0% NaCl. A total of 6,170 differentially expressed genes (DEGs) were identified, of which 4,005 were upregulated and 2,165 were downregulated (Fig. S8). GO analysis revealed that the differentially expressed genes (DEGs) were significantly enriched in molecular functions related to ‘ATP binding’ and ‘electron carrier activity’, cellular components including ‘membrane’, ‘plasma membrane’, ‘vacuole’, and ‘cytoplasmic membrane‑associated vesicles’, and biological processes such as ‘oxidation‑reduction process’ and ‘transmembrane transport’ (Fig. S9A and Table 2). These enriched terms suggest that the mutant may enhance salt tolerance by regulating Na⁺ sequestration and transmembrane transport, which is consistent with the altered Na⁺ levels observed in its roots, stems, and leaves (Fig. 2A-C). A substantial number of differentially expressed genes (DEGs) in both the WT and all mutant lines under salt stress relative to normal growth conditions (Fig. S9B). Intersection analysis of differentially expressed genes identified with 1,177 shared DEGs between the triple mutant and all other genotypes (Fig. S10). This core set of genes likely underlies the markedly enhanced salt tolerance observed in the triple mutant. Furthermore, by intersecting the differentially expressed genes between each mutant and the WT under salt stress and normal conditions, we identified a substantial number of overlapping genes across these comparison groups (Fig. S10). These overlapping genes, along with those unique to each mutant, may also be key factors contributing to the significant increase in salt tolerance observed in the corresponding mutants. To identify the downstream regulatory components of the salt stress response in the triple‑gene mutant, we examined 1,175 differentially expressed genes (DEGs) that were common to the triple mutant and other genotypes under salt stress. This analysis showed that the transcript level of the vacuolar Na⁺/H⁺ antiporter OsNHX1 ( LOC_Os07g47100 ) was significantly upregulated in the triple mutant (Fig. S12). Previous studies have demonstrated that activation of OsNHX1 under salt stress promotes Na⁺ sequestration into vacuoles, thereby reducing cytosolic Na⁺ toxicity [ 31 ] . We further analyzed the expression of Na⁺‑related transporter genes—including OsNHX1 , OsHKT2;1 , OsHKT1;1 , OsHKT1;5 , OsAKT1 , OsHAK1 , and OsSOS1 —as well as antioxidant‑related genes ( OsCatB and OsPOD1 ). Under non‑stress conditions, the basal expression levels of these genes were significantly upregulated or downregulated in each mutant compared with the WT (Fig. 6). This observation may be attributed to the loss of function of OsWRKY53 , OsARF18 , and OsRR22 , as RNA‑seq analysis prior to salt stress treatment revealed a large number of DEGs in all mutant plants relative to the WT (Fig. 13). After salt stress treatment, these genes also exhibited significant up‑ or down‑regulation in the WT and the various mutants (Fig. S14). Notably, compared with the WT, the expression changes of OsNHX1 , OsHKT2;1 , OsHKT1;1 , OsCatB , and OsPOD1 were most pronounced in the triple mutant (Fig. 6A, B, F, H, I), which is consistent with the GO pathways specifically enriched by the DEGs. These results suggest that the mechanism by which OsWRKY53 ‑ OsARF18 ‑ OsRR22 substantially enhances salt tolerance may involve regulating intracellular and extracellular Na⁺ homeostasis through vacuolar Na⁺ sequestration, together with a marked increase in antioxidant enzyme activity to improve salt resistance. In addition, the relative expression levels of OsNHX1 , OsHKT1;1 , OsCatB , and OsPOD1 were significantly higher in the double mutant than in the single mutants (Fig. 6B, F, H, I). Further GO enrichment analysis of differentially expressed genes between the triple mutant and other genotypes under salt stress revealed that, overall, these differentially expressed genes were enriched in the two most significant pathways: redox processes and heme binding. Two most significant genes were identified: OsrbohH ( LOC_Os12g35610 ) and OsPgb1.2 ( LOC_Os03g12510 ) (Table. 3–8). OsrbohH encodes an NADPH oxidase, its overexpression has been shown to lower reactive oxygen species (ROS) accumulation in plants, along with reduced malondialdehyde (MDA) content and increased catalase (CAT) and peroxidase (POD) activities, thereby enhancing tolerance to high‑temperature and drought stress [ 32 ] . OsPgb1.2 encodes a hemoglobin that is induced under salt, drought, and cold stress. Overexpression of OsPgb1.2 improves plant tolerance to K⁺ deficiency stress and is associated with decreased H₂O₂ and ROS levels [ 33 ] . These results align well with the observation that the triple mutant displayed the strongest salt tolerance, the highest antioxidant enzyme activities, and the lowest accumulations of H₂O₂, O₂⁻, and MDA. Discussion Salt stress severely hampers rice production. The identification of salt‑tolerance genes and the development of high‑quality, salt‑tolerant rice varieties are therefore critical for enhancing the utilization of saline‑alkali soils and ensuring national food security [ 1 ] . To date, although scientists have identified and cloned numerous salt‑tolerance‑related genes in rice, progress in enhancing salt tolerance through conventional breeding methods remains slow. Improving or introducing only one or a few related genes often yields limited effects, making it difficult to develop salt‑tolerant varieties suitable for field application. Therefore, it is essential to integrate multiple favorable genes to accelerate the genetic improvement of salt‑tolerant rice cultivars [ 8 ] . This study found that under 1.0% NaCl treatment, the triple‑gene knockout mutant OsWRKY53 ‑ OsARF18 ‑ OsRR22 exhibited a significantly higher survival rate compared with single‑gene mutants, double‑gene mutants, and the WT, reaching approximately 80% (Fig. 1A, B), even under 1.2% NaCl stress, the survival rate remained around 20% (Fig. 1C, D), indicating that the combined action of two or three of these genes substantially enhances salt tolerance in the rice variety Shuanghui 459, without compromising overall plant growth and development (Fig. S6). Furthermore, OsARF18 conferred stronger salt tolerance than OsWRKY53 and OsRR22 , and OsWRKY53 showed greater tolerance than OsRR22 . Previous studies have shown that OsWRKY53 and OsRR22 mediate Na⁺ efflux in roots [ 10 , 13 ] , whereas OsARF18 is involved in Na⁺ removal from aerial tissues [ 11 ] . Before salt treatment in this study, Na⁺ content in stems and leaves showed no significant differences among the plant lines, although root Na⁺ levels were substantially higher than those in shoots (Fig. S7B). After salt treatment, significant differences in Na⁺ content emerged in both stems and leaves across all genotypes (Fig. S7C), and all mutants accumulated more Na⁺ in roots, stems, and leaves compared with the WT. Except for the OsARF18 - OsRR22 double mutant, Na⁺ content in the roots, stems, and leaves of the other genotypes generally displayed a decreasing trend, which was most pronounced in the triple mutant (Fig. 2A-C). Notably, Na⁺ content in the roots and stems of the OsARF18 single mutant and the OsWRKY53 - OsRR22 double mutant did not differ significantly from the WT (Fig. 2A-B), this may be due to the relatively short treatment duration and high salt solution concentration, which have not yet fully activated the salt tolerance-related mechanisms (Fig. 2A-C). The changes in Na⁺ content observed in the roots, stems, and leaves of all mutants under salt treatment clearly suggest that they may resist the toxic effects of Na⁺ accumulation by storing Na⁺ and regulating intracellular and extracellular Na⁺ balance. Notably, the OsARF18 single mutant accumulated significantly more Na⁺ in roots and stems, whereas the OsRR22 single mutant showed pronounced Na⁺ accumulation in leaves. The OsARF18 - OsRR22 double mutant exhibited markedly elevated Na⁺ levels in roots, stems, and leaves, with Na⁺ content in leaves being comparable to or even exceeding that in stems (Fig. 2A-B), While the triple mutant displayed consistently higher Na⁺ levels in roots and stems than the OsARF18 - OsRR22 double mutant, its leaf Na⁺ content was significantly lower than OsARF18 - OsRR22 double mutant (Fig. 2A-C). These results indicate that the triple-gene mutant exhibits a progressive change in function based on the single- and double-gene mutants, suggesting that OsWRKY53 - OsARF18 - OsRR22 may have a certain special mechanism jointly responding to salt stress. Furthermore, the results from DAB and NBT staining, as well as measurements of antioxidant enzyme activities (CAT, SOD, POD) and malondialdehyde (MDA) content, all showed trends consistent with a salt-tolerant phenotype. Specifically, the local accumulation of H₂O₂ and O₂⁻ in the leaves of WT, single-gene, double-gene, and triple-gene mutants decreased successively (Fig. 2D). Correspondingly, antioxidant enzyme activities increased sequentially, while the MDA content decreased (Fig. 2E-H). Collectively, these results suggest that although Na⁺ gradually accumulates in the mutants, coordinated changes in endogenous metabolites and enhanced antioxidant capacity enable more effective scavenging of reactive oxygen species (ROS) generated under excessive Na⁺ stress. Transcription factors (TFs) play pivotal roles in regulating plant growth and development, as well as in responding to diverse biotic and abiotic stresses, by activating or repressing multiple genes within downstream regulatory networks [ 34 , 35 ] . A single transcription factor (TF) can regulate the expression of numerous genes within a pathway [ 36 ] , while multiple TFs—such as NAC, MYB, AP2/ERF, bZIP, and WRKY—form intricate regulatory networks. Changes in their gene expression directly influence downstream gene expression within these regulatory networks, triggering a cascade of pleiotropic responses such as functional redundancy or functional overlap [ 37 , 38 ] . Previous studies have shown that WRKY transcription factors bind specifically to the W‑box elements within the promoters of target genes [ 30 ] . In this study, we demonstrated that under non‑stress conditions, OsWRKY53 specifically binds to the W‑box motifs in the promoters of OsARF18 and OsRR22 and represses their expression (Fig. 3 ). Subsequent RT‑qPCR assays corroborated these findings: the loss of OsWRKY53 function released its transcriptional repression of OsARF18 and OsRR22 , resulting in their upregulation relative to the WT (Fig. 4 A, C). Conversely, loss of function in OsARF18 or OsRR22 led to downregulation of OsWRKY53 expression compared with the WT (Fig. 4 B). After salt treatment, the WT exhibited upregulation of OsRR22 and OsARF18 but downregulation of OsWRKY53 relative to the pre‑treatment levels (Fig. 4 D, F), In the OsRR22 single mutant, both OsWRKY53 and OsARF18 were significantly upregulated (Fig. 4 E, F), suggesting an antagonistic interaction between OsWRKY53 and OsARF18 in this background. Notably, however, OsWRKY53 expression remained lower than in the WT (Fig. 4 B), indicating that even with its antagonistic role in the OsRR22 mutant, OsWRKY53 still confers stronger salt tolerance than the WT. Meanwhile, OsARF18 expression remained significantly higher than in the WT (Fig. 4 C). This indicates that under the combined effects of OsRR22 functional loss and partial antagonism by OsWRKY53 in the OsRR22 monohybrid mutant, the elevated expression of OsARF18 partially suppresses the enhanced salt tolerance. In the OsWRKY53 single mutant, the expression levels of OsRR22 and OsARF18 were significantly elevated compared with the WT before salt treatment (Fig. 4 A, C). Following salt stress, both genes were significantly downregulated relative to their pre‑stress levels (Fig. 4 D, F) yet remained higher than in the WT (Fig. 4 A, C), these results suggest that OsRR22 and OsARF18 still retained partial antagonistic activity under these conditions. In the OsRR22 single mutant, OsWRKY53 and OsARF18 expressions were significantly upregulated (Fig. 4 E, F). Conversely, in the OsWRKY53 single mutant, OsRR22 and OsARF18 were significantly downregulated after salt treatment (Fig. 4 D, F). These opposing expression patterns may contribute to the stronger salt tolerance observed in the OsWRKY53 mutant compared to the OsRR22 mutant. In the OsARF18 single mutant, OsRR22 showed no significant change relative to the WT before salt stress (Fig. 4 A), whereas OsWRKY53 was significantly lower (Fig. 4 B). Following salt treatment, OsRR22 expression increased but not significantly (Fig. 4 D), while OsWRKY53 decreased further (Fig. 4 E). Compared to the WT, OsRR22 remained lower (Fig. 4 A), and OsWRKY53 was the most suppressed (Fig. 4 B). These results indicate that OsRR22 likely exerts only minimal antagonistic effects in this background. The pronounced downregulation of OsWRKY53 may be a key factor underlying the markedly stronger salt tolerance of the OsARF18 single mutant relative to the OsWRKY53 and OsRR22 single mutants. In the OsARF18 - OsRR22 double mutant, the expression of OsWRKY53 increased after salt treatment but did not reach statistical significance and remained markedly lower than in the WT (Fig. 4 B, E), this likely represents one of the key reasons why its salt tolerance is improved yet remains lower than that of the triple mutant. In the OsWRKY53 - OsRR22 double mutant, OsARF18 expression was significantly higher than in the WT before salt treatment but declined following stress exposure (Fig. 4 C, F), this may be one reason why the salt tolerance of the OsWRKY53 - OsRR22 double mutant shows no significant difference from that of the OsARF18 - OsRR22 double mutant. The above results indicate that OsWRKY53 , OsARF18 , and OsRR22 form a mutually antagonistic regulatory network. The loss of function in all three genes completely disrupts this antagonistic interplay, which in turn induces transcriptional reprogramming within the downstream regulatory networks of these transcription factors, ultimately leading to a further enhancement of salt tolerance in the triple mutant. In addition, proteins from different transcription factor families can interact to form protein complexes that collaboratively regulate the expression of downstream genes. Recent studies reveal that the cold stress‑upregulated VQ protein OsVQ3 interacts with the transcriptional repressor OsWRKY7 , inhibiting its transcriptional activity and DNA‑binding capacity, this interaction further enhances the expression of downstream target genes, thereby improving cold tolerance in rice [ 35 ] . In tomato, SiVQ10 physically interacts with SiWRKY51 to synergistically activate SiP5CS1 expression, promoting proline accumulation and conferring resistance to cold stress [ 39 ] . Similarly, in rice, the transcription factors OsbZIP61 and OsRF2b interact physically to form a heterodimer, which strengthens the DNA‑binding activity of OsRF2b . This complex directly suppresses the expression of OsNRT1.1B , leading to improved yield and nitrogen use efficiency [ 40 ] . This study shows that OsWRKY53 and OsRR22 directly interact and co-localize in the nucleus (Fig. 5A-C). Protein–protein interaction prediction and yeast two‑hybrid assays further suggest that OsWRKY53 may also physically interact with OsARF18 (Fig. S15). These results imply that OsWRKY53 not only transcriptionally regulates OsARF18 and OsRR22 but could also form a protein complex with them within a common regulatory network, where they cooperate to repress downstream target genes. Loss of function in the double or triple mutants relieves this repression, leading to the upregulation of positively regulated downstream genes and consequently enhancing salt tolerance. These findings reveal the sophisticated genetic interplay among OsWRKY53 , OsARF18 , and OsRR22 . Therefore, future studies will aim to further delineate this genetic network to uncover the regulatory mechanisms underlying the salt stress response in the OsWRKY53 - OsARF18 - OsRR22 triple mutant. Salt stress primarily results from excessive accumulation of soluble sodium ions (Na⁺) in the soil, plants passively absorb surplus Na⁺, leading to disruption of cellular ion homeostasis and subsequently triggering a series of physiological injuries [ 41 – 43 ] . To maintain Na⁺ balance, rice employs multiple adaptive strategies, including limiting Na⁺ uptake, promoting Na⁺ efflux, and compartmentalizing Na⁺ [ 44 – 47 ] . To further identify key targets in the downstream regulatory network altered by the OsWRKY53 - OsARF18 - OsRR22 triple knockout, we collected roots from each mutant and the WT under salt stress and normal conditions for RNA-seq analysis. We then intersected the differentially expressed genes between the triple mutant and the other mutants and the WT. The results revealed that the vacuolar Na⁺/H⁺ antiporter OsNHX1 was significantly up regulated in the triple mutant (Fig. S12). RT‑qPCR further confirmed these observations: after 24 h of salt treatment, OsNHX1 expression was significantly elevated in both the OsARF18 ‑ OsRR22 double mutant and the triple mutant (Fig. 6). OsNHX1 encodes a vacuolar Na⁺/H⁺ antiporter that sequesters Na⁺ into vacuoles, thereby alleviating the toxicity caused by excessive cytoplasmic Na⁺ accumulation. Previous studies have shown that Arabidopsis plants overexpressing AtNHX1 accumulate more Na⁺ than WT plants under salt stress, yet exhibit less leaf chlorosis and better growth [ 48 ] . Similarly, in Brassica napus, overexpression of AtNHX1 leads to higher Na⁺ accumulation in leaves compared to the WT but reduces Na⁺‑related toxicity [ 49 ] . In rice, both OsNHX1 -overexpressing lines and the WT showed progressive increases in intracellular Na⁺ as salt concentration rose. At high salinity, OsNHX1 -overexpressing plants maintained higher intracellular Na⁺ levels than the WT yet grew significantly better, this was attributed to OsNHX1 -mediated compartmentalization of Na⁺ into vacuoles for storage, thereby reducing cytoplasmic Na⁺ accumulation and its toxic effects [ 50 ] . These results are consistent with the higher Na⁺ content in mutant roots, stems, and leaves compared to the WT, yet the mutants exhibited significantly better growth than the WT. Furthermore, the substantial upregulation of OsrbohH and OsPgb1.2 in the triple mutant endows it with potent antioxidant enzyme activity and ROS scavenging capacity. Moreover, expression of OsNHX1 , OsHKT2;1 , OsHKT1;1 , OsCatB , and OsPOD1 was significantly elevated in the triple mutant relative to the WT (Fig. 6A, B, F, H, I). These findings indicate that the triple mutant enhances salt tolerance by sequestering Na⁺ into vacuoles for storage, regulating intracellular and extracellular Na⁺ balance, and significantly boosting antioxidant enzyme activity. Conclusions This study found that the aggregation of OsWRKY53 - OsRR22 and OsARF18 - OsRR22 in both dual and triple gene combinations significantly enhances salt tolerance in rice varieties, with the triple gene aggregation exhibiting the strongest salt tolerance. The loss of OsWRKY53 function relieved transcriptional repression on OsARF18 and OsRR22 , leading to upregulation of the latter two under normal conditions and downregulation under salt stress while remaining higher than WT. Conversely, loss of OsARF18 or OsRR22 function resulted in down‑regulation of OsWRKY53 . These interactions indicate that OsWRKY53 , OsARF18 , and OsRR22 form a mutually antagonistic regulatory loop. Double‑gene combinations partially alleviated this antagonism, whereas the triple‑gene combination completely disrupted it. This disruption led to significant up‑regulation of key genes such as OsNHX1 , OsrbohH , OsPgb1.2 , OsHKT2;1 , OsHKT1;1 , OsCatB , and OsPOD1 . These changes promote vacuolar sequestration of Na⁺, improve intra‑ and extracellular Na⁺ homeostasis, enhance leaf antioxidant enzyme activities (CAT, SOD, POD), and reduce malondialdehyde (MDA) content, collectively contributing to markedly enhanced salt tolerance. Importantly, pyramiding OsWRKY53 , OsARF18 , and OsRR22 did not compromise normal plant growth and development. These findings provide new strategies for polygenic aggregation to improve rice salt tolerance. Abbreviations ROS Reactive oxygen species H₂O₂ Hydrogen Peroxide O₂⁻ Superoxide Anion SOD Superoxide Dismutase CAT Catalase MDA Malondialdehyde DEGs Differentially expressed genes GO Gene ontology Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Availability of data and materials All data generated or analysed during this study are included in this article and its supplementary information files. The raw data used in this study has been uploaded into China National center for Bioinformation system with the primary accession code GSA: CRA062376 . Competing interests The authors declare no competing interests. Funding This research was supported by the Top Ten Technical Breakthrough Projects of Hunan Province (Grant No. 2025QK1006). Authors’ contributions P.S., H.D., F.S., W.Z. and Q.H. conceptualized the research;Y.Z. performed the experiments; Y.Z., P.S., P.F., J.Z. and X.L. analyzed the data; P.S. and Y.Z. wrote the paper; all authors reviewed the manuscript and agreed to its submission. Acknowledgements Not applicable. Author details 1 State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, China 2 College of Tropical Agriculture and Forestry, Hainan University, Danzhou, Hainan 571737, China 3 Longping Agricultural College, Hunan University, Changsha 410125, China 4 Yuelu Shan Laboratory, Changsha 410128, China References Liang XY, Li JF, Yang YQ, Jiang CF, Guo Y. Designing salt stress‐resilient crops: Current progress and future challenges. Journal of Integrative Plant Biology. 2024;66(3):303-329. Hernández JA. Salinity Tolerance in Plants: Trends and Perspectives. International Journal of Molecular Sciences. 2019;20(10):2408. Lutts J, Kinet JM, Bouharmont J. 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SupplementaryData2.xlsx Supplementary Material 2. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 04 May, 2026 Reviews received at journal 19 Apr, 2026 Reviews received at journal 17 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviewers agreed at journal 04 Apr, 2026 Reviews received at journal 03 Apr, 2026 Reviewers agreed at journal 02 Apr, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers agreed at journal 27 Mar, 2026 Reviewers agreed at journal 26 Mar, 2026 Reviewers invited by journal 26 Mar, 2026 Editor invited by journal 20 Mar, 2026 Editor assigned by journal 04 Feb, 2026 Submission checks completed at journal 04 Feb, 2026 First submitted to journal 02 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-8764411","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":613371117,"identity":"34f88805-1687-4320-aa82-ecef910f0160","order_by":0,"name":"Yi Zhou","email":"","orcid":"","institution":"State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Zhou","suffix":""},{"id":613371118,"identity":"07fdefd2-5b42-43f2-bd7c-de16fe7b0b8b","order_by":1,"name":"Pengpeng Fang","email":"","orcid":"","institution":"Longping Agricultural College, Hunan University","correspondingAuthor":false,"prefix":"","firstName":"Pengpeng","middleName":"","lastName":"Fang","suffix":""},{"id":613371122,"identity":"40c5de6b-2f36-4440-88e9-71e13d11977a","order_by":2,"name":"Jia Zeng","email":"","orcid":"","institution":"Longping Agricultural College, Hunan University","correspondingAuthor":false,"prefix":"","firstName":"Jia","middleName":"","lastName":"Zeng","suffix":""},{"id":613371125,"identity":"b1f24ec8-60c2-4e95-a94b-257b7fcdf384","order_by":3,"name":"Xinpeng Li","email":"","orcid":"","institution":"Yuelu Shan Laboratory","correspondingAuthor":false,"prefix":"","firstName":"Xinpeng","middleName":"","lastName":"Li","suffix":""},{"id":613371135,"identity":"f3271c0c-7aad-406a-bb73-d71679377382","order_by":4,"name":"Qiang He","email":"","orcid":"","institution":"State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center","correspondingAuthor":false,"prefix":"","firstName":"Qiang","middleName":"","lastName":"He","suffix":""},{"id":613371139,"identity":"786d02c7-a0d6-4888-b3d5-27eb15b94f65","order_by":5,"name":"Wuhan Zhang","email":"","orcid":"","institution":"State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center","correspondingAuthor":false,"prefix":"","firstName":"Wuhan","middleName":"","lastName":"Zhang","suffix":""},{"id":613371142,"identity":"6232b9e9-00d7-452e-ac40-d10a01735862","order_by":6,"name":"Huafeng Deng","email":"","orcid":"","institution":"State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center","correspondingAuthor":false,"prefix":"","firstName":"Huafeng","middleName":"","lastName":"Deng","suffix":""},{"id":613371144,"identity":"aaf073ec-1663-4741-a2c6-5532def0936b","order_by":7,"name":"Fu Shu","email":"","orcid":"","institution":"State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center","correspondingAuthor":false,"prefix":"","firstName":"Fu","middleName":"","lastName":"Shu","suffix":""},{"id":613371145,"identity":"a38cd76d-cd19-41af-a2b0-221ed004736e","order_by":8,"name":"Pingyong Sun","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIie3PsQrCMBCA4cSD63I4FyrGR7AU7OvEqUu7d+hWSBfxCUTfwjldOtUncCkUurl3Eltw99wE8w9HhvvgIoTL9Yt588iLNXqlZRKYR9tES2r0F0Qa2J/9dMMTqvSGnhATI1Ihxvz6mciS4oholRlxs/LQ3hl3AWFAPmZGHjVIwyAI3hDQFhKEabIIgdgFpEEjcokPFIcn24SGUNesv6iqGrrHs1Dq0tfdmDPIFC7o/bKs/ZnIkbvqcrlc/9kLzCMxFTnNKGIAAAAASUVORK5CYII=","orcid":"","institution":"State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center","correspondingAuthor":true,"prefix":"","firstName":"Pingyong","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2026-02-02 11:38:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8764411/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8764411/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105744882,"identity":"c00813bc-3d28-4391-93e2-399ac05f7781","added_by":"auto","created_at":"2026-03-30 14:05:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":813764,"visible":true,"origin":"","legend":"\u003cp\u003eLoss of function of \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e significantly enhances rice salt tolerance. \u003cstrong\u003e(A, B)\u003c/strong\u003e Phenotype and survival rate of WT and mutant plants following treatment with 1.0 % NaCl. \u003cstrong\u003e(C, D)\u003c/strong\u003e Phenotype and survival rate of WT and mutant plants following treatment with 1.2 % NaCl. Data are presented as mean ± SD of three biological replicates. Significant differences between different genotypes were determined by one‑way ANOVA. \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001; ns, not significant.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/20f0b58097631f2e1dcf3ec3.png"},{"id":105744883,"identity":"1e4c7e85-a413-482a-92cd-9639c55c01ee","added_by":"auto","created_at":"2026-03-30 14:05:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":370547,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of endogenous substance content in each genotype. \u003cstrong\u003e(A-C)\u003c/strong\u003e Na⁺ content in the roots, stems, and leaves of WT and mutant plants after treatment with 1.0 % NaCl for 0 and 24 h, respectively. \u003cstrong\u003e(D)\u003c/strong\u003e Histochemical staining of H₂O₂ (DAB) and O₂⁻ (NBT) in leaves sampled at 0 and 24 h after 1.0 % NaCl treatment. \u003cstrong\u003e(E-H)\u003c/strong\u003e Antioxidant enzyme activities (CAT, SOD, and POD) and malondialdehyde (MDA) content after 0h and 24 h NaCl treatment. Data are presented as mean ± standard deviation (SD) of three independent biological replicates. Significant differences between mutants and the WT were determined by one-way ANOVA and are indicated as follows: \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/0656a2c1463772c3ec2d6d24.png"},{"id":105744890,"identity":"2abfe260-9624-46ce-a67c-d162a0bffb06","added_by":"auto","created_at":"2026-03-30 14:05:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":469121,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOsWRKY53 \u003c/em\u003eregulates the expression of \u003cem\u003eOsARF18 \u003c/em\u003eand \u003cem\u003eOsRR22.\u003c/em\u003e\u003cstrong\u003e (A) \u003c/strong\u003eSchematic representation of the binding of the \u003cem\u003eOsWRKY53\u003c/em\u003eWRKY domain to promoter regions containing the W‑box cis‑element. \u003cstrong\u003e(B)\u003c/strong\u003e Yeast one‑hybrid assays were performed to examine the binding of \u003cem\u003eOsWRKY53\u003c/em\u003e to the promoter of \u003cem\u003eOsRR22\u003c/em\u003e. Yeast cells co‑transformed with the bait construct (pHIS2‑\u003cem\u003eOsRR22\u003c/em\u003e) and the prey construct (pGADT7‑\u003cem\u003eOsWRKY53\u003c/em\u003e) were cultured on SD/–Trp/–Leu medium containing 3‑AT, along with the following controls: a positive control (p53‑HIS2 + pGAD‑Rec53), a negative control (pGADT7 + p53‑HIS2), and a self‑activation control (pHIS2‑\u003cem\u003eOsRR22\u003c/em\u003e + pGADT7‑Rec2). Plates were incubated for 3–5 days before observation. \u003cstrong\u003e(C)\u003c/strong\u003e LUC signal in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves infiltrated with specified effector proteins and reporter genes. \u003cstrong\u003e(D)\u003c/strong\u003e Yeast one‑hybrid assays were performed to examine the binding of \u003cem\u003eOsWRKY53\u003c/em\u003e to the promoter of \u003cem\u003eOsARF18\u003c/em\u003e. Yeast cells co‑transformed with the bait construct (pHIS2‑\u003cem\u003eOsARF18\u003c/em\u003e) and the prey construct (pGADT7‑\u003cem\u003eOsWRKY53\u003c/em\u003e) were cultured on SD/–Trp/–Leu medium containing 3‑AT, along with the following controls: a positive control (p53‑HIS2 + pGAD‑Rec53), a negative control (pGADT7 + p53‑HIS2), and a self‑activation control (pHIS2‑\u003cem\u003eOsARF18\u003c/em\u003e + pGADT7‑Rec2). Plates were incubated for 3–5 days before observation. (E) LUC signal in \u003cem\u003eN. benthamiana\u003c/em\u003eleaves infiltrated with specified effector proteins and reporter genes.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/da80f45da2b91a43f077809c.png"},{"id":105912116,"identity":"175c51d1-d507-4d25-9b89-79aa4af98786","added_by":"auto","created_at":"2026-04-01 10:58:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":149906,"visible":true,"origin":"","legend":"\u003cp\u003eExpression Analysis of OsWRKY53, OsARF18, and OsRR22. \u003cstrong\u003e(A-C)\u003c/strong\u003e Transcript levels of \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsRR22\u003c/em\u003e, and \u003cem\u003eOsARF18\u003c/em\u003e in corresponding mutant lines and WT plants before (0 h) and after 24 h of treatment with 1.0 % NaCl, with comparisons shown across genotypes. \u003cstrong\u003e(D-F)\u003c/strong\u003e Transcript levels of \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsRR22\u003c/em\u003e, and \u003cem\u003eOsARF18\u003c/em\u003e in the corresponding mutant lines and WT plants before (0 h) and after 24 h of treatment with 1.0 % NaCl. Data are presented as mean ± standard deviation (SD) of three independent biological replicates. Significant differences between mutants and the WT were determined by one-way ANOVA and are indicated as follows: \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/b6b1f353dd6cc223d88fd340.png"},{"id":105752194,"identity":"15a4a436-5af5-40a6-a038-56bf8c6e259f","added_by":"auto","created_at":"2026-03-30 15:55:41","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":386876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eOsWRKY53 \u003c/em\u003ephysically interacts with \u003cem\u003eOsRR22\u003c/em\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Potential amino acid binding sites between \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e, with dark blue and light blue representing the three-dimensional protein structures of \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e, respectively. \u003cstrong\u003e(B)\u003c/strong\u003e Yeast two‑hybrid analysis of the \u003cem\u003eOsWRKY53\u003c/em\u003e–\u003cem\u003eOsRR22\u003c/em\u003einteraction. Yeast cells co‑expressing pGBKT7‑\u003cem\u003eOsRR22\u003c/em\u003e (bait) and pGADT7‑\u003cem\u003eOsWRKY53\u003c/em\u003e(prey) were selected on SD/–Trp/–Leu medium and then grown on SD/–Trp/–Leu/–His/–Ade medium supplemented with 3‑AT and X‑α‑Gal. Controls included a positive pair (pGBKT7‑53 + pGADT7‑T), a negative pair (pGBKT7‑Lam + pGADT7‑T), and a self‑activation control (pGBKT7‑\u003cem\u003eOsRR22 \u003c/em\u003e+ pGADT7). Plates were incubated at 30 °C for 3–5 days. \u003cstrong\u003e(C)\u003c/strong\u003e A bimolecular fluorescence complementation (BiFC) assay confirmed the interaction between \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e fusion proteins in rice protoplasts, using GHD7‑mCherry as a nuclear marker. Scale bar:10 um.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/621f94e19db26e21f2564c9a.png"},{"id":105744884,"identity":"e424ab0c-5331-4982-acd3-4d145630e1c8","added_by":"auto","created_at":"2026-03-30 14:05:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":223161,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of Na⁺-associated transporter genes in roots of WT and various mutants treated with 1.0% NaCl solution for 0h and 24h. \u003cstrong\u003e(A-C)\u003c/strong\u003e Members of the rice high‑affinity K⁺ transporter (HKT) family: \u003cem\u003eOsHKT2;1\u003c/em\u003e, \u003cem\u003eOsHKT1;1\u003c/em\u003e, and \u003cem\u003eOsHKT1;5\u003c/em\u003e. \u003cstrong\u003e(D, E)\u003c/strong\u003e K⁺ transporters \u003cem\u003eOsAKT1\u003c/em\u003e and \u003cem\u003eOsHAK1\u003c/em\u003e. \u003cstrong\u003e(F)\u003c/strong\u003e Vacuolar Na⁺/H⁺ antiporter \u003cem\u003eOsNHX1\u003c/em\u003e. (G) Plasma membrane Na⁺/H⁺ antiporter \u003cem\u003eOsSOS1\u003c/em\u003e. \u003cstrong\u003e(H, I)\u003c/strong\u003e Antioxidant enzyme activity genes \u003cem\u003eOsCatB\u003c/em\u003e and \u003cem\u003eOsPOD1\u003c/em\u003e. Data are presented as mean ± SD of three independent biological replicates. Significant differences between mutants and the WT were assessed by one‑way ANOVA and are indicated as follows: \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e***\u003c/sup\u003eP \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/693aaa0457099718e860a736.png"},{"id":107705095,"identity":"f2e5649a-b58c-4956-884b-aed51f769776","added_by":"auto","created_at":"2026-04-24 09:08:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3145538,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/c3b01907-9477-40df-9a8b-2fb8d5c84af5.pdf"},{"id":105744887,"identity":"e682791b-84dd-480a-82ee-1632c9ec863d","added_by":"auto","created_at":"2026-03-30 14:05:45","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4282666,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material 1.\u003c/p\u003e","description":"","filename":"SupplementaryData1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/35fe52bdab770398497230e6.docx"},{"id":105744888,"identity":"5c1265df-6f12-4d5d-8976-171dff372635","added_by":"auto","created_at":"2026-03-30 14:05:46","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1995908,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Material 2.\u003c/p\u003e","description":"","filename":"SupplementaryData2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8764411/v1/9bf2f209da7790c423f1aafd.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil salinity stress constitutes a primary global abiotic constraint that severely limits crop growth, development, and yield\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Among cereal crops, rice (Oryza sativa L.) is particularly sensitive to salinity stress\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e, yet progress in developing salt‑tolerant rice cultivars has been slow. This is primarily because salt tolerance in rice entails the coordinated action of numerous physiological and biochemical processes, constituting a polygenic quantitative trait with a complex genetic architecture that is strongly influenced by environmental factors\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Previous attempts at single‑gene‑based improvement have yielded modest outcomes, and the repertoire of validated genes available for genetic enhancement remains constrained \u003csup\u003e[\u003cspan additionalcitationids=\"CR6 CR7\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e, therefore, innovation and breakthroughs in genetic improvement methods are needed.\u003c/p\u003e \u003cp\u003ePrevious studies have identified \u003cem\u003eOsRR22\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eOsWRKY53\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eOsARF18\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e as key negative regulators of salt tolerance in rice. \u003cem\u003eOsRR22\u003c/em\u003e encodes a B‑type response regulator of 696 amino acids\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, predominantly expressed in the root cortex and vascular tissues, where it directly upregulates \u003cem\u003eOsHKT2;1\u003c/em\u003e expression. \u003cem\u003eOsSLR1\u003c/em\u003e functions as a transcriptional co‑activator in this process. Loss‑of‑function mutations in either \u003cem\u003eOsSLR1\u003c/em\u003e or \u003cem\u003eOsRR22\u003c/em\u003e disrupt \u003cem\u003eOsHKT2;1\u003c/em\u003e activation, leading to its downregulation and reduced Na⁺ accumulation in roots\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Yu et al.\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003eidentified \u003cem\u003eOsWRKY53\u003c/em\u003e as a pivotal negative regulator of salt tolerance in rice via genome‑wide association analysis. Their study revealed that \u003cem\u003eOsWRKY53\u003c/em\u003e specifically binds to W‑box cis‑elements in the promoters of \u003cem\u003eOsMKK10.2\u003c/em\u003e and \u003cem\u003eOsHKT1;5\u003c/em\u003e, leading to transcriptional repression of both genes. Loss‑of‑function of \u003cem\u003eOsWRKY53\u003c/em\u003e relieved this repression, resulting in upregulated expression of \u003cem\u003eOsMKK10.2\u003c/em\u003e and \u003cem\u003eOsHKT1;5\u003c/em\u003e. This regulatory change promotes Na⁺ efflux from xylem and phloem parenchyma cells, thereby enhancing salinity tolerance. Furthermore, in the rice cultivar Zhonghua 11, mutation of \u003cem\u003eOsWRKY53\u003c/em\u003e upregulates \u003cem\u003eOsMYB63\u003c/em\u003e expression, which contributes to stronger resistance against bacterial leaf blight compared with the WT during the booting stage\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Tang et al.\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003eexposed the rice variety LJ11 to low‑temperature (15\u0026deg;C) treatment during the booting stage. They reported that loss‑of‑function of \u003cem\u003eOsWRKY53\u003c/em\u003e upregulated gibberellin biosynthesis genes, leading to increased accumulation of bioactive gibberellins in anthers. This enhancement in cold tolerance at booting occurred without compromising grain yield. In a separate study, Deng et al.\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e identified RST1 as \u003cem\u003eOsARF18\u003c/em\u003e. Analysis of an \u003cem\u003eOsARF18\u003c/em\u003e loss‑of‑function mutant derived from a Nipponbare population demonstrated that the mutation promotes asparagine synthesis, reduces excessive NH₄⁺ accumulation in shoots, and maintains elevated K⁺/Na⁺ ratios in aerial tissues, collectively contributing to enhanced salt tolerance. Furthermore, in the Zhonghua 11 background, \u003cem\u003eOsARF18\u003c/em\u003e loss‑of‑function elevates transcript levels of \u003cem\u003eOsGS1;1\u003c/em\u003e and \u003cem\u003eOsGS1;2\u003c/em\u003e, increases glutamine synthetase (GS) activity, and upregulates detoxification‑related genes, conferring stronger glufosinate resistance compared to the WT\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCRISPR/Cas9 is the third-generation gene editing technology following zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs)\u003csup\u003e[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In contrast to conventional breeding methods, which are often time‑consuming and labor‑intensive, CRISPR/Cas9‑mediated editing enables precise, simultaneous modification of multiple target genes within a relatively short timeframe, thereby facilitating targeted trait enhancement. This technology significantly accelerates the breeding process and shortens variety development cycles\u003csup\u003e[\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. To date, the CRISPR/Cas9 system has been successfully implemented for genome editing in a broad range of organisms, including rice\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. For instance, Zhang et al.\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003esubstantially improved salt tolerance in rice by targeting \u003cem\u003eOsRR22\u003c/em\u003e using CRISPR/Cas9. In another study, Zhou et al.\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003eapplied CRISPR/Cas9 to the widely used two‑line male‑sterile line Longke 638S, simultaneously editing three known broad‑spectrum blast resistance genes\u0026mdash;\u003cem\u003eBsr‑d1\u003c/em\u003e, \u003cem\u003ePi21\u003c/em\u003e, and \u003cem\u003eERF922\u003c/em\u003e\u0026mdash;to generate triple‑gene and corresponding single‑gene mutants. Phenotypic assessment revealed that the triple-gene mutant and the \u003cem\u003eERF922\u003c/em\u003e single-gene mutant exhibited the most pronounced disease resistance among all mutant lines and the WT control.\u003c/p\u003e \u003cp\u003e \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e serve as key regulators not only in the salt‑stress response but also in the growth and development of rice\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan additionalcitationids=\"CR25 CR26\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. To date, the relative contributions of these three genes to salt tolerance and whether their aggregation within the same rice variety exhibits an additive effect remain unknown. In this study, CRISPR/Cas9-mediated genome editing was employed to simultaneously disrupt these three salt-tolerance repressors in the rice cultivar Shuanghui 459, successfully generating single-gene, double-gene (excluding the \u003cem\u003eOsWRKY53\u003c/em\u003e\u0026ndash;\u003cem\u003eOsRR22\u003c/em\u003e combination), and triple-gene mutant lines. The results showed that after treatment with 1.0% NaCl, the survival rates of single-gene, double-gene, and triple-gene mutants increased sequentially, with the survival rate of the triple-gene mutant reaching about 80%. More importantly, under non‑stress conditions, loss of \u003cem\u003eOsWRKY53\u003c/em\u003e function increased the transcript levels of \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e compared with the WT. Conversely, loss of function of \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e decreased \u003cem\u003eOsWRKY53\u003c/em\u003e expression. These results indicate that \u003cem\u003eOsWRKY53\u003c/em\u003e forms a mutually antagonistic negative‑feedback loop with \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e. The improved salt tolerance of the double and triple mutants is likely attributable to an additive effect resulting from the disruption of this antagonistic interaction. These findings provide new strategies for improving salt tolerance in rice through multi‑gene pyramiding.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of transgenic plants\u003c/h2\u003e \u003cp\u003eBased on the principle of CRISPR/Cas9-mediated genome editing, a specific protospacer adjacent motif (PAM) sequence was selected within the first exon of each target gene (\u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e). The corresponding single-guide RNA (sgRNA) was assembled and cloned into the binary vector pEGCas9Pubi-H. The resulting construct was introduced into rice cultivar Shuanghui 459 via Agrobacterium tumefaciens (strain EHA105)-mediated transformation. Mutations at the target loci in transgenic plants were validated by Sanger sequencing. The following mutant lines were successfully generated: single-gene knockout mutants of \u003cem\u003eOsWRKY53\u003c/em\u003e (\u003cem\u003eoswrky53-1\u003c/em\u003e, \u003cem\u003eoswrky53-2\u003c/em\u003e), \u003cem\u003eOsARF18\u003c/em\u003e (\u003cem\u003eosarf18-1\u003c/em\u003e, \u003cem\u003eosarf18-2\u003c/em\u003e), and \u003cem\u003eOsRR22\u003c/em\u003e (\u003cem\u003eosrr22-1\u003c/em\u003e, \u003cem\u003eosrr22-2\u003c/em\u003e), double-gene knockout mutants of \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e (\u003cem\u003eoswrky53\u003c/em\u003e-\u003cem\u003eosrr22-1\u003c/em\u003e, \u003cem\u003eoswrky53\u003c/em\u003e-\u003cem\u003eosrr22-2\u003c/em\u003e) and \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e (\u003cem\u003eosarf18\u003c/em\u003e-\u003cem\u003eosrr22-1\u003c/em\u003e, \u003cem\u003eosarf18\u003c/em\u003e-\u003cem\u003eosrr22\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003e), and the triple-gene knockout mutant of \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e (\u003cem\u003eoswrky53\u003c/em\u003e-\u003cem\u003eosarf18\u003c/em\u003e-\u003cem\u003eosrr22-1\u003c/em\u003e, \u003cem\u003eoswrky53\u003c/em\u003e-\u003cem\u003eosarf18\u003c/em\u003e-\u003cem\u003eosrr22-2\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSalt tolerance treatment\u003c/h3\u003e\n\u003cp\u003eSeeds of rice cultivar Shuanghui 459 and transgenic materials were incubated at 37\u0026deg;C for 48 h to break dormancy. They were then surface-sterilized with 15% sodium hypochlorite for 15 min, rinsed thoroughly five times with distilled water, and soaked in distilled water at room temperature for 48 h. Subsequently, seeds were returned to 37\u0026deg;C for an additional 48 h to promote germination. Germinated seeds were transferred to a perforated cultivation plate and grown in a controlled-environment chamber under a 14-h-light/10-h-dark photoperiod. Day/night temperatures were maintained at 28\u0026deg;C/26\u0026deg;C, with a light intensity of 30,000 lx and 70% relative humidity. Two-week-old seedlings were subjected to salt stress by treatment with 1.0% or 1.2% NaCl solution for 7 d, followed by a 7‑d recovery period in Yoshida nutrient solution without NaCl. Each treatment consisted of three biological replicates, with 48 seedlings per replicate. Salt tolerance was quantified by comparing the survival rates of salt-treated seedlings to those of the untreated control group.\u003c/p\u003e \u003cp\u003eAfter 7 days of treatment with 1.0% NaCl and a subsequent 7-day recovery period, whole seedlings from both control and treated groups were harvested for fresh weight measurement. Following this, shoot and root lengths were determined using five randomly selected seedlings per biological replicate, and mean values were calculated.\u003c/p\u003e\n\u003ch3\u003eDAB and NBT staining\u003c/h3\u003e\n\u003cp\u003eLeaves were harvested from 14‑day‑old seedlings that had been treated with 1.0% NaCl for 0 or 24 h. The experiment consisted of three biological replicates, each comprising a pool of leaves from five seedlings. Histochemical staining to visualize H₂O₂ and O₂⁻ accumulation was performed using 3,3\u0026rsquo;‑diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) kits, respectively, according to the manufacturer\u0026rsquo;s instructions (Leagene Biotechnology, China). Briefly, leaf samples were vacuum‑infiltrated with the corresponding staining working solution in the dark for 30 min. After staining, samples were rinsed 3\u0026ndash;5 times with distilled water to remove excess dye. Chlorophyll was then removed by incubating the samples in 95% ethanol in an 80\u0026deg;C water bath; the ethanol was replaced every 10 min until the leaf tissue became completely clear.\u003c/p\u003e\n\u003ch3\u003eQuantification of Na⁺\u003c/h3\u003e\n\u003cp\u003eRoots, stems, and leaves were harvested from seedlings treated with 1.0% NaCl for 0 h or 24 h. Each treatment consisted of three biological replicates, each comprising tissues pooled from 5\u0026ndash;7 seedlings. Samples were transferred to pre‑cleaned polytetrafluoroethylene (PTFE) microwave digestion vessels. Then, 10 mL of high‑purity concentrated HNO₃ was added to each vessel, followed by vortex mixing for 1 min. An additional 5 mL of ultra‑pure concentrated HNO₃ was added before sealing. Digestion was performed in a closed‑vessel microwave system using a stepped temperature program: 120\u0026deg;C (5 min), 150\u0026deg;C (5 min), and 180\u0026deg;C (20 min). After digestion, the vessels were transferred to an acid evaporation cabinet and held at 180\u0026deg;C for 30 min to reduce the residual volume to approximately 1 mL. The digestates were subsequently diluted threefold with ultra‑pure water and finally adjusted to a final volume of 50 mL in centrifuge tubes. After thorough mixing (1 min), samples were filtered through a 0.22‑\u0026micro;m membrane. The filtrates were appropriately diluted with 2% HNO₃ as required and analyzed for Na⁺ content using inductively coupled plasma mass spectrometry (ICP‑MS).\u003c/p\u003e\n\u003ch3\u003eQuantification of endogenous compounds\u003c/h3\u003e\n\u003cp\u003eLeaves were harvested from 14-day-old seedlings exposed to 1.0% NaCl for 0 h or 24 h. The experiment included three biological replicates, with each replicate consisting of leaves pooled from 5 to 7 seedlings. The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), as well as the malondialdehyde (MDA) content, were quantified in leaf tissues. All measurements were conducted using fluorescence spectrophotometry following the protocols provided with the respective commercial assay kits (BOXBIO, Beijing, China).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReverse transcription quantitative PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the roots of mutant plants treated with 1.0% NaCl for 0 or 24 h, using the Eastep Super Total RNA Extraction Kit (Promega, China). Subsequently, cDNA was synthesized from equal amounts of total RNA using the HiScript IV All-in-One Ultra RT SuperMix for qPCR (Novizan, China). QPCR was performed on a Quant Studio 3 Real-Time PCR System. Each reaction contained TB Green Premix Ex Taq II (Tli RNaseH Plus) and gene-specific primers. The actin gene was used as an internal control for normalization. Relative gene expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, with the WT sample at 0 h serving as the calibrator.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eYeast one-hybrid assay\u003c/h3\u003e\n\u003cp\u003eAnalysis of the \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e promoter regions identified two and one W‑box elements within the 1500 bp and 1000 bp upstream sequences of their respective ATG start codons. The corresponding promoter fragments were PCR‑amplified and cloned into the pHis2 reporter vector. Separately, the full‑length coding sequence (CDS) of \u003cem\u003eOsWRKY53\u003c/em\u003e was inserted into the pGADT7 vector to generate the \u003cem\u003eOsWRKY53\u003c/em\u003e-CDS effector construct. After sequence verification, the reporter and effector plasmids were co‑transformed into the Saccharomyces cerevisiae strain Y187. Transformants were first selected on synthetic dropout (SD) medium lacking leucine and tryptophan (SD/\u0026ndash;Leu/\u0026ndash;Trp). Positive clones were then screened on SD/\u0026ndash;Leu/\u0026ndash;Trp/\u0026ndash;His plates supplemented with 3‑amino‑1, 2, 4‑triazole (3‑AT). Plates were incubated at 30\u0026deg;C for 4 days to assess the protein‑DNA interactions.\u003c/p\u003e\n\u003ch3\u003eDual-luciferase reporter in vivo imaging\u003c/h3\u003e\n\u003cp\u003eTo analyze the transcriptional activity of \u003cem\u003eOsWRKY53\u003c/em\u003e on the promoters of \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e, the effector and reporter vectors were constructed. The effector vector 62-SK-\u003cem\u003eOsWRKY53\u003c/em\u003e expressed the full-length \u003cem\u003eOsWRKY53\u003c/em\u003e protein, while the reporter vectors pro\u003cem\u003eOsRR22\u003c/em\u003e-0800-Luc and pro\u003cem\u003eOsARF18\u003c/em\u003e-0800-Luc contained the respective promoter fragments driving the firefly luciferase gene. Each construct was introduced into Agrobacterium tumefaciens strain GV3101 (pSoup-p19). Bacterial cultures carrying the effector and a reporter were mixed and co-infiltrated into the leaves of 4-week-old tobacco plants for transient expression. Luminescence imaging was performed 72 h post-infiltration using an LB985 Night SHADE Plant Molecular Imaging System.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid assay\u003c/h2\u003e \u003cp\u003eThe full-length coding sequences (CDS) of \u003cem\u003eOsWRKY53\u003c/em\u003e were cloned into the pGADT7 vector (activation domain), while the CDS of \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e were cloned into the pGBKT7 vector (DNA-binding domain). After sequence verification, the resulting constructs were pairwise co-transformed (pGADT7-\u003cem\u003eOsWRKY53\u003c/em\u003e with pGBKT7-\u003cem\u003eOsRR22\u003c/em\u003e, and pGADT7-\u003cem\u003eOsWRKY53\u003c/em\u003e with pGBKT7-\u003cem\u003eOsARF18\u003c/em\u003e) into the Saccharomyces cerevisiae strain Y2HGold. Transformants were first selected on synthetic dropout (SD) medium lacking leucine and tryptophan (SD/\u0026ndash;Leu/\u0026ndash;Trp). Positive colonies were then screened on SD medium additionally lacking adenine and histidine (SD/\u0026ndash;Ade/\u0026ndash;Leu/\u0026ndash;Trp/\u0026ndash;His), supplemented with 3-amino-1,2,4-triazole (3-AT). Plates were incubated at 30\u0026deg;C for 4 days to assess protein-protein interactions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBifc assay\u003c/h2\u003e \u003cp\u003eTo test for protein-protein interaction in vivo, the full-length coding sequences (CDSs) of \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e were cloned into the pUC‑SPYNE and pUC‑SPYCE vectors, respectively, to generate \u003cem\u003eOsWRKY53\u003c/em\u003e‑nYFP and \u003cem\u003eOsRR22\u003c/em\u003e‑cYFP fusion constructs. The known interacting pair \u003cem\u003eBZIP63\u003c/em\u003e‑nYFP/\u003cem\u003eBZIP63\u003c/em\u003e‑cYFP served as a positive control, while empty nYFP and cYFP vectors were used as negative controls. Different plasmid combinations were co‑transfected into rice protoplasts. Fluorescence signals were captured 16\u0026ndash;24 h after transfection using a Leica SP8 confocal laser‑scanning microscope equipped with appropriate filter sets (excitation: 488 nm and 587 nm).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eRNA-seq analysis\u003c/h2\u003e \u003cp\u003eRoot tissues were harvested from WT and mutant plants treated with 1.0% NaCl for 0 or 30 h, with each treatment performed in biological triplicate. Total RNA was extracted using the MagPure Universal RNA LQ Kit 2 (Magen, China). Sequencing libraries were constructed with the VAHTS Universal V8 RNA-seq Library Prep Kit (Vazyme, China) and sequenced on an Illumina NovaSeq platform to generate paired-end reads. Differentially expressed genes (DEGs) were identified using the DESeq2 package with an adjusted P-value (padj) cutoff of \u0026le;\u0026thinsp;0.05, using the WT samples as the reference group. Gene Ontology (GO) enrichment analysis of the DEGs was subsequently performed using the GOSeq software (v1.34.1) with default parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Differences among groups were assessed by one-way analysis of variance (ANOVA) followed by post-hoc comparisons, using SPSS 27.0. Significance levels are denoted as follows: \u003csup\u003e*\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003csup\u003e**\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01, \u003csup\u003e***\u003c/sup\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.001. All oligonucleotide primers used in this study for vector construction, yeast one-hybrid (Y1H), yeast two-hybrid (Y2H), dual-luciferase reporter (LUC), bimolecular fluorescence complementation (BiFC), and quantitative real-time PCR (qPCR) assays are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eLoss of function of\u003c/strong\u003e \u003cstrong\u003eOsWRKY53\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eOsARF18\u003c/strong\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003cstrong\u003eOsRR22\u003c/strong\u003e \u003cstrong\u003esignificantly enhances rice salt tolerance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e have additive effects on salt tolerance in rice, we generated single, double, and triple mutants using CRISPR/Cas9. Each mutant line comprised two independent alleles (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), all of which carried frameshift mutations leading to disruption of the functional protein domains (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u0026ndash;4). Subsequently, plants were subjected to salt stress. After a 7‑day treatment with 1.0% NaCl followed by 7‑day recovery, all WT plants died. In contrast, the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e triple mutant exhibited the strongest tolerance, with survival rates of 79.86% and 83.33%, significantly exceeding all other genotypes (Fig. 1A, B). The \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutants showed comparable survival, both significantly higher than the three single mutants (Fig. 1A, B). Among single mutants, a survival gradient was observed: \u003cem\u003eOsARF18\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eOsWRKY53\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;\u003cem\u003eOsRR22\u003c/em\u003e (Fig. 1A, B). Similarly, under 1.2% NaCl treatment, Consistent with the trend observed at 1.0% NaCl, the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e triple mutant remained the most tolerant under 1.2% NaCl stress, with survival rates of 20.83% and 21.53% (Fig. 1C, D), and the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant outperformed all other mutant combinations (Fig. 1C, D).\u003c/p\u003e\n\u003cp\u003eFollowing treatment with 1.0% NaCl, root length, shoot length, and fresh weight were measured for all plant materials. With the exception of the \u003cem\u003eOsRR22\u003c/em\u003e single mutant, which did not differ significantly from the WT in shoot or root length, all other mutants showed significant increases in all three traits relative to the WT. Among them, the triple mutant (\u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e) had the longest roots, whereas the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant and the \u003cem\u003eOsARF18\u003c/em\u003e single mutant exhibited the greatest shoot length and the highest fresh weight, respectively (Fig. S5A-C). Under normal field conditions, no significant differences were observed between the mutants and the WT in key agronomic traits such as plant height, tiller number, and grain-setting rate (Fig. S6). Collectively, these results demonstrate that the combined loss of function of \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e significantly enhances salt tolerance in Shuanghui 459 without compromising normal growth and development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe collective action of\u003c/strong\u003e \u003cstrong\u003eOsWRKY53\u003c/strong\u003e, \u003cstrong\u003eOsRR22\u003c/strong\u003e, \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eOsARF18\u003c/strong\u003e \u003cstrong\u003eenhances rice salt tolerance by elevating antioxidant enzyme activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnder salt stress, excessive uptake and accumulation of Na⁺ in plant tissues leads to ionic imbalance and cytotoxicity\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Quantitative analysis of Na⁺ in roots, stems, and leaves showed that, under non‑saline conditions, all mutants maintained comparable, low Na⁺ levels to the WT (Fig. S7A). Across all genotypes, Na⁺ content was significantly higher in roots than in stems or leaves, with no significant difference between the latter two tissues (Fig. S7B). However, following 24 h of treatment with 1.0% NaCl, distinct accumulation patterns emerged. The \u003cem\u003eOsWRKY53\u003c/em\u003e single mutant, the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant, and the triple mutant all accumulated significantly more Na⁺ in roots, stems, and leaves than the WT. In a tissue‑specific manner, the \u003cem\u003eOsRR22\u003c/em\u003e single mutant showed higher Na⁺ content specifically in roots, while the \u003cem\u003eOsARF18\u003c/em\u003e single mutant and the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant accumulated more Na⁺ predominantly in leaves (Fig. 2A-C). Compared to pre-salt treatment, significant differences in Na⁺ content were observed in stems and leaves across all treated plants. Na⁺ content in roots, stems, and leaves of WT, single-gene, and \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double-gene and triple-gene mutants decreased significantly in that order, with the triple-gene mutant exhibiting the most pronounced trend of change. Na⁺ content in leaves of the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant was significantly higher than in other mutants. Furthermore, the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant exhibited uniquely high Na⁺ content in leaves compared to other mutants. The \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant exhibited higher Na⁺ content in leaves compared with the other mutants, and in the \u003cem\u003eosarf18\u003c/em\u003e-\u003cem\u003eosrr22\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003e mutant, leaf Na⁺ levels were even significantly higher than those in stems. (Fig. S7C). Collectively, these results suggest that these mutants alter Na⁺ partitioning, likely to enhance its retention in roots and stems to limit flux into young leaves, while promoting vacuolar sequestration to mitigate cellular toxicity.\u003c/p\u003e\n\u003cp\u003eSalt stress triggers an overproduction of reactive oxygen species (ROS), resulting in oxidative stress that disrupts cellular metabolism and can lead to cytotoxic effects\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. DAB and NBT staining revealed that WT leaves exhibited significantly higher localized production of H₂O₂ and O₂⁻ compared to mutants. The accumulation of H₂O₂ and O₂⁻ progressively decreased in monogenic, bigene, and trigenic mutants, respectively (Fig. 2D). Simultaneously, the activities of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) increased sequentially and were significantly higher than those in WT (Fig. 2E-G), while malondialdehyde (MDA) levels decreased sequentially and were significantly lower than those in WT (Fig. 2H). This result indicates that the enhanced antioxidant enzyme activity in the mutant significantly improves salt tolerance. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsWRKY53\u003c/strong\u003e \u003cstrong\u003eantagonizes\u003c/strong\u003e \u003cstrong\u003eOsARF18\u003c/strong\u003e \u003cstrong\u003eand\u003c/strong\u003e \u003cstrong\u003eOsRR22\u003c/strong\u003e \u003cstrong\u003ein response to salt stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWRKY transcription factors recognize and bind to the W-box cis-element in promoter regions via their conserved WRKY domain to regulate gene expression\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, we identified W-box motifs within the promoters of both \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eA), suggesting they are potential direct targets of \u003cem\u003eOsWRKY53\u003c/em\u003e. Yeast one-hybrid analysis confirmed the direct binding of \u003cem\u003eOsWRKY53\u003c/em\u003e to these promoters, supporting its role as a transcriptional regulator. Dual‑luciferase reporter assays further showed that \u003cem\u003eOsWRKY53\u003c/em\u003e represses the transcriptional activity of the \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e promoters under non‑stress conditions (Fig. \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-E). Consistent with this, RT‑qPCR analysis revealed that loss of \u003cem\u003eOsWRKY53\u003c/em\u003e function relieved its repression, leading to significantly elevated transcript levels of both \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e compared with the WT (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). Moreover, in the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant, \u003cem\u003eOsARF18\u003c/em\u003e expression was also markedly upregulated (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Compared with the pre‑treatment levels, \u003cem\u003eOsRR22\u003c/em\u003e expression was upregulated in both the WT and \u003cem\u003eOsARF18\u003c/em\u003e single‑gene mutant after salt treatment, although not statistically significant, whereas it was significantly downregulated in the \u003cem\u003eOsWRKY53\u003c/em\u003e single‑gene mutant (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). \u003cem\u003eOsWRKY53\u003c/em\u003e expression was significantly downregulated in the WT and \u003cem\u003eOsARF18\u003c/em\u003e single‑gene mutant, significantly upregulated in the \u003cem\u003eOsRR22\u003c/em\u003e single‑gene mutant, and showed a non‑significant increasing trend in the \u003cem\u003eOsARF18\u003c/em\u003e\u0026ndash;\u003cem\u003eOsRR22\u003c/em\u003e double‑gene mutant (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). \u003cem\u003eOsARF18\u003c/em\u003e was significantly upregulated in the WT and \u003cem\u003eOsRR22\u003c/em\u003e single‑gene mutant, and significantly downregulated in the \u003cem\u003eOsWRKY53\u003c/em\u003e single‑gene mutant and the \u003cem\u003eOsWRKY53\u003c/em\u003e\u0026ndash;\u003cem\u003eOsRR22\u003c/em\u003e double‑gene mutant (Fig. \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).Considering the salt tolerance phenotypes (Fig. 1), \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOSRR22\u003c/em\u003e may not exhibit a linear regulatory relationship but rather involve complex feedback and antagonistic interactions. Therefore, functional redundancy may exist among these three genes under salt stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsWRKY53\u003c/strong\u003e \u003cstrong\u003ephysically interacts with\u003c/strong\u003e \u003cstrong\u003eOsRR22\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further elucidate the molecular mechanism underlying the salt stress response mediated by the \u003cem\u003eOsWRKY53\u003c/em\u003e\u0026ndash;\u003cem\u003eOsARF18\u003c/em\u003e\u0026ndash;\u003cem\u003eOsRR22\u003c/em\u003e module, protein-protein interaction visualization analysis revealed a potential binding site between \u003cem\u003eOsWRKY53\u003c/em\u003e and amino acids 28\u0026ndash;409 of the \u003cem\u003eOsRR22\u003c/em\u003e protein (Fig. 5A). This suggests that \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e may physically interact. Yeast two‑hybrid assays demonstrated that co‑transformation with \u003cem\u003eOsWRKY53\u003c/em\u003e‑pGADT7 and \u003cem\u003eOsRR22\u003c/em\u003e‑pGBKT7 supported normal growth and produced blue colonies on SD-ALWH/\u0026ndash;Ade/\u0026ndash;Leu/\u0026ndash;Trp/\u0026ndash;His medium supplemented with 10 mM 3‑AT and X‑\u0026alpha;‑Gal. In contrast, the negative control (pGADT7\u0026thinsp;+\u0026thinsp;\u003cem\u003eOsRR22\u003c/em\u003e‑pGBKT7) showed effective suppression of self‑activation on the same selective medium, confirming a specific interaction between \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e (Fig. 5B). Bimolecular fluorescence complementation (BiFC) assays showed that co‑expression of \u003cem\u003eOsWRKY53\u003c/em\u003e‑nYFP and \u003cem\u003eOsRR22\u003c/em\u003e‑cYFP in rice protoplasts produced a reconstituted YFP signal localized in the nucleus, while control combinations yielded no detectable fluorescence (Fig. 5C). These results further confirm that \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e interact specifically within the nuclear compartment. Thus, \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e likely form a protein complex that functions as a transcriptional repressor of downstream salt‑stress‑responsive target genes. Loss‑of‑function mutations in either \u003cem\u003eOsWRKY53\u003c/em\u003e or \u003cem\u003eOsRR22\u003c/em\u003e, or their combined knockout, relieves this repression, resulting in the upregulation of these target genes and thereby enhancing salt tolerance.\u003c/p\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003eRNA-seq Comparative Analysis\u003c/h2\u003e\n \u003cp\u003eTo comprehensively dissect the downstream signaling pathway of the \u003cem\u003eOsWRKY53\u003c/em\u003e\u0026ndash;\u003cem\u003eOsARF18\u003c/em\u003e\u0026ndash;\u003cem\u003eOsRR22\u003c/em\u003e module in response to salt stress, transcriptome sequencing was performed on roots of two‑week‑old WT and mutant plants treated with 1.0% NaCl. A total of 6,170 differentially expressed genes (DEGs) were identified, of which 4,005 were upregulated and 2,165 were downregulated (Fig. S8). GO analysis revealed that the differentially expressed genes (DEGs) were significantly enriched in molecular functions related to \u0026lsquo;ATP binding\u0026rsquo; and \u0026lsquo;electron carrier activity\u0026rsquo;, cellular components including \u0026lsquo;membrane\u0026rsquo;, \u0026lsquo;plasma membrane\u0026rsquo;, \u0026lsquo;vacuole\u0026rsquo;, and \u0026lsquo;cytoplasmic membrane‑associated vesicles\u0026rsquo;, and biological processes such as \u0026lsquo;oxidation‑reduction process\u0026rsquo; and \u0026lsquo;transmembrane transport\u0026rsquo; (Fig. S9A and Table 2). These enriched terms suggest that the mutant may enhance salt tolerance by regulating Na⁺ sequestration and transmembrane transport, which is consistent with the altered Na⁺ levels observed in its roots, stems, and leaves (Fig. 2A-C). A substantial number of differentially expressed genes (DEGs) in both the WT and all mutant lines under salt stress relative to normal growth conditions (Fig. S9B). Intersection analysis of differentially expressed genes identified with 1,177 shared DEGs between the triple mutant and all other genotypes (Fig. S10). This core set of genes likely underlies the markedly enhanced salt tolerance observed in the triple mutant. Furthermore, by intersecting the differentially expressed genes between each mutant and the WT under salt stress and normal conditions, we identified a substantial number of overlapping genes across these comparison groups (Fig. S10). These overlapping genes, along with those unique to each mutant, may also be key factors contributing to the significant increase in salt tolerance observed in the corresponding mutants.\u003c/p\u003e\n \u003cp\u003eTo identify the downstream regulatory components of the salt stress response in the triple‑gene mutant, we examined 1,175 differentially expressed genes (DEGs) that were common to the triple mutant and other genotypes under salt stress. This analysis showed that the transcript level of the vacuolar Na⁺/H⁺ antiporter \u003cem\u003eOsNHX1\u003c/em\u003e (\u003cem\u003eLOC_Os07g47100\u003c/em\u003e) was significantly upregulated in the triple mutant (Fig. S12). Previous studies have demonstrated that activation of \u003cem\u003eOsNHX1\u003c/em\u003e under salt stress promotes Na⁺ sequestration into vacuoles, thereby reducing cytosolic Na⁺ toxicity\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. We further analyzed the expression of Na⁺‑related transporter genes\u0026mdash;including \u003cem\u003eOsNHX1\u003c/em\u003e, \u003cem\u003eOsHKT2;1\u003c/em\u003e, \u003cem\u003eOsHKT1;1\u003c/em\u003e, \u003cem\u003eOsHKT1;5\u003c/em\u003e, \u003cem\u003eOsAKT1\u003c/em\u003e, \u003cem\u003eOsHAK1\u003c/em\u003e, and \u003cem\u003eOsSOS1\u003c/em\u003e\u0026mdash;as well as antioxidant‑related genes (\u003cem\u003eOsCatB\u003c/em\u003e and \u003cem\u003eOsPOD1\u003c/em\u003e). Under non‑stress conditions, the basal expression levels of these genes were significantly upregulated or downregulated in each mutant compared with the WT (Fig. 6). This observation may be attributed to the loss of function of \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e, as RNA‑seq analysis prior to salt stress treatment revealed a large number of DEGs in all mutant plants relative to the WT (Fig. 13). After salt stress treatment, these genes also exhibited significant up‑ or down‑regulation in the WT and the various mutants (Fig. S14). Notably, compared with the WT, the expression changes of \u003cem\u003eOsNHX1\u003c/em\u003e, \u003cem\u003eOsHKT2;1\u003c/em\u003e, \u003cem\u003eOsHKT1;1\u003c/em\u003e, \u003cem\u003eOsCatB\u003c/em\u003e, and \u003cem\u003eOsPOD1\u003c/em\u003e were most pronounced in the triple mutant (Fig. 6A, B, F, H, I), which is consistent with the GO pathways specifically enriched by the DEGs. These results suggest that the mechanism by which \u003cem\u003eOsWRKY53\u003c/em\u003e‑\u003cem\u003eOsARF18\u003c/em\u003e‑\u003cem\u003eOsRR22\u003c/em\u003e substantially enhances salt tolerance may involve regulating intracellular and extracellular Na⁺ homeostasis through vacuolar Na⁺ sequestration, together with a marked increase in antioxidant enzyme activity to improve salt resistance. In addition, the relative expression levels of \u003cem\u003eOsNHX1\u003c/em\u003e, \u003cem\u003eOsHKT1;1\u003c/em\u003e, \u003cem\u003eOsCatB\u003c/em\u003e, and \u003cem\u003eOsPOD1\u003c/em\u003e were significantly higher in the double mutant than in the single mutants (Fig. 6B, F, H, I).\u003c/p\u003e\n \u003cp\u003eFurther GO enrichment analysis of differentially expressed genes between the triple mutant and other genotypes under salt stress revealed that, overall, these differentially expressed genes were enriched in the two most significant pathways: redox processes and heme binding. Two most significant genes were identified: \u003cem\u003eOsrbohH\u003c/em\u003e (\u003cem\u003eLOC_Os12g35610\u003c/em\u003e) and \u003cem\u003eOsPgb1.2\u003c/em\u003e (\u003cem\u003eLOC_Os03g12510\u003c/em\u003e) (Table. 3\u0026ndash;8). \u003cem\u003eOsrbohH\u003c/em\u003e encodes an NADPH oxidase, its overexpression has been shown to lower reactive oxygen species (ROS) accumulation in plants, along with reduced malondialdehyde (MDA) content and increased catalase (CAT) and peroxidase (POD) activities, thereby enhancing tolerance to high‑temperature and drought stress\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eOsPgb1.2\u003c/em\u003e encodes a hemoglobin that is induced under salt, drought, and cold stress. Overexpression of \u003cem\u003eOsPgb1.2\u003c/em\u003e improves plant tolerance to K⁺ deficiency stress and is associated with decreased H₂O₂ and ROS levels\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. These results align well with the observation that the triple mutant displayed the strongest salt tolerance, the highest antioxidant enzyme activities, and the lowest accumulations of H₂O₂, O₂⁻, and MDA.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSalt stress severely hampers rice production. The identification of salt‑tolerance genes and the development of high‑quality, salt‑tolerant rice varieties are therefore critical for enhancing the utilization of saline‑alkali soils and ensuring national food security\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. To date, although scientists have identified and cloned numerous salt‑tolerance‑related genes in rice, progress in enhancing salt tolerance through conventional breeding methods remains slow. Improving or introducing only one or a few related genes often yields limited effects, making it difficult to develop salt‑tolerant varieties suitable for field application. Therefore, it is essential to integrate multiple favorable genes to accelerate the genetic improvement of salt‑tolerant rice cultivars \u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. This study found that under 1.0% NaCl treatment, the triple‑gene knockout mutant \u003cem\u003eOsWRKY53\u003c/em\u003e‑\u003cem\u003eOsARF18\u003c/em\u003e‑\u003cem\u003eOsRR22\u003c/em\u003e exhibited a significantly higher survival rate compared with single‑gene mutants, double‑gene mutants, and the WT, reaching approximately 80% (Fig.\u0026nbsp;1A, B), even under 1.2% NaCl stress, the survival rate remained around 20% (Fig.\u0026nbsp;1C, D), indicating that the combined action of two or three of these genes substantially enhances salt tolerance in the rice variety Shuanghui 459, without compromising overall plant growth and development (Fig. S6). Furthermore, \u003cem\u003eOsARF18\u003c/em\u003e conferred stronger salt tolerance than \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e, and \u003cem\u003eOsWRKY53\u003c/em\u003e showed greater tolerance than \u003cem\u003eOsRR22\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e mediate Na⁺ efflux in roots\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, whereas \u003cem\u003eOsARF18\u003c/em\u003e is involved in Na⁺ removal from aerial tissues\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Before salt treatment in this study, Na⁺ content in stems and leaves showed no significant differences among the plant lines, although root Na⁺ levels were substantially higher than those in shoots (Fig. S7B). After salt treatment, significant differences in Na⁺ content emerged in both stems and leaves across all genotypes (Fig. S7C), and all mutants accumulated more Na⁺ in roots, stems, and leaves compared with the WT. Except for the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant, Na⁺ content in the roots, stems, and leaves of the other genotypes generally displayed a decreasing trend, which was most pronounced in the triple mutant (Fig.\u0026nbsp;2A-C). Notably, Na⁺ content in the roots and stems of the \u003cem\u003eOsARF18\u003c/em\u003e single mutant and the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant did not differ significantly from the WT (Fig.\u0026nbsp;2A-B), this may be due to the relatively short treatment duration and high salt solution concentration, which have not yet fully activated the salt tolerance-related mechanisms (Fig.\u0026nbsp;2A-C). The changes in Na⁺ content observed in the roots, stems, and leaves of all mutants under salt treatment clearly suggest that they may resist the toxic effects of Na⁺ accumulation by storing Na⁺ and regulating intracellular and extracellular Na⁺ balance. Notably, the \u003cem\u003eOsARF18\u003c/em\u003e single mutant accumulated significantly more Na⁺ in roots and stems, whereas the \u003cem\u003eOsRR22\u003c/em\u003e single mutant showed pronounced Na⁺ accumulation in leaves. The \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant exhibited markedly elevated Na⁺ levels in roots, stems, and leaves, with Na⁺ content in leaves being comparable to or even exceeding that in stems (Fig.\u0026nbsp;2A-B), While the triple mutant displayed consistently higher Na⁺ levels in roots and stems than the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant, its leaf Na⁺ content was significantly lower than \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant (Fig.\u0026nbsp;2A-C). These results indicate that the triple-gene mutant exhibits a progressive change in function based on the single- and double-gene mutants, suggesting that \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e may have a certain special mechanism jointly responding to salt stress. Furthermore, the results from DAB and NBT staining, as well as measurements of antioxidant enzyme activities (CAT, SOD, POD) and malondialdehyde (MDA) content, all showed trends consistent with a salt-tolerant phenotype. Specifically, the local accumulation of H₂O₂ and O₂⁻ in the leaves of WT, single-gene, double-gene, and triple-gene mutants decreased successively (Fig.\u0026nbsp;2D). Correspondingly, antioxidant enzyme activities increased sequentially, while the MDA content decreased (Fig.\u0026nbsp;2E-H). Collectively, these results suggest that although Na⁺ gradually accumulates in the mutants, coordinated changes in endogenous metabolites and enhanced antioxidant capacity enable more effective scavenging of reactive oxygen species (ROS) generated under excessive Na⁺ stress.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) play pivotal roles in regulating plant growth and development, as well as in responding to diverse biotic and abiotic stresses, by activating or repressing multiple genes within downstream regulatory networks\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. A single transcription factor (TF) can regulate the expression of numerous genes within a pathway\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e, while multiple TFs\u0026mdash;such as NAC, MYB, AP2/ERF, bZIP, and WRKY\u0026mdash;form intricate regulatory networks. Changes in their gene expression directly influence downstream gene expression within these regulatory networks, triggering a cascade of pleiotropic responses such as functional redundancy or functional overlap\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. Previous studies have shown that WRKY transcription factors bind specifically to the W‑box elements within the promoters of target genes\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In this study, we demonstrated that under non‑stress conditions, \u003cem\u003eOsWRKY53\u003c/em\u003e specifically binds to the W‑box motifs in the promoters of \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e and represses their expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Subsequent RT‑qPCR assays corroborated these findings: the loss of \u003cem\u003eOsWRKY53\u003c/em\u003e function released its transcriptional repression of \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e, resulting in their upregulation relative to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). Conversely, loss of function in \u003cem\u003eOsARF18\u003c/em\u003e or \u003cem\u003eOsRR22\u003c/em\u003e led to downregulation of \u003cem\u003eOsWRKY53\u003c/em\u003e expression compared with the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). After salt treatment, the WT exhibited upregulation of \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e but downregulation of \u003cem\u003eOsWRKY53\u003c/em\u003e relative to the pre‑treatment levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F), In the \u003cem\u003eOsRR22\u003c/em\u003e single mutant, both \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e were significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F), suggesting an antagonistic interaction between \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e in this background. Notably, however, \u003cem\u003eOsWRKY53\u003c/em\u003e expression remained lower than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that even with its antagonistic role in the \u003cem\u003eOsRR22\u003c/em\u003e mutant, \u003cem\u003eOsWRKY53\u003c/em\u003e still confers stronger salt tolerance than the WT. Meanwhile, \u003cem\u003eOsARF18\u003c/em\u003e expression remained significantly higher than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This indicates that under the combined effects of \u003cem\u003eOsRR22\u003c/em\u003e functional loss and partial antagonism by \u003cem\u003eOsWRKY53\u003c/em\u003e in the \u003cem\u003eOsRR22\u003c/em\u003e monohybrid mutant, the elevated expression of \u003cem\u003eOsARF18\u003c/em\u003e partially suppresses the enhanced salt tolerance. In the \u003cem\u003eOsWRKY53\u003c/em\u003e single mutant, the expression levels of \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e were significantly elevated compared with the WT before salt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C). Following salt stress, both genes were significantly downregulated relative to their pre‑stress levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F) yet remained higher than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, C), these results suggest that \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e still retained partial antagonistic activity under these conditions. In the \u003cem\u003eOsRR22\u003c/em\u003e single mutant, \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e expressions were significantly upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F). Conversely, in the \u003cem\u003eOsWRKY53\u003c/em\u003e single mutant, \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e were significantly downregulated after salt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, F). These opposing expression patterns may contribute to the stronger salt tolerance observed in the \u003cem\u003eOsWRKY53\u003c/em\u003e mutant compared to the \u003cem\u003eOsRR22\u003c/em\u003e mutant. In the \u003cem\u003eOsARF18\u003c/em\u003e single mutant, \u003cem\u003eOsRR22\u003c/em\u003e showed no significant change relative to the WT before salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), whereas \u003cem\u003eOsWRKY53\u003c/em\u003e was significantly lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Following salt treatment, \u003cem\u003eOsRR22\u003c/em\u003e expression increased but not significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), while \u003cem\u003eOsWRKY53\u003c/em\u003e decreased further (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Compared to the WT, \u003cem\u003eOsRR22\u003c/em\u003e remained lower (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), and \u003cem\u003eOsWRKY53\u003c/em\u003e was the most suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results indicate that \u003cem\u003eOsRR22\u003c/em\u003e likely exerts only minimal antagonistic effects in this background. The pronounced downregulation of \u003cem\u003eOsWRKY53\u003c/em\u003e may be a key factor underlying the markedly stronger salt tolerance of the \u003cem\u003eOsARF18\u003c/em\u003e single mutant relative to the \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e single mutants. In the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant, the expression of \u003cem\u003eOsWRKY53\u003c/em\u003e increased after salt treatment but did not reach statistical significance and remained markedly lower than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, E), this likely represents one of the key reasons why its salt tolerance is improved yet remains lower than that of the triple mutant. In the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant, \u003cem\u003eOsARF18\u003c/em\u003e expression was significantly higher than in the WT before salt treatment but declined following stress exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, F), this may be one reason why the salt tolerance of the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant shows no significant difference from that of the \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e double mutant. The above results indicate that \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e form a mutually antagonistic regulatory network. The loss of function in all three genes completely disrupts this antagonistic interplay, which in turn induces transcriptional reprogramming within the downstream regulatory networks of these transcription factors, ultimately leading to a further enhancement of salt tolerance in the triple mutant. In addition, proteins from different transcription factor families can interact to form protein complexes that collaboratively regulate the expression of downstream genes. Recent studies reveal that the cold stress‑upregulated VQ protein \u003cem\u003eOsVQ3\u003c/em\u003e interacts with the transcriptional repressor \u003cem\u003eOsWRKY7\u003c/em\u003e, inhibiting its transcriptional activity and DNA‑binding capacity, this interaction further enhances the expression of downstream target genes, thereby improving cold tolerance in rice\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. In tomato, \u003cem\u003eSiVQ10\u003c/em\u003e physically interacts with \u003cem\u003eSiWRKY51\u003c/em\u003e to synergistically activate SiP5CS1 expression, promoting proline accumulation and conferring resistance to cold stress\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. Similarly, in rice, the transcription factors \u003cem\u003eOsbZIP61\u003c/em\u003e and \u003cem\u003eOsRF2b\u003c/em\u003e interact physically to form a heterodimer, which strengthens the DNA‑binding activity of \u003cem\u003eOsRF2b\u003c/em\u003e. This complex directly suppresses the expression of \u003cem\u003eOsNRT1.1B\u003c/em\u003e, leading to improved yield and nitrogen use efficiency\u003csup\u003e[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]\u003c/sup\u003e. This study shows that \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e directly interact and co-localize in the nucleus (Fig.\u0026nbsp;5A-C). Protein\u0026ndash;protein interaction prediction and yeast two‑hybrid assays further suggest that \u003cem\u003eOsWRKY53\u003c/em\u003e may also physically interact with \u003cem\u003eOsARF18\u003c/em\u003e (Fig. S15). These results imply that \u003cem\u003eOsWRKY53\u003c/em\u003e not only transcriptionally regulates \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e but could also form a protein complex with them within a common regulatory network, where they cooperate to repress downstream target genes. Loss of function in the double or triple mutants relieves this repression, leading to the upregulation of positively regulated downstream genes and consequently enhancing salt tolerance. These findings reveal the sophisticated genetic interplay among \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e. Therefore, future studies will aim to further delineate this genetic network to uncover the regulatory mechanisms underlying the salt stress response in the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e triple mutant.\u003c/p\u003e \u003cp\u003eSalt stress primarily results from excessive accumulation of soluble sodium ions (Na⁺) in the soil, plants passively absorb surplus Na⁺, leading to disruption of cellular ion homeostasis and subsequently triggering a series of physiological injuries\u003csup\u003e[\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]\u003c/sup\u003e. To maintain Na⁺ balance, rice employs multiple adaptive strategies, including limiting Na⁺ uptake, promoting Na⁺ efflux, and compartmentalizing Na⁺\u003csup\u003e[\u003cspan additionalcitationids=\"CR45 CR46\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]\u003c/sup\u003e. To further identify key targets in the downstream regulatory network altered by the \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e triple knockout, we collected roots from each mutant and the WT under salt stress and normal conditions for RNA-seq analysis. We then intersected the differentially expressed genes between the triple mutant and the other mutants and the WT. The results revealed that the vacuolar Na⁺/H⁺ antiporter \u003cem\u003eOsNHX1\u003c/em\u003e was significantly up regulated in the triple mutant (Fig. S12). RT‑qPCR further confirmed these observations: after 24 h of salt treatment, \u003cem\u003eOsNHX1\u003c/em\u003e expression was significantly elevated in both the \u003cem\u003eOsARF18\u003c/em\u003e‑\u003cem\u003eOsRR22\u003c/em\u003e double mutant and the triple mutant (Fig.\u0026nbsp;6). \u003cem\u003eOsNHX1\u003c/em\u003e encodes a vacuolar Na⁺/H⁺ antiporter that sequesters Na⁺ into vacuoles, thereby alleviating the toxicity caused by excessive cytoplasmic Na⁺ accumulation. Previous studies have shown that Arabidopsis plants overexpressing \u003cem\u003eAtNHX1\u003c/em\u003e accumulate more Na⁺ than WT plants under salt stress, yet exhibit less leaf chlorosis and better growth\u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. Similarly, in Brassica napus, overexpression of \u003cem\u003eAtNHX1\u003c/em\u003e leads to higher Na⁺ accumulation in leaves compared to the WT but reduces Na⁺‑related toxicity\u003csup\u003e[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]\u003c/sup\u003e. In rice, both \u003cem\u003eOsNHX1\u003c/em\u003e-overexpressing lines and the WT showed progressive increases in intracellular Na⁺ as salt concentration rose. At high salinity, \u003cem\u003eOsNHX1\u003c/em\u003e-overexpressing plants maintained higher intracellular Na⁺ levels than the WT yet grew significantly better, this was attributed to \u003cem\u003eOsNHX1\u003c/em\u003e-mediated compartmentalization of Na⁺ into vacuoles for storage, thereby reducing cytoplasmic Na⁺ accumulation and its toxic effects\u003csup\u003e[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]\u003c/sup\u003e. These results are consistent with the higher Na⁺ content in mutant roots, stems, and leaves compared to the WT, yet the mutants exhibited significantly better growth than the WT. Furthermore, the substantial upregulation of \u003cem\u003eOsrbohH\u003c/em\u003e and \u003cem\u003eOsPgb1.2\u003c/em\u003e in the triple mutant endows it with potent antioxidant enzyme activity and ROS scavenging capacity. Moreover, expression of \u003cem\u003eOsNHX1\u003c/em\u003e, \u003cem\u003eOsHKT2;1\u003c/em\u003e, \u003cem\u003eOsHKT1;1\u003c/em\u003e, \u003cem\u003eOsCatB\u003c/em\u003e, and \u003cem\u003eOsPOD1\u003c/em\u003e was significantly elevated in the triple mutant relative to the WT (Fig.\u0026nbsp;6A, B, F, H, I). These findings indicate that the triple mutant enhances salt tolerance by sequestering Na⁺ into vacuoles for storage, regulating intracellular and extracellular Na⁺ balance, and significantly boosting antioxidant enzyme activity.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study found that the aggregation of \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e in both dual and triple gene combinations significantly enhances salt tolerance in rice varieties, with the triple gene aggregation exhibiting the strongest salt tolerance. The loss of \u003cem\u003eOsWRKY53\u003c/em\u003e function relieved transcriptional repression on \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e, leading to upregulation of the latter two under normal conditions and downregulation under salt stress while remaining higher than WT. Conversely, loss of \u003cem\u003eOsARF18\u003c/em\u003e or \u003cem\u003eOsRR22\u003c/em\u003e function resulted in down‑regulation of \u003cem\u003eOsWRKY53\u003c/em\u003e. These interactions indicate that \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e form a mutually antagonistic regulatory loop. Double‑gene combinations partially alleviated this antagonism, whereas the triple‑gene combination completely disrupted it. This disruption led to significant up‑regulation of key genes such as \u003cem\u003eOsNHX1\u003c/em\u003e, \u003cem\u003eOsrbohH\u003c/em\u003e, \u003cem\u003eOsPgb1.2\u003c/em\u003e, \u003cem\u003eOsHKT2;1\u003c/em\u003e, \u003cem\u003eOsHKT1;1\u003c/em\u003e, \u003cem\u003eOsCatB\u003c/em\u003e, and \u003cem\u003eOsPOD1\u003c/em\u003e. These changes promote vacuolar sequestration of Na⁺, improve intra‑ and extracellular Na⁺ homeostasis, enhance leaf antioxidant enzyme activities (CAT, SOD, POD), and reduce malondialdehyde (MDA) content, collectively contributing to markedly enhanced salt tolerance. Importantly, pyramiding \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e did not compromise normal plant growth and development. These findings provide new strategies for polygenic aggregation to improve rice salt tolerance.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eH₂O₂ \u0026nbsp; \u0026nbsp; \u0026nbsp; Hydrogen Peroxide\u003c/p\u003e\n\u003cp\u003eO₂⁻ \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Superoxide Anion\u003c/p\u003e\n\u003cp\u003eSOD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Superoxide Dismutase\u003c/p\u003e\n\u003cp\u003eCAT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Catalase\u003c/p\u003e\n\u003cp\u003eMDA \u0026nbsp; \u0026nbsp; \u0026nbsp; Malondialdehyde\u003c/p\u003e\n\u003cp\u003eDEGs \u0026nbsp; \u0026nbsp; \u0026nbsp;Differentially expressed genes\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eGO \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gene ontology\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this article and its supplementary information files. The raw data used in this study has been uploaded into China National center for Bioinformation system with the primary accession code \u003cem\u003eGSA: CRA062376\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Top Ten Technical Breakthrough Projects of Hunan Province (Grant No. 2025QK1006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.S., H.D., F.S., W.Z. and Q.H. conceptualized the research;Y.Z. performed the experiments; Y.Z., P.S., P.F., J.Z. and X.L. analyzed the data; P.S. and Y.Z. wrote the paper; all authors reviewed the manuscript and agreed to its submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eState Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125, China\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eCollege of Tropical Agriculture and Forestry, Hainan University, Danzhou, Hainan 571737, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003eLongping Agricultural College, Hunan University, Changsha 410125, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003eYuelu Shan Laboratory, Changsha 410128, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLiang XY, Li JF, Yang YQ, Jiang CF, Guo Y. 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Function, Intracellular Localization and the Importance in Salt Tolerance of a Vacuolar Na\u003csup\u003e+\u003c/sup\u003e/H\u003csup\u003e+\u003c/sup\u003e Antiporter from Rice. Plant and Cell Physiology. 2004;45(2)146-159.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Salt stress tolerance, OsWRKY53, OsARF18, OsRR22, Genome editing","lastPublishedDoi":"10.21203/rs.3.rs-8764411/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8764411/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSalt stress is a key abiotic stress factor limiting rice growth and development. Previous studies have shown that \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e not only serve as important negative regulators of salt tolerance in rice but are also crucial for growth and development. However, the relative strengths of salt tolerance among these three genes and their combined effects within the same rice variety have not yet been reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we employed CRISPR/Cas9-mediated genome editing to simultaneously disrupt \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e in the rice cultivar Shuanghui 459. Salt tolerance increases sequentially in single-gene, double-gene, and triple-gene mutants. Under 1.0% NaCl stress, the triple mutant exhibited approximately 80% survival versus complete lethality in the wild type (WT). Physiologically, ROS accumulation progressively declined, while key antioxidant enzyme activities (CAT, SOD, POD) significantly increased. More importantly, molecular analyses revealed that the \u003cem\u003eOsWRKY53\u003c/em\u003e WRKY domain binds W-box elements in the \u003cem\u003eOsRR22\u003c/em\u003e and \u003cem\u003eOsARF18\u003c/em\u003e promoters, repressing their transcription. Loss function of \u003cem\u003eOsWRKY53\u003c/em\u003e depresses both genes. Reciprocally, \u003cem\u003eOsARF18\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e knockout downregulated \u003cem\u003eOsWRKY53\u003c/em\u003e. Furthermore, \u003cem\u003eOsWRKY53\u003c/em\u003e and \u003cem\u003eOsRR22\u003c/em\u003e directly interact at the protein level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTaken together, our results reveal that \u003cem\u003eOsWRKY53\u003c/em\u003e, \u003cem\u003eOsARF18\u003c/em\u003e, and \u003cem\u003eOsRR22\u003c/em\u003e constitute a reciprocal negative-feedback loop, wherein these three transcriptional regulators mutually antagonize each other. Disruption of this antagonistic network likely represents a core mechanism responsible for the robust salt tolerance observed in the triple mutant. Importantly, the polygenic aggregation of \u003cem\u003eOsWRKY53\u003c/em\u003e-\u003cem\u003eOsARF18\u003c/em\u003e-\u003cem\u003eOsRR22\u003c/em\u003e not only significantly enhances rice salt tolerance but also does not affect normal plant growth and development. These findings provide new strategies for polygenic aggregation-based genetic improvement of salt-tolerant rice varieties.\u003c/p\u003e","manuscriptTitle":"Loss of function of OsWRKY53-OsARF18-OsRR22 significantly enhances rice salt tolerance","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-30 14:05:40","doi":"10.21203/rs.3.rs-8764411/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-04T06:10:15+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T08:41:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-17T16:50:36+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-16T06:10:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"239124201201660927733098886749202270128","date":"2026-04-04T09:13:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-03T13:36:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"235022806948428742403841918724225448570","date":"2026-04-03T00:42:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16606051149841212685991852662007470556","date":"2026-03-27T09:43:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62934579687858597580576664640254874652","date":"2026-03-27T04:20:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"156448252858393170714746810197805060014","date":"2026-03-27T04:14:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101221487784023762273850220291952898525","date":"2026-03-27T03:17:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-27T02:42:03+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-20T05:30:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-04T15:55:11+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-04T15:49:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2026-02-02T11:03:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"621d073a-e4b3-47e6-93b1-f5397c0493d1","owner":[],"postedDate":"March 30th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-04T06:10:15+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T06:24:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-30 14:05:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8764411","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8764411","identity":"rs-8764411","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}