Natural variations of OsALDH2B1 contribute geographical adaptation to soil pH in rice

Natural variations of OsALDH2B1 contribute geographical adaptation to soil pH in rice | 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 Article Natural variations of OsALDH2B1 contribute geographical adaptation to soil pH in rice yinggen Ke, zemin ma, Xuanlin Gao, Shuaizu An, Mengyuan Chen, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7346407/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Enhancing the resilience of crops to ensure stable and high yields under adverse environmental conditions has long been a key objective in rice breeding, yet it remains challenging due to inherent trade-off mechanisms. Here, we report that OsALDH2B1 significantly improves both grain length and alkaline tolerance. Specifically, OsALDH2B1 enhances grain size by suppressing the expression of a grain size and alkaline tolerance related gene GS3 , while it positively regulates alkaline tolerance by reducing reactive oxygen species (ROS) accumulation in a manner that is partially independent of GS3 . Moreover, somatic embryogenesis receptor kinase like 1 (SERL1) phosphorylates and stabilizes OsALDH2B1 in response to alkaline stress. Additionally, the alkaline tolerant allele of OsALDH2B1 is predominantly distributed in high soil pH level regions. This study defines a previously unknown pathway by which the OsALDH2B1-centered module regulates alkaline tolerance for high soil pH value adaptation in rice. Biological sciences/Plant sciences/Plant stress responses/Salt Biological sciences/Plant sciences/Plant breeding Alkaline tolerance Grain length Catalase gene Rice (Oryza sativa L.) Geographical adaptation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Soil salinization and alkalization, now intensified by rapid climate change, have become urgent global threats. These combined stresses impose ion toxicity, osmotic shock, oxidative burst, and high-pH injury on crops, sharply curtailing where and how much we can grow 1 , 2 . Meanwhile, the human population is projected to reach 11 billion by 2100 3 . Rising numbers demand not only more calories but also more nutritious diets, yet urbanization, industrialization, and unsustainable farming continue to shrink the arable land base. Feeding the planet from an ever-smaller patch of arable land is therefore the defining agricultural challenge of the 21st century. Meeting this challenge demands a multi-pronged strategy, one of whose pillars must be the creation of high-yielding, stress-resilient cultivars capable of thriving on previously unusable sodic soils. However, yield and environmental robustness have traded off against each other 4 – 7 . Discovering genes that can simultaneously boost productivity and alkaline tolerance is thus a prerequisite for next-generation breeding. Rice ( Oryza sativa L.), the staple of nearly half of humanity, offers an exceptional opportunity. Its vast collection of landraces carries genetic variants adapted to soils of widely differing pH, providing an unmatched reservoir for climate-smart breeding 8 – 11 . Recent years have witnessed landmark advances in dissecting rice alkaline-stress biology: GS3, an atypical Gγ protein, was shown to limit tolerance by promoting aquaporin phosphorylation and hydrogen peroxide (H 2 O 2 ) accumulation 12 , while optimal gibberellin levels were found to balance both alkaline-thermal resilience and yield via ROS control and chromatin modification 13 . Yet the genetic architecture that links regional soil pH to the geographic distribution of rice accessions remains largely uncharted. Grain size is a major determinant of grain yield and has been a key target during rice domestication 14 . This complex, polygenic trait is orchestrated by a network of quantitative trait loci (QTLs) 15 . During the past decade, numerous QTLs and genes have been isolated and mechanistically dissected, revealing a circuitry that spans hormone signalling, G-protein cascades, ubiquitin-proteasome degradation, kinase relays and transcriptional control 14 , 16 , 17 . We previously identified a multifunctional aldehyde dehydrogenase OsALDH2B1 as a positive regulator of grain size that acts by repressing GS3 16,18–20 . Here we reveal that OsALDH2B1 simultaneously lengthens grains and confers robust alkaline tolerance. Natural variation in OsALDH2B1 tracks global soil pH gradients, and the derived functional allele frequency mirrors zones of historic sodicity. Mechanistically, OsALDH2B1 represses GS3 and trans-activates catalase genes, curbing ROS accumulation. Upon alkaline challenge, the plasma-membrane receptor-like kinase SERL1 phosphorylates and stabilizes OsALDH2B1, thereby locking the protective response in place. Our findings establish OsALDH2B1 as a central node in rice geographic adaptation and provide breeders with a single, dual-purpose target to simultaneously enhance yield and alkaline resilience. Results OsALDH2B1 promotes alkaline tolerance To identify transcription factors controlling grain size and potentially conferring alkaline tolerance in rice, we re-analyzed the alkaline-stress microarray dataset 21 . Among 21 known grain-size regulated transcription factors 16 – 17 , seven exhibited differential expression under alkaline stress (Fig. 1 A), prompting us to focus on OsALDH2B1. Further analysis showed that OsALDH2B1 expression was significantly upregulated and peaked at 6 h after alkaline treatment (Fig. 1 F). Consistently, OsALDH2B1 protein accumulation and phosphorylation is activated by this stress (Fig. 1 H, I). To further explore the potential correlation between OsALDH2B1 transcripts and alkaline tolerance, we examined its transcript levels across ten rice varieties, comprising five japonica varieties and five indica varieties. The indica varieties were more susceptible to alkaline stress while the japonica varieties, which demonstrated greater tolerance (Extended Data Fig. 1 ). The transcript levels of OsALDH2B1 were significantly higher in the resistant varieties compared to the susceptible ones, both before and after treatment (Fig. 1 G). These findings suggest that higher OsALDH2B1 transcript levels are associated with enhanced alkaline tolerance. To elucidate the genetic role of OsALDH2B1 in alkaline tolerance, we subjected the OsALDH2B1 knockout mutant osaldh2b1 ( 2b1 ) and its complementary plants ( 2B1-C ) 16 to stress. The mutant exhibited a marked reduction in alkaline tolerance compared to WT; however, the 2B1-C plants restored the tolerance (Fig. 1 B, C), thereby confirming that OsALDH2B1 mutation compromises alkaline tolerance. To investigate whether overexpression of OsALDH2B1 could increase alkaline tolerance, we generated OsALDH2B1 -overexpressing plants ( 2B1-oe ) in ZH11 (Extended Data Fig. 2 A). Subsequently, the 2B1-oe seedlings were subjected to treatment and exhibited enhanced tolerance (Fig. 1 D, E). Collectively, these data underscored the positive role of OsALDH2B1 in rice alkaline tolerance. Alkaline stress damages plant cells largely through the over-accumulation of ROS 12 , 13 . After alkaline treatment, the 2b1 mutant accumulated significantly more H 2 O 2 , whereas 2B1-oe seedlings showed lower H 2 O 2 levels than WT (Extended Data Fig. 3 A, B). Concomitantly, catalase activity decreased in 2b1 but increased in 2B1-oe relative to WT (Extended Data Fig. 3 C). Together, these data indicate that OsALDH2B1 enhances alkaline tolerance by activating catalase and thus suppressing H 2 O 2 accumulation. To address the impact of OsALDH2B1 on grain yield, we conducted small-scale field trials. Given that the 2b1 mutant is completely male sterile and fails to set seeds 16 , we focused on testing the grain yield of 2B1-oe plants. The field trials revealed that the grain yield of 2B1-oe plants was comparable to that of WT on a per-plant and per-plot basis in normal field (Fig. 1 J, L, N, P). We further explored the role of OsALDH2B1 in yield traits under alkaline conditions. WT and 2B1-oe plants grew throughout their development in alkaline pools with the pH value maintained at approximately 9.1 for plant yield, and solic fields with the pH value maintained at approximately 9.0 for plot yield. Under alkaline conditions, 2B1-oe plants exhibited 28.8% and 31.9% increase in grain yield per plant, and 20.5% and 22.7% increase in grain yield per plot compared to WT (Fig. 1 K, M, O, Q), indicating that overexpressing OsALDH2B1 can markedly enhance alkaline tolerance and grain yield in rice exposed to alkaline conditions. SERL1 physically associates with OsALDH2B1 and regulates alkaline tolerance Alkaline stress rapidly elevated the transcripts, protein abundance and phosphorylation of OsALDH2B1, indicating that the gene is transcriptionally and post-transcriptionally responsive to this stress, implying that the stress stabilizes the OsALDH2B1 protein (Fig. 1 F–I). To uncover the mechanism underlying this stabilization, we purified OsALDH2B1 immunocomplex with anti-OsALDH2B1 antibody and subjected the complex to liquid chromatography-tandem mass spectrometry. Among the co-purified proteins, we identified somatic embryogenesis receptor kinase like 1 (SERL1) (Extended Data Fig. 4 ) 22 . The physical association between OsALDH2B1 and SERL1 was corroborated by yeast two hybrid, in vitro pull-down, in vivo co-immunoprecipitation and bimolecular fluorescence complementation assays, demonstrating that the two proteins interact in vitro and in vivo (Fig. 2 A–D). To assess the physiological relevance of this interaction, we generated two independent SERL1 knockout lines ( serl1-1 and serl1-2 ) using CRISPR/Cas9-mediated genome editing (Extended Data Fig. 5 ) 23 and tested their alkaline tolerance. Both mutants displayed a severe reduction in tolerance relative to WT controls (Fig. 2 E, H). The complementary plants ( SERL1-C ) fully restored alkaline tolerance, confirming that SERL1 is essential for alkaline-stress survival (Fig. 2 F, I). SERL1 phosphorylates and stabilizes OsALDH2B1 under alkaline stress Bioinformatic analysis predicts that SERL1 harbors an N-terminal signal peptide, tandem leucine-rich repeats, a single transmembrane helix, and a C-terminal cytoplasmic kinase domain (Fig. 2 G) 22 . Consistent with this topology, BiFC assay localized the SERL1-OsALDH2B1 interaction to the plasma membrane, suggesting that SERL1 itself resides at this site (Fig. 2 D). Transient expression of SERL1-GFP in rice protoplasts yielded a strong ring-like fluorescence at the cell periphery, confirming plasma-membrane localization (Extended Data Fig. 6 A). This pattern was reproduced in stable SERL1-C transgenic lines, where SERL1-GFP was consistently detected at the plasma membrane of root epidermal cells (Fig. 3 A; Extended Data Fig. 6 B). Together, these data establish SERL1 as a plasma-membrane protein. We next evaluated SERL1 kinase activity in vitro. Recombinant MBP-tagged SERL1 JMK (MBP-SERL1 JMK ) and the constitutively active variant MBP-SERL1 JMK/K310D displayed robust autophosphorylation, whereas the kinase-dead variant MBP-SERL1 JMK/K310R was catalytically inactive (Fig. 2 G, 3 B). Thus, SERL1 possesses intrinsic kinase activity, confirming SERL1 as an active receptor-like kinase. Prompted by the SERL1-OsALDH2B1 interaction, we tested whether OsALDH2B1 is a direct SERL1 substrate. In-vitro phosphorylation assay showed that both MBP-SERL1 JMK and MBP-SERL1 JMK/K310D but not MBP-SERL1 JMK/K310R phosphorylate OsALDH2B1 (Fig. 3 C). We then examined whether SERL1 influences OsALDH2B1 phosphorylation in vivo. Alkaline stress rapidly induced SERL1 accumulation and phosphorylation (Fig. 3 D, E), and subsequent analysis revealed that OsALDH2B1 phosphorylation was markedly reduced, though not abolished, in serl1 seedlings under alkaline stress, implying SERL1 regulating OsALDH2B1 phosphorylation in vivo and the existence of additional kinases (Fig. 3 F). Because phosphorylation often modulates substrate stability 24 , we asked whether SERL1 affects OsALDH2B1 abundance. Immunoblotting showed that alkaline stress-induced accumulation of OsALDH2B1 was attenuated in serl1 seedlings compared with WT (Fig. 3 G), indicating that SERL1-mediated phosphorylation stabilizes OsALDH2B1 under alkaline conditions. To dissect the genetic relationship between SERL1 and OsALDH2B1 in alkaline tolerance, we generated serl1-1/2B1-oe7 plants by crossing. After alkaline treatment, their survival rate was significantly lower than that of 2B1-oe7 , but modestly higher than that of WT and serl1-1 (Fig. 3 H, I). Collectively, these results demonstrate that SERL1 acts both genetically and biochemically as a positive regulator required for full OsALDH2B1 function in rice alkaline tolerance. OsALDH2B1 acts upstream of GS3 Our earlier work established that OsALDH2B1 directly represses GS3 transcription and mutation in OsALDH2B1 reduces grain size 16 . Because the 2b1 mutant is male sterile, we field-examined the grain phenotype of 2B1-oe plants. These lines produced markedly larger, heavier grains (Fig. 4 A, D, E), accompanied by reduced GS3 expression (Extended Data Fig. 2 B), confirming that OsALDH2B1 promotes grain enlargement. To position OsALDH2B1 genetically relative to GS3 , we crossed 2B1-oe7 with the short-grain line GS3-1-flag 20 and generated the 2B1-oe7/GS3-1-flag plants. Seeds from the hybrid were significantly smaller and lighter than those of WT or 2B1-oe7 , and indistinguishable from GS3-1-flag (Fig. 4 B, F, G), demonstrating that OsALDH2B1 functions genetically upstream of GS3 in controlling grain size. GS3 homologs have been reported to heighten alkaline sensitivity, and GS3-1-flag seedlings display increased susceptibility 12 . We therefore asked whether GS3 mediates the alkaline tolerance conferred by OsALDH2B1 . Under alkaline stress, 2B1-oe7/GS3-1-flag seedlings were as sensitive as GS3-1-flag and significantly more sensitive than WT or 2B1-oe7 (Fig. 4 C, I). This epistasis was mirrored by GS3 expression patterns in 2b1 and 2B1-oe lines following alkaline treatment (Fig. 4 H). Consistently, 2B1-oe7/GS3-1-flag seedlings accumulated more H 2 O 2 than WT and 2B1-oe7 , less than GS3-1-flag (Extended Data Fig. 7A, B). Surprisingly, catalase activity was indistinguishable between 2B1-oe7/GS3-1-flag and 2B1-oe7 seedlings, and GS3-1-flag seedlings showed wild-type catalase levels (Extended Data Fig. 7C). Together, these data indicate that GS3 promotes alkaline sensitivity through ROS over-accumulation, independently of catalase activity. OsALDH2B1 thus acts genetically upstream of GS3 in regulating both grain size and alkaline tolerance, while its modulation of catalase activity proceeds via a GS3 -independent pathway. OsALDH2B1 directly targets Cats As a transcription factor, OsALDH2B1 is known to bind to the promoters of target genes and differentially regulate their expression 16 . This prompted us to investigate whether OsALDH2B1 modulates catalase activity by regulating the expression of catalase genes ( Cat s). To this end, we measured the expression patterns of Cat s in 2b1 mutant and 2B1-oe seedlings in response to alkaline treatment. We found that all three Cat s, Catalase A ( CatA ), CatB and CatC , exhibited similar expression patterns. Specifically, the 2b1 mutant accumulated lower levels of Cat s’ transcripts, whereas the 2B1-oe seedlings exhibited higher levels of Cat s’ transcripts (Fig. 5 I; Extended Data Fig. 8). To determine whether OsALDH2B1 can directly bind to the promoters of Cat s, we first searched for OsALDH2B1 binding sequences within these promoters and discovered that all three promoters contained multiple sequences similar to OsALDH2B1 binding motifs (Fig. 5 E). ChIP–qPCR and EMSA demonstrate that OsALDH2B1 can bind to the promoters of Cat s both in vitro and in vivo (Fig. 5 F, G). The transient expression assay in rice protoplasts indicated that OsALDH2B1 activates CatA expression (Fig. 5 H). These results indicate that OsALDH2B1 binds to the promoters of Cat s and activates their expression. It has been well-established that Cats play crucial roles in scavenging ROS in rice stresses response 25 – 28 . To examine the role of Cats in alkaline tolerance, the catc mutants 27 , 29 , 30 were first subjected to alkaline treatment exhibited increased sensitivity indicating that CatC mutation increases alkaline sensitivity (Extended Data Fig. 9). We further created CatA knockout mutants ( cata-1 and cata-2 ) in the ZH11 background using CRISPR/Cas9 technology Extended Data Fig. 10) 23 . The cata mutants showed alkaline sensitive phenotypes (Fig. 5 A, B). Thus, similar to CatC , CatA also functions as a positive regulator of alkaline tolerance in rice. The 2B1-oe7/cata-2 line was developed by crossing 2B1-oe7 with cata-2. After treatment, the relative survival rates of 2B1-oe7/cata-2 plants were comparable to those of the cata-2 mutant but were significantly lower than those of WT and the 2B1-oe7 line (Fig. 5 C, D). The H 2 O 2 accumulation in 2B1-oe7/cata-2 plants was attenuated relative to cata-2 plants yet exceeded that of WT and 2B1-oe7 plants. In parallel, catalase activity in 2B1-oe7/cata-2 plants surpassed that of cata-2 plants but lagged behind WT and 2B1-oe7 plants (Extended Data Fig. 11). The reduced H 2 O 2 accumulation and heightened catalase activity in 2B1-oe7/cata-2 plants, as contrasted with cata-2 plants, likely stem from the activation of CatB and CatC expression by OsALDH2B1. Collectively, these observations signify that OsALDH2B1 operates genetically upstream of CatA . In summary, these results demonstrate that OsALDH2B1 directly activates the expression of Cat s, thereby promoting catalase activity to scavenge H 2 O 2 and enhance alkaline tolerance. Natural variations in OsALDH2B1 are associated with rice alkaline tolerance and geographical distribution The differential accumulation of OsALDH2B1 transcripts among rice varieties suggests the existence of natural variations in the promoter. To pinpoint these variations, we analyzed insertion-deletions (InDels) and single-nucleotide polymorphisms (SNPs) within the OsALDH2B1 promoter region (a 2.0-kb upstream region from the translation start site) using genomic variation data from 918 accessions sourced from RiceVarMap2.0 (Extended Data Table 1;) 31 . Based on polymorphism patterns, these accessions were categorized into four high-confidence haplotypes (Fig. 6 A; Extended Data Fig. 12). Notably, 94.7% of the accessions in Hap1 were indica, while 98.2% of those in Hap4 were japonica. Meanwhile, 92.9% of the accessions in Hap3 were aus, and 66.4% and 20.9% of those in Hap2 were japonica and indica, respectively (Fig. 6 C). To elucidate the impact of these haplotypes on alkaline tolerance, we randomly selected 170 from 918 accessions and subjected them to treatment. Notably, Hap1 demonstrated the highest sensitivity to alkaline stress, while Hap4 exhibited the most remarkable tolerance (Fig. 6 A, B; Extended Data Table 2). Further investigation revealed that japonica rice generally exhibited tolerance to alkaline stress, while indica rice was relatively sensitive, corroborating our previous results (Extended Data Fig. 1 , 13). We also examined the geographical distribution of these accessions relative to the most sensitive and tolerant haplotypes. Accessions in the most sensitive haplotype OsALDH2B1- Hap1 were predominantly found in low-soil-pH regions, whereas those in the most tolerant haplotype OsALDH2B1- Hap4 were widely distributed in high-soil-pH regions (Fig. 6 D). These findings imply that rice accessions with different OsALDH2B1 haplotypes exhibit distinct soil pH distribution tendencies. Discussion Alkalinization of arable soils is an escalating abiotic stress that threatens global food security. Deciphering the genetic and molecular basis of alkaline tolerance is therefore imperative for breeding crops that can thrive on marginal lands. In this study, we reveal that natural allelic variation in the promoter of OsALDH2B1 shapes rice geographical distribution by modulating its expression in response to local soil pH. The OsALDH2B1 -Hap4 haplotype, enriched in japonica, drives higher transcript abundance and confers markedly greater alkaline tolerance, whereas OsALDH2B1 -Hap1, prevalent in indica, is associated with lower expression and heightened sensitivity. Post-transcriptionally, the alkaline-activated kinase SERL1 phosphorylates and stabilizes OsALDH2B1, reinforcing its downstream regulatory circuitry (Fig. 6 E). Rice domestication across contrasting climates has produced two major subspecies that differ in both thermal and pH adaptation: indica, heat-tolerant but alkali-sensitive, dominates low-latitude, low-pH regions, while japonica, cold-tolerant and alkali-resilient, prevails at higher latitudes with alkaline soils (Extended Data Fig. 13;) 9,32 . The selective retention of OsALDH2B1 -Hap4 in high-pH zones underscores its adaptive value and positions japonica germplasm as a strategic resource for enhancing alkaline tolerance in modern cultivars. OsALDH2B1 orchestrates a regulatory cascade that couples stress signalling to developmental output. Alkaline stress triggers ROS over-accumulation; accordingly, 2b1 mutants show elevated H 2 O 2 , whereas 2B1-oe lines exhibit reduced ROS. Mechanistically, OsALDH2B1 represses GS3 , encoding a G-protein component that negatively regulates both grain size and alkaline tolerance 12 , 18 – 20 , 33 , while directly activates the Cats . Enhanced catalase activity accelerates H 2 O 2 detoxification, thereby mitigating oxidative damage. The SERL1-OsALDH2B1- Cats module thus converts an environmental pH cue into precise control of ROS homeostasis. Intriguingly, OsALDH2B1’s ROS-scavenging role is echoed in salt stress, where it is integrated into the OsHDAC1-OsALDH2B1- OsGR3 pathway 34 . Whether ROS itself feeds back on OsALDH2B1 activity, establishing a self-balancing loop, remains an open question. What is clear is that OsALDH2B1 sits at the nexus of multiple stress-response networks, making it a prime target for simultaneous improvement of yield and resilience. In summary, we establish OsALDH2B1 as a dual-function regulator that enlarges grains and fortifies alkaline tolerance. Its natural variants provide a ready genetic toolkit for marker-assisted breeding, and its overexpression elevates yield under stress without the typical trade-off. Deploying OsALDH2B1 -Hap4 or engineered alleles promises rice varieties that maintain productivity on saline–alkaline soils, directly contributing to global food security. Future work should (i) dissect the promoter polymorphisms governing expression, (ii) clarify feedback between ROS and OsALDH2B1 activity, and (iii) explore additional signalling pathways and the enzymatic contribution of its aldehyde-dehydrogenase to alkaline resilience. Materials and methods Plant materials All the 918 accessions use for are listed in Extended Data Table 5. Five japonica rice ( O . sativa L.) varieties, NIP, ZH11, Taichung 65, Dongjin, and Mudanjiang 8, and five indica rice varieties, 9311, Minghui 63, Nanjing 11, Zhenshan 97, and IR64 are wild types. The 2B1-oe lines, serl1 and cata were derived from ZH11. The 2b1 , 2B1-C , GS3-1-flag, catc and catc-cr lines used in this study have been described in detail in previous studies 16 , 20 , 29 , 30 . Phenotypic evaluation To examine alkali tolerance at the seedling stage, seeds were germinated and cultivated in Yoshida solution (pH 5.8) at 28°C, 60 ± 5% relative humidity under a 14 h light/10 h dark photoperiod in a growth chamber (RuihuaHP1000GS-LED/H11). One week after germination, seedlings were treated with 75 mM mixed alkali salts (62.5 mM NaHCO 3 and 12.5 mM Na 2 CO 3 ) 12 for 3–5 days. To determine the survival rate after alkali and heat treatment, seedlings were allowed to recover under normal growth conditions in a timely manner to prevent over-treatment. After 7 days of recovery, the death of young leaves was used as an indicator of seedling death. The survival rate was then calculated as the ratio of the number of surviving seedlings to the total number of seedlings, and survival phenotypes were photographed. For identifying rice alkali tolerance at the reproductive stage, rice plants were grown throughout their development in alkaline pools and alkaline soil plot with the pH value maintained at approximately 9.1 and 9.0 adjusted by mixed alkali (NaHCO 3 :Na 2 CO 3 with a molar ratio of 5:1), respectively. RNA extraction and reverse transcription quantitative PCR Total RNA was extracted from fresh tissues using TRIzol reagent (Invitrogen). cDNA was synthesized with HiScript III RT SuperMix (Vazyme, China) according to the manufacturer’s protocol. For gene expression analysis, reverse transcription quantitative PCR (RT-qPCR) was performed using LightCycler 480 SYBR Green I Master (Roche, Switzerland) in the ABI 7500 Real-Time PCR System (Applied Biosystems, USA) with gene-specific primers (Extended Data Table 3). The rice actin gene was used as inner control. Plasmid construction and transformation The OsALDH2B1 overexpression ( 2B1-oe ) construct was prepared by amplifying and inserting the full-length coding sequences of OsALDH2B1 derived from ZH11 into pCambia1301-HA-cYFP vector with a HA tag located at the N-terminal and C-terminal of yellow fluorescent protein (cYFP) located at the C-terminal and driven by the Cauliflower mosaic virus 35S promoter. The cata and serl1 mutants were constructed using a genome-editing system 23 . Two 19-bp–specific single-guide RNA (sgRNA) target sequences of the gene were synthesized and ligated to the pYLgRNA-OsU3 and YLgRNA-OsU6a vectors, respectively. The purified guide RNA (gRNA) expression cassette was subsequently inserted into the binary pYLCRISPR–Cas9-MH vector. The sgRNAs were designed using the website http://crispr.hzau.edu.cn/CRISPR2/ . 2B1-oe and these recombinant pYLCRISPR–Cas9-MH plasmids were each introduced into ZH11 through Agrobacterium-mediated transformation by Boyuan Biotechnology Co., Ltd, Wuhan China. The positive transgenic plants of the T 0 generation were identified by PCR. Gene expression levels of OsALDH2B1 in transgenic overexpression plants were detected by qPCR. Homozygous mutants of the T 1 generation were confirmed in the sgRNA target sequence by PCR and sequence. The complementary construct was generated by introducing full length SERL1 cDNA fused with Green fluorescent protein ( GFP) under the control of SERL1 native promoter ( SERL1-C ) and was inserted into pCambia1300 vector and transformed in the serl1-1 mutant. The P CatA : LUC construct was made by fusing the luciferase ( LUC ) to a ∼2 kb putative promoter region of CatA in pGreenⅡ0800 vector. All related primers are shown in Extended Data Table 4. DAB staining and measurement of H 2 O 2 content The formation of H 2 O 2 in the leaves was investigated by the 3,3′-diaminobenzidine (DAB) staining and quantification methods 27 . Two-week-old seedlings were used for physiological measurements. Leaves under treatment were collected and soaked in 50 ml of staining buffer (1 mg mL − 1 DAB, 20 mM pH7.5 phosphate buffer). After vacuuming, the samples were incubated overnight in the dark at room temperature. Samples were transferred to an eluent (anhydrous ethanol:glacial acetic acid:glycerol = 3:1:1) and boiled for 10 min to remove chlorophyll, and then the images of samples were taken under the same condition. For H 2 O 2 quantification, the leaves from different plants for each sample were homogenized in liquid nitrogen, and H 2 O 2 were extracted and determined using an H 2 O 2 assay kit (Sangon, China) according to the manufacturer’s instructions. Catalase activity assay To analyze catalase activity, leaves from different plants for each sample were homogenized in liquid nitrogen and were extracted in 1 mL extraction buffer (150 mM NaCl, 50 mM phosphate buffer, pH7.5, 0.5% [v/v] Triton X-100, 10% [v/v] glycerol, and 1% [w/v] protease inhibitor cocktail). The supernatant was collected after being centrifuged at 12,000 rpm for 10 min at 4°C, and protein concentration was estimated by the Bradford method according to the manufacturer’s protocol and standardized using a BSA standard curve (0.25 to 4 µg µL − 1 ). The supernatant was used for catalase activity analysis with the Catalase Assay Kit (Beyotime, China) according to previous study 27 . Chromatin immunoprecipitation (ChIP)–qPCR assay The ChIP–qPCR assay was conducted according to the ChIP Assay method for rice 16 . Three grams of 12-day-old rice seedlings were rapidly fixed by infiltration with 1% (v/v) formaldehyde under vacuum at 25°C for 15 min, after which they were ground to a powder in liquid nitrogen. The cell nuclei were separated and lysed. Chromatin was extracted and fragmented via ultrasound to 200–800 bp. Anti-OsALDH2B1 antibody (abclonal, Cat# A20663) and IgG (Abcam, Cat# ab171870, control) were incubated with 40 µL protein A Dynabeads (Invitrogen, Cat# 10001D) at 4°C for 4 h after washing the beads, 100 µL fragmented chromatin suspension was added, followed by incubation at 4°C overnight. After extensive washing and de-crosslink, the precipitated and input DNA samples were amplified by qPCR. Primers for ChIP-qPCR are listed in Extended Data Table 5. Recombinant protein expression and purification The MBP-OsALDH2B1 , MBP-SERL1 JMK , MBP-SERL1 JMK/K310D , MBP-SERL1 JMK/K310R and GST-SERL1 JMK constructs were prepared and transformed into Escherichia coli strain BL21 to express recombinant proteins. The recombinant proteins were induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 16°C for 20 h. MBP-tagged recombinant proteins were purified with Amylose Resin (New England Biolabs, Cat# E8021L), and GST and GST-SERL1 JMK recombinant protein were purified with was purified with Pierce Glutathione Agarose Resin (Thermo Scientific, Cat# 16101) according to the manufacturer’s instructions. Primers used for plasmid construction are listed in Extended Data Table 4. Electrophoretic mobility shift assay (EMSA) EMSA were performed as previously described 16 . Recombinant MBP-OsALDH2B1 protein was produced in E.coli as described in the section ‘Recombinant protein expression and purification’ above. DNA probes were synthesized and labelled with biotin. DNA gel-shift assays were performed using the chemiluminescence EMSA kit (Beyotime, GS005) and detection and image capture were captured by the Tanon-5200 image system (Tanon, China). The relevant probe sequence is shown in Extended Data Table 6. Transient expression assay in protoplasts To determine the transcriptional regulation activity, the effector construct was prepared by amplifying and inserting the full-length coding sequences of OsALDH2B1 derived from ZH11 fused with Green fluorescent protein (GFP) driven by the ubiquitin promoter and the reporter construct was prepared by amplifying and inserting the CatA promoter sequence into the pGreenⅡ -0800 vector. The effector and reporter constructs were then co-transfected into rice protoplasts. The transfected protoplasts were cultured for 12 ~ 16 h at 25°C in the dark. The luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega, Cat# E1910) according to the manufacturer’s instructions. The relative reporter gene expression levels were expressed as the ratio of firefly luciferase to the Renilla luciferase. Primers used for plasmid construction are listed in Extended Data Table 4. Liquid chromatography-tandem mass spectrometry ( LC-MS/MS) assay Total proteins were extracted from SERL1-C plants expressing SERL1-GFP using protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl 2 , 0.2% NP-40, 5 mM DTT, and 1× protease inhibitor cocktail) and incubated with Dynabeads (Invitrogen, Cat# 10001D) pre-conjugated with anti-GFP antibody (Clonetech, Cat# JL-8) for 2 h. The immunoprecipitated samples were washed five times with protein extraction buffer, and then the proteins were eluted from the beads before proceeding with the LC-MS/MS assay. Yeast two-hybrid assay The constructs were co-transformed into AH109 yeast cells, with the empty vector pGADT7 and pGBKT7 serving as negative controls. Yeast cells that contain both pGBKT7-p53 and pGADT7-T and should grow on SD-LWHA were set as a positive control. The interaction was assessed on synthetically defined (SD) medium lacking Ade, His, Leu and Trp, according to the protocols provided by the manufacturer (Clontech). Co-immunoprecipitation (Co-IP) assay Co-IP assay was performed as described previously 24 . Total proteins from SERL1-C plants were extracted with IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 5 mM DTT and 1× protease inhibitor cocktail) and incubated with anti-GFP antibody conjugated Dynabeads for 2 h. The immunoprecipitated samples were washed five times with washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40), then separated on SDS-PAGE and subjected to immunoblot analysis with anti-OsALDH2B1 antibody (Abclonal, Cat# A20663) 34 . Bimolecular fluorescence complementation ( BiFC) assay BiFC assay was performed as described previously 27 . The constructs P 35S : OsALDH2B1-cYFP , P 35S : SERL1-nYFP , P 35S : GUS-cYFP , and P 35S : GUS-nYFP were transformed into A. tumefaciens strain GV1301 and co-expressed in N. benthamiana leaves for 48 h. YFP fluorescence was visualized by confocal microscopy (TCS SP2; Leica) with an excitation wavelength of 514 nm. In vitro pull-down assay The in vitro pull-down assay was performed as previously described 27 . Recombinant proteins GST-SERL1 JMK and MBP-OsALDH2B1 were purified from E. coli . GST-SERL1 JMK or GST were incubated with Glutathione beads (GE Healthcare) at 4C for 2 h, followed by incubation with MBP-OsALDH2B1 for an additional hour. After elution from the beads, the proteins were analyzed by immunoblotting with anti-GST (ABclonal, Cat# AE001) and anti-MBP antibodies (New England Biolabs, Cat# E8032L) to detect GST-SERL1 JMK and MBP-OsALDH2B1, respectively. Phosphorylation assay The phosphorylation assay was performed as previously described 35 . For in vitro phosphorylation assays followed by p-Nitrobenzyl mesylate (PNBM) alkylation, MBP-OsALDH2B1 was incubated with MBP-SERL1 JMK , MBP-SERL1 JMK/S310R , MBP-SERL1 JMK/S310D or in 30 µl of reaction buffer [50 mM tris-HCl, pH 7.5, 10 mM MgCl2, and 1 Mm adenosine 5′-(γ-thio)triphosphate (ATPγS; Abcam, Cat# ab138911)] at room temperature for 30 min (flick and centrifuge briefly). PNBM (2.5 mM; Abcam, Cat# ab138910) was then added to the reaction mixture. After incubation at room temperature for 1 hour, the proteins were separated by SDS-PAGE and detected using anti-thiophosphateester antibody (Abcam, Cat# ab92570). The Coomassie brilliant blue–stained gel was used as a loading control. For in vivo phosphorylation assays, 10-day-old seedlings were treated with alkali for the indicated times and collected immediately. Total proteins were extracted with buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 5 mM DTT, 1× protease inhibitor cocktail and 1× PhosStop) and followed by digestion with or without λPP (λ alkaline protein phosphatase; New England Biolabs, Cat# P0753S) at 30°C for 30 min, then separated on SDS-PAGE with 50 µM Phos-tag (APExBIO, F4002) and subjected to immunoblot analysis with anti-GFP and anti-OsALDH2B1 antibodies, respectively. Rice geographical distribution and soil pH value analysis For the geographical distribution of OsALDH2B1 haplotypes, rice varieties with confirmed location information were projected onto the map based on their genetic structure and origin information from RiceVarMap2.0 31 . For the analysis of soil pH values in rice-planting areas, the global soil pH distribution data (1:5 soil:water suspension) and regional averages were obtained from the World Soil Information Service 2023 Snapshot 36 . Statistical analysis All data plotting and statistical analyses were performed with GraphPad Prism 8.0 software ( https://www.graphpad.com/ ). Details about the statistical parameters, such as the means ± SEM, are shown in the figure legends. A two-tailed Student’s t test for two groups or a one-way analysis of variance (ANOVA) with Dunnett’s or Tukey’s multiple comparisons test for multiple groups were carried out. The number of samples is represented by n. Asterisks indicate statistical significance: * P < 0.05; ** P < 0.01. Different letters above bars indicate differences at P < 0.05. Declarations Funding information This work was supported by the Hubei Provincial Natural Science Foundation (2024AFB917, 2023AFA016), and Hubei Provincial Key Research and Development Projects (2024BBB001). Acknowledgments We sincerely thank Dr. Guangcun He from Wuhan University, Dr. Shengyuan Sun from Yangzhou University and Dr. Liyong Cao from China National Rice Research Institute for their providing rice germplasm accessions, GS3-1-flag and catc-cr lines, respectively. Data availability All data are included in the manuscript and supplemental files. Conflict of interest The authors declare no conflict of interest. Author contributions Z.M., X.G., S.A. performed the experiments. Z.M., X.G., S.A., M.C., J.L., B.Z., F.Y., M.L., Y.K., P.Y. performed some of the experiments and data analysis. Z.M., X.G., S.A., Y.K., P.Y. designed the experiments, analyzed data, and wrote the paper. Y.K., P.Y. review, editing and supervision. References Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444:139–158 Zhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167:313–324 Adam D (2021) How far will global population rise? Researchers can’t agree. Nature 597:462–465 Peleg Z, Blumwald E (2011) Hormone balance and abiotic stress tolerance in crop plants. Curr Opin Plant Biol 14:290–295 Qian Q, Guo LB, Smith SM, Li JY (2016) Breeding high-yield superior quality hybrid super rice by rational design. Natl Sci Rev 3:283–294 Bailey-Serres J, Parker JE, Ainsworth EA, Oldroyd GED, Schroeder J (2019) I. Genetic strategies for improving crop yields. Nature 575:109–118 Salse J, Barnard RL, Veneault-Fourrey C, Rouached H (2024) Strategies for breeding crops for future environments. Trends Plant Sci 29:303–318 Savolainen O (2011) The genomic basis of local climatic adaptation. Science 334:49–50 Li XM et al (2015) Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of african rice. Nat Genet 47:827–833 Russell J et al (2016) Exome sequencing of geographically diverse barley landraces and wild relatives gives insights into environmental adaptation. Nat Genet 48:1024–1030 Romero Navarro JA et al (2017) A study of allelic diversity underlying flowering-time adaptation in maize landraces. Nat Genet 49:476–480 Zhang HL et al (2023) A Gγ protein regulates alkaline sensitivity in crops. Science 379 Guo S-Q et al (2025) Fine-tuning gibberellin improves rice alkali–thermal tolerance and yield. Nature 639:162–171 Li N, Xu R, Li YH (2019) Molecular networks of seed size control in plants. Annu Rev Plant Biol 70:435–463 Xing YZ, Zhang QF (2010) Genetic and molecular bases of rice yield. Annu Rev Plant Biol 61:421–442 Ke YG et al (2020) The versatile functions of OsALDH2B1 provide a genic basis for growth–defense trade-offs in rice. Proc. Natl. Acad. Sci. 117, 3867–3873 Ren DY, Ding CQ, Qian Q (2023) Molecular bases of rice grain size and quality for optimized productivity. Sci Bull 68:314–350 Fan CC et al (2006) GS3 , a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor Appl Genet 112:1164–1171 Mao HL et al (2010) Linking differential domain functions of the GS3 protein to natural variation of grain size in rice. Proc. Natl. Acad. Sci. 107, 19579–19584 Sun SY et al (2018) A G-protein pathway determines grain size in rice. Nat Commun 9 Li N et al (2018) Transcriptome analysis of two contrasting rice cultivars during alkaline stress. Sci Rep 8 Singla B, Khurana JP, Khurana P (2009) Structural characterization and expression analysis of the SERK/SERL gene family in rice ( Oryza sativa ). Int. J. Plant Genomics (2009) Ma XL et al (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284 Zeng R et al (2025) A natural variant of COOL1 gene enhances cold tolerance for high-latitude adaptation in maize. Cell 188:1315–1329e13 Gao MJ et al (2021) Ca 2+ sensor-mediated ROS scavenging suppresses rice immunity and is exploited by a fungal effector. Cell 184:5391–5404e17 You XM et al (2022) Rice catalase OsCATC is degraded by E3 ligase APIP6 to negatively regulate immunity. Plant Physiol 190:1095–1099 Liao M et al (2023) ENHANCED DISEASE SUSCEPTIBILITY 1 promotes hydrogen peroxide scavenging to enhance rice thermotolerance. Plant Physiol 192:3106–3119 Wang JJ et al (2023) SEMI-ROLLED LEAF 10 stabilizes catalase isozyme B to regulate leaf morphology and thermotolerance in rice ( Oryza sativa L). Plant Biotechnol J 21:819–838 Lin AH et al (2011) Nitric oxide and Protein S-nitrosylation are integral to hydrogen peroxide-induced leaf cell death in rice. Plant Physiol 158:451–464 Wang BF et al (2024) OsCPK12 phosphorylates OsCATA and OsCATC to regulate H 2 O 2 homeostasis and improve oxidative stress tolerance in rice. Plant Commun 5:100780 Zhao H et al (2014) RiceVarMap: a comprehensive database of rice genomic variations. Nucleic Acids Res 43:D1018–D1022 Xu YF et al (2020) Natural variations of SLG1 confer high-temperature tolerance in indica rice. Nat Commun 11 Sun SY et al (2025) Novel repetitive elements in plant-specific tails of Gγ proteins as the functional unit in G-protein signaling in crops. Plant Cell 37 Wu YQ et al (2025) OsHDAC1 deacetylates the aldehyde dehydrogenase OsALDH2B1, repressing OsGR3 and decreasing salt tolerance in rice. Plant Physiol 198 Xu L et al (2024) An AP2/ERF transcription factor confers chilling tolerance in rice. Sci Adv 10 Batjes NH, Calisto L, de Sousa LM (2024) Providing quality-assessed and standardised soil data to support global mapping and modelling (WoSIS snapshot 2023). Earth Syst Sci Data 16:4735–4765 Additional Declarations There is NO Competing Interest. Supplementary Files MZMSERL1OsALDH2B1ExtendedDataFig.113.docx Figure 1, Figure 2,Figure 3, Figure 4,Figure 5, Figure 6,Figure 7, Figure 8,Figure 9, Figure 10,Figure 11, Figure 12,Figure 13 MZMOsALDH2B1ExtendedDataTable36.xlsx Dataset 3,Dataset 4,Dataset 5,Dataset 6. MZMOsALDH2B1ExtendedDataTable1.xlsx Table 1 MZMOsALDH2B1ExtendedDataTable2.xlsx Table 2 ExtendedDatainformationLegends.docx Cite Share Download PDF Status: Under Review Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-7346407","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":505741690,"identity":"9f9d7774-1937-4292-abbe-248aa84d5bc0","order_by":0,"name":"yinggen Ke","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFACNgaGjw0MCSCmBNFaGGeSrIWZlyQtBueXJT623XE4z+AA88HbPAx2eYS13Hh22Dj3zOFigwNsydY8DMnFRGg53iad23Y4ccMBHjNpHoYDiQ1EaGn/bQnWwv+NSC3n244xM0JsYSNOi+QNtmTJ3rb0YsnDbMaWcwySCWvhO3/M8MPPNus8vuPND2+8qbAjrEXhRgKUxQx2JyH1QCDff4AIVaNgFIyCUTCyAQBGuEGhrTo4jQAAAABJRU5ErkJggg==","orcid":"","institution":"Hubei University","correspondingAuthor":true,"prefix":"","firstName":"yinggen","middleName":"","lastName":"Ke","suffix":""},{"id":505741691,"identity":"8f33c941-09a9-4c20-a282-3aedca6da41b","order_by":1,"name":"zemin ma","email":"","orcid":"https://orcid.org/0009-0007-3660-9992","institution":"","correspondingAuthor":false,"prefix":"","firstName":"zemin","middleName":"","lastName":"ma","suffix":""},{"id":505741692,"identity":"109509a4-b449-40a6-a387-cb5807020845","order_by":2,"name":"Xuanlin Gao","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Xuanlin","middleName":"","lastName":"Gao","suffix":""},{"id":505741693,"identity":"eb40d331-bfa6-4e82-8dff-0db14f463ce9","order_by":3,"name":"Shuaizu An","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Shuaizu","middleName":"","lastName":"An","suffix":""},{"id":505741694,"identity":"c2d484d7-947f-47d3-a355-d669927ab5ac","order_by":4,"name":"Mengyuan Chen","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Mengyuan","middleName":"","lastName":"Chen","suffix":""},{"id":505741695,"identity":"ef3bba2c-e167-46a6-8cd8-aaa3b97dedad","order_by":5,"name":"Jun Lv","email":"","orcid":"","institution":"Hubei University of Medicine,Taihe Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Lv","suffix":""},{"id":505741696,"identity":"9ccbc61a-6b63-44ea-82b3-6658021ced84","order_by":6,"name":"Biaoming Zhang","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Biaoming","middleName":"","lastName":"Zhang","suffix":""},{"id":505741697,"identity":"274fb27f-2390-459e-9718-22c88d7df384","order_by":7,"name":"Feng Yu","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Feng","middleName":"","lastName":"Yu","suffix":""},{"id":505741698,"identity":"bf15d4ff-5f19-4abc-8168-467f61cbd757","order_by":8,"name":"Ming Li","email":"","orcid":"","institution":"Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Li","suffix":""},{"id":505741699,"identity":"358d0ebb-3acb-477d-8f1d-3d4a6d922e46","order_by":9,"name":"Pingfang Yang","email":"","orcid":"https://orcid.org/0000-0003-3526-4543","institution":"State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University","correspondingAuthor":false,"prefix":"","firstName":"Pingfang","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-08-11 12:35:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7346407/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7346407/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92237560,"identity":"32ae94ff-dc11-4d7a-aca4-e8745485820c","added_by":"auto","created_at":"2025-09-26 07:49:21","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":297370,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsALDH2B1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promotes alkaline tolerance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe expression of rice grain size-regulated transcription factor genes in response to alkaline treatment. The data were analyzed according to the public microarray database (\u003ca href=\"https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104928\"\u003eGSE104928\u003c/a\u003e). Blue and red asterisks indicate down- and up-regulation (|log2 fold change| ≥ 1, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05), respectively. \u003cstrong\u003e(B and C) \u003c/strong\u003eRepresentative phenotypes \u003cstrong\u003e(B)\u003c/strong\u003eand survival rates \u003cstrong\u003e(C) \u003c/strong\u003eafter alkaline stress of ZH11, \u003cem\u003e2b1\u003c/em\u003e and \u003cem\u003e2B1-C\u003c/em\u003eseedlings. n = 64 plants from four biological repeats. Bars, 2 cm. \u003cstrong\u003e(D and E)\u003c/strong\u003eRepresentative phenotypes \u003cstrong\u003e(D)\u003c/strong\u003e and survival rates \u003cstrong\u003e(E) \u003c/strong\u003eafter alkaline stress of ZH11 and \u003cem\u003e2B1-oe7\u003c/em\u003e seedlings. n = 48 plants from three biological repeats. Bars, 2 cm. \u003cstrong\u003e(F)\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eOsALDH2B1 \u003c/em\u003eexpression patterns in response to alkaline stress. n = 3. \u003cstrong\u003e(G)\u003c/strong\u003e The expression of \u003cem\u003eOsALDH2B1\u003c/em\u003ein different rice varieties before and 3 h after alkaline treatment. Expression level of \u003cem\u003eOsALDH2B1 \u003c/em\u003ein Nipponbare was set to 1. n = 3. \u003cstrong\u003e(H and I)\u003c/strong\u003eIn vivo analysis showing that OsALDH2B1 protein accumulation \u003cstrong\u003e(H)\u003c/strong\u003e and phosphorylation \u003cstrong\u003e(I)\u003c/strong\u003e is activated by alkaline stress. 10-day-old WT seedlings were treated with alkaline for the indicated times. OsALDH2B1 proteins were detected with anti-OsALDH2B1 antibody. The numbers indicate the ratio of OsALDH2B1/RbcL \u003cstrong\u003e(H)\u003c/strong\u003e and phosphorylated OsALDH2B1/unphosphorylated OsALDH2B1\u003cstrong\u003e (I)\u003c/strong\u003e, respectively. The ratio of ck was set to 1. The lower panel shows the loading control. λPP, λalkaline protein phosphatase. \u003cstrong\u003e(J and K)\u003c/strong\u003e Plant architecture of ZH11 and \u003cem\u003e2B1-oe\u003c/em\u003elines under normal \u003cstrong\u003e(J)\u003c/strong\u003e and alkali \u003cstrong\u003e(K) \u003c/strong\u003estress conditions. Bars, 20 cm. \u003cstrong\u003e(L and M)\u003c/strong\u003e Panicle morphology of ZH11 and \u003cem\u003e2B1-oe\u003c/em\u003e lines under normal \u003cstrong\u003e(L)\u003c/strong\u003e and alkaline stress \u003cstrong\u003e(M)\u003c/strong\u003e conditions. Bars, 5 cm. \u003cstrong\u003e(N and O)\u003c/strong\u003e Plant grain yield of ZH11 and \u003cem\u003e2B1-oe\u003c/em\u003e lines under normal \u003cstrong\u003e(N)\u003c/strong\u003eand alkaline stress \u003cstrong\u003e(O)\u003c/strong\u003e conditions. n = 20 plants. \u003cstrong\u003e(P and Q)\u003c/strong\u003e Plot grain yield of ZH11 and \u003cem\u003e2B1-oe\u003c/em\u003e lines under normal \u003cstrong\u003e(P)\u003c/strong\u003e and alkaline stress \u003cstrong\u003e(Q)\u003c/strong\u003e conditions. n = 3 biological repeats and each repeat has 40 plants.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SE. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 and *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 analyzed by Student’s \u003cem\u003et-test\u003c/em\u003e. Different letters above bars indicate differences by Multiple range test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/1b0f0facc8ab2d3900a3cd81.png"},{"id":92236438,"identity":"e0e48ff9-3373-4ced-a195-de87599a1dca","added_by":"auto","created_at":"2025-09-26 07:33:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":205570,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSERL1 interacts with OsALDH2B1 and is indispensable for rice alkaline tolerance\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Yeast two hybrid assays showing the interaction of OsALDH2B1 and SERL1. Transformed yeast were serially diluted and placed on SD (synthetic dropout medium). SD-LW, SD-Leu/-Trp. SD-LWHA, SD-Leu/-Trp/-His/-Ade. pGADT7 (AD) and pGBKT7 (BD) are empty vectors. Yeast cells that contain both pGBKT7-p53 and pGADT7-T and should grow on SD-LWHA were set as a positive control. \u003cstrong\u003e(B)\u003c/strong\u003e Co-immunoprecipitation (Co-IP) assay showing the interaction between OsALDH2B1 and SERL1. Total proteins were immunoprecipitated with anti-GFP antibody in \u003cem\u003eSERL1-C\u003c/em\u003e and \u003cem\u003eGFP\u003c/em\u003e plants. Co-immunoprecipitated OsALDH2B1 was detected with anti-OsALDH2B1 antibody. \u003cstrong\u003e(C) \u003c/strong\u003eIn vitro MBP pull-down assay showing the interaction of OsALDH2B1 and SERL1. Recombinant GST-SERL1\u003csup\u003eJMK\u003c/sup\u003e or GST were immobilized on glutathione agarose beads and incubated with MBP-OsALDH2B1 (MBP-2B1). GST-SERL1\u003csup\u003eJMK \u003c/sup\u003ewas detected with anti-GST antibody. \u003cstrong\u003e(D)\u003c/strong\u003e Bimolecular fluorescence complementation (BiFC) assay showing the interaction between OsALDH2B1 and SERL1 in \u003cem\u003eN. benthamiana \u003c/em\u003eleaf cells. The Yellow Fluorescent Protein (YFP) signal was visualized by confocal microscopy Bars, 10 µm. \u003cstrong\u003e(E and H)\u003c/strong\u003e Representative phenotypes \u003cstrong\u003e(E)\u003c/strong\u003e and survival rates \u003cstrong\u003e(H)\u003c/strong\u003e after alkaline stress of ZH11 and \u003cem\u003eserl1\u003c/em\u003e seedlings. Bars, 2 cm. \u003cstrong\u003e(F and I) \u003c/strong\u003eRepresentative phenotypes \u003cstrong\u003e(F)\u003c/strong\u003e and survival rates\u003cstrong\u003e (I)\u003c/strong\u003e after alkaline stress of ZH11, \u003cem\u003eserl1\u003c/em\u003e and \u003cem\u003eSERL1-C\u003c/em\u003e seedlings. Bars, 2 cm. \u003cstrong\u003e(G) \u003c/strong\u003eSchematic diagram of SERL1 protein. SP, signal peptide. LRR, leucine-rich repeat. TM, transmembrane region. KD, kinase domain. JMK, juxtamembrane and kinase domain. K310R and K310D mark the lysine to arginine and aspartate mutation in the kinase domain resulting in a kinase-dead and a kinase-active of SERL1, respectively.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SE. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 and *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 analyzed by Student’s \u003cem\u003et-test\u003c/em\u003e. Different letters above bars indicate differences by Multiple range test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. n = 48 plants from three biological repeats.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/f77338fed24c059bb7e76f2d.png"},{"id":92237383,"identity":"e4771325-8724-48c4-a155-bd4596c2c4ae","added_by":"auto","created_at":"2025-09-26 07:41:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":154244,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSERL1 phosphorylates and stabilizes OsALDH2B1.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eAnalysis the subcellular localization of SERL1 protein in \u003cem\u003eSERL1-C\u003c/em\u003e plants. The root tips of \u003cem\u003eSERL1-C\u003c/em\u003e plants at three day after germination were used for analysis. The cell wall was stained with propidium iodide (PI). Bars, 10 µm. \u003cstrong\u003e(B)\u003c/strong\u003e Autophosphorylation assay of the SERL1\u003csup\u003eJMK\u003c/sup\u003e isoforms. Phosphorylated proteins were detected by an anti-thiophosphateester antibody. \u003cstrong\u003e(C)\u003c/strong\u003e Analysis the in vitro phosphorylation of OsALDH2B1 by the SERL1\u003csup\u003eJMK\u003c/sup\u003e isoforms. Phosphorylated protein was detected by an anti-thiophosphateester antibody. \u003cstrong\u003e(D and E)\u003c/strong\u003e In vivo assay showing that SERL1 accumulation \u003cstrong\u003e(D)\u003c/strong\u003e and SERL1 phosphorylation \u003cstrong\u003e(E)\u003c/strong\u003e is activated by alkaline. Total proteins were extracted from 10-day-old seedlings of \u003cem\u003eSERL1-C\u003c/em\u003e plants at the indicated time. SERL1-GFP proteins were detected by anti-GFP antibody. The numbers indicate the ratio of SERL1/RbcL\u003cstrong\u003e (D)\u003c/strong\u003e and phosphorylated SERL1/unphosphorylated SERL1\u003cstrong\u003e (E)\u003c/strong\u003e. \u003cstrong\u003e(F and G)\u003c/strong\u003e In vivo assay showing that OsALDH2B1 phosphorylation \u003cstrong\u003e(F) \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003eaccumulation \u003cstrong\u003e(G)\u003c/strong\u003e is reduced in \u003cem\u003eserl1\u003c/em\u003e plants under alkaline stress. Total proteins were extracted from 10-day-old seedlings of WT and \u003cem\u003eserl1-1\u003c/em\u003e plants at the indicated time. OsALDH2B1 proteins were detected by anti-OsALDH2B1 antibody. The numbers indicate the ratio of phosphorylated OsALDH2B1/unphosphorylated OsALDH2B1\u003cstrong\u003e (F)\u003c/strong\u003e and OsALDH2B1/RbcL\u003cstrong\u003e (G)\u003c/strong\u003e. The lower panel shows the loading control. λPP, λ alkaline protein phosphatase. \u003cstrong\u003e(H and I) \u003c/strong\u003eRepresentative phenotypes \u003cstrong\u003e(H)\u003c/strong\u003e and survival rates \u003cstrong\u003e(I) \u003c/strong\u003eafter alkaline stress of ZH11, \u003cem\u003e2B1-oe7, serl1-1/2B1-oe7\u003c/em\u003e and \u003cem\u003eserl1-1 \u003c/em\u003eseedlings.\u003cstrong\u003e \u003c/strong\u003en = 48 plants from three biological repeats. Bars, 2 cm.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/33f7bd385ac103dc411e135a.png"},{"id":92236433,"identity":"01d5b828-cbbd-4dd2-8da7-afe6de8d8d9c","added_by":"auto","created_at":"2025-09-26 07:33:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":201731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsALDH2B1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e functions genetically upstream of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eGS3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and B)\u003c/strong\u003e Representative grain morphology of ZH11 and \u003cem\u003e2B1-oe\u003c/em\u003e \u003cstrong\u003e(A)\u003c/strong\u003e, ZH11, \u003cem\u003e2B1-oe\u003c/em\u003e,\u003cem\u003e2B1-oe/GS3-flag\u003c/em\u003e and \u003cem\u003eGS3-flag\u003c/em\u003e plants \u003cstrong\u003e(B)\u003c/strong\u003e. Bars, 1 cm. \u003cstrong\u003e(C and I) \u003c/strong\u003eRepresentative phenotypes \u003cstrong\u003e(C)\u003c/strong\u003e and survival rates\u003cstrong\u003e (I) \u003c/strong\u003eof ZH11, \u003cem\u003e2B1-oe\u003c/em\u003e,\u003cem\u003e 2B1-oe/GS3-flag\u003c/em\u003e and \u003cem\u003eGS3-flag\u003c/em\u003e seedlings after alkaline treatment. n = 48 from three biological repeats. Bars, 2 cm. \u003cstrong\u003e(D and E) \u003c/strong\u003eGrain length \u003cstrong\u003e(D) \u003c/strong\u003eand grain weight \u003cstrong\u003e(E)\u003c/strong\u003e of ZH11 and \u003cem\u003e2B1-oe \u003c/em\u003eplants. n = 50 for \u003cstrong\u003eD \u003c/strong\u003eand 6 for \u003cstrong\u003eE\u003c/strong\u003e. \u003cstrong\u003e(F and G) \u003c/strong\u003eGrain length \u003cstrong\u003e(F) \u003c/strong\u003eand grain weight \u003cstrong\u003e(G)\u003c/strong\u003e of ZH11, \u003cem\u003e2B1-oe\u003c/em\u003e,\u003cem\u003e2B1-oe/GS3-flag\u003c/em\u003e and \u003cem\u003eGS3-flag\u003c/em\u003e plants. n = 50 for \u003cstrong\u003eF \u003c/strong\u003eand 6 for \u003cstrong\u003eG\u003c/strong\u003e. \u003cstrong\u003e(H)\u003c/strong\u003e \u003cem\u003eGS3\u003c/em\u003e expression in ZH11, \u003cem\u003e2b1\u003c/em\u003e and \u003cem\u003e2B1\u003c/em\u003e-oe plants in response to alkaline treatment. 10-day-old WT seedlings were treated with alkaline for the indicated times. n = 3.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SE. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 and *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 analyzed by Student’s \u003cem\u003et-test\u003c/em\u003e. Different letters above bars indicate differences by Multiple range test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/a7feda90c424288a1951ce44.png"},{"id":92236440,"identity":"c9cbc40e-041d-4d5d-ab5b-f52ce614a2e4","added_by":"auto","created_at":"2025-09-26 07:33:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":169374,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eOsALDH2B1 \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003efunctions genetically upstream of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eCatA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A and B)\u003c/strong\u003e Representative phenotypes \u003cstrong\u003e(A)\u003c/strong\u003e and survival rates after alkaline treatment \u003cstrong\u003e(B)\u003c/strong\u003e of ZH11 and\u003cem\u003e cata\u003c/em\u003e seedlings.\u003cstrong\u003e \u003c/strong\u003en = 48 from three biological repeats. \u003cstrong\u003e(C and D)\u003c/strong\u003e Representative phenotypes \u003cstrong\u003e(C)\u003c/strong\u003e and survival rates after alkaline treatment \u003cstrong\u003e(D)\u003c/strong\u003e of ZH11, \u003cem\u003e2B1-oe7\u003c/em\u003e, \u003cem\u003e2B1-oe7/cata-2\u003c/em\u003eand \u003cem\u003ecata-2 \u003c/em\u003eseedlings. n = 64 from four biological repeats. \u003cstrong\u003e(E)\u003c/strong\u003eIdentifying binding sites in promoters by OsALDH2B1. PA indicates the probe used in EMSA bound by OsALDH2B1. 1–4 indicate sequences tested in the chromatin immunoprecipitation (ChIP)–qPCRassay bound by OsALDH2B1. \u003cstrong\u003e(F)\u003c/strong\u003e Binding assays of OsALDH2B1 to the promoters \u003cem\u003eCatA\u003c/em\u003e, \u003cem\u003eCatB\u003c/em\u003e and \u003cem\u003eCatC\u003c/em\u003eby ChIP–qPCR with \u003cem\u003e2B1-oe\u003c/em\u003e plants using anti-OsALDH2B1 antibody. n = 3. \u003cstrong\u003e(G)\u003c/strong\u003eElectrophoretic mobility shift assay (EMSA) assay on binding of OsALDH2B1 to \u003cem\u003eCatA \u003c/em\u003epromoter. \u003cstrong\u003e(H) \u003c/strong\u003eActivity assay of OsALDH2B1 in regulating \u003cem\u003eCatA\u003c/em\u003eexpression in a transient expression analysis in rice protoplast. n = 6.\u003cstrong\u003e (I)\u003c/strong\u003e \u003cem\u003eCatA\u003c/em\u003e expression in ZH11, \u003cem\u003e2b1\u003c/em\u003e and \u003cem\u003e2B1\u003c/em\u003e-oe plants in response to alkaline treatment. 10-day-old WT seedlings were treated with alkaline for the indicated times. n = 3.\u003c/p\u003e\n\u003cp\u003eData represent mean ± SE. **\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 and *\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05 analyzed by Student’s \u003cem\u003et-test\u003c/em\u003e. Different letters above bars indicate differences by Multiple range test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Bars, 2 cm.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/6a650a566c658b7586c1f1a8.png"},{"id":92237385,"identity":"482e321d-8560-42b3-bd4e-9e175c2a203a","added_by":"auto","created_at":"2025-09-26 07:41:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":177785,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNatural variations of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsALDH2B1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e were associated with rice alkaline tolerance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eHaplotype analysis of the \u003cem\u003eOsALDH2B1\u003c/em\u003e promoter regions. \u003cstrong\u003e(B) \u003c/strong\u003eComparison of alkaline tolerance among accessions carrying different \u003cem\u003eOsALDH2B1 \u003c/em\u003ehaplotypes. Data represent mean ± SE. Different letters above bars indicate differences by Multiple range test at \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. \u003cstrong\u003e(C)\u003c/strong\u003e Distribution frequency of different cultivated rice collections in the four \u003cem\u003eOsALDH2B1\u003c/em\u003e haplotypes. The number of cultivars for each haplotype was given from left to right below each haplotype. The subpopulation with the largest number was highlighted in red. \u003cstrong\u003e(D) \u003c/strong\u003eGeographic distribution of accessions in \u003cem\u003eOsALDH2B1 \u003c/em\u003eHap1 and Hap4. \u003cstrong\u003e(E)\u003c/strong\u003e A proposed model for the function of the OsALDH2B1-mediated and alkaline tolerance regulation. Under alkaline stress, SERL1 is activated and phosphorylates OsALDH2B1, thereby enhancing its stability. OsALDH2B1 then binds to the promoters of \u003cem\u003eGS3\u003c/em\u003e and \u003cem\u003eCats\u003c/em\u003eto regulate their expression and promote rice alkaline tolerance. Natural variations within the \u003cem\u003eOsALDH2B1\u003c/em\u003e promoter leads to its differential expression. The pie charts represent the frequency of \u003cem\u003eOsALDH2B1\u003c/em\u003e alleles across populations, with the alkali tolerant \u003cem\u003eOsALDH2B1 \u003c/em\u003eHap4 allele being more prevalent in high soil pH regions. This prevalence suggests that \u003cem\u003eOsALDH2B1 \u003c/em\u003eHap4 plays a significant role in soil pH adaptation.\u003c/p\u003e","description":"","filename":"Picture6.png","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/13b891e2a231d8b670735c90.png"},{"id":92238618,"identity":"67e78298-507a-4945-9c3a-b70fcfaa3175","added_by":"auto","created_at":"2025-09-26 08:05:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2615148,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/abeb808f-35c8-4d09-8280-a08be67e6479.pdf"},{"id":92236443,"identity":"f7478710-7f43-4d2b-91e3-b1d90095f981","added_by":"auto","created_at":"2025-09-26 07:33:21","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15629686,"visible":true,"origin":"","legend":"Figure 1, Figure 2,Figure 3, Figure 4,Figure 5, Figure 6,Figure 7, Figure 8,Figure 9, Figure 10,Figure 11, Figure 12,Figure 13","description":"","filename":"MZMSERL1OsALDH2B1ExtendedDataFig.113.docx","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/79521f9fc85a3d136a60d98f.docx"},{"id":92238456,"identity":"ed504f9a-c25d-41b5-a440-91b2112c00a1","added_by":"auto","created_at":"2025-09-26 07:57:21","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":71683,"visible":true,"origin":"","legend":"Dataset 3,Dataset 4,Dataset 5,Dataset 6.","description":"","filename":"MZMOsALDH2B1ExtendedDataTable36.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/c022ebd10b633bcaf8207b22.xlsx"},{"id":92236434,"identity":"f8af1053-5cc5-478e-ac5e-394cb9e9737f","added_by":"auto","created_at":"2025-09-26 07:33:21","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":53601,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"MZMOsALDH2B1ExtendedDataTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/e537f2302f7f6d3d3818793c.xlsx"},{"id":92237559,"identity":"3a5ad8a3-d852-4f0b-a527-5e96bf8b5d3d","added_by":"auto","created_at":"2025-09-26 07:49:21","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":19440,"visible":true,"origin":"","legend":"Table 2","description":"","filename":"MZMOsALDH2B1ExtendedDataTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/b9e451391fc1de5f043ea2c2.xlsx"},{"id":92236442,"identity":"4cb2dd7f-dd68-4551-9eb7-af490a607f7f","added_by":"auto","created_at":"2025-09-26 07:33:21","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15128,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDatainformationLegends.docx","url":"https://assets-eu.researchsquare.com/files/rs-7346407/v1/a76fcadc3b7393030c435c30.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Natural variations of OsALDH2B1 contribute geographical adaptation to soil pH in rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSoil salinization and alkalization, now intensified by rapid climate change, have become urgent global threats. These combined stresses impose ion toxicity, osmotic shock, oxidative burst, and high-pH injury on crops, sharply curtailing where and how much we can grow\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Meanwhile, the human population is projected to reach 11\u0026nbsp;billion by 2100\u003csup\u003e3\u003c/sup\u003e. Rising numbers demand not only more calories but also more nutritious diets, yet urbanization, industrialization, and unsustainable farming continue to shrink the arable land base. Feeding the planet from an ever-smaller patch of arable land is therefore the defining agricultural challenge of the 21st century.\u003c/p\u003e\u003cp\u003eMeeting this challenge demands a multi-pronged strategy, one of whose pillars must be the creation of high-yielding, stress-resilient cultivars capable of thriving on previously unusable sodic soils. However, yield and environmental robustness have traded off against each other\u003csup\u003e\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Discovering genes that can simultaneously boost productivity and alkaline tolerance is thus a prerequisite for next-generation breeding.\u003c/p\u003e\u003cp\u003eRice (\u003cem\u003eOryza sativa\u003c/em\u003e L.), the staple of nearly half of humanity, offers an exceptional opportunity. Its vast collection of landraces carries genetic variants adapted to soils of widely differing pH, providing an unmatched reservoir for climate-smart breeding \u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Recent years have witnessed landmark advances in dissecting rice alkaline-stress biology: GS3, an atypical Gγ protein, was shown to limit tolerance by promoting aquaporin phosphorylation and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) accumulation\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, while optimal gibberellin levels were found to balance both alkaline-thermal resilience and yield via ROS control and chromatin modification\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Yet the genetic architecture that links regional soil pH to the geographic distribution of rice accessions remains largely uncharted.\u003c/p\u003e\u003cp\u003eGrain size is a major determinant of grain yield and has been a key target during rice domestication\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. This complex, polygenic trait is orchestrated by a network of quantitative trait loci (QTLs)\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. During the past decade, numerous QTLs and genes have been isolated and mechanistically dissected, revealing a circuitry that spans hormone signalling, G-protein cascades, ubiquitin-proteasome degradation, kinase relays and transcriptional control\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. We previously identified a multifunctional aldehyde dehydrogenase OsALDH2B1 as a positive regulator of grain size that acts by repressing \u003cem\u003eGS3\u003c/em\u003e\u003csup\u003e16,18\u0026ndash;20\u003c/sup\u003e. Here we reveal that OsALDH2B1 simultaneously lengthens grains and confers robust alkaline tolerance. Natural variation in OsALDH2B1 tracks global soil pH gradients, and the derived functional allele frequency mirrors zones of historic sodicity. Mechanistically, OsALDH2B1 represses \u003cem\u003eGS3\u003c/em\u003e and trans-activates \u003cem\u003ecatalase\u003c/em\u003e genes, curbing ROS accumulation. Upon alkaline challenge, the plasma-membrane receptor-like kinase SERL1 phosphorylates and stabilizes OsALDH2B1, thereby locking the protective response in place. Our findings establish \u003cem\u003eOsALDH2B1\u003c/em\u003e as a central node in rice geographic adaptation and provide breeders with a single, dual-purpose target to simultaneously enhance yield and alkaline resilience.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eOsALDH2B1\u003c/b\u003e \u003cb\u003epromotes alkaline tolerance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify transcription factors controlling grain size and potentially conferring alkaline tolerance in rice, we re-analyzed the alkaline-stress microarray dataset\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Among 21 known grain-size regulated transcription factors\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e, seven exhibited differential expression under alkaline stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), prompting us to focus on OsALDH2B1. Further analysis showed that \u003cem\u003eOsALDH2B1\u003c/em\u003e expression was significantly upregulated and peaked at 6 h after alkaline treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Consistently, OsALDH2B1 protein accumulation and phosphorylation is activated by this stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, I). To further explore the potential correlation between \u003cem\u003eOsALDH2B1\u003c/em\u003e transcripts and alkaline tolerance, we examined its transcript levels across ten rice varieties, comprising five japonica varieties and five indica varieties. The indica varieties were more susceptible to alkaline stress while the japonica varieties, which demonstrated greater tolerance (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The transcript levels of \u003cem\u003eOsALDH2B1\u003c/em\u003e were significantly higher in the resistant varieties compared to the susceptible ones, both before and after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eG). These findings suggest that higher \u003cem\u003eOsALDH2B1\u003c/em\u003e transcript levels are associated with enhanced alkaline tolerance.\u003c/p\u003e\u003cp\u003eTo elucidate the genetic role of \u003cem\u003eOsALDH2B1\u003c/em\u003e in alkaline tolerance, we subjected the \u003cem\u003eOsALDH2B1\u003c/em\u003e knockout mutant \u003cem\u003eosaldh2b1\u003c/em\u003e (\u003cem\u003e2b1\u003c/em\u003e) and its complementary plants (\u003cem\u003e2B1-C\u003c/em\u003e)\u003csup\u003e16\u003c/sup\u003e to stress. The mutant exhibited a marked reduction in alkaline tolerance compared to WT; however, the \u003cem\u003e2B1-C\u003c/em\u003e plants restored the tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, C), thereby confirming that \u003cem\u003eOsALDH2B1\u003c/em\u003e mutation compromises alkaline tolerance. To investigate whether overexpression of \u003cem\u003eOsALDH2B1\u003c/em\u003e could increase alkaline tolerance, we generated \u003cem\u003eOsALDH2B1\u003c/em\u003e-overexpressing plants (\u003cem\u003e2B1-oe\u003c/em\u003e) in ZH11 (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Subsequently, the \u003cem\u003e2B1-oe\u003c/em\u003e seedlings were subjected to treatment and exhibited enhanced tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). Collectively, these data underscored the positive role of \u003cem\u003eOsALDH2B1\u003c/em\u003e in rice alkaline tolerance.\u003c/p\u003e\u003cp\u003eAlkaline stress damages plant cells largely through the over-accumulation of ROS\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. After alkaline treatment, the \u003cem\u003e2b1\u003c/em\u003e mutant accumulated significantly more H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, whereas \u003cem\u003e2B1-oe\u003c/em\u003e seedlings showed lower H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels than WT (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Concomitantly, catalase activity decreased in \u003cem\u003e2b1\u003c/em\u003e but increased in \u003cem\u003e2B1-oe\u003c/em\u003e relative to WT (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Together, these data indicate that \u003cem\u003eOsALDH2B1\u003c/em\u003e enhances alkaline tolerance by activating catalase and thus suppressing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation.\u003c/p\u003e\u003cp\u003eTo address the impact of \u003cem\u003eOsALDH2B1\u003c/em\u003e on grain yield, we conducted small-scale field trials. Given that the \u003cem\u003e2b1\u003c/em\u003e mutant is completely male sterile and fails to set seeds\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, we focused on testing the grain yield of \u003cem\u003e2B1-oe\u003c/em\u003e plants. The field trials revealed that the grain yield of \u003cem\u003e2B1-oe\u003c/em\u003e plants was comparable to that of WT on a per-plant and per-plot basis in normal field (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eJ, L, N, P). We further explored the role of \u003cem\u003eOsALDH2B1\u003c/em\u003e in yield traits under alkaline conditions. WT and \u003cem\u003e2B1-oe\u003c/em\u003e plants grew throughout their development in alkaline pools with the pH value maintained at approximately 9.1 for plant yield, and solic fields with the pH value maintained at approximately 9.0 for plot yield. Under alkaline conditions, \u003cem\u003e2B1-oe\u003c/em\u003e plants exhibited 28.8% and 31.9% increase in grain yield per plant, and 20.5% and 22.7% increase in grain yield per plot compared to WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eK, M, O, Q), indicating that overexpressing \u003cem\u003eOsALDH2B1\u003c/em\u003e can markedly enhance alkaline tolerance and grain yield in rice exposed to alkaline conditions.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSERL1 physically associates with OsALDH2B1 and regulates alkaline tolerance\u003c/h2\u003e\u003cp\u003eAlkaline stress rapidly elevated the transcripts, protein abundance and phosphorylation of OsALDH2B1, indicating that the gene is transcriptionally and post-transcriptionally responsive to this stress, implying that the stress stabilizes the OsALDH2B1 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003eF\u0026ndash;I). To uncover the mechanism underlying this stabilization, we purified OsALDH2B1 immunocomplex with anti-OsALDH2B1 antibody and subjected the complex to liquid chromatography-tandem mass spectrometry. Among the co-purified proteins, we identified somatic embryogenesis receptor kinase like 1 (SERL1) (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. The physical association between OsALDH2B1 and SERL1 was corroborated by yeast two hybrid, in vitro pull-down, in vivo co-immunoprecipitation and bimolecular fluorescence complementation assays, demonstrating that the two proteins interact in vitro and in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u0026ndash;D).\u003c/p\u003e\u003cp\u003eTo assess the physiological relevance of this interaction, we generated two independent \u003cem\u003eSERL1\u003c/em\u003e knockout lines (\u003cem\u003eserl1-1\u003c/em\u003e and \u003cem\u003eserl1-2\u003c/em\u003e) using CRISPR/Cas9-mediated genome editing (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003e)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e and tested their alkaline tolerance. Both mutants displayed a severe reduction in tolerance relative to WT controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, H). The complementary plants (\u003cem\u003eSERL1-C\u003c/em\u003e) fully restored alkaline tolerance, confirming that \u003cem\u003eSERL1\u003c/em\u003e is essential for alkaline-stress survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, I).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eSERL1 phosphorylates and stabilizes OsALDH2B1 under alkaline stress\u003c/h3\u003e\n\u003cp\u003eBioinformatic analysis predicts that SERL1 harbors an N-terminal signal peptide, tandem leucine-rich repeats, a single transmembrane helix, and a C-terminal cytoplasmic kinase domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eG)\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Consistent with this topology, BiFC assay localized the SERL1-OsALDH2B1 interaction to the plasma membrane, suggesting that SERL1 itself resides at this site (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Transient expression of SERL1-GFP in rice protoplasts yielded a strong ring-like fluorescence at the cell periphery, confirming plasma-membrane localization (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This pattern was reproduced in stable \u003cem\u003eSERL1-C\u003c/em\u003e transgenic lines, where SERL1-GFP was consistently detected at the plasma membrane of root epidermal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eA; Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Together, these data establish SERL1 as a plasma-membrane protein.\u003c/p\u003e\u003cp\u003eWe next evaluated SERL1 kinase activity in vitro. Recombinant MBP-tagged SERL1\u003csup\u003eJMK\u003c/sup\u003e (MBP-SERL1\u003csup\u003eJMK\u003c/sup\u003e) and the constitutively active variant MBP-SERL1\u003csup\u003eJMK/K310D\u003c/sup\u003e displayed robust autophosphorylation, whereas the kinase-dead variant MBP-SERL1\u003csup\u003eJMK/K310R\u003c/sup\u003e was catalytically inactive (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eG, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Thus, SERL1 possesses intrinsic kinase activity, confirming SERL1 as an active receptor-like kinase. Prompted by the SERL1-OsALDH2B1 interaction, we tested whether OsALDH2B1 is a direct SERL1 substrate. In-vitro phosphorylation assay showed that both MBP-SERL1\u003csup\u003eJMK\u003c/sup\u003e and MBP-SERL1\u003csup\u003eJMK/K310D\u003c/sup\u003e but not MBP-SERL1\u003csup\u003eJMK/K310R\u003c/sup\u003e phosphorylate OsALDH2B1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). We then examined whether SERL1 influences OsALDH2B1 phosphorylation in vivo. Alkaline stress rapidly induced SERL1 accumulation and phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, E), and subsequent analysis revealed that OsALDH2B1 phosphorylation was markedly reduced, though not abolished, in \u003cem\u003eserl1\u003c/em\u003e seedlings under alkaline stress, implying SERL1 regulating OsALDH2B1 phosphorylation in vivo and the existence of additional kinases (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Because phosphorylation often modulates substrate stability\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we asked whether SERL1 affects OsALDH2B1 abundance. Immunoblotting showed that alkaline stress-induced accumulation of OsALDH2B1 was attenuated in \u003cem\u003eserl1\u003c/em\u003e seedlings compared with WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), indicating that SERL1-mediated phosphorylation stabilizes OsALDH2B1 under alkaline conditions.\u003c/p\u003e\u003cp\u003eTo dissect the genetic relationship between \u003cem\u003eSERL1\u003c/em\u003e and \u003cem\u003eOsALDH2B1\u003c/em\u003e in alkaline tolerance, we generated \u003cem\u003eserl1-1/2B1-oe7\u003c/em\u003e plants by crossing. After alkaline treatment, their survival rate was significantly lower than that of \u003cem\u003e2B1-oe7\u003c/em\u003e, but modestly higher than that of WT and \u003cem\u003eserl1-1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e3\u003c/span\u003eH, I). Collectively, these results demonstrate that SERL1 acts both genetically and biochemically as a positive regulator required for full OsALDH2B1 function in rice alkaline tolerance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsALDH2B1\u003c/b\u003e \u003cb\u003eacts upstream of\u003c/b\u003e \u003cb\u003eGS3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur earlier work established that OsALDH2B1 directly represses \u003cem\u003eGS3\u003c/em\u003e transcription and mutation in \u003cem\u003eOsALDH2B1\u003c/em\u003e reduces grain size\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Because the \u003cem\u003e2b1\u003c/em\u003e mutant is male sterile, we field-examined the grain phenotype of \u003cem\u003e2B1-oe\u003c/em\u003e plants. These lines produced markedly larger, heavier grains (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, D, E), accompanied by reduced \u003cem\u003eGS3\u003c/em\u003e expression (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), confirming that \u003cem\u003eOsALDH2B1\u003c/em\u003e promotes grain enlargement. To position \u003cem\u003eOsALDH2B1\u003c/em\u003e genetically relative to \u003cem\u003eGS3\u003c/em\u003e, we crossed \u003cem\u003e2B1-oe7\u003c/em\u003e with the short-grain line \u003cem\u003eGS3-1-flag\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e and generated the \u003cem\u003e2B1-oe7/GS3-1-flag\u003c/em\u003e plants. Seeds from the hybrid were significantly smaller and lighter than those of WT or \u003cem\u003e2B1-oe7\u003c/em\u003e, and indistinguishable from \u003cem\u003eGS3-1-flag\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, F, G), demonstrating that \u003cem\u003eOsALDH2B1\u003c/em\u003e functions genetically upstream of \u003cem\u003eGS3\u003c/em\u003e in controlling grain size.\u003c/p\u003e\u003cp\u003eGS3 homologs have been reported to heighten alkaline sensitivity, and \u003cem\u003eGS3-1-flag\u003c/em\u003e seedlings display increased susceptibility\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. We therefore asked whether \u003cem\u003eGS3\u003c/em\u003e mediates the alkaline tolerance conferred by \u003cem\u003eOsALDH2B1\u003c/em\u003e. Under alkaline stress, \u003cem\u003e2B1-oe7/GS3-1-flag\u003c/em\u003e seedlings were as sensitive as \u003cem\u003eGS3-1-flag\u003c/em\u003e and significantly more sensitive than WT or \u003cem\u003e2B1-oe7\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, I). This epistasis was mirrored by \u003cem\u003eGS3\u003c/em\u003e expression patterns in \u003cem\u003e2b1\u003c/em\u003e and \u003cem\u003e2B1-oe\u003c/em\u003e lines following alkaline treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Consistently, \u003cem\u003e2B1-oe7/GS3-1-flag\u003c/em\u003e seedlings accumulated more H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e than WT and \u003cem\u003e2B1-oe7\u003c/em\u003e, less than \u003cem\u003eGS3-1-flag\u003c/em\u003e (Extended Data Fig.\u0026nbsp;7A, B). Surprisingly, catalase activity was indistinguishable between \u003cem\u003e2B1-oe7/GS3-1-flag\u003c/em\u003e and \u003cem\u003e2B1-oe7\u003c/em\u003e seedlings, and \u003cem\u003eGS3-1-flag\u003c/em\u003e seedlings showed wild-type catalase levels (Extended Data Fig.\u0026nbsp;7C). Together, these data indicate that \u003cem\u003eGS3\u003c/em\u003e promotes alkaline sensitivity through ROS over-accumulation, independently of catalase activity. \u003cem\u003eOsALDH2B1\u003c/em\u003e thus acts genetically upstream of \u003cem\u003eGS3\u003c/em\u003e in regulating both grain size and alkaline tolerance, while its modulation of catalase activity proceeds via a \u003cem\u003eGS3\u003c/em\u003e-independent pathway.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOsALDH2B1 directly targets\u003c/b\u003e \u003cb\u003eCats\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs a transcription factor, OsALDH2B1 is known to bind to the promoters of target genes and differentially regulate their expression\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. This prompted us to investigate whether OsALDH2B1 modulates catalase activity by regulating the expression of \u003cem\u003ecatalase\u003c/em\u003e genes (\u003cem\u003eCat\u003c/em\u003es). To this end, we measured the expression patterns of \u003cem\u003eCat\u003c/em\u003es in \u003cem\u003e2b1\u003c/em\u003e mutant and \u003cem\u003e2B1-oe\u003c/em\u003e seedlings in response to alkaline treatment. We found that all three \u003cem\u003eCat\u003c/em\u003es, \u003cem\u003eCatalase A\u003c/em\u003e (\u003cem\u003eCatA\u003c/em\u003e), \u003cem\u003eCatB\u003c/em\u003e and \u003cem\u003eCatC\u003c/em\u003e, exhibited similar expression patterns. Specifically, the \u003cem\u003e2b1\u003c/em\u003e mutant accumulated lower levels of \u003cem\u003eCat\u003c/em\u003es\u0026rsquo; transcripts, whereas the \u003cem\u003e2B1-oe\u003c/em\u003e seedlings exhibited higher levels of \u003cem\u003eCat\u003c/em\u003es\u0026rsquo; transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eI; Extended Data Fig.\u0026nbsp;8).\u003c/p\u003e\u003cp\u003eTo determine whether OsALDH2B1 can directly bind to the promoters of \u003cem\u003eCat\u003c/em\u003es, we first searched for OsALDH2B1 binding sequences within these promoters and discovered that all three promoters contained multiple sequences similar to OsALDH2B1 binding motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). ChIP\u0026ndash;qPCR and EMSA demonstrate that OsALDH2B1 can bind to the promoters of \u003cem\u003eCat\u003c/em\u003es both in vitro and in vivo (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eF, G). The transient expression assay in rice protoplasts indicated that OsALDH2B1 activates \u003cem\u003eCatA\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). These results indicate that OsALDH2B1 binds to the promoters of \u003cem\u003eCat\u003c/em\u003es and activates their expression.\u003c/p\u003e\u003cp\u003eIt has been well-established that \u003cem\u003eCats\u003c/em\u003e play crucial roles in scavenging ROS in rice stresses response\u003csup\u003e\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. To examine the role of \u003cem\u003eCats\u003c/em\u003e in alkaline tolerance, the \u003cem\u003ecatc\u003c/em\u003e mutants\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e were first subjected to alkaline treatment exhibited increased sensitivity indicating that \u003cem\u003eCatC\u003c/em\u003e mutation increases alkaline sensitivity (Extended Data Fig.\u0026nbsp;9). We further created \u003cem\u003eCatA\u003c/em\u003e knockout mutants (\u003cem\u003ecata-1\u003c/em\u003e and \u003cem\u003ecata-2\u003c/em\u003e) in the ZH11 background using CRISPR/Cas9 technology Extended Data Fig.\u0026nbsp;10)\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. The \u003cem\u003ecata\u003c/em\u003e mutants showed alkaline sensitive phenotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Thus, similar to \u003cem\u003eCatC\u003c/em\u003e, \u003cem\u003eCatA\u003c/em\u003e also functions as a positive regulator of alkaline tolerance in rice.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003e2B1-oe7/cata-2\u003c/em\u003e line was developed by crossing \u003cem\u003e2B1-oe7\u003c/em\u003e with \u003cem\u003ecata-2.\u003c/em\u003e After treatment, the relative survival rates of \u003cem\u003e2B1-oe7/cata-2\u003c/em\u003e plants were comparable to those of the \u003cem\u003ecata-2\u003c/em\u003e mutant but were significantly lower than those of WT and the \u003cem\u003e2B1-oe7\u003c/em\u003e line (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, D). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation in \u003cem\u003e2B1-oe7/cata-2\u003c/em\u003e plants was attenuated relative to \u003cem\u003ecata-2\u003c/em\u003e plants yet exceeded that of WT and \u003cem\u003e2B1-oe7\u003c/em\u003e plants. In parallel, catalase activity in \u003cem\u003e2B1-oe7/cata-2\u003c/em\u003e plants surpassed that of \u003cem\u003ecata-2\u003c/em\u003e plants but lagged behind WT and \u003cem\u003e2B1-oe7\u003c/em\u003e plants (Extended Data Fig.\u0026nbsp;11). The reduced H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation and heightened catalase activity in \u003cem\u003e2B1-oe7/cata-2\u003c/em\u003e plants, as contrasted with \u003cem\u003ecata-2\u003c/em\u003e plants, likely stem from the activation of \u003cem\u003eCatB\u003c/em\u003e and \u003cem\u003eCatC\u003c/em\u003e expression by OsALDH2B1. Collectively, these observations signify that \u003cem\u003eOsALDH2B1\u003c/em\u003e operates genetically upstream of \u003cem\u003eCatA\u003c/em\u003e. In summary, these results demonstrate that OsALDH2B1 directly activates the expression of \u003cem\u003eCat\u003c/em\u003es, thereby promoting catalase activity to scavenge H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and enhance alkaline tolerance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eNatural variations in\u003c/b\u003e \u003cb\u003eOsALDH2B1\u003c/b\u003e \u003cb\u003eare associated with rice alkaline tolerance and geographical distribution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe differential accumulation of \u003cem\u003eOsALDH2B1\u003c/em\u003e transcripts among rice varieties suggests the existence of natural variations in the promoter. To pinpoint these variations, we analyzed insertion-deletions (InDels) and single-nucleotide polymorphisms (SNPs) within the \u003cem\u003eOsALDH2B1\u003c/em\u003e promoter region (a 2.0-kb upstream region from the translation start site) using genomic variation data from 918 accessions sourced from RiceVarMap2.0 (Extended Data Table\u0026nbsp;1;)\u003csup\u003e31\u003c/sup\u003e. Based on polymorphism patterns, these accessions were categorized into four high-confidence haplotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; Extended Data Fig.\u0026nbsp;12). Notably, 94.7% of the accessions in Hap1 were indica, while 98.2% of those in Hap4 were japonica. Meanwhile, 92.9% of the accessions in Hap3 were aus, and 66.4% and 20.9% of those in Hap2 were japonica and indica, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo elucidate the impact of these haplotypes on alkaline tolerance, we randomly selected 170 from 918 accessions and subjected them to treatment. Notably, Hap1 demonstrated the highest sensitivity to alkaline stress, while Hap4 exhibited the most remarkable tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B; Extended Data Table\u0026nbsp;2). Further investigation revealed that japonica rice generally exhibited tolerance to alkaline stress, while indica rice was relatively sensitive, corroborating our previous results (Extended Data Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e, 13). We also examined the geographical distribution of these accessions relative to the most sensitive and tolerant haplotypes. Accessions in the most sensitive haplotype \u003cem\u003eOsALDH2B1-\u003c/em\u003eHap1 were predominantly found in low-soil-pH regions, whereas those in the most tolerant haplotype \u003cem\u003eOsALDH2B1-\u003c/em\u003eHap4 were widely distributed in high-soil-pH regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings imply that rice accessions with different \u003cem\u003eOsALDH2B1\u003c/em\u003e haplotypes exhibit distinct soil pH distribution tendencies.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlkalinization of arable soils is an escalating abiotic stress that threatens global food security. Deciphering the genetic and molecular basis of alkaline tolerance is therefore imperative for breeding crops that can thrive on marginal lands. In this study, we reveal that natural allelic variation in the promoter of \u003cem\u003eOsALDH2B1\u003c/em\u003e shapes rice geographical distribution by modulating its expression in response to local soil pH. The \u003cem\u003eOsALDH2B1\u003c/em\u003e-Hap4 haplotype, enriched in japonica, drives higher transcript abundance and confers markedly greater alkaline tolerance, whereas \u003cem\u003eOsALDH2B1\u003c/em\u003e-Hap1, prevalent in indica, is associated with lower expression and heightened sensitivity. Post-transcriptionally, the alkaline-activated kinase SERL1 phosphorylates and stabilizes OsALDH2B1, reinforcing its downstream regulatory circuitry (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003eRice domestication across contrasting climates has produced two major subspecies that differ in both thermal and pH adaptation: indica, heat-tolerant but alkali-sensitive, dominates low-latitude, low-pH regions, while japonica, cold-tolerant and alkali-resilient, prevails at higher latitudes with alkaline soils (Extended Data Fig.\u0026nbsp;13;)\u003csup\u003e9,32\u003c/sup\u003e. The selective retention of \u003cem\u003eOsALDH2B1\u003c/em\u003e-Hap4 in high-pH zones underscores its adaptive value and positions japonica germplasm as a strategic resource for enhancing alkaline tolerance in modern cultivars.\u003c/p\u003e\u003cp\u003eOsALDH2B1 orchestrates a regulatory cascade that couples stress signalling to developmental output. Alkaline stress triggers ROS over-accumulation; accordingly, \u003cem\u003e2b1\u003c/em\u003e mutants show elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, whereas \u003cem\u003e2B1-oe\u003c/em\u003e lines exhibit reduced ROS. Mechanistically, OsALDH2B1 represses \u003cem\u003eGS3\u003c/em\u003e, encoding a G-protein component that negatively regulates both grain size and alkaline tolerance\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, while directly activates the \u003cem\u003eCats\u003c/em\u003e. Enhanced catalase activity accelerates H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e detoxification, thereby mitigating oxidative damage. The SERL1-OsALDH2B1-\u003cem\u003eCats\u003c/em\u003e module thus converts an environmental pH cue into precise control of ROS homeostasis.\u003c/p\u003e\u003cp\u003eIntriguingly, OsALDH2B1\u0026rsquo;s ROS-scavenging role is echoed in salt stress, where it is integrated into the OsHDAC1-OsALDH2B1-\u003cem\u003eOsGR3\u003c/em\u003e pathway\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Whether ROS itself feeds back on OsALDH2B1 activity, establishing a self-balancing loop, remains an open question. What is clear is that \u003cem\u003eOsALDH2B1\u003c/em\u003e sits at the nexus of multiple stress-response networks, making it a prime target for simultaneous improvement of yield and resilience.\u003c/p\u003e\u003cp\u003eIn summary, we establish \u003cem\u003eOsALDH2B1\u003c/em\u003e as a dual-function regulator that enlarges grains and fortifies alkaline tolerance. Its natural variants provide a ready genetic toolkit for marker-assisted breeding, and its overexpression elevates yield under stress without the typical trade-off. Deploying \u003cem\u003eOsALDH2B1\u003c/em\u003e-Hap4 or engineered alleles promises rice varieties that maintain productivity on saline\u0026ndash;alkaline soils, directly contributing to global food security. Future work should (i) dissect the promoter polymorphisms governing expression, (ii) clarify feedback between ROS and OsALDH2B1 activity, and (iii) explore additional signalling pathways and the enzymatic contribution of its aldehyde-dehydrogenase to alkaline resilience.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003eAll the 918 accessions use for are listed in Extended Data Table\u0026nbsp;5. Five \u003cem\u003ejaponica\u003c/em\u003e rice (\u003cem\u003eO\u003c/em\u003e. \u003cem\u003esativa\u003c/em\u003e L.) varieties, NIP, ZH11, Taichung 65, Dongjin, and Mudanjiang 8, and five \u003cem\u003eindica\u003c/em\u003e rice varieties, 9311, Minghui 63, Nanjing 11, Zhenshan 97, and IR64 are wild types. The \u003cem\u003e2B1-oe\u003c/em\u003e lines, \u003cem\u003eserl1\u003c/em\u003e and \u003cem\u003ecata\u003c/em\u003e were derived from ZH11. The \u003cem\u003e2b1\u003c/em\u003e, \u003cem\u003e2B1-C\u003c/em\u003e, \u003cem\u003eGS3-1-flag, catc\u003c/em\u003e and \u003cem\u003ecatc-cr\u003c/em\u003e lines used in this study have been described in detail in previous studies\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePhenotypic evaluation\u003c/h2\u003e\u003cp\u003eTo examine alkali tolerance at the seedling stage, seeds were germinated and cultivated in Yoshida solution (pH 5.8) at 28\u0026deg;C, 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5% relative humidity under a 14 h light/10 h dark photoperiod in a growth chamber (RuihuaHP1000GS-LED/H11). One week after germination, seedlings were treated with 75 mM mixed alkali salts (62.5 mM NaHCO\u003csub\u003e3\u003c/sub\u003e and 12.5 mM Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e)\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e for 3\u0026ndash;5 days. To determine the survival rate after alkali and heat treatment, seedlings were allowed to recover under normal growth conditions in a timely manner to prevent over-treatment. After 7 days of recovery, the death of young leaves was used as an indicator of seedling death. The survival rate was then calculated as the ratio of the number of surviving seedlings to the total number of seedlings, and survival phenotypes were photographed. For identifying rice alkali tolerance at the reproductive stage, rice plants were grown throughout their development in alkaline pools and alkaline soil plot with the pH value maintained at approximately 9.1 and 9.0 adjusted by mixed alkali (NaHCO\u003csub\u003e3\u003c/sub\u003e:Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e with a molar ratio of 5:1), respectively.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNA extraction and reverse transcription quantitative PCR\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from fresh tissues using TRIzol reagent (Invitrogen). cDNA was synthesized with HiScript III RT SuperMix (Vazyme, China) according to the manufacturer\u0026rsquo;s protocol. For gene expression analysis, reverse transcription quantitative PCR (RT-qPCR) was performed using LightCycler 480 SYBR Green I Master (Roche, Switzerland) in the ABI 7500 Real-Time PCR System (Applied Biosystems, USA) with gene-specific primers (Extended Data Table\u0026nbsp;3). The rice \u003cem\u003eactin\u003c/em\u003e gene was used as inner control.\u003c/p\u003e\n\u003ch3\u003ePlasmid construction and transformation\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eOsALDH2B1\u003c/em\u003e overexpression (\u003cem\u003e2B1-oe\u003c/em\u003e) construct was prepared by amplifying and inserting the full-length coding sequences of \u003cem\u003eOsALDH2B1\u003c/em\u003e derived from ZH11 into pCambia1301-HA-cYFP vector with a HA tag located at the N-terminal and C-terminal of yellow fluorescent protein (cYFP) located at the C-terminal and driven by the \u003cem\u003eCauliflower mosaic virus 35S\u003c/em\u003e promoter. The \u003cem\u003ecata\u003c/em\u003e and \u003cem\u003eserl1\u003c/em\u003e mutants were constructed using a genome-editing system\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Two 19-bp\u0026ndash;specific single-guide RNA (sgRNA) target sequences of the gene were synthesized and ligated to the pYLgRNA-OsU3 and YLgRNA-OsU6a vectors, respectively. The purified guide RNA (gRNA) expression cassette was subsequently inserted into the binary pYLCRISPR\u0026ndash;Cas9-MH vector. The sgRNAs were designed using the website \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.hzau.edu.cn/CRISPR2/\u003c/span\u003e\u003cspan address=\"http://crispr.hzau.edu.cn/CRISPR2/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cem\u003e2B1-oe\u003c/em\u003e and these recombinant pYLCRISPR\u0026ndash;Cas9-MH plasmids were each introduced into ZH11 through Agrobacterium-mediated transformation by Boyuan Biotechnology Co., Ltd, Wuhan China. The positive transgenic plants of the T\u003csub\u003e0\u003c/sub\u003e generation were identified by PCR. Gene expression levels of \u003cem\u003eOsALDH2B1\u003c/em\u003e in transgenic overexpression plants were detected by qPCR. Homozygous mutants of the T\u003csub\u003e1\u003c/sub\u003e generation were confirmed in the sgRNA target sequence by PCR and sequence. The complementary construct was generated by introducing full length \u003cem\u003eSERL1\u003c/em\u003e cDNA fused with \u003cem\u003eGreen fluorescent protein\u003c/em\u003e (\u003cem\u003eGFP)\u003c/em\u003e under the control of SERL1 native promoter (\u003cem\u003eSERL1-C\u003c/em\u003e) and was inserted into pCambia1300 vector and transformed in the \u003cem\u003eserl1-1\u003c/em\u003e mutant. The \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003eCatA\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eLUC\u003c/em\u003e construct was made by fusing the \u003cem\u003eluciferase\u003c/em\u003e (\u003cem\u003eLUC\u003c/em\u003e) to a \u0026sim;2 kb putative promoter region of \u003cem\u003eCatA\u003c/em\u003e in pGreenⅡ0800 vector. All related primers are shown in Extended Data Table\u0026nbsp;4.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eDAB staining and measurement of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content\u003c/h2\u003e\u003cp\u003eThe formation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in the leaves was investigated by the 3,3\u0026prime;-diaminobenzidine (DAB) staining and quantification methods\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Two-week-old seedlings were used for physiological measurements. Leaves under treatment were collected and soaked in 50 ml of staining buffer (1 mg mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e DAB, 20 mM pH7.5 phosphate buffer). After vacuuming, the samples were incubated overnight in the dark at room temperature. Samples were transferred to an eluent (anhydrous ethanol:glacial acetic acid:glycerol\u0026thinsp;=\u0026thinsp;3:1:1) and boiled for 10 min to remove chlorophyll, and then the images of samples were taken under the same condition. For H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e quantification, the leaves from different plants for each sample were homogenized in liquid nitrogen, and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e were extracted and determined using an H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e assay kit (Sangon, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCatalase activity assay\u003c/h2\u003e\u003cp\u003eTo analyze catalase activity, leaves from different plants for each sample were homogenized in liquid nitrogen and were extracted in 1 mL extraction buffer (150 mM NaCl, 50 mM phosphate buffer, pH7.5, 0.5% [v/v] Triton X-100, 10% [v/v] glycerol, and 1% [w/v] protease inhibitor cocktail). The supernatant was collected after being centrifuged at 12,000 rpm for 10 min at 4\u0026deg;C, and protein concentration was estimated by the Bradford method according to the manufacturer\u0026rsquo;s protocol and standardized using a BSA standard curve (0.25 to 4 \u0026micro;g \u0026micro;L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). The supernatant was used for catalase activity analysis with the Catalase Assay Kit (Beyotime, China) according to previous study\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003eChromatin immunoprecipitation (ChIP)\u0026ndash;qPCR assay\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eThe ChIP\u0026ndash;qPCR assay was conducted according to the ChIP Assay method for rice\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Three grams of 12-day-old rice seedlings were rapidly fixed by infiltration with 1% (v/v) formaldehyde under vacuum at 25\u0026deg;C for 15 min, after which they were ground to a powder in liquid nitrogen. The cell nuclei were separated and lysed. Chromatin was extracted and fragmented via ultrasound to 200\u0026ndash;800 bp. Anti-OsALDH2B1 antibody (abclonal, Cat# A20663) and IgG (Abcam, Cat# ab171870, control) were incubated with 40 \u0026micro;L protein A Dynabeads (Invitrogen, Cat# 10001D) at 4\u0026deg;C for 4 h after washing the beads, 100 \u0026micro;L fragmented chromatin suspension was added, followed by incubation at 4\u0026deg;C overnight. After extensive washing and de-crosslink, the precipitated and input DNA samples were amplified by qPCR. Primers for ChIP-qPCR are listed in Extended Data Table\u0026nbsp;5.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eRecombinant protein expression and purification\u003c/h2\u003e\u003cp\u003eThe \u003cem\u003eMBP-OsALDH2B1\u003c/em\u003e, \u003cem\u003eMBP-SERL1\u003c/em\u003e\u003csup\u003e\u003cem\u003eJMK\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eMBP-SERL1\u003c/em\u003e\u003csup\u003e\u003cem\u003eJMK/K310D\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eMBP-SERL1\u003c/em\u003e\u003csup\u003e\u003cem\u003eJMK/K310R\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eGST-SERL1\u003c/em\u003e\u003csup\u003e\u003cem\u003eJMK\u003c/em\u003e\u003c/sup\u003e constructs were prepared and transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e strain BL21 to express recombinant proteins. The recombinant proteins were induced by 1 mM isopropyl β-D-1-thiogalactopyranoside at 16\u0026deg;C for 20 h. MBP-tagged recombinant proteins were purified with Amylose Resin (New England Biolabs, Cat# E8021L), and GST and GST-SERL1\u003csup\u003eJMK\u003c/sup\u003e recombinant protein were purified with was purified with Pierce Glutathione Agarose Resin (Thermo Scientific, Cat# 16101) according to the manufacturer\u0026rsquo;s instructions. Primers used for plasmid construction are listed in Extended Data Table\u0026nbsp;4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eElectrophoretic mobility shift assay (EMSA)\u003c/h2\u003e\u003cp\u003eEMSA were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Recombinant MBP-OsALDH2B1 protein was produced in \u003cem\u003eE.coli\u003c/em\u003e as described in the section \u0026lsquo;Recombinant protein expression and purification\u0026rsquo; above. DNA probes were synthesized and labelled with biotin. DNA gel-shift assays were performed using the chemiluminescence EMSA kit (Beyotime, GS005) and detection and image capture were captured by the Tanon-5200 image system (Tanon, China). The relevant probe sequence is shown in Extended Data Table\u0026nbsp;6.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eTransient expression assay in protoplasts\u003c/h2\u003e\u003cp\u003eTo determine the transcriptional regulation activity, the effector construct was prepared by amplifying and inserting the full-length coding sequences of \u003cem\u003eOsALDH2B1\u003c/em\u003e derived from ZH11 fused with Green fluorescent protein (GFP) driven by the \u003cem\u003eubiquitin\u003c/em\u003e promoter and the reporter construct was prepared by amplifying and inserting the \u003cem\u003eCatA\u003c/em\u003e promoter sequence into the pGreenⅡ -0800 vector. The effector and reporter constructs were then co-transfected into rice protoplasts. The transfected protoplasts were cultured for 12\u0026thinsp;~\u0026thinsp;16 h at 25\u0026deg;C in the dark. The luciferase activities were measured using the Dual Luciferase Reporter Assay System (Promega, Cat# E1910) according to the manufacturer\u0026rsquo;s instructions. The relative reporter gene expression levels were expressed as the ratio of firefly luciferase to the Renilla luciferase. Primers used for plasmid construction are listed in Extended Data Table\u0026nbsp;4.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLiquid chromatography-tandem mass spectrometry\u003c/b\u003e (\u003cb\u003eLC-MS/MS) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotal proteins were extracted from \u003cem\u003eSERL1-C\u003c/em\u003e plants expressing SERL1-GFP using protein extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 0.2% NP-40, 5 mM DTT, and 1\u0026times; protease inhibitor cocktail) and incubated with Dynabeads (Invitrogen, Cat# 10001D) pre-conjugated with anti-GFP antibody (Clonetech, Cat# JL-8) for 2 h. The immunoprecipitated samples were washed five times with protein extraction buffer, and then the proteins were eluted from the beads before proceeding with the LC-MS/MS assay.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eYeast two-hybrid assay\u003c/h2\u003e\u003cp\u003eThe constructs were co-transformed into AH109 yeast cells, with the empty vector pGADT7 and pGBKT7 serving as negative controls. Yeast cells that contain both pGBKT7-p53 and pGADT7-T and should grow on SD-LWHA were set as a positive control. The interaction was assessed on synthetically defined (SD) medium lacking Ade, His, Leu and Trp, according to the protocols provided by the manufacturer (Clontech).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eCo-immunoprecipitation (Co-IP) assay\u003c/h2\u003e\u003cp\u003eCo-IP assay was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Total proteins from \u003cem\u003eSERL1-C\u003c/em\u003e plants were extracted with IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 5 mM DTT and 1\u0026times; protease inhibitor cocktail) and incubated with anti-GFP antibody conjugated Dynabeads for 2 h. The immunoprecipitated samples were washed five times with washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40), then separated on SDS-PAGE and subjected to immunoblot analysis with anti-OsALDH2B1 antibody (Abclonal, Cat# A20663)\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBimolecular fluorescence complementation\u003c/b\u003e (\u003cb\u003eBiFC) assay\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBiFC assay was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. The constructs \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e35S\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eOsALDH2B1-cYFP\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e35S\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eSERL1-nYFP\u003c/em\u003e, \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e35S\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eGUS-cYFP\u003c/em\u003e, and \u003cem\u003eP\u003c/em\u003e\u003csub\u003e\u003cem\u003e35S\u003c/em\u003e\u003c/sub\u003e:\u003cem\u003eGUS-nYFP\u003c/em\u003e were transformed into \u003cem\u003eA. tumefaciens\u003c/em\u003e strain GV1301 and co-expressed in \u003cem\u003eN. benthamiana\u003c/em\u003e leaves for 48 h. YFP fluorescence was visualized by confocal microscopy (TCS SP2; Leica) with an excitation wavelength of 514 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eIn vitro pull-down assay\u003c/h2\u003e\u003cp\u003eThe in vitro pull-down assay was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Recombinant proteins GST-SERL1\u003csup\u003eJMK\u003c/sup\u003e and MBP-OsALDH2B1 were purified from \u003cem\u003eE. coli\u003c/em\u003e. GST-SERL1\u003csup\u003eJMK\u003c/sup\u003e or GST were incubated with Glutathione beads (GE Healthcare) at 4C for 2 h, followed by incubation with MBP-OsALDH2B1 for an additional hour. After elution from the beads, the proteins were analyzed by immunoblotting with anti-GST (ABclonal, Cat# AE001) and anti-MBP antibodies (New England Biolabs, Cat# E8032L) to detect GST-SERL1\u003csup\u003eJMK\u003c/sup\u003e and MBP-OsALDH2B1, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003ePhosphorylation assay\u003c/h2\u003e\u003cp\u003eThe phosphorylation assay was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. For in vitro phosphorylation assays followed by p-Nitrobenzyl mesylate (PNBM) alkylation, MBP-OsALDH2B1 was incubated with MBP-SERL1\u003csup\u003eJMK\u003c/sup\u003e, MBP-SERL1\u003csup\u003eJMK/S310R\u003c/sup\u003e, MBP-SERL1\u003csup\u003eJMK/S310D\u003c/sup\u003e or in 30 \u0026micro;l of reaction buffer [50 mM tris-HCl, pH 7.5, 10 mM MgCl2, and 1 Mm adenosine 5\u0026prime;-(γ-thio)triphosphate (ATPγS; Abcam, Cat# ab138911)] at room temperature for 30 min (flick and centrifuge briefly). PNBM (2.5 mM; Abcam, Cat# ab138910) was then added to the reaction mixture. After incubation at room temperature for 1 hour, the proteins were separated by SDS-PAGE and detected using anti-thiophosphateester antibody (Abcam, Cat# ab92570). The Coomassie brilliant blue\u0026ndash;stained gel was used as a loading control.\u003c/p\u003e\u003cp\u003eFor in vivo phosphorylation assays, 10-day-old seedlings were treated with alkali for the indicated times and collected immediately. Total proteins were extracted with buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 5 mM DTT, 1\u0026times; protease inhibitor cocktail and 1\u0026times; PhosStop) and followed by digestion with or without λPP (λ alkaline protein phosphatase; New England Biolabs, Cat# P0753S) at 30\u0026deg;C for 30 min, then separated on SDS-PAGE with 50 \u0026micro;M Phos-tag (APExBIO, F4002) and subjected to immunoblot analysis with anti-GFP and anti-OsALDH2B1 antibodies, respectively.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eRice geographical distribution and soil pH value analysis\u003c/h2\u003e\u003cp\u003eFor the geographical distribution of \u003cem\u003eOsALDH2B1\u003c/em\u003e haplotypes, rice varieties with confirmed location information were projected onto the map based on their genetic structure and origin information from RiceVarMap2.0\u003csup\u003e31\u003c/sup\u003e. For the analysis of soil pH values in rice-planting areas, the global soil pH distribution data (1:5 soil:water suspension) and regional averages were obtained from the World Soil Information Service 2023 Snapshot\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data plotting and statistical analyses were performed with GraphPad Prism 8.0 software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.graphpad.com/\u003c/span\u003e\u003cspan address=\"https://www.graphpad.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Details about the statistical parameters, such as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, are shown in the figure legends. A two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e test for two groups or a one-way analysis of variance (ANOVA) with Dunnett\u0026rsquo;s or Tukey\u0026rsquo;s multiple comparisons test for multiple groups were carried out. The number of samples is represented by n. Asterisks indicate statistical significance: *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Different letters above bars indicate differences at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Hubei Provincial Natural Science Foundation (2024AFB917, 2023AFA016), and Hubei Provincial Key Research and Development Projects (2024BBB001).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank Dr. Guangcun He from Wuhan University, Dr. Shengyuan Sun from Yangzhou University and Dr. Liyong Cao from China National Rice Research Institute for their providing rice germplasm accessions, \u003cem\u003eGS3-1-flag\u003c/em\u003e and \u003cem\u003ecatc-cr\u003c/em\u003e lines, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are included in the manuscript and supplemental files.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZ.M., X.G., S.A. performed the experiments. Z.M., X.G., S.A., M.C., J.L., B.Z., F.Y., M.L., Y.K., P.Y. performed some of the experiments and data analysis. Z.M., X.G., S.A., Y.K., P.Y. designed the experiments, analyzed data, and wrote the paper. Y.K., P.Y. review, editing and supervision.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444:139\u0026ndash;158\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167:313\u0026ndash;324\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAdam D (2021) How far will global population rise? Researchers can\u0026rsquo;t agree. Nature 597:462\u0026ndash;465\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePeleg Z, Blumwald E (2011) Hormone balance and abiotic stress tolerance in crop plants. 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Earth Syst Sci Data 16:4735\u0026ndash;4765\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Alkaline tolerance, Grain length, Catalase gene, Rice (Oryza sativa L.), Geographical adaptation","lastPublishedDoi":"10.21203/rs.3.rs-7346407/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7346407/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEnhancing the resilience of crops to ensure stable and high yields under adverse environmental conditions has long been a key objective in rice breeding, yet it remains challenging due to inherent trade-off mechanisms. Here, we report that \u003cem\u003eOsALDH2B1\u003c/em\u003e significantly improves both grain length and alkaline tolerance. Specifically, OsALDH2B1 enhances grain size by suppressing the expression of a grain size and alkaline tolerance related gene \u003cem\u003eGS3\u003c/em\u003e, while it positively regulates alkaline tolerance by reducing reactive oxygen species (ROS) accumulation in a manner that is partially independent of \u003cem\u003eGS3\u003c/em\u003e. Moreover, somatic embryogenesis receptor kinase like 1 (SERL1) phosphorylates and stabilizes OsALDH2B1 in response to alkaline stress. Additionally, the alkaline tolerant allele of \u003cem\u003eOsALDH2B1\u003c/em\u003e is predominantly distributed in high soil pH level regions. This study defines a previously unknown pathway by which the OsALDH2B1-centered module regulates alkaline tolerance for high soil pH value adaptation in rice.\u003c/p\u003e","manuscriptTitle":"Natural variations of OsALDH2B1 contribute geographical adaptation to soil pH in rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-26 07:33:17","doi":"10.21203/rs.3.rs-7346407/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bcdece2d-c868-48ab-9db5-8461e092026f","owner":[],"postedDate":"September 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53715200,"name":"Biological sciences/Plant sciences/Plant stress responses/Salt"},{"id":53715201,"name":"Biological sciences/Plant sciences/Plant breeding"}],"tags":[],"updatedAt":"2025-09-26T07:33:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-26 07:33:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7346407","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7346407","identity":"rs-7346407","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}