The miPEP156e-miR156-SPL2 module functions in rice salt tolerance by regulating ion and ROS homeostasis

The miPEP156e-miR156-SPL2 module functions in rice salt tolerance by regulating ion and ROS homeostasis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The miPEP156e-miR156-SPL2 module functions in rice salt tolerance by regulating ion and ROS homeostasis long li, Shunjiao Qiu, Chenlu Kong, Yuan Wang, Yanyan Sun, Rongjun Zhang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8884320/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 12 You are reading this latest preprint version Abstract Salt stress severely constrains crop growth and yield, posing a significant threat to global food security. Although primary transcripts of microRNAs (pri-miRNAs) are known to encode regulatory peptides (miPEPs), their functions in salt tolerance remain poorly understood. Here, we report that miPEP156e, a small peptide encoded by pri-miR156e in rice, acts as a positive regulator of salt tolerance. Application or overexpression of miPEP156e markedly enhances salt tolerance in rice, while loss-of-function mutants exhibit increased sensitivity. Transcriptomic and physiological analyses reveal that miPEP156e modulates genes involved in ion transport, ROS scavenging, and osmotic adjustment. Under salt stress, miPEP156e maintains ion balance by limiting Na⁺ accumulation and preserving K⁺, while concurrently strengthening ROS scavenging capacity. Further analysis demonstrates that miPEP156e exerts these effects by regulating the miR156-SPL2 module. Collectively, our study establishes miPEP156e as a key regulatory peptide in rice salt tolerance, providing new insights into how miPEPs help plants cope with environmental stress. miPEP156e salt stress rice ion homeostasis ROS Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key message miPEP156e enhances salt tolerance without compromising yield-related traits, showing its potential as a novel strategy for crop improvement. Introduction Salt stress is one of the major abiotic stresses affecting global crop growth and yield. High salinity disrupts cellular homeostasis and physiological functions in plants by causing osmotic stress, ion toxicity, and oxidative stress (Zhu 2016 ). Plants have evolved multi-layered and interconnected regulatory mechanisms to cope with salt stress. Among these, the Salt Overly Sensitive (SOS) signaling pathway helps maintain cellular ion balance by regulating ion transporters. The ROS signaling pathway counteracts oxidative stress by activating antioxidant systems to scavenge excess reactive oxygen species. Phytohormone signaling pathways also contribute significantly, which balance growth and stress resistance by regulating the expression of stress-responsive genes. In addition to these pathways, microRNA (miRNA)-mediated post-transcriptional regulation plays a critical role in the plant's response to salt stress (Geng et al. 2013 ; Ma et al. 2015 ; Zhu 2002 ). miRNAs are endogenous non-coding small RNAs, typically 20–24 nucleotides (nt) in length, which function by guiding the cleavage or translational inhibition of target mRNAs (Brant et al. 2018; Cai et al. 2009 ). Numerous studies have demonstrated that various miRNAs are involved in plant salt tolerance. For instance, rice miR172 enhances salt tolerance by regulating its target gene IDS1 and integrating ROS scavenging pathways with phytohormone signaling (Cheng et al. 2021 ; Cheng et al. 2018 ; Lu et al. 2025 ). Maize miR408 influences salt tolerance by targeting LAC9 and regulating secondary cell wall development (Qin et al. 2023 ). In soybean, the miR160a- GmARF16 module acts as an upstream regulator, activating GmMYC2 to promote proline biosynthesis, alleviate osmotic stress-induced damage, and thereby improve salt tolerance (Wang et al. 2024 ). These findings underscore the importance of miRNAs as key subjects for deciphering plant salt stress response mechanisms and identifying regulatory targets for stress resistance. Primary transcripts of miRNAs (Pri-miRNAs) are traditionally considered non-coding. However, recent studies have discovered that they can encode a class of small regulatory peptides (miPEPs) (Erokhina et al. 2025 ; Hou et al. 2024 ; Lauressergues et al. 2015 ; Lauressergues et al. 2022 ; Zhang et al. 2024 ). These peptides can specifically promote the transcription of their own pri-miRNAs, thereby modulating the accumulation of corresponding mature miRNAs and associated phenotypes. To date, the functions of miPEPs have been demonstrated in several species, such as Arabidopsis thaliana , soybean ( Glycine max ), and grapevine ( Vitis vinifera ). Arabidopsis miPEP165a enhances root growth by promoting miR165a expression and increases root meristem cell number (Lauressergues et al. 2015 ). In soybean, miPEP172c regulates nodulation through inhibition of the AP2-type transcription factor NNC (Couzigou et al. 2016 ) while miPEP171d promotes adventitious root formation in grapevine (Chen et al. 2020 ). Beyond developmental roles, miPEPs are increasingly recognized for their functions in stress responses. For instance, Arabidopsis miPEP169c, miPEP169h, and miPEP396b can induce PR1 expression and enhance resistance to pathogen infection. Similarly, miPEP169d can improve broad-spectrum resistance against pathogens in tomato (Ormancey et al. 2024 ). Arabidopsis miPEP408 enhances arsenic stress tolerance by modulating the sulfur assimilation pathway (Kumar et al. 2023 ). Our previous study identified miPEP156e as a functional small peptide encoded by pri-miR156e, which significantly enhances cadmium tolerance and limits cadmium accumulation in rice by modulating miR156 and its downstream genes, including cadmium transporters and ROS scavenging-related genes. (Lu et al. 2024 ). Despite these advances, the roles of miPEPs in rice, a major staple crop, remain largely unexplored, particularly their functions and regulatory mechanisms under salt stress. Among the numerous miRNA families involved in plant stress responses, the miR156 family has attracted significant attention due to its "dual function" in regulating both development and stress resistance. Previous studies have confirmed that the miR156- SPL module can regulate salt tolerance in maize ( Zea mays ) and apple ( Malus domestica ) (Ding et al. 2009 ; Ma et al. 2021 ). miR156b regulates anthocyanin biosynthesis in grape berries under drought stress by targeting VvSBP8/13 (Guo et al. 2024 ). The miR156- SPL -miR528- PPO cascade module regulates cold stress tolerance in banana (Kong et al. 2025 ). The miR156- SPL pathway can mediate alkaline tolerance in alfalfa ( Medicago sativa ) (Yao et al. 2025 ). These findings suggest a potentially conserved function for the miR156- SPL module in abiotic stress response. However, the specific role and mechanism of this module in regulating salt tolerance in rice are not well defined. In this study, we revealed that miPEP156e, a regulatory peptide encoded by pri-miR156e, plays an essential role in regulating salt tolerance in rice. Combined analyses based on genetic, physiological, and transcriptomic evidence showed that miPEP156e enhances rice salt tolerance by fine-tuning the miR156-SPL2 module to coordinate ROS and ion homeostasis. Our findings give insights into the regulatory mechanism of miRNA-encoded peptides in abiotic stress adaptation. Materials and methods Plant Materials and Growth Conditions The rice ( Oryza sativa ) cultivars Nipponbare (Nip) and Zhonghua 11 (ZH11) were used as wild type (WT) in this study. miPEP156e -OE, miPEP156e -Cr, and miR156 -OE were described in previous studies (Liu et al. 2019 ; Lu et al. 2024 ). The spl2 loss-of-function mutants were generated via CRISPR/Cas9-mediated gene editing, in which a single T or A base insertion was introduced into the first exon of SPL2 . All rice seedlings were grown in a growth chamber set at 28°C with a 14-h light/10-h dark photoperiod. To evaluate salt tolerance, three-week-old hydroponically grown rice seedlings were exposed to 120 mM NaCl for 6–7 days. Following a 5-day recovery period, phenotypic analysis was conducted on at least 15 seedlings per group. Agrobacterium tumefaciens-mediated genetic transformation To generate the spl2 mutant lines, a single guide RNA (sgRNA) targeting the SPL2 gene (sequence: 5'-GCCTTCCTGGGACCTCGGCACGG-3') was designed using the online tool CRISPR-GE ( http://skl.scau.edu.cn/ ). This sgRNA was cloned into the pBUE411 vector to construct the CRISPR/Cas9 binary vector. The recombinant plasmid was introduced into the ZH11 genetic background via Agrobacterium -mediated transformation. Positive transformants were selected on medium containing 100 mg L⁻¹ hygromycin. Mutations were confirmed by Sanger sequencing, and homozygous mutant lines were used for detailed analysis. RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) analysis Two-week-old rice seedlings were transferred to a nutrient solution containing either Mock or 120 mM NaCl for 24 hours. Root tissues were then collected, immediately frozen in liquid nitrogen, and stored for total RNA extraction. Total RNA was extracted using the TRNzol Universal Reagent (Tiangen Biotech, China) following the manufacturer's instructions. First-strand cDNA was synthesized from the RNA using the First-Strand Synthesis Master Mix (Lablead, China). RT-qPCR was performed using the 2×Realab Green PCR Fast mixture (Lablead) on an Applied Biosystems QuantStudio 1 Plus Real-Time PCR System. The OsActin gene was used as an internal control. All RT-qPCR experiments included three biological replicates. Primer sequences used for gene expression analysis are listed in Supplementary Table S1 . Functional validation with a synthetic peptide The miPEP156e peptide (sequence:MHGAQPPRQTPTNPPDHRDGNPSPAPFPVELAAATC, purity > 95%) was synthesized by Sangon Biotech (Shanghai, China). It was dissolved in sterile water to make a 5 mM stock solution. Rice seedlings subjected to salt stress were treated with different concentrations (0.1, 0.5, 0.7, 1 µM) of the synthetic peptide. Phenotypic parameters, including root length, fresh weight, and survival rate, were measured after treatment. Transcriptome analysis Root samples were collected from two-week-old wild-type (Nip) and miPEP156e -OE transgenic seedlings. Three biological replicates were included for each genotype. Total RNA was extracted using TRNzol Universal Reagent (Tiangen). RNA sequencing services were provided by the Beijing Genomics Institute (BGI, China). After removing adapters and performing quality filtering, the clean reads were aligned to the Nipponbare reference genome using Bowtie2 software, and gene expression levels were quantified using RSEM. Differential expression gene (DEG) analysis was performed using the DESeq2 software. Genes with |log₂ (fold change) | > 0.5 and an adjusted P -value (Q value) < 0.05 were identified as DEGs. Measurement of Na⁺ and K⁺ content Dried rice samples were ground into a fine powder. Approximately 0.1 g of the powder was accurately weighed and digested in 4 mL of 65% nitric acid at 200°C for 2 hours. Near the end of digestion, 1 mL of 30% H₂O₂ was added to remove residual acids until the solution became clear and transparent. After cooling to room temperature, the digest was transferred to a 10 mL centrifuge tube, diluted to 6 mL with 2% nitric acid, and filtered through a 0.45 µm aqueous filter membrane. The Na⁺ and K⁺ contents in the shoots and roots of the seedlings were determined using an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, PE OPTIMA 8000DV, USA). ROS detection The accumulation of superoxide anion (O 2 •− ) was detected using Nitroblue Tetrazolium (NBT) staining, as described previously (Jabs et al. 1996 ). The level of hydrogen peroxide (H₂O₂) in rice leaves and roots was detected using 3,3'-Diaminobenzidine (DAB) staining, following a previously described method(Daudi et al. 2012). Statistical analysis Statistical analyses were performed using SPSS software (Version 26.0 for Windows, SPSS, Chicago, IL, USA), and the significance of differences was determined using Student’s t -test. Results Expression patterns of MIR156 family members under salt stress To investigate the effect of salt stress on the expression of MIR156 family members, we examined the transcript levels of various MIR156 genes in rice roots under salt stress. The results indicated differential responses to salt stress among MIR156 family members. Most members exhibited dynamic increases in expression following stress exposure, with MIR156e showing the most rapid upregulation (Figure S1 A). Its expression peaked at 1 hour after stress initiation, remained elevated until 3 hours, and then gradually declined, returning to initial levels by 12 hours. In contrast, the expression of MIR156g and MIR156k showed a decreasing trend (Figure S1 A). Further analysis of miR156e accumulation under salt stress revealed that its expression was also significantly upregulated after salt treatment (Figure S1 B). These results suggest that salt stress significantly influences the dynamic expression of MIR156e , indicating its potential key role in rice salt tolerance. Exogenous application of miPEP156e enhances rice salt tolerance Our previous study demonstrated that pri-miR156 e encodes a small regulatory peptide designated as miPEP156e (Lu et al. 2024 ). To further elucidate the regulatory mechanism of miPEP156e on miR156e, dual-luciferase reporter assays were performed. The results showed that luciferase activity driven by the miR156e promoter was significantly induced by miPEP156e, establishing that miPEP156e can transcriptionally activate the miR156e promoter (Figure S2 A). To characterize the bioactivity of miPEP156e, we evaluated its regulatory impact on the MIR156e locus. Exogenous peptide application elicited a dose-dependent increase in the abundance of both pri-miR156e and mature miR156e, with maximal induction peaking at 0.7 µM (Figure S2 B). We further explored the regulatory interplay between miPEP156e and salinity stress (Figure S2 C). While NaCl treatment independently triggered a pronounced upregulation of miR156e , the combined application of miPEP156e and NaCl resulted in a synergistic potentiation of pri-miR156e transcripts, significantly exceeding the levels of either individual treatment (Figure S2 C). Therefore, we assessed the effect of synthesized miPEP156e on rice salt tolerance. The results showed that exogenous application of miPEP156e significantly reduced the sensitivity of rice seedlings to salt stress (Fig. 1 A). Application of miPEP156e at concentrations of 0.5, 0.7, and 1 µM significantly increased root length and fresh weight of the seedlings (Fig. 1 B and 1 C). In addition, the survival rate of seedlings treated with 0.5 and 0.7 µM miPEP156e was significantly higher than that of the control group (Fig. 1 D and 1 E). At these concentrations, the malondialdehyde (MDA) content in seedling leaf sheaths and roots significantly decreased, while superoxide dismutase (SOD) activity significantly increased (Figure S3 A and S3B). These results suggest that exogenous miPEP156e effectively enhances salt tolerance in rice. Overexpression of miPEP156e and miR156 enhances rice salt tolerance To further elucidate the functions of miPEP156e and miR156 in regulating rice salt tolerance, we evaluated the phenotypes of miPEP156e -OE and miR156 -OE lines under salt stress. qRT-PCR analysis indicated that the accumulation of miR156 is significantly higher in miPEP156e -OE lines (Figure S4 A). We also conducted a Western blot analysis using an endogenous antibody against miPEP156e to verify its production in vivo . The results showed a significant increase in miPEP156e protein levels in the miPEP156e -OE lines compared to the wild type (Figure S4 B).Under normal growth conditions, no obvious phenotypic differences were observed between the overexpression lines and the WT. However, under salt stress conditions, both miPEP156e -OE and miR156 -OE lines exhibited significantly enhanced salt tolerance (Fig. 2 A and 2 D). After five days of recovery post-salt stress, the survival rate and fresh weight of the overexpression lines were significantly higher than those of the WT (Fig. 2 B, 2 C, 2 E, and 2 F). These results demonstrate the important role of the miPEP156e-miR156 pathway in regulating rice salt tolerance. miPEP156e -Cr mutants display enhanced sensitivity to salt stress To further investigate the involvement of miPEP156e in salt tolerance, we evaluated the phenotype of the miPEP156e -Cr mutants under salinity conditions. qRT-PCR analysis confirmed that the abundance of miR156 was significantly reduced in the miPEP156e -Cr lines (Figure S4 A). Furthermore, Western blot analysis revealed that miPEP156e protein accumulation was completely abolished in these mutants (Figure S4 B). Under normal conditions, the mutants and the WT exhibited no significant phenotypic differences. However, under salt stress, the miPEP156e -Cr mutants displayed a more sensitive phenotype (Fig. 3 A). After five days of recovery, the survival rate and fresh weight of the mutants were significantly lower than those of the WT (Fig. 3 B and 3 C). To further determine whether the salt-sensitive phenotype of miPEP156e- Cr was directly attributable to the deficiency of the miPEP156e peptide, we performed a functional rescue experiment. Exogenous application of the synthetic miPEP156e peptide significantly mitigated the growth inhibition of miPEP156e- Cr seedlings under salt stress, restoring their survival rates to levels comparable to those of the wild-type plants (Figure S5 A and S5B). These results further demonstrate that miPEP156e positively regulates salt tolerance in rice. Identification of miPEP156e-regulated pathway by transcriptome profiling To gain deeper insights into the molecular mechanism by which miPEP156e regulates rice salt tolerance, we performed high-throughput transcriptome sequencing, comparing WT and miPEP156e -OE plants. Volcano plot analysis showed that miPEP156e overexpression led to significant downregulation of 1072 genes and significant upregulation of 1207 genes (Fig. 4 A). A Venn diagram revealed an overlap of 824 genes between salt stress-responsive genes and miPEP156e-regulated genes (Fig. 4 B), indicating that miPEP156e participates extensively in the gene regulatory network responding to salt stress in rice. KEGG enrichment analysis indicated that the differentially expressed genes (DEGs) were significantly enriched in pathways such as terpenoid backbone biosynthesis, circadian rhythm-plant, and phenylpropanoid biosynthesis (Fig. 4 C). GO enrichment analysis showed that the DEGs were significantly enriched in biological processes, including hydrogen peroxide catabolic process, terpenoid metabolic process, response to oxidative stress, glutathione metabolic process, and sodium ion transport (Fig. 4 D). To corroborate the transcriptomic results, we performed RT-qPCR analysis on representative genes involved in reactive oxygen species (ROS) and ion homeostasis. The expression patterns of these genes, determined by RT qPCR, were consistent with the transcriptomic data. The results demonstrated that under salt stress, the transcript levels of ROS-detoxifying genes PRX102 and IRL were significantly higher in the miPEP156e -OE lines than in the wild-type (Figure S6). Conversely, the expression of pro-oxidant genes, including RbohA , RbohB , PAO3 , and PAO4 , was markedly repressed in the overexpression lines (Figure S6). Regarding ion homeostasis, the high-affinity potassium transporter gene HKT1;5 was significantly upregulated, whereas the expression of HKT2;1 was notably diminished in miPEP156e -OE plants (Figure S6). These findings reveal that miPEP156e may confer salt tolerance in rice through the collective regulation of multiple pathways, including antioxidant defense, secondary metabolism, and ion homeostasis. miPEP156e maintains ion homeostasis under salt stress Transcriptomic data suggested a potential role for miPEP156e in regulating ion transport in rice. To investigate the effect of miPEP156e on ion homeostasis in rice, we measured Na⁺, K⁺ levels, and the Na⁺/K⁺ ratio in WT, miPEP156e -OE, and miPEP156e -Cr mutants following salt treatment. In shoots, the miPEP156e -OE lines exhibited significantly lower Na⁺ content compared to the WT, without a significant change in K⁺ content, resulting in a significantly decreased Na⁺/K⁺ ratio. In contrast, the mutants accumulated more Na⁺, less K⁺, and showed a higher Na⁺/K⁺ ratio (Fig. 5 A). In roots, the miPEP156e -OE lines showed no significant difference in Na⁺ content compared to the WT, but had higher K⁺ content and a lower Na⁺/K⁺ ratio. Conversely, the mutants had a marked accumulation of Na⁺, a decrease in K⁺, and an elevated Na⁺/K⁺ ratio (Fig. 5 B). Together, these results suggest that miPEP156e may maintain ion homeostasis in rice shoots and roots by regulating the accumulation and distribution of Na⁺ and K⁺, thereby enhancing salt tolerance in rice seedlings. We further analyzed the expression of the high-affinity K + transporter (HKT) genes HKT2;1 and HKT1;5 in different genotypes. The results showed that under salt stress, the miPEP156e -Cr mutant had significantly higher HKT2;1 expression but lower HKT1;5 expression compared to the WT (Fig. 5 C), suggesting that miPEP156e likely affects rice salt tolerance by regulating ion transport processes. miPEP156e regulates ROS homeostasis under salt stress Given that transcriptome data suggested miPEP156e regulates ROS scavenging-related gene expression, we further investigated the function of miPEP156e on the ROS accumulation under salt stress conditions. The levels of hydrogen peroxide (H₂O₂) and superoxide anion (O 2 •− ) in roots and leaves were assessed using DAB and NBT staining, respectively. Under control conditions, ROS levels were similar among different rice materials. However, under NaCl stress, DAB and NBT staining revealed that compared to the WT, the accumulation of H₂O₂ and O 2 •− was reduced in miPEP156e -OE and miR156 -OE plants, but higher in the miPEP156e -Cr mutants (Fig. 6 A and 6 B). We further analyzed the expression of ROS-related genes in the miPEP156e -Cr lines under salt stress. Compared with the WT, the expression of ROS scavenging genes PRX102 and IRL was down-regulated in miPEP156e -Cr, while the expression of ROS-producing genes RbohA , RbohB , PAO3 , and PAO4 was up-regulated (Fig. 6 C). These findings suggest that miPEP156e and miR156 mediate salt tolerance by modulating ROS homeostasis. SPL2 is a negative regulator of salt tolerance in rice To understand the mechanistic basis of miPEP156e-mediated salt tolerance, we investigated the miR156 target genes, particularly the SPL transcription factors (Lu et al. 2024 ; Miao et al. 2019 ). We therefore analyzed the expression patterns of SPL family genes in the miPEP156e -Cr mutants and the WT before and after salt stress, revealing significant changes in the expression of several SPL genes, with SPL2 being the most prominent. Under normal conditions, SPL2 expression was significantly higher in the mutants than in the WT. After treatment with 120 mM NaCl for 6 hours, SPL2 expression decreased significantly in the WT (Fig. 7 A). Although it also decreased in the mutants, its level remained significantly higher than in the WT (Fig. 7 A). Following salt stress, the expression of the five genes, SPL2 , SPL3 , SPL4 , SPL14 , and SPL18 , was significantly downregulated in wild-type plants (Fig. 7 A and S7A). Among them, only SPL2 expression remained notably higher in the miPEP156e mutants under the same conditions (Fig. 7 A). To evaluate the effect of the small peptide miPEP156e on SPL2 expression, we measured SPL2 transcript levels following exogenous application of miPEP156e at varying concentrations. The results showed that SPL2 expression decreased in a dose-dependent manner with increasing miPEP156e concentration, with the most significant inhibition observed at 0.7 µM (Figure S7B). Furthermore, compared to their respective wild-type controls, SPL2 transcript levels were significantly downregulated in both miR156 -OE and miPEP156e -OE plants (Figure S7C). To further investigate whether miPEP156e regulates OsSPL2 through a miR156-dependent mechanism, we performed a dual-luciferase reporter assay in rice protoplasts. We fused the OsSPL2 CDS (CDS SPL2 ) sequence containing the miR156 target site (5'-TGTGCTCTCTCTCTTCTGTCA-3') to the LUC reporter gene (Figure S8A). Overexpression of either miPEP156e or miR156 significantly suppressed the luciferase activity of the LUC-CDS SPL2 reporter (Figure S8B). Notably, when a mutated miR156 target site was used CDS mspl2 (5'-TaTGaTaTaTcTTTaCaGTtA − 3', mutated bases are shown in lowercase) (Figure S8A), the inhibitory effects of both miPEP156e and miR156 were completely abolished (Figure S8C). These findings demonstrate that the suppression of OsSPL2 by miPEP156e is entirely dependent on the recognition and binding of miR156 to its target site. Therefore, we propose that SPL2 plays a critical role in the salt stress response. To elucidate the function of OsSPL2 in rice salt tolerance, we generated two spl2 mutants using CRISPR/Cas9 technology, each with a single base insertion (T or A) in the coding region (Fig. 7 B). Phenotypic analysis showed that under normal conditions, the spl2 mutants were not significantly different from the WT. However, under salt stress, the spl2 mutants exhibited significantly enhanced salt tolerance (Fig. 7 C). After five days of recovery, the survival rate and fresh weight of the spl2 mutants were significantly higher than those of the WT (Fig. 7 D and 7 E). Subsequently, we examined the expression of ROS-related genes and ion transporter genes in the WT and spl2 mutants. After salt stress treatment, the relative expression levels of PRX102 , IRL , and HKT1;5 were significantly higher, while the expression of RbohA , RbohB , PAO3 , PAO4 , and HKT2;1 was lower in the spl2 mutants compared to the WT (Fig. 7 F). To substantiate the physiological basis for the enhanced salt tolerance observed in spl2 mutants, we performed histochemical staining with DAB and NBT to visualize ROS accumulation. Under salt stress, spl2 leaves exhibited markedly lighter staining compared to the wild type, indicating significantly lower accumulation of H₂O₂ and O 2 •− (Figure S9A and S9B). Under 120 mM NaCl stress, spl2 mutants exhibited significantly lower Na + accumulation and higher K + content in both shoots and roots compared to WT (Figure S9C and S9D). Consequently, the Na + /K + ratio was markedly reduced in spl2 plants. These results indicate that the spl2 mutation enhances salt tolerance by maintaining ion homeostasis. These findings suggest that SPL2 regulates rice salt tolerance by modulating the pathways involved in ROS and ion homeostasis, indicating that miPEP156e likely enhances rice salt tolerance via the miR156-SPL2 module. The agronomic performance of miPEP156e -OE lines Finally, to investigate the impact of miPEP156e overexpression on rice agronomic traits, we examined the agronomic performance of miPEP156e -OE lines at the mature stage. Compared to the WT, miPEP156e -OE rice had a significantly higher tiller number and significantly reduced plant height (Fig. 8 A, 8 C and 8 D). Notably, compared to the previously reported phenotype of miR156 -OE, which led to a substantial increase in tiller number but decreased seed setting rate or even sterility (Chen et al. 2015 ; Dai et al. 2018 ), the increase in tillering caused by miPEP156e -OE was more limited and moderate. As a result, the 1000-grain weight of miPEP156e -OE lines showed no significant difference from the WT (Fig. 8 B and 8 E), indicating that miPEP156e has little effect on this trait. Similarly, no significant differences were observed in grain width and length between the WT and miPEP156e -OE lines (Fig. 8 F and 8 G), suggesting miPEP156e has minimal impact on grain morphology. Collectively, these results indicate that overexpression of miPEP156e enhances salt tolerance without adversely affecting yield-related traits. Discussion While microRNAs (miRNAs) are well-documented regulators of plant abiotic stress responses, the upstream mechanisms governing their expression remain largely elusive. Building on the identification of miPEP156e (Lu et al. 2024 ), we demonstrate that this small peptide, which is encoded by pri-miR156e, acts as a pivotal upstream regulator. It functions by promoting miR156 accumulation and suppressing its target gene SPL2 , which fine-tunes ion and ROS homeostasis and consequently improves seedling survival and physiological adaptation under salinity stress (Fig. 1 – 7 ). The miR156 family is a highly conserved regulator of plant development and stress responses (Wu et al. 2006; Yu et al. 2016 ). Here, we found that salt stress significantly induced multiple MIR156 genes, with MIR156e responding most rapidly and strongly, suggesting its specific function in this process (Figure S1 A). Dual-luciferase reporter assays demonstrated that miPEP156e activates the miR156e promoter activity (Figure S2 A), and exogenous peptide treatment further confirmed that miPEP156e promotes miR156e accumulation (Figure S2 B). Within this pathway, the miR156 target SPL2 occupies an essential position. Our expression analysis showed that SPL2 is significantly downregulated in miPEP156e -OE lines (Figure S7C), and Dual-LUC assays confirmed that SPL2 is a direct target of miR156 (Figure S8). SPL proteins are SBP-domain transcription factors that have long been recognized as key regulators of plant growth and environmental adaptation(Chen et al. 2020 ; Cui et al. 2020 ; Miura et al. 2010 ; Shikata et al. 2009 ). For instance, OsSPL8 directly represses the expression of salt tolerance-related genes such as OsHKT1;1 and OsTPP1 , and loss of OsSPL8 function enhances tolerance to both drought and salt stress in rice (He et al. 2025 ). OsSPL10 negatively regulates salt tolerance while positively regulating trichome formation(Lan et al. 2019 ). Additionally, OsSPL14 , a target of miR529a, acts as a negative regulator of rice tolerance to salt and oxidative stress (Jia et al. 2022 ; Yue et al. 2017 ). Our genetic evidence confirms that knockout of the SPL2 gene significantly enhances salt tolerance in rice (Fig. 7 C). Moreover, SPL2 expression is directly suppressed by miR156 (Figure S8). Mechanistically, this regulation maintains a lower Na⁺/K⁺ ratio to alleviate ionic toxicity by coordinating the expression of HKT1;5 and HKT2;1 , while also rebalancing redox homeostasis through upregulation of ROS-scavenging genes and suppression of ROS-producing genes (Fig. 7 F and Figure S9). Given our previous finding that miPEP156e also confers cadmium tolerance by promoting miR156 accumulation and mitigating cellular damage (Lu et al. 2024 ). We propose that miPEP156e may function as a broad-spectrum regulator that enhances multi-stress resistance in rice. Maintaining cellular ion homeostasis is crucial for plant salt tolerance. Our results showed that under salt stress, the miPEP156e -OE lines exhibited a significantly lower Na⁺/K⁺ ratio in both shoots and roots. In contrast, the miPEP156 -Cr mutants displayed the opposite phenotype with a higher Na⁺/K⁺ ratio (Fig. 5 A and 5 B). A low Na⁺/K⁺ ratio is essential for plant survival in saline environments, as K⁺ is indispensable for osmotic adjustment, protein synthesis, enzyme catalysis, and photosynthesis (Lin et al. 2004 ). Our findings suggest that miPEP156e alleviates Na⁺ toxicity by modulating ion uptake and transport systems. Transcriptome data indicated significant changes in the expression of several ion transporter genes, including OsHKT2;1 and OsHKT1;5 in miPEP156e -OE (Fig. 4 and S6). HKT family members play vital roles in salt stress response by maintaining Na⁺/K⁺ balance. In rice, OsHKT1;5 is specifically expressed in roots and mediates the reverse transport of Na⁺ from the xylem to surrounding cells. This process reduces the Na⁺ concentration in the xylem sap and prevents long-distance transport of Na⁺ to the shoots. By effectively restricting Na⁺ transport, OsHKT1;5 alleviates the antagonistic effect of Na⁺ on K⁺ uptake, indirectly helping to maintain higher K⁺ levels in the shoots (Kobayashi et al. 2017 ). By contrast, OsHKT2;1 is a transporter with high affinity for Na⁺. Its expression is induced by K⁺ starvation but rapidly downregulated by toxic levels of Na⁺. Therefore, under K⁺ deficiency, OsHKT2;1 mediates Na⁺ uptake in roots to partially substitute for K⁺. However, under persistent high salt stress, OsHKT2 ;1 expression is strongly suppressed to avoid excessive Na⁺ influx into root cells, causing ion toxicity (Garciadeblas et al. 2003 ; Horie et al. 2007 ; Jabnoune et al. 2009 ). Our study suggests that miPEP156e may upregulate OsHKT1;5 and suppress OsHKT2;1 expression, which collectively enhances Na⁺ sequestration in roots, reduces shoot Na⁺ translocation to shoots, and optimizes root Na⁺ dynamics (Fig. 5 ). This coordinated regulation ultimately maintains whole-plant ion homeostasis and imp roves salt tolerance. Salt stress often induces a ROS burst, leading to oxidative damage to cellular components. Excessive ROS, such as H₂O₂ and O 2 •− , can trigger membrane lipid peroxidation, oxidize proteins and nucleic acids, and inactivate enzymes, thereby disrupting cellular structure and function(Apel et al. 2004; Li et al. 2025 ; Mittler 2017 ; Sofo et al. 2005 ; Suzuki et al. 2005). To counteract these effects, plants activate their antioxidant system to scavenge excessively accumulated ROS. We found that under salt stress, miPEP156e -OE and miR156 -OE plants exhibited reduced accumulation of H₂O₂ and O 2 •− , whereas miPEP156e -Cr mutants displayed elevated levels of both ROS (Fig. 6 and S6). These results demonstrate that the miPEP156e-miR156-SPL2 module functions to alleviate oxidative stress by modulating ROS accumulation. KEGG and GO enrichment analyses further support this conclusion, showing that DEGs were significantly enriched in pathways such as "response to oxidative stress," "hydrogen peroxide catabolic process," and "glutathione metabolic process"(Fig. 4 C and 4 D), which are important for maintaining ROS homeostasis (Sharma et al. 2007; Sofo et al. 2005 ). Similar to miPEP172b, which improves rice salt tolerance by integrating ROS scavenging pathways and phytohormone signaling through the miR172- IDS1 module (Lu et al. 2025 ), our study suggests that miPEP156e may systematically enhance the oxidative stress tolerance of rice by regulating a set of antioxidant-related genes. In terms of yield and agronomic traits, our results demonstrate that miPEP156e exhibits a striking advantage over conventional miRNA manipulation through its precise regulatory potential. Observations at the mature stage revealed that although miPEP156e -OE plants showed a significant increase in tiller number and reduced plant height (Fig. 8 A, 8 C, and 8 D), these alterations were moderate and well-controlled. Unlike the severe reduction in seed setting rate or even sterility often associated with miR156 overexpression in previous studies(Chen et al. 2015 ; Dai et al. 2018 ), key yield-related traits—including 1000-grain weight, grain length, and grain width—did not differ significantly between miPEP156e -OE lines and the wild type (Fig. 8 B, 8 E, 8 F amd 8G). These findings indicate that miPEP156e can substantially enhance salt tolerance in rice while effectively avoiding negative impacts on core yield components, thereby successfully coordinating stress resilience with growth and development. This offers a valuable molecular strategy for breeding high-yielding and stress-tolerant rice varieties. In summary, our findings provide molecular evidence that miPEP156e is a key regulator of salt tolerance in rice. It functions by fine-tuning the miR156-SPL2 module, which coordinates the re-establishment of ion homeostasis and the suppression of oxidative stress. Notably, this regulation enhances salt tolerance without compromising yield-related traits, offering new insights into the functions of miRNA-encoded peptides and their potential application in crop improvement. Declarations Conflict of interest The authors declare no conflict of interest. Author Contribution LL and SJQ performed most of the work and initiated the draft. CLK and YW helped to conduct phenotypic analysis of transgenic rice plants. YYS and RJZ participated in rice cultivation. SRC and YKZ helped with some experiments and data analysis. RSZ, YYS, and LL conceived the study, obtained funding, and revised the final version of the manuscript. All authors read and approved the final article. Acknowledgements This work was supported by the National Key R&D Program (2023YFC2812300), the National Natural Science Foundation of China (32471590 and 32371588), and the Fujian Agriculture and Forestry University Natural Science Funds for Distinguished Young Scholar (xjq21001). Data Availability The data that support the findings of this study are available from the corresponding author upon reasonable request. References Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55, 373–399. http://doi.org/10.1146/annurev.arplant.55.031903.141701 Brant E J, Budak H (2018) Plant Small Non-coding RNAs and Their Roles in Biotic Stresses. Front Plant Sci 9, 1038. http://doi.org/10.3389/fpls.2018.01038 Cai Y, Yu X, Hu S, Yu J (2009) A brief review on the mechanisms of miRNA regulation. 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legend.\u003c/p\u003e","description":"","filename":"Slide6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/f7ef7e4cb1a11df228c35455.jpg"},{"id":103942784,"identity":"273189d3-3412-4ccd-aea7-008005366e04","added_by":"auto","created_at":"2026-03-04 19:52:10","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":166000,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Slide7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/3b4de3d2c804642acef8836a.jpg"},{"id":103942781,"identity":"f2bb8f04-8ac8-4197-be83-993db9a91009","added_by":"auto","created_at":"2026-03-04 19:52:10","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":127060,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Slide8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/d6e95d2da7a6cb9b1b791dc3.jpg"},{"id":104410750,"identity":"045beb6c-ea74-404f-81e3-413af400410c","added_by":"auto","created_at":"2026-03-11 12:53:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2001730,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/0ed526d0-e42c-4558-97cc-b237f6c359dd.pdf"},{"id":104401881,"identity":"2695eefa-7eff-4906-a394-57aa7e9245ce","added_by":"auto","created_at":"2026-03-11 12:13:48","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":10695,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/95514cb3d2e6de78dc470919.xlsx"},{"id":103942782,"identity":"1ace6a08-294d-4e2a-9ac6-e83ef7332bb7","added_by":"auto","created_at":"2026-03-04 19:52:10","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":11202,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/4c44025e9a8ba68ec43cc1d2.xlsx"},{"id":103942785,"identity":"91c966c1-7668-4ab8-afb7-49ec6ac4bd25","added_by":"auto","created_at":"2026-03-04 19:52:10","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":321593,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/0702ed10e3f17b89d7887355.xlsx"},{"id":104402032,"identity":"d16ddcbc-e849-4d41-b471-b0275a649dda","added_by":"auto","created_at":"2026-03-11 12:14:04","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":314041,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/3080b4d56de0c08f877fb764.xlsx"},{"id":103942786,"identity":"d92ab5ef-cad2-4d1a-9106-fc9d2ece893d","added_by":"auto","created_at":"2026-03-04 19:52:11","extension":"pptx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":27645483,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.pptx","url":"https://assets-eu.researchsquare.com/files/rs-8884320/v1/5d5e050f152a1ae05993d9f5.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The miPEP156e-miR156-SPL2 module functions in rice salt tolerance by regulating ion and ROS homeostasis","fulltext":[{"header":"Key message","content":"\u003cp\u003emiPEP156e enhances salt tolerance without compromising yield-related traits, showing its potential as a novel strategy for crop improvement.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSalt stress is one of the major abiotic stresses affecting global crop growth and yield. High salinity disrupts cellular homeostasis and physiological functions in plants by causing osmotic stress, ion toxicity, and oxidative stress (Zhu \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Plants have evolved multi-layered and interconnected regulatory mechanisms to cope with salt stress. Among these, the Salt Overly Sensitive (SOS) signaling pathway helps maintain cellular ion balance by regulating ion transporters. The ROS signaling pathway counteracts oxidative stress by activating antioxidant systems to scavenge excess reactive oxygen species. Phytohormone signaling pathways also contribute significantly, which balance growth and stress resistance by regulating the expression of stress-responsive genes. In addition to these pathways, microRNA (miRNA)-mediated post-transcriptional regulation plays a critical role in the plant's response to salt stress (Geng et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhu \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). miRNAs are endogenous non-coding small RNAs, typically 20\u0026ndash;24 nucleotides (nt) in length, which function by guiding the cleavage or translational inhibition of target mRNAs (Brant et al. 2018; Cai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Numerous studies have demonstrated that various miRNAs are involved in plant salt tolerance. For instance, rice miR172 enhances salt tolerance by regulating its target gene \u003cem\u003eIDS1\u003c/em\u003e and integrating ROS scavenging pathways with phytohormone signaling (Cheng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Maize miR408 influences salt tolerance by targeting \u003cem\u003eLAC9\u003c/em\u003e and regulating secondary cell wall development (Qin et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In soybean, the miR160a-\u003cem\u003eGmARF16\u003c/em\u003e module acts as an upstream regulator, activating \u003cem\u003eGmMYC2\u003c/em\u003e to promote proline biosynthesis, alleviate osmotic stress-induced damage, and thereby improve salt tolerance (Wang et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These findings underscore the importance of miRNAs as key subjects for deciphering plant salt stress response mechanisms and identifying regulatory targets for stress resistance.\u003c/p\u003e \u003cp\u003ePrimary transcripts of miRNAs (Pri-miRNAs) are traditionally considered non-coding. However, recent studies have discovered that they can encode a class of small regulatory peptides (miPEPs) (Erokhina et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Hou et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Lauressergues et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lauressergues et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These peptides can specifically promote the transcription of their own pri-miRNAs, thereby modulating the accumulation of corresponding mature miRNAs and associated phenotypes. To date, the functions of miPEPs have been demonstrated in several species, such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, soybean (\u003cem\u003eGlycine max\u003c/em\u003e), and grapevine (\u003cem\u003eVitis vinifera\u003c/em\u003e). \u003cem\u003eArabidopsis\u003c/em\u003e miPEP165a enhances root growth by promoting miR165a expression and increases root meristem cell number (Lauressergues et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In soybean, miPEP172c regulates nodulation through inhibition of the AP2-type transcription factor \u003cem\u003eNNC\u003c/em\u003e (Couzigou et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) while miPEP171d promotes adventitious root formation in grapevine (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Beyond developmental roles, miPEPs are increasingly recognized for their functions in stress responses. For instance, \u003cem\u003eArabidopsis\u003c/em\u003e miPEP169c, miPEP169h, and miPEP396b can induce \u003cem\u003ePR1\u003c/em\u003e expression and enhance resistance to pathogen infection. Similarly, miPEP169d can improve broad-spectrum resistance against pathogens in tomato (Ormancey et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). \u003cem\u003eArabidopsis\u003c/em\u003e miPEP408 enhances arsenic stress tolerance by modulating the sulfur assimilation pathway (Kumar et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Our previous study identified miPEP156e as a functional small peptide encoded by pri-miR156e, which significantly enhances cadmium tolerance and limits cadmium accumulation in rice by modulating miR156 and its downstream genes, including cadmium transporters and ROS scavenging-related genes. (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Despite these advances, the roles of miPEPs in rice, a major staple crop, remain largely unexplored, particularly their functions and regulatory mechanisms under salt stress.\u003c/p\u003e \u003cp\u003eAmong the numerous miRNA families involved in plant stress responses, the miR156 family has attracted significant attention due to its \"dual function\" in regulating both development and stress resistance. Previous studies have confirmed that the miR156-\u003cem\u003eSPL\u003c/em\u003e module can regulate salt tolerance in maize (\u003cem\u003eZea mays\u003c/em\u003e) and apple (\u003cem\u003eMalus domestica\u003c/em\u003e) (Ding et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ma et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). miR156b regulates anthocyanin biosynthesis in grape berries under drought stress by targeting \u003cem\u003eVvSBP8/13\u003c/em\u003e (Guo et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The miR156-\u003cem\u003eSPL\u003c/em\u003e-miR528-\u003cem\u003ePPO\u003c/em\u003e cascade module regulates cold stress tolerance in banana (Kong et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The miR156-\u003cem\u003eSPL\u003c/em\u003e pathway can mediate alkaline tolerance in alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e) (Yao et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These findings suggest a potentially conserved function for the miR156-\u003cem\u003eSPL\u003c/em\u003e module in abiotic stress response. However, the specific role and mechanism of this module in regulating salt tolerance in rice are not well defined.\u003c/p\u003e \u003cp\u003eIn this study, we revealed that miPEP156e, a regulatory peptide encoded by pri-miR156e, plays an essential role in regulating salt tolerance in rice. Combined analyses based on genetic, physiological, and transcriptomic evidence showed that miPEP156e enhances rice salt tolerance by fine-tuning the miR156-SPL2 module to coordinate ROS and ion homeostasis. Our findings give insights into the regulatory mechanism of miRNA-encoded peptides in abiotic stress adaptation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant Materials and Growth Conditions\u003c/h2\u003e \u003cp\u003eThe rice (\u003cem\u003eOryza sativa\u003c/em\u003e) cultivars Nipponbare (Nip) and Zhonghua 11 (ZH11) were used as wild type (WT) in this study. \u003cem\u003emiPEP156e\u003c/em\u003e-OE, \u003cem\u003emiPEP156e\u003c/em\u003e-Cr, and \u003cem\u003emiR156\u003c/em\u003e-OE were described in previous studies (Liu et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The \u003cem\u003espl2\u003c/em\u003e loss-of-function mutants were generated via CRISPR/Cas9-mediated gene editing, in which a single T or A base insertion was introduced into the first exon of \u003cem\u003eSPL2\u003c/em\u003e. All rice seedlings were grown in a growth chamber set at 28\u0026deg;C with a 14-h light/10-h dark photoperiod. To evaluate salt tolerance, three-week-old hydroponically grown rice seedlings were exposed to 120 mM NaCl for 6\u0026ndash;7 days. Following a 5-day recovery period, phenotypic analysis was conducted on at least 15 seedlings per group.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAgrobacterium tumefaciens-mediated genetic transformation\u003c/h3\u003e\n\u003cp\u003eTo generate the \u003cem\u003espl2\u003c/em\u003e mutant lines, a single guide RNA (sgRNA) targeting the \u003cem\u003eSPL2\u003c/em\u003e gene (sequence: 5'-GCCTTCCTGGGACCTCGGCACGG-3') was designed using the online tool CRISPR-GE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://skl.scau.edu.cn/\u003c/span\u003e\u003cspan address=\"http://skl.scau.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). This sgRNA was cloned into the pBUE411 vector to construct the CRISPR/Cas9 binary vector. The recombinant plasmid was introduced into the ZH11 genetic background via \u003cem\u003eAgrobacterium\u003c/em\u003e-mediated transformation. Positive transformants were selected on medium containing 100 mg L⁻\u0026sup1; hygromycin. Mutations were confirmed by Sanger sequencing, and homozygous mutant lines were used for detailed analysis.\u003c/p\u003e\n\u003ch3\u003eRNA extraction and reverse transcription-quantitative PCR (RT-qPCR) analysis\u003c/h3\u003e\n\u003cp\u003eTwo-week-old rice seedlings were transferred to a nutrient solution containing either Mock or 120 mM NaCl for 24 hours. Root tissues were then collected, immediately frozen in liquid nitrogen, and stored for total RNA extraction. Total RNA was extracted using the TRNzol Universal Reagent (Tiangen Biotech, China) following the manufacturer's instructions. First-strand cDNA was synthesized from the RNA using the First-Strand Synthesis Master Mix (Lablead, China). RT-qPCR was performed using the 2\u0026times;Realab Green PCR Fast mixture (Lablead) on an Applied Biosystems QuantStudio 1 Plus Real-Time PCR System. The \u003cem\u003eOsActin\u003c/em\u003e gene was used as an internal control. All RT-qPCR experiments included three biological replicates. Primer sequences used for gene expression analysis are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eFunctional validation with a synthetic peptide\u003c/h3\u003e\n\u003cp\u003eThe miPEP156e peptide (sequence:MHGAQPPRQTPTNPPDHRDGNPSPAPFPVELAAATC, purity\u0026thinsp;\u0026gt;\u0026thinsp;95%) was synthesized by Sangon Biotech (Shanghai, China). It was dissolved in sterile water to make a 5 mM stock solution. Rice seedlings subjected to salt stress were treated with different concentrations (0.1, 0.5, 0.7, 1 \u0026micro;M) of the synthetic peptide. Phenotypic parameters, including root length, fresh weight, and survival rate, were measured after treatment.\u003c/p\u003e\n\u003ch3\u003eTranscriptome analysis\u003c/h3\u003e\n\u003cp\u003eRoot samples were collected from two-week-old wild-type (Nip) and \u003cem\u003emiPEP156e\u003c/em\u003e-OE transgenic seedlings. Three biological replicates were included for each genotype. Total RNA was extracted using TRNzol Universal Reagent (Tiangen). RNA sequencing services were provided by the Beijing Genomics Institute (BGI, China). After removing adapters and performing quality filtering, the clean reads were aligned to the Nipponbare reference genome using Bowtie2 software, and gene expression levels were quantified using RSEM. Differential expression gene (DEG) analysis was performed using the DESeq2 software. Genes with |log₂ (fold change) | \u0026gt; 0.5 and an adjusted \u003cem\u003eP\u003c/em\u003e-value (Q value)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were identified as DEGs.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMeasurement of Na⁺ and K⁺ content\u003c/h2\u003e \u003cp\u003eDried rice samples were ground into a fine powder. Approximately 0.1 g of the powder was accurately weighed and digested in 4 mL of 65% nitric acid at 200\u0026deg;C for 2 hours. Near the end of digestion, 1 mL of 30% H₂O₂ was added to remove residual acids until the solution became clear and transparent. After cooling to room temperature, the digest was transferred to a 10 mL centrifuge tube, diluted to 6 mL with 2% nitric acid, and filtered through a 0.45 \u0026micro;m aqueous filter membrane. The Na⁺ and K⁺ contents in the shoots and roots of the seedlings were determined using an Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES, PE OPTIMA 8000DV, USA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eROS detection\u003c/h3\u003e\n\u003cp\u003eThe accumulation of superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) was detected using Nitroblue Tetrazolium (NBT) staining, as described previously (Jabs et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). The level of hydrogen peroxide (H₂O₂) in rice leaves and roots was detected using 3,3'-Diaminobenzidine (DAB) staining, following a previously described method(Daudi et al. 2012).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS software (Version 26.0 for Windows, SPSS, Chicago, IL, USA), and the significance of differences was determined using Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExpression patterns of\u003c/b\u003e \u003cb\u003eMIR156\u003c/b\u003e \u003cb\u003efamily members under salt stress\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the effect of salt stress on the expression of \u003cem\u003eMIR156\u003c/em\u003e family members, we examined the transcript levels of various \u003cem\u003eMIR156\u003c/em\u003e genes in rice roots under salt stress. The results indicated differential responses to salt stress among \u003cem\u003eMIR156\u003c/em\u003e family members. Most members exhibited dynamic increases in expression following stress exposure, with \u003cem\u003eMIR156e\u003c/em\u003e showing the most rapid upregulation (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Its expression peaked at 1 hour after stress initiation, remained elevated until 3 hours, and then gradually declined, returning to initial levels by 12 hours. In contrast, the expression of \u003cem\u003eMIR156g\u003c/em\u003e and \u003cem\u003eMIR156k\u003c/em\u003e showed a decreasing trend (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Further analysis of miR156e accumulation under salt stress revealed that its expression was also significantly upregulated after salt treatment (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). These results suggest that salt stress significantly influences the dynamic expression of \u003cem\u003eMIR156e\u003c/em\u003e, indicating its potential key role in rice salt tolerance.\u003c/p\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eExogenous application of miPEP156e enhances rice salt tolerance\u003c/h2\u003e \u003cp\u003eOur previous study demonstrated that pri-miR156\u003cem\u003ee\u003c/em\u003e encodes a small regulatory peptide designated as miPEP156e (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). To further elucidate the regulatory mechanism of miPEP156e on miR156e, dual-luciferase reporter assays were performed. The results showed that luciferase activity driven by the miR156e promoter was significantly induced by miPEP156e, establishing that miPEP156e can transcriptionally activate the miR156e promoter (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). To characterize the bioactivity of miPEP156e, we evaluated its regulatory impact on the \u003cem\u003eMIR156e\u003c/em\u003e locus. Exogenous peptide application elicited a dose-dependent increase in the abundance of both pri-miR156e and mature miR156e, with maximal induction peaking at 0.7 \u0026micro;M (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). We further explored the regulatory interplay between miPEP156e and salinity stress (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). While NaCl treatment independently triggered a pronounced upregulation of \u003cem\u003emiR156e\u003c/em\u003e, the combined application of miPEP156e and NaCl resulted in a synergistic potentiation of pri-miR156e transcripts, significantly exceeding the levels of either individual treatment (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Therefore, we assessed the effect of synthesized miPEP156e on rice salt tolerance. The results showed that exogenous application of miPEP156e significantly reduced the sensitivity of rice seedlings to salt stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Application of miPEP156e at concentrations of 0.5, 0.7, and 1 \u0026micro;M significantly increased root length and fresh weight of the seedlings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In addition, the survival rate of seedlings treated with 0.5 and 0.7 \u0026micro;M miPEP156e was significantly higher than that of the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). At these concentrations, the malondialdehyde (MDA) content in seedling leaf sheaths and roots significantly decreased, while superoxide dismutase (SOD) activity significantly increased (Figure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA and S3B). These results suggest that exogenous miPEP156e effectively enhances salt tolerance in rice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOverexpression of\u003c/b\u003e \u003cb\u003emiPEP156e\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003emiR156\u003c/b\u003e \u003cb\u003eenhances rice salt tolerance\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further elucidate the functions of miPEP156e and miR156 in regulating rice salt tolerance, we evaluated the phenotypes of \u003cem\u003emiPEP156e\u003c/em\u003e-OE and \u003cem\u003emiR156\u003c/em\u003e-OE lines under salt stress. qRT-PCR analysis indicated that the accumulation of miR156 is significantly higher in \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). We also conducted a Western blot analysis using an endogenous antibody against miPEP156e to verify its production \u003cem\u003ein vivo\u003c/em\u003e. The results showed a significant increase in miPEP156e protein levels in the \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines compared to the wild type (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB).Under normal growth conditions, no obvious phenotypic differences were observed between the overexpression lines and the WT. However, under salt stress conditions, both \u003cem\u003emiPEP156e\u003c/em\u003e-OE and \u003cem\u003emiR156\u003c/em\u003e-OE lines exhibited significantly enhanced salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). After five days of recovery post-salt stress, the survival rate and fresh weight of the overexpression lines were significantly higher than those of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results demonstrate the important role of the miPEP156e-miR156 pathway in regulating rice salt tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003emiPEP156e\u003c/b\u003e \u003cb\u003e-Cr mutants display enhanced sensitivity to salt stress\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the involvement of miPEP156e in salt tolerance, we evaluated the phenotype of the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutants under salinity conditions. qRT-PCR analysis confirmed that the abundance of miR156 was significantly reduced in the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr lines (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA). Furthermore, Western blot analysis revealed that miPEP156e protein accumulation was completely abolished in these mutants (Figure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB). Under normal conditions, the mutants and the WT exhibited no significant phenotypic differences. However, under salt stress, the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutants displayed a more sensitive phenotype (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). After five days of recovery, the survival rate and fresh weight of the mutants were significantly lower than those of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). To further determine whether the salt-sensitive phenotype of \u003cem\u003emiPEP156e-\u003c/em\u003eCr was directly attributable to the deficiency of the miPEP156e peptide, we performed a functional rescue experiment. Exogenous application of the synthetic miPEP156e peptide significantly mitigated the growth inhibition of \u003cem\u003emiPEP156e-\u003c/em\u003eCr seedlings under salt stress, restoring their survival rates to levels comparable to those of the wild-type plants (Figure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eA and S5B). These results further demonstrate that miPEP156e positively regulates salt tolerance in rice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of miPEP156e-regulated pathway by transcriptome profiling\u003c/h2\u003e \u003cp\u003eTo gain deeper insights into the molecular mechanism by which miPEP156e regulates rice salt tolerance, we performed high-throughput transcriptome sequencing, comparing WT and \u003cem\u003emiPEP156e\u003c/em\u003e-OE plants. Volcano plot analysis showed that \u003cem\u003emiPEP156e\u003c/em\u003e overexpression led to significant downregulation of 1072 genes and significant upregulation of 1207 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). A Venn diagram revealed an overlap of 824 genes between salt stress-responsive genes and miPEP156e-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), indicating that miPEP156e participates extensively in the gene regulatory network responding to salt stress in rice. KEGG enrichment analysis indicated that the differentially expressed genes (DEGs) were significantly enriched in pathways such as terpenoid backbone biosynthesis, circadian rhythm-plant, and phenylpropanoid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). GO enrichment analysis showed that the DEGs were significantly enriched in biological processes, including hydrogen peroxide catabolic process, terpenoid metabolic process, response to oxidative stress, glutathione metabolic process, and sodium ion transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To corroborate the transcriptomic results, we performed RT-qPCR analysis on representative genes involved in reactive oxygen species (ROS) and ion homeostasis. The expression patterns of these genes, determined by RT qPCR, were consistent with the transcriptomic data. The results demonstrated that under salt stress, the transcript levels of ROS-detoxifying genes \u003cem\u003ePRX102\u003c/em\u003e and \u003cem\u003eIRL\u003c/em\u003e were significantly higher in the \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines than in the wild-type (Figure S6). Conversely, the expression of pro-oxidant genes, including \u003cem\u003eRbohA\u003c/em\u003e, \u003cem\u003eRbohB\u003c/em\u003e, \u003cem\u003ePAO3\u003c/em\u003e, and \u003cem\u003ePAO4\u003c/em\u003e, was markedly repressed in the overexpression lines (Figure S6). Regarding ion homeostasis, the high-affinity potassium transporter gene \u003cem\u003eHKT1;5\u003c/em\u003e was significantly upregulated, whereas the expression of \u003cem\u003eHKT2;1\u003c/em\u003e was notably diminished in \u003cem\u003emiPEP156e\u003c/em\u003e-OE plants (Figure S6). These findings reveal that miPEP156e may confer salt tolerance in rice through the collective regulation of multiple pathways, including antioxidant defense, secondary metabolism, and ion homeostasis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003emiPEP156e maintains ion homeostasis under salt stress\u003c/h2\u003e \u003cp\u003eTranscriptomic data suggested a potential role for miPEP156e in regulating ion transport in rice. To investigate the effect of miPEP156e on ion homeostasis in rice, we measured Na⁺, K⁺ levels, and the Na⁺/K⁺ ratio in WT, \u003cem\u003emiPEP156e\u003c/em\u003e-OE, and \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutants following salt treatment. In shoots, the \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines exhibited significantly lower Na⁺ content compared to the WT, without a significant change in K⁺ content, resulting in a significantly decreased Na⁺/K⁺ ratio. In contrast, the mutants accumulated more Na⁺, less K⁺, and showed a higher Na⁺/K⁺ ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In roots, the \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines showed no significant difference in Na⁺ content compared to the WT, but had higher K⁺ content and a lower Na⁺/K⁺ ratio. Conversely, the mutants had a marked accumulation of Na⁺, a decrease in K⁺, and an elevated Na⁺/K⁺ ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Together, these results suggest that miPEP156e may maintain ion homeostasis in rice shoots and roots by regulating the accumulation and distribution of Na⁺ and K⁺, thereby enhancing salt tolerance in rice seedlings.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further analyzed the expression of the high-affinity K\u003csup\u003e+\u003c/sup\u003e transporter (HKT) genes \u003cem\u003eHKT2;1\u003c/em\u003e and \u003cem\u003eHKT1;5\u003c/em\u003e in different genotypes. The results showed that under salt stress, the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutant had significantly higher \u003cem\u003eHKT2;1\u003c/em\u003e expression but lower \u003cem\u003eHKT1;5\u003c/em\u003e expression compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting that miPEP156e likely affects rice salt tolerance by regulating ion transport processes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003emiPEP156e regulates ROS homeostasis under salt stress\u003c/h2\u003e \u003cp\u003eGiven that transcriptome data suggested miPEP156e regulates ROS scavenging-related gene expression, we further investigated the function of miPEP156e on the ROS accumulation under salt stress conditions. The levels of hydrogen peroxide (H₂O₂) and superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e) in roots and leaves were assessed using DAB and NBT staining, respectively. Under control conditions, ROS levels were similar among different rice materials. However, under NaCl stress, DAB and NBT staining revealed that compared to the WT, the accumulation of H₂O₂ and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e was reduced in \u003cem\u003emiPEP156e\u003c/em\u003e-OE and \u003cem\u003emiR156\u003c/em\u003e-OE plants, but higher in the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe further analyzed the expression of ROS-related genes in the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr lines under salt stress. Compared with the WT, the expression of ROS scavenging genes \u003cem\u003ePRX102\u003c/em\u003e and \u003cem\u003eIRL\u003c/em\u003e was down-regulated in \u003cem\u003emiPEP156e\u003c/em\u003e-Cr, while the expression of ROS-producing genes \u003cem\u003eRbohA\u003c/em\u003e, \u003cem\u003eRbohB\u003c/em\u003e, \u003cem\u003ePAO3\u003c/em\u003e, and \u003cem\u003ePAO4\u003c/em\u003e was up-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These findings suggest that miPEP156e and miR156 mediate salt tolerance by modulating ROS homeostasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSPL2 is a negative regulator of salt tolerance in rice\u003c/h2\u003e \u003cp\u003eTo understand the mechanistic basis of miPEP156e-mediated salt tolerance, we investigated the miR156 target genes, particularly the SPL transcription factors (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Miao et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). We therefore analyzed the expression patterns of \u003cem\u003eSPL\u003c/em\u003e family genes in the \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutants and the WT before and after salt stress, revealing significant changes in the expression of several \u003cem\u003eSPL\u003c/em\u003e genes, with \u003cem\u003eSPL2\u003c/em\u003e being the most prominent. Under normal conditions, \u003cem\u003eSPL2\u003c/em\u003e expression was significantly higher in the mutants than in the WT. After treatment with 120 mM NaCl for 6 hours, \u003cem\u003eSPL2\u003c/em\u003e expression decreased significantly in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Although it also decreased in the mutants, its level remained significantly higher than in the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Following salt stress, the expression of the five genes, \u003cem\u003eSPL2\u003c/em\u003e, \u003cem\u003eSPL3\u003c/em\u003e, \u003cem\u003eSPL4\u003c/em\u003e, \u003cem\u003eSPL14\u003c/em\u003e, and \u003cem\u003eSPL18\u003c/em\u003e, was significantly downregulated in wild-type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and S7A). Among them, only \u003cem\u003eSPL2\u003c/em\u003e expression remained notably higher in the \u003cem\u003emiPEP156e\u003c/em\u003e mutants under the same conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). To evaluate the effect of the small peptide miPEP156e on \u003cem\u003eSPL2\u003c/em\u003e expression, we measured \u003cem\u003eSPL2\u003c/em\u003e transcript levels following exogenous application of miPEP156e at varying concentrations. The results showed that \u003cem\u003eSPL2\u003c/em\u003e expression decreased in a dose-dependent manner with increasing miPEP156e concentration, with the most significant inhibition observed at 0.7 \u0026micro;M (Figure S7B). Furthermore, compared to their respective wild-type controls, \u003cem\u003eSPL2\u003c/em\u003e transcript levels were significantly downregulated in both \u003cem\u003emiR156\u003c/em\u003e-OE and \u003cem\u003emiPEP156e\u003c/em\u003e-OE plants (Figure S7C). To further investigate whether miPEP156e regulates \u003cem\u003eOsSPL2\u003c/em\u003e through a miR156-dependent mechanism, we performed a dual-luciferase reporter assay in rice protoplasts. We fused the \u003cem\u003eOsSPL2\u003c/em\u003e CDS (CDS\u003csub\u003e\u003cem\u003eSPL2\u003c/em\u003e\u003c/sub\u003e) sequence containing the miR156 target site (5'-TGTGCTCTCTCTCTTCTGTCA-3') to the \u003cem\u003eLUC\u003c/em\u003e reporter gene (Figure S8A). Overexpression of either \u003cem\u003emiPEP156e\u003c/em\u003e or \u003cem\u003emiR156\u003c/em\u003e significantly suppressed the luciferase activity of the LUC-CDS\u003csub\u003e\u003cem\u003eSPL2\u003c/em\u003e\u003c/sub\u003e reporter (Figure S8B). Notably, when a mutated miR156 target site was used CDS\u003csub\u003e\u003cem\u003emspl2\u003c/em\u003e\u003c/sub\u003e (5'-TaTGaTaTaTcTTTaCaGTtA\u0026thinsp;\u0026minus;\u0026thinsp;3', mutated bases are shown in lowercase) (Figure S8A), the inhibitory effects of both \u003cem\u003emiPEP156e\u003c/em\u003e and \u003cem\u003emiR156\u003c/em\u003e were completely abolished (Figure S8C). These findings demonstrate that the suppression of \u003cem\u003eOsSPL2\u003c/em\u003e by miPEP156e is entirely dependent on the recognition and binding of miR156 to its target site.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, we propose that \u003cem\u003eSPL2\u003c/em\u003e plays a critical role in the salt stress response. To elucidate the function of \u003cem\u003eOsSPL2\u003c/em\u003e in rice salt tolerance, we generated two \u003cem\u003espl2\u003c/em\u003e mutants using CRISPR/Cas9 technology, each with a single base insertion (T or A) in the coding region (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Phenotypic analysis showed that under normal conditions, the \u003cem\u003espl2\u003c/em\u003e mutants were not significantly different from the WT. However, under salt stress, the \u003cem\u003espl2\u003c/em\u003e mutants exhibited significantly enhanced salt tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). After five days of recovery, the survival rate and fresh weight of the \u003cem\u003espl2\u003c/em\u003e mutants were significantly higher than those of the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). Subsequently, we examined the expression of ROS-related genes and ion transporter genes in the WT and \u003cem\u003espl2\u003c/em\u003e mutants. After salt stress treatment, the relative expression levels of \u003cem\u003ePRX102\u003c/em\u003e, \u003cem\u003eIRL\u003c/em\u003e, and \u003cem\u003eHKT1;5\u003c/em\u003e were significantly higher, while the expression of \u003cem\u003eRbohA\u003c/em\u003e, \u003cem\u003eRbohB\u003c/em\u003e, \u003cem\u003ePAO3\u003c/em\u003e, \u003cem\u003ePAO4\u003c/em\u003e, and \u003cem\u003eHKT2;1\u003c/em\u003e was lower in the \u003cem\u003espl2\u003c/em\u003e mutants compared to the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). To substantiate the physiological basis for the enhanced salt tolerance observed in \u003cem\u003espl2\u003c/em\u003e mutants, we performed histochemical staining with DAB and NBT to visualize ROS accumulation. Under salt stress, \u003cem\u003espl2\u003c/em\u003e leaves exhibited markedly lighter staining compared to the wild type, indicating significantly lower accumulation of H₂O₂ and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e (Figure S9A and S9B). Under 120 mM NaCl stress, \u003cem\u003espl2\u003c/em\u003e mutants exhibited significantly lower Na\u003csup\u003e+\u003c/sup\u003e accumulation and higher K\u003csup\u003e+\u003c/sup\u003e content in both shoots and roots compared to WT (Figure S9C and S9D). Consequently, the Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ratio was markedly reduced in \u003cem\u003espl2\u003c/em\u003e plants. These results indicate that the \u003cem\u003espl2\u003c/em\u003e mutation enhances salt tolerance by maintaining ion homeostasis.\u003c/p\u003e \u003cp\u003eThese findings suggest that SPL2 regulates rice salt tolerance by modulating the pathways involved in ROS and ion homeostasis, indicating that miPEP156e likely enhances rice salt tolerance via the miR156-SPL2 module.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe agronomic performance of\u003c/b\u003e \u003cb\u003emiPEP156e\u003c/b\u003e\u003cb\u003e-OE lines\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFinally, to investigate the impact of \u003cem\u003emiPEP156e\u003c/em\u003e overexpression on rice agronomic traits, we examined the agronomic performance of \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines at the mature stage. Compared to the WT, \u003cem\u003emiPEP156e\u003c/em\u003e-OE rice had a significantly higher tiller number and significantly reduced plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Notably, compared to the previously reported phenotype of \u003cem\u003emiR156\u003c/em\u003e-OE, which led to a substantial increase in tiller number but decreased seed setting rate or even sterility (Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), the increase in tillering caused by \u003cem\u003emiPEP156e\u003c/em\u003e-OE was more limited and moderate. As a result, the 1000-grain weight of \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines showed no significant difference from the WT (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE), indicating that miPEP156e has little effect on this trait. Similarly, no significant differences were observed in grain width and length between the WT and \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG), suggesting miPEP156e has minimal impact on grain morphology. Collectively, these results indicate that overexpression of miPEP156e enhances salt tolerance without adversely affecting yield-related traits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWhile microRNAs (miRNAs) are well-documented regulators of plant abiotic stress responses, the upstream mechanisms governing their expression remain largely elusive. Building on the identification of miPEP156e (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), we demonstrate that this small peptide, which is encoded by pri-miR156e, acts as a pivotal upstream regulator. It functions by promoting miR156 accumulation and suppressing its target gene \u003cem\u003eSPL2\u003c/em\u003e, which fine-tunes ion and ROS homeostasis and consequently improves seedling survival and physiological adaptation under salinity stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe miR156 family is a highly conserved regulator of plant development and stress responses (Wu et al. 2006; Yu et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Here, we found that salt stress significantly induced multiple \u003cem\u003eMIR156\u003c/em\u003e genes, with \u003cem\u003eMIR156e\u003c/em\u003e responding most rapidly and strongly, suggesting its specific function in this process (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Dual-luciferase reporter assays demonstrated that miPEP156e activates the miR156e promoter activity (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA), and exogenous peptide treatment further confirmed that miPEP156e promotes miR156e accumulation (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). Within this pathway, the miR156 target \u003cem\u003eSPL2\u003c/em\u003e occupies an essential position. Our expression analysis showed that \u003cem\u003eSPL2\u003c/em\u003e is significantly downregulated in \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines (Figure S7C), and Dual-LUC assays confirmed that \u003cem\u003eSPL2\u003c/em\u003e is a direct target of miR156 (Figure S8). SPL proteins are SBP-domain transcription factors that have long been recognized as key regulators of plant growth and environmental adaptation(Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Cui et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Miura et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Shikata et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). For instance, OsSPL8 directly represses the expression of salt tolerance-related genes such as \u003cem\u003eOsHKT1;1\u003c/em\u003e and \u003cem\u003eOsTPP1\u003c/em\u003e, and loss of \u003cem\u003eOsSPL8\u003c/em\u003e function enhances tolerance to both drought and salt stress in rice (He et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). OsSPL10 negatively regulates salt tolerance while positively regulating trichome formation(Lan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Additionally, \u003cem\u003eOsSPL14\u003c/em\u003e, a target of miR529a, acts as a negative regulator of rice tolerance to salt and oxidative stress (Jia et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Yue et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our genetic evidence confirms that knockout of the \u003cem\u003eSPL2\u003c/em\u003e gene significantly enhances salt tolerance in rice (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Moreover, \u003cem\u003eSPL2\u003c/em\u003e expression is directly suppressed by miR156 (Figure S8). Mechanistically, this regulation maintains a lower Na⁺/K⁺ ratio to alleviate ionic toxicity by coordinating the expression of \u003cem\u003eHKT1;5\u003c/em\u003e and \u003cem\u003eHKT2;1\u003c/em\u003e, while also rebalancing redox homeostasis through upregulation of ROS-scavenging genes and suppression of ROS-producing genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF and Figure S9). Given our previous finding that miPEP156e also confers cadmium tolerance by promoting miR156 accumulation and mitigating cellular damage (Lu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). We propose that miPEP156e may function as a broad-spectrum regulator that enhances multi-stress resistance in rice.\u003c/p\u003e \u003cp\u003eMaintaining cellular ion homeostasis is crucial for plant salt tolerance. Our results showed that under salt stress, the \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines exhibited a significantly lower Na⁺/K⁺ ratio in both shoots and roots. In contrast, the \u003cem\u003emiPEP156\u003c/em\u003e-Cr mutants displayed the opposite phenotype with a higher Na⁺/K⁺ ratio (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). A low Na⁺/K⁺ ratio is essential for plant survival in saline environments, as K⁺ is indispensable for osmotic adjustment, protein synthesis, enzyme catalysis, and photosynthesis (Lin et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Our findings suggest that miPEP156e alleviates Na⁺ toxicity by modulating ion uptake and transport systems. Transcriptome data indicated significant changes in the expression of several ion transporter genes, including \u003cem\u003eOsHKT2;1\u003c/em\u003e and \u003cem\u003eOsHKT1;5\u003c/em\u003e in \u003cem\u003emiPEP156e\u003c/em\u003e-OE (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and S6). HKT family members play vital roles in salt stress response by maintaining Na⁺/K⁺ balance. In rice, OsHKT1;5 is specifically expressed in roots and mediates the reverse transport of Na⁺ from the xylem to surrounding cells. This process reduces the Na⁺ concentration in the xylem sap and prevents long-distance transport of Na⁺ to the shoots. By effectively restricting Na⁺ transport, OsHKT1;5 alleviates the antagonistic effect of Na⁺ on K⁺ uptake, indirectly helping to maintain higher K⁺ levels in the shoots (Kobayashi et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). By contrast, OsHKT2;1 is a transporter with high affinity for Na⁺. Its expression is induced by K⁺ starvation but rapidly downregulated by toxic levels of Na⁺. Therefore, under K⁺ deficiency, OsHKT2;1 mediates Na⁺ uptake in roots to partially substitute for K⁺. However, under persistent high salt stress, \u003cem\u003eOsHKT2\u003c/em\u003e;1 expression is strongly suppressed to avoid excessive Na⁺ influx into root cells, causing ion toxicity (Garciadeblas et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Horie et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Jabnoune et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Our study suggests that miPEP156e may upregulate \u003cem\u003eOsHKT1;5\u003c/em\u003e and suppress \u003cem\u003eOsHKT2;1\u003c/em\u003e expression, which collectively enhances Na⁺ sequestration in roots, reduces shoot Na⁺ translocation to shoots, and optimizes root Na⁺ dynamics (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This coordinated regulation ultimately maintains whole-plant ion homeostasis and imp roves salt tolerance.\u003c/p\u003e \u003cp\u003eSalt stress often induces a ROS burst, leading to oxidative damage to cellular components. Excessive ROS, such as H₂O₂ and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, can trigger membrane lipid peroxidation, oxidize proteins and nucleic acids, and inactivate enzymes, thereby disrupting cellular structure and function(Apel et al. 2004; Li et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Mittler \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sofo et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Suzuki et al. 2005). To counteract these effects, plants activate their antioxidant system to scavenge excessively accumulated ROS. We found that under salt stress, \u003cem\u003emiPEP156e\u003c/em\u003e-OE and \u003cem\u003emiR156\u003c/em\u003e-OE plants exhibited reduced accumulation of H₂O₂ and O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e\u0026bull;\u0026minus;\u003c/sup\u003e, whereas \u003cem\u003emiPEP156e\u003c/em\u003e-Cr mutants displayed elevated levels of both ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and S6). These results demonstrate that the miPEP156e-miR156-SPL2 module functions to alleviate oxidative stress by modulating ROS accumulation. KEGG and GO enrichment analyses further support this conclusion, showing that DEGs were significantly enriched in pathways such as \"response to oxidative stress,\" \"hydrogen peroxide catabolic process,\" and \"glutathione metabolic process\"(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), which are important for maintaining ROS homeostasis (Sharma et al. 2007; Sofo et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Similar to miPEP172b, which improves rice salt tolerance by integrating ROS scavenging pathways and phytohormone signaling through the miR172-\u003cem\u003eIDS1\u003c/em\u003e module (Lu et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), our study suggests that miPEP156e may systematically enhance the oxidative stress tolerance of rice by regulating a set of antioxidant-related genes.\u003c/p\u003e \u003cp\u003eIn terms of yield and agronomic traits, our results demonstrate that miPEP156e exhibits a striking advantage over conventional miRNA manipulation through its precise regulatory potential. Observations at the mature stage revealed that although \u003cem\u003emiPEP156e\u003c/em\u003e-OE plants showed a significant increase in tiller number and reduced plant height (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD), these alterations were moderate and well-controlled. Unlike the severe reduction in seed setting rate or even sterility often associated with \u003cem\u003emiR156\u003c/em\u003e overexpression in previous studies(Chen et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Dai et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), key yield-related traits\u0026mdash;including 1000-grain weight, grain length, and grain width\u0026mdash;did not differ significantly between \u003cem\u003emiPEP156e\u003c/em\u003e-OE lines and the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF amd 8G). These findings indicate that miPEP156e can substantially enhance salt tolerance in rice while effectively avoiding negative impacts on core yield components, thereby successfully coordinating stress resilience with growth and development. This offers a valuable molecular strategy for breeding high-yielding and stress-tolerant rice varieties.\u003c/p\u003e \u003cp\u003eIn summary, our findings provide molecular evidence that miPEP156e is a key regulator of salt tolerance in rice. It functions by fine-tuning the miR156-SPL2 module, which coordinates the re-establishment of ion homeostasis and the suppression of oxidative stress. Notably, this regulation enhances salt tolerance without compromising yield-related traits, offering new insights into the functions of miRNA-encoded peptides and their potential application in crop improvement.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLL and SJQ performed most of the work and initiated the draft. CLK and YW helped to conduct phenotypic analysis of transgenic rice plants. YYS and RJZ participated in rice cultivation. SRC and YKZ helped with some experiments and data analysis. RSZ, YYS, and LL conceived the study, obtained funding, and revised the final version of the manuscript. All authors read and approved the final article.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program (2023YFC2812300), the National Natural Science Foundation of China (32471590 and 32371588), and the Fujian Agriculture and Forestry University Natural Science Funds for Distinguished Young Scholar (xjq21001).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eApel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. 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Annu Rev Plant Biol 53, 247\u0026ndash;273.\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://doi.org/10.1146/annurev.arplant.53.091401.143329\u003c/span\u003e\u003cspan address=\"10.1146/annurev.arplant.53.091401.143329\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"miPEP156e, salt stress, rice, ion homeostasis, ROS","lastPublishedDoi":"10.21203/rs.3.rs-8884320/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8884320/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Salt stress severely constrains crop growth and yield, posing a significant threat to global food security. Although primary transcripts of microRNAs (pri-miRNAs) are known to encode regulatory peptides (miPEPs), their functions in salt tolerance remain poorly understood. Here, we report that miPEP156e, a small peptide encoded by pri-miR156e in rice, acts as a positive regulator of salt tolerance. Application or overexpression of miPEP156e markedly enhances salt tolerance in rice, while loss-of-function mutants exhibit increased sensitivity. Transcriptomic and physiological analyses reveal that miPEP156e modulates genes involved in ion transport, ROS scavenging, and osmotic adjustment. Under salt stress, miPEP156e maintains ion balance by limiting Na⁺ accumulation and preserving K⁺, while concurrently strengthening ROS scavenging capacity. Further analysis demonstrates that miPEP156e exerts these effects by regulating the miR156-SPL2 module. Collectively, our study establishes miPEP156e as a key regulatory peptide in rice salt tolerance, providing new insights into how miPEPs help plants cope with environmental stress.","manuscriptTitle":"The miPEP156e-miR156-SPL2 module functions in rice salt tolerance by regulating ion and ROS homeostasis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-04 19:52:04","doi":"10.21203/rs.3.rs-8884320/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-03T13:48:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T06:07:26+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T09:50:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157678028975825168209576566731704703109","date":"2026-04-22T07:33:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87931886210608153920482984096010852150","date":"2026-04-01T14:43:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-13T07:47:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"63377756755112694987498004560765437815","date":"2026-02-27T13:37:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"295421063771684207025202751407448308680","date":"2026-02-27T00:20:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-26T23:58:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T06:31:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-17T06:24:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-02-15T07:11:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4019b1bb-c103-420d-9742-9e2cb8f0958c","owner":[],"postedDate":"March 4th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-03T13:48:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-02T06:07:26+00:00","index":24,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-03T13:55:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-04 19:52:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8884320","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8884320","identity":"rs-8884320","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}