<scp>OsMYB4P</scp> regulates serotonin biosynthesis and <scp>ROS</scp> homeostasis to confer cold tolerance in rice

Cold stress (CS) significantly hampers rice production, a staple crop for over half of the global population (Adel khatab et al., 2025; Wu et al., 2025; Yamamori et al., 2025). Rice (Oryza sativa L.), originating in tropical regions, is highly sensitive to cold (Li et al., 2024, 2025, 2026). Serotonin, known for its role in mammals, also affects plant stress responses (Sun et al., 2025). The tryptamine 5-hydroxylase (OsT5H) gene encodes a cytochrome P450 mono-oxygenase with 5-hydroxylase activity, converting tryptamine to serotonin (Sun et al., 2025). Its mutation or overexpression affect pests resistance (Lu et al., 2018; Wang et al., 2023), salt stress tolerance (Lu et al., 2022), and nitrogen deficiency adaptation (Jiang et al., 2023). However, the role of serotonin in cold stress tolerance and its molecular regulatory mechanism remain to be elucidated. To explore its role in cold response, we observed that OsT5H expression and serotonin content increased with prolonged CS (Supporting Information Fig. S1). Furthermore, we utilized different rice lines, including wild-type (WT), its OsT5H knockout (KO) line, or overexpressed (OE) line, namely, Zhonghua11 (ZH11) and its KO line ost5h-1 (Jiang et al., 2023), JiazheB (JZ-WT) and its KO line JZ-LM (Lu et al., 2018), Xidao#1 (XD-WT), XD-KO, and XD-OE (Wang et al., 2023). For CS treatment, 14-d-old seedlings were subjected to CS (10°C) for 5 d, followed by a recovery phase under normal condition (NC, 28°C) for 7 d (Fig. S2a). KO lines showed increased sensitivity to CS treatment, with significantly lower survival rate, plant height, and dry weight, and increased ion leakage, compared with their respective WT parental line (Figs 1a–c, S3, S4). To test whether the observed inferior cold tolerance resulted from OsT5H KO and subsequent serotonin deficiency (ost5h-1) or abolition (XD-KO, JZ-LM), exogenous serotonin was applied to these lines before CS. Partial rescue of cold tolerance was observed in all KO lines, with significantly increased survival rates compared with the control (Figs 1a–c, S3, S4). Although serotonin application did not improve the survival of WT and overexpression lines (Figs 1a–c, S3, S4), it did improve their growth, as reflected by significantly increased plant height and biomass weight, similar to the KO lines (Figs 1a–c, S3, S4). The above results demonstrated that serotonin is vital for cold tolerance. To further explore how CS increases serotonin biosynthesis to confer cold tolerance in rice, we attempted to identify transcription factor(s) that regulate OsT5H transcription. By employing yeast one-hybrid (Y1H) assay, OsMYB4P, a R2R3-MYB-type transcription factor, was identified as such a regulator. By dividing MYB into three regions (1–330, 1–120, and 1–14 amino acids), we found that OsMYB4P, via its MYB domain, bound to OsT5H promoter, to the fragment with multiple MYB-binding elements (Figs 1d, S5, S6). A dual-luciferase reporter (DLR) assay confirmed that OsMYB4P could activate OsT5H transcription under both normal condition and CS (Fig. 1e). Chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays (Fig. 1f) and electrophoretic mobility shift assay (EMSA) (Fig. 1g) also confirmed OsMYB4P physically bound to OsT5H promoter. The above results demonstrate that OsMYB4P acts as a direct transcriptional regulator of OsT5H. To determine whether OsMYB4P plays a role in cold tolerance, we first analyzed OsMYB4P expression in rice seedlings subjected to CS. Strikingly, OsMYB4P mRNA abundance and protein levels were markedly increased within 6 h of CS exposure (Fig. S7), demonstrating that OsMYB4P is a cold-inducible regulator. To examine its role in cold tolerance, loss-of-function OsMYB4P mutants (osmyb4p-1 and osmyb4p-2) (Fig. S8) and overexpressing lines (OsMYB4P-OE-1 and OsMYB4P-OE-2) (Fig. S10a) were generated. Phenotypic analysis revealed that the osmyb4p mutants had significantly increased ion leakage, along with reduced other phenotypic traits (Figs 1h,j, S9). Conversely, OsMYB4P overexpression enhanced cold tolerance (Fig. S10b–f). Interestingly, OsT5H expression and serotonin content were greatly decreased in osmyb4p mutants (Fig. 1i,j), but notably increased in OsMYB4P-OE plants (Figs 1k,l, S11), indicating that the OsMYB4P-OsT5H module is integral to the establishment of cold tolerance. To further elucidate the genetic relationship between OsMYB4P and OsT5H, we generated an osmyb4p-1 ost5h-1 double mutant and overexpression of OsMYB4P in ost5h-1 (ost5h-1 OE-OsMYB4P). The double mutant line exhibited comparable cold sensitivity with the single mutant lines (ost5h-1 and osmyb4p-1) in terms of survival rate and ion leakage, whereas the ost5h-1 OE-OsMYB4P line had intermediate performance between the single/double mutant and WT (Fig. 1m–o). These results indicate that, on one hand, OsMYB4P and OsT5H function in the same genetic pathway, and on the other hand, OsMYB4P may modulate other pathway(s) parallel to the OsMYB4P-serotonin module to enhance cold tolerance. To identify other OsMYB4P regulated cold tolerance pathway(s), comparative transcriptome sequencing was performed on 2-wk-old seedlings of ZH11, osmyb4p-1, and OsMYB4P-OE-1 subjected to CS. A total of 1487 differentially expressed genes (DEGs) (654 up- and 833 downregulated) were identified in ZH11 vs osmyb4p-1 (Fig. 2a), and 2988 up- and 3528 downregulated DEGs in OsMYB4P-OE-1 vs ZH11 (Fig. 2b). To identify genes potentially regulated by OsMYB4P, the upregulated DEGs in both ZH11 vs osmyb4p-1 and OsMYB4P-OE-1 vs ZH11 was compared to a set of 212 reported cold-responsive genes (Fig. 2c). The fact that OsT5H exhibited the lowest expression in osmyb4p-1 and the highest in OsMYB4P-OE-1 (Fig. S12a) demonstrated that such analysis was effective to identify genes under OsMYB4P regulation. Eventually, two genes, LOC_Os03g20680 (OsLEA3-2, a late embryogenesis abundant gene) and LOC_Os03g17790, were identified to have transcription profiles similar to OsT5H (Fig. S12b). Since OsLEA3-2 is known to be involved in abiotic stress tolerance (Duan & Cai, 2012), it was subjected to further analysis. Y1H assay showed that OsMYB4P through its MYB-domain, could bind to the OsLEA3-2 promoter (Figs 2d, S13, S14), which consists of multiple MYB-binding elements (Fig. S13). DLR assay further proved that OsMYB4P could enhance OsLEA3-2 transcription under both NC and CS (Fig. 2e). While ChIP-qPCR results showed the interaction also exists in vivo (Fig. 2f), EMSA confirmed OsMYB4P physically binds to the OsLEA3-2 promoter (Fig. 2g). Consistent with its positive role in transactivating OsLEA3-2, the transcription of OsLEA3-2 was significantly suppressed in the osmyb4p mutant lines, but increased in OsMYB4P-OE plants under CS (Fig. S15). Although OsLEA3-2 is known as a stress responsive gene (Duan & Cai, 2012), no functional analysis of cold response has been reported; hence, two loss-of-function mutants (oslea3-2-1 and oslea3-2-2) were generated (Fig. S16) for cold tolerance evaluation. The two oslea3-2 mutants exhibited obvious inferior growth and recovery (Figs 2h,i, S17) and significantly reduced survival rates (Fig. 2h) compared to the WT after subject to CS. On the opposite, ion leakage was significantly greater in the two mutants compared to the WT, an indicator of CS caused damage to plant cells (Fig. 2j). To examine the genetic relationship between OsMYB4P and OsLEA3-2, an osmyb4p-1 oslea3-2-1 double mutant line, and an OsMYB4P overexpression line in the oslea3-2-1 background (oslea3-2-1 OE-OsMYB4P) were generated for comparison of their cold tolerance, together with other lines. The double mutant exhibited comparable cold sensitivity with the single mutants, whereas the oslea3-2-1 OE-OsMYB4P line had an intermediate performance between the single/double mutant and the WT line (Fig. 2k–m). Furthermore, we found that the ost5h-1 oslea3-2-1 double mutant exhibits enhanced sensitivity to cold compared to the oslea3-2-1 single mutant (Fig. 2k–m). These results indicate OsMYB4P is in the genetic pathway as OsLEA3-2 for cold tolerance regulation. Additionally, OsLEA3-2 and OsT5H operate in distinct genetic pathways governing this process. To understand OsMYB4P-OsLEA3-2 module basis under CS, we assessed key physiological parameters related to reactive oxygen species (ROS) homeostasis under NC and CS over 5 d. Under NC, no significant differences were observed in the physiological indices among ZH11, osmyb4p-1, and oslea3-2-1 (Figs 2n, S18). However, under CS, the superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities were significantly lower in the mutants compared with ZH11, while the hydrogen peroxide (H2O2) content was significantly higher (Figs 2n, S18). These findings imply that OsMYB4P-OsLEA3-2 module is crucial for maintaining ROS homeostasis during rice cold response. To elucidate the relationship between serotonin and ROS homeostasis, we observed a significant reduction in SOD, POD, and CAT activities in the ost5h-1 mutant compared with ZH11 under CS, along with elevated H2O2 level (Fig. S19). The application of serotonin by spraying led to a substantial increase in the activities of ROS scavenging enzymes and a considerable decrease in H2O2 levels under cold stress. These results suggest that serotonin is pivotal in enhancing rice resilience to cold stress by promoting the maintenance of ROS homeostasis. In summary, our study identifies OsMYB4P as a pivotal regulator in the upstream pathway of the OsT5H and OsLEA3-2 cascade, which is activated in response to cold stress (Fig. 2o). Specifically, under cold stress conditions, OsMYB4P plays a crucial role in upregulating the expression of OsT5H and OsLEA3-2. This, in turn, leads to an increase in serotonin levels and a reduction in ROS. Moreover, serotonin itself contributes to lowering ROS levels, thereby enhancing the cold tolerance of rice plants. The KO mutants of rice (Oryza sativa L.), including ost5h-1 (Jiang et al., 2023), JZ-LM (Lu et al., 2018), and XD-KO, as well as the overexpression line XD-OE (Lu et al., 2022), were previously generated. The osmyb4p (osmyb4p-1 and osmyb4p-2) and oslea3-2 (oslea3-2-1 and oslea3-2-2) mutants were created using CRISPR/Cas9 technology. Transgenic plants were generated by introducing the respective plasmids into rice calli (ZH11) via Agrobacterium-mediated transformation. The rice variety ZH11 was used as the WT control. The overexpression lines OsMYB4P-OE-1 and OsMYB4P-OE-2 were generated by inserting the OsMYB4P coding sequence (CDS) into vector pCAMBIA1301-Ubi. The double mutants (F3 or higher generations) of osmyb4p-1 ost5h-1, osmyb4p-1 oslea3-2-1, and ost5h-1 oslea3-2-1 were generated by crossing the mutants of osmyb4p-1 and ost5h-1, osmyb4p-1 and oslea3-2-1, and ost5h-1 and oslea3-2-1. The ost5h-1 OE-OsMYB4P and oslea3-2-1 OE-OsMYB4P transgenic line was generated by Agrobacterium-mediated transformation of the pCAMBIA1301-Ubi-OsMYB4P construct into calli derived from the ost5h-1 and oslea3-2-1 mutant. Cold tolerance was evaluated in 14-d-old seedlings, which were exposed to 10°C or 28°C for 7 d, followed by a 7-d recovery period at 28°C to assess survival rates. Each treatment included at least three to six biological replicates, with 30–40 plants per replicate. To assess the stress-induced expression of OsT5H and OsMYB4P, seedlings were cultured in ½-strength Murashige & Skoog (½MS) liquid medium and subjected to 10°C or 28°C for different time points (0, 1, 3, 6, 12, 18, and 24 h). All experiments were performed in a growth chamber with the following conditions: light intensity of 500 μmol photons m2 s−1, a 14 h : 10 h, light : dark cycle, and relative humidity ranging from 65% to 75%. For dry weight measurements, seedlings were dried at 105°C for 120 min and then at 85°C for 24 h. Ion leakage was quantified following Li et al. (2024). Approximately 0.1 g of leaf tissue from 10°C-treated seedlings was placed in 15 ml tubes with 5 ml deionized water. The samples were shaken gently at room temperature for 1 h at 200 rpm, and the initial conductivity (A1) was recorded. The samples were then heated in boiling water for 20 min, followed by another 1-h incubation at room temperature, and the final conductivity (A2) was measured. Deionized water conductivity (A0) served as the baseline. Ion leakage was calculated as: ion leakage (%) = (A1 − A0)/(A2 − A0). Total RNA was isolated using the RNAprep Pure Plant Kit (Tiangen, Beijing, China), and qRT-PCR was performed with the AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) according to the manufacturer's protocol, employing gene-specific primers (Table S1). Transcript levels were normalized to the ACTIN gene using the 2−ΔΔCt method. For western blot analysis, 200 mg of plant tissue was ground to a fine powder under liquid nitrogen. The powdered tissue was resuspended in 1 ml of extraction buffer (2% (w/v) SDS, 12% (w/v) sucrose, 50 mM dithiothreitol, 50 mM Na2CO3, 2 mM EDTA, pH 8.0, 0.1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aldrich)) and incubated at 65°C for 1 h with gentle agitation. The protein concentration of the resulting lysate was quantified using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). Antibody against OsMYB4P (AbP80423-A-SE) was custom-generated by Beijing Protein Innovation (China). Following SDS-PAGE separation, the proteins were analyzed via immunoblotting. RNA was extracted from the leaves of 14-d-old rice seedlings treated at 10°C for 24 h using TRIzol reagent (Invitrogen) for RNA-sequencing (RNA-seq). The extracted RNA was then used to construct sequencing libraries, which were subsequently sequenced on the NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Three independent biological replicates were performed. Quality control of the raw data was carried out using Cutadapt (v.1.15). Clean reads were aligned to the Oryza sativa reference genome (MSU_v.7.0) using HISAT2 (v.2.0.5) with default parameters. Differentially expressed genes (DEGs) were determined using DESeq (v.1.30.0), with a significance threshold of P < 0.05 and |log2 FoldChange| > 0.9. Venn diagrams were constructed using VENNY (v.2.1, https://bioinfogp.cnb.csic.es/tools/venny/). Y1H assays were performed according to Lin et al. (2007). The full-length or truncated coding region of OsMYB4P was inserted into the pB42AD vector, generating the AD-OsMYB4P construct (Table S1). The c. 2 kb and truncated OsT5H and OsLEA3-2 promoter fragments were amplified (Table S1) and cloned into the pLacZi reporter vector. These constructs were cotransformed into the yeast strain EGY48. Transformants were selected on SD-Trp/-Ura (SD-TU) plates and grown at 30°C for 3 d, then transferred to plates for β-galactosidase activity detection with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Yeast strains containing the empty pB42AD vector and LacZ reporter constructs served as negative controls. ChIP assays were performed as previously described (Li et al., 2025) with minor modifications. The full-length CDS of OsMYB4P was cloned into a pGreen-35S vector using Escherichia coli DH5α and transformed into Agrobacterium tumefaciens GV3101. For transient expression, Nicotiana benthamiana leaves (4–6-wk-old plants) were infiltrated with agrobacterial suspensions (OD600 = 0.6–0.8) in infiltration buffer (10 mM MES, 10 mM MgCl2, 150 μM acetosyringone, pH 5.6). After 48–72 h of incubation under light, total protein was extracted from infiltrated leaf tissues using RIPA buffer supplemented with protease inhibitors, followed by quantification via BCA assay. For chromatin immunoprecipitation (ChIP), crosslinking was performed by vacuum infiltration of leaf tissues with 1% formaldehyde for 15 min, followed by quenching with 0.125 M glycine. Chromatin was sheared by sonication (30% amplitude) with 10 min (10 s ON and 30 s OFF) to 200–500 bp fragments. Immunoprecipitation was carried out overnight at 4°C with anti-GFP antibody (5 μg/100 μg chromatin) and Protein A/G magnetic beads. After stringent washes, precipitated DNA was reverse-crosslinked (65°C, 4 h), purified by phenol–chloroform extraction, and analyzed by qPCR using specific primers (Table S1) using SYBR Green Master Mix (Vazyme). The EMSA was performed as previously described by Jiang et al. (2023). The CDS of OsMYB4P was cloned into the PET-32a vector, and the resultant combined vector was transformed into E. coli for the generation of His-OsMYB4P protein. The purification of recombinant proteins was conducted as previously described (Li et al., 2025). The purified protein was incubated with various biotin-labeled probes at 20°C for 20 min using a commercial Light Shift Chemiluminescent EMSA Kit (Promega). The probes' details are provided in Table S1. The c. 2-kb promoter regions of OsT5H and OsLEA3-2 were amplified using the LUC/REN-proOsT5H and LUC/REN-proOsLEA3-2 primer pairs (Table S1) and inserted into the upstream region of the LUC reporter gene, generating the LUC/REN-proOsT5H and LUC/REN-proOsLEA3-2 construct. The full-length coding sequence of OsMYB4P was amplified (Table S1) and inserted into the pGreen-35S vector to produce the pGreen-35S-OsMYB4P construct. These reporter and effector constructs were cotransformed as previously outlined (Li et al., 2026). Dual-luciferase activity was quantified using the Dual-Luciferase Reporter Assay System (Promega), and the relative LUC activity was calculated as the ratio of LUC to REN. Serotonin levels in WT and mutant plants were analyzed and quantified at baseline (0 h) and following cold stress treatment using high-performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) on an Agilent 1290 Infinity II system (Agilent Technologies, Deutschland GmbH, Waldbronn, Germany). The analytical procedure was adapted from the protocol described by Kang et al. (2007) for with for serotonin tissue samples were in liquid nitrogen and extracted with ml of at 4°C for 10 min with gentle agitation. The was at 500 g for 5 min at The was through a and 2 ml of water was to the the was purified using a which had been with and water. The was with 10 ml of and the containing serotonin was The combined was to and in 500 of For separation, serotonin was using with a (Agilent 5 The phase of in water containing at a rate of ml and quantification were carried out with a (Agilent 1290 Infinity II set at was on an from serotonin at ranging from 50 to 150 Under the described serotonin at a time of c. serotonin were determined by the at this time with the of H2O2 were quantified using a Assay Kit Beijing, In mg of tissue was with 500 of and at g for 10 min at The resulting was combined with an of hydrogen peroxide detection reagent and incubated at for 5 at was and H2O2 concentration was calculated on a leaf samples were ground in 50 mM buffer containing 1% using a and The resulting was at 15 g for 20 min at The was and the activities of SOD, POD, and CAT were according to the protocol outlined by et al. study was by the and the of and the and the of the Innovation of and the from of and the and performed the experiments and data analysis. and the and and the final All and the final and to this All data that the findings of this study are within the and the Information (Figs and Table S1). ZH11 vs osmyb4p-1 and OsMYB4P-OE-1 vs is in the at the Beijing of of under Fig. OsT5H expression and serotonin content were by cold stress in Fig. of rice treatment Fig. OsT5H cold stress in Fig. The of cold stress on ZH11, ost5h-1, XD-KO, and Fig. of the promoter region of OsT5H. Fig. OsMYB4P protein binds promoter of OsT5H. Fig. OsMYB4P expression is by cold stress in Fig. of OsMYB4P for Fig. OsMYB4P cold stress in Fig. OsMYB4P plays a key role in rice cold response. Fig. OsMYB4P the expression of OsT5H. Fig. of differentially expressed genes in ZH11, osmyb4p-1, and OsMYB4P-OE-1 under cold stress. Fig. of the promoter regions of OsLEA3-2. Fig. of OsMYB4P protein that binds to the promoter region of OsLEA3-2. Fig. levels of OsLEA3-2 in ZH11, and OsMYB4P-OE under cold stress. Fig. of OsLEA3-2 for Fig. The of cold stress on ZH11, oslea3-2-1, and Fig. The of cold stress on ZH11, osmyb4p-1, and oslea3-2-1. 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