Fig. 5: The regulation of cold tolerance in rice by OsCACT and OsPTR.

a, b Box plots of OsCACT (a) and OsPTR (b) expression levels at different cold treatment time points. The different colors represent cold-sensitive (CS) strains, which include six indica accessions, and cold-tolerant (CT) varieties, consisting of four japonica accessions. In the box plots, the center line represents the median, the box bounds indicate the 25th and 75th percentiles, and the whiskers extend to the minimum and maximum values. For CS (n = 6) and CT (n = 4), each accession was assessed with 3 biological replicates (total n = 18 and 12, respectively). Statistical analysis was performed using a two-tailed Wilcoxon test (*P < 0.05, ***P < 0.001). c Bar plots of OsCACT relative expression levels over time under cold, drought, and salt stress. For each time point, n = 3 biological replicates. Statistical analysis was performed using a two-tailed Student’s t-test, with 0 day as the reference (**P < 0.01, ***P < 0.001, ns: P > 0.05). Error bars present mean values  ±  SD. d Representative images of the transgenic lines (OsCACT and OsPTR knockout mutants and overexpression lines) and their corresponding control of Zhonghua 11 (ZH11) after 5 days of cold treatment followed by 3 days of recovery at the two-leaf and four-leaf stages. Scale bar: 5 cm. e Bar plots of plant survival rates after 5 days of cold treatment. For each time point, n = 3 biological replicates. Statistical analysis was performed using a two-tailed Student’s t-test, with ZH11 as the reference (*P < 0.05, ***P < 0.001). Error bars present mean values  ±  SD. f DAB and NBT staining images of leaves from ZH11 and two genes knockout mutants at different cold treatment time points at the two-leaf stage. g Line plots of relative electrolyte leakage rates of two genes knockout mutants and ZH11 over during cold treatment at the two-leaf stage. h Bar plots of MDA content in ZH11, OsCACT knockout mutants, and OsCACT overexpression lines after 5 days of cold treatment at the four-leaf stage. For each time point, n = 3 biological replicates. Statistical analysis was performed using a two-tailed Student’s t-test, with ZH11 as the reference (***P < 0.001, ns: P > 0.05). Error bars present mean values  ±  SD. i Model diagram of the mechanism by which OsCACT enhances cold tolerance in rice. The expression of OsCACT in the mitochondrial inner membrane affects the carnitine content in cells, which in turn influences rice cold tolerance through three distinct pathways. oscact-ko1 OsCACT knockout mutant-1, oscact-ko2 OsCACT knockout mutant-2; OsCACT-OE-1 OsCACT overexpression line-1; OsCACT-OE-2 OsCACT overexpression line-2, osptr-ko OsPTR knockout mutant, OsPTR-OE-1 OsPTR overexpression line-1, OsPTR-OE-2 OsPTR overexpression line-2, DAB, 3, 3’-diaminobenzidine; NBT nitro blue tetrazolium, MDA malondialdehyde. Source data are provided as a Source Data file.