OsNAL11 and OsGASR9 Regulate the Low-Temperature Germination of Rice Seeds by Affecting GA Content

Low temperatures cause serious threat to rice seed emergence, which has become one of the main limiting factors in the production of direct seeding rice. It is of great importance to study the genes controlling low-temperature tolerance during seed germination and to mine the possible regulatory mechanism for developing new rice varieties with immense low-temperature germination ability. In the current research study, two types of mutants of nal11 and gasr9, derived from the WT (wild type) ZH11, were used for the analysis of low-temperature germinability. The results showed that the nal11 and gasr9 mutants displayed no significant difference in germination rate with ZH11 at room temperature, but the mutants showed significantly lower germination rates, germination potential and germination index, and slowed seedling growth in the simulated direct seeding experiments at low temperatures compared to ZH11. Additionally, the activity of POD, SOD, CAT, and anti-superoxide anion radial activity were significantly reduced, but the levels of MDA and H₂O₂ were significantly higher in the nal11 and gasr9 mutant seeds that were germinated at low temperatures compared to ZH11. Further analysis revealed that the levels of total active GA, especially GA4 and GA7, were significantly lower in the nal11 and gasr9 mutants than that in ZH11 during low-temperature germination. Based on qRT-PCR analysis, the expression levels of some GA synthesis-related genes were higher, whereas some were lower in the nal11 and gasr9 mutants than those in ZH11, however, the GA metabolism-related genes OsGA2ox8 and OsGA2ox10 and the GA signaling negative regulator gene SLR1 were significantly up-regulated in both nal11 and gasr9 mutants at several time points during low-temperature germination. This may explain the lower GA levels in the nal11 and gasr9 mutants. Furthermore, the interaction between the OsNAL11 and OsGASR9 proteins was confirmed by Y2H, LUC, and Co-IP assays. This study provides preliminary insights into the regulatory mechanism of the OsNAL11 and OsGASR9 genes, which control the low-temperature germination of rice seeds by affecting the GA pathway. Our study will provide the basis for further mining the molecular mechanisms of low-temperature germination in rice and valuable theoretical reference for breeding varieties with strong low-temperature germinability.