Molecular Basis Of OsCYP71D8L Regulating Plant Height And Stress Tolerance In Rice

As an important food crop,the high yield,high quality and stress tolerance of rice are controlled by environmental conditions and genotypes.Plant height is an important agronomic trait closely related to yield and stress resistance in rice,which is usually controlled by major genes.Therefore,it is of great significance to explore and identify the key genes controlling plant height for genetic improvement of high yield,high quality and stress tolerance in rice varieties.Preliminary studies have shown that plant hormones such as gibberellin,cytokinin and auxin play an important role in the regulation of rice plant height.Cytochrome P450 s are an important family of proteases in plants,which regulate plant growth and development and stress response by participating in the synthesis and metabolism of a variety of plant hormones.Through bioinformatics analysis,we found that there are eight tandem repeat CYP450 s genes on chromosome 2 of rice genome,among which Os CYP71D7,OsCYP71D8L and Os CYP71D8 belong to the CYP71 family.Phylogenetic tree analysis showed that OsCYP71D8L and Os CYP71D8 had 91% amino acid similarity.Our previous studies showed that Os CYP71D8 was differentially expressed in different rice varieties,and the expression level of Os CYP71D8 was significantly increased in drought tolerant varieties.However,the physiological functions of these genes in rice have not been reported.In this study,we used OsCYP71D8L overexpression transgenic rice(OsCYP71D8L-OE),function acquired T-DNA mutant(cyp71d8l)and gene editing to explore the physiological function and molecular basis of OsCYP71D8L in regulating rice growth,development and stress response.The phenotype observation showed that the growth and development of OsCYP71D8L-OE and cyp71d8 l were inhibited compared with the corresponding wild type,which were characterized by plant dwarfing,shorter tassel length,fewer grains per tassel and poor root development.Hormone analysis showed that the content of active gibberellin and precursor in OsCYP71D8L-OE was significantly lower than that of wild type,while the content of active gibberellin GA3 in cyp71d8 l was significantly lower than that of wild type;meanwhile,the content of active cytokinin(isopentenyl adenine)in OsCYP71D8LOE was about twice that of wild type.Further studies confirmed that exogenous GA3 could restore the growth of aboveground parts of seedlings,while low level of cytokinin synthesis inhibitor could restore root growth.These results suggest that OsCYP71D8L inhibits rice growth and development by changing the homeostasis of endogenous gibberellin and cytokinin.In rice,the homeostasis of gibberellin and cytokinin not only affects the growth and development,but also affects the tolerance of rice to abiotic stress.OsCYP71D8L-OE and cyp71d8 l had stronger stress tolerance than the corresponding wild type.The results showed that the leaf curl,wilt and yellowing degree of wild type were more serious after drought or high salt treatment,the stress phenotype was more obvious,and the survival rate was lower than OsCYP71D8L-OE and cyp71d8 l after rehydration culture.In terms of plant physiological indexes,the contents of soluble sugar and chlorophyll of OsCYP71D8LOE and cyp71d8 l were higher,and the accumulation of hydrogen peroxide was less,which indicated that OsCYP71D8L improved the stress tolerance of rice from the physiological and biochemical level.Furthermore,the expression levels of genes related to ROS scavenging enzymes were analyzed.The genes encoding peroxidase and ascorbic acid peroxidase were up-regulated in OsCYP71D8L-OE and cyp71d8 l.These results indicate that OsCYP71D8L can positively regulate rice tolerance to drought and salt stress at transcriptional level.In order to further study the physiological function and molecular basis of OsCYP71D8L,we constructed different mutants by CRISPR.We identified that cyp71d8l-92 and cyp71d8l-93,which mutated at about 380 bp,were more sensitive to drought,which further proved the regulation of OsCYP71D8L on stress tolerance of rice.In conclusion,our results suggest that OsCYP71D8L,as a member of CYP450 s protease family,can inhibit the growth and development of rice by regulating the endogenous homeostasis of gibberellin and cytokinin,and improve the salt tolerance and drought resistance of rice.