Preliminary Study Of OsMYB3 On Regulating Dormancy And Germination In Rice Seeds

Seed dormancy is an important agronomic trait of crops and is closely related to crop quality and yield.The seeds from most of the plants germinate after a period of dormancy after harvest.This phenomenon is known as the dormant nature of seeds.Seeds with low dormancy are prone to pre-harvest sprouting,and seeds with high dormancy will cause low germination rate in the sowing season.Therefore,the optimal dormancy level of seeds is crucial to agricultural production.The seed germination is affected by both genetic control and the environment(such as high temperature and high humidity),and the molecular mechanism of its regulation is still unclear.Therefore,the molecular mechanism of seed dormancy and germination is analyzed through genetics,molecular biology and other methods to provide a basis for the prevention and control of crop pre-harvest sprouting and the creation of germplasm resources resistant to pre-harvest sprouting.This will become the key to solving the problem of crop pre-harvest sprouting in the future.Earlier in our laboratory,the RNA-seq method was used to obtain OsMYB3,a transcription factor that is inhibited by high temperature during the filling period.Furthermore,many genetic materials of OsMYB3,such as overexpression lines and RNAi lines,were obtained for further study.Here,genetic,biochemical and molecular biological methods were carried out for functional study of the OsMYB3 gene on this basis,and following research progress were made:1.Regardless of field or greenhouse treatment under high humidity,the seeds harvested from the overexpressed plants(OsMYB3-OE)showed obviously viviparous phenotype,while the RNAi(OsMYB3-RNAi)transgenic lines had no significant difference sprouting rate compared with the wild type.2.Using the CISPR / Cas9 gene editing method,loss of function mutant myb3-1was obtained.Seed germination experiments were carried out by using either the freshly harvested seeds from myb3-1,OsMYB3-RNAi and OsMYB3-OE lines,or the mature seeds after ripening.The results showed that the germination rates of myb3-1and OsMYB3-RNAi were significantly lower than that of wild type,while it was opposite between OsMYB3-OE and WT.These data indicated that OsMYB3 may positively regulate seed germination.3.Previous data in our lab showed that the expression of OsMYB3 gene was regulated by rhythm and had high expression in developing seeds.In this study,we further investigated the expression pattern of OsMYB3 at different germination stages and found that it was constitutively upregulated during seed germination.4.The starch hydrolase gene is a marker gene for seed germination.In this study,we tested the expression of α-starch hydrolase gene at different time point of during seed germination in WT,myb3-1 and OsMYB3-OE lines.Results showed that the presence of OsMYB3 can promote the expression of some starch hydrolases AMY1 A,AMY1C,AMY3 C,which indicated that OsMYB3 may positively regulate seed germination in rice.5.As ABA and GA are the two most important hormones involved in the regulation of seed dormancy.ABA and GA were exogenously applied to the seeds for germination in our study.Results showed that myb3-1 mutant was not sensitive to GA while OsMYB3-OE seeds were super sensitive.However,there is no significant difference in the sensitivity of the above seeds to ABA,indicating that the OsMYB3 gene may regulate seed germination of seeds through the GA signaling pathway.6.Earlier studies in our lab indicated that OsMYB3 protein had no transcriptional activation function.In this paper,we used yeast two-hybrid to screen OsMYB3 interaction protein in rice endosperm cDNA library.Kinase SnRK1 A was isolated in this screening.Furthermore,cDNA library of rice transcription factors was screened,and two transcription factors were found to be interacted with OsMYB3.7.Although SnRK1 A was reported to be involved in the low-glucose-induced seed germination,we failed to detect that the expression of OsMYB3 was induced bylow-glucose or suppressed by high-glucose in protoplasts in our study.8.Using the Dual-LUC system,we found that OsMYB3 alone can significantly enhance the expression of the reporter gene driven by starch hydrolase α-AMY1 A.However,the enhancement was obviously inhibited by expressing SnRK1 A protein together.It is speculated that SnRK1 A may inhibit the transcription activation function of OsMYB3,and the exact regulatory mechanism needs to be further verified.In summary,we speculated that OsMYB3 is a positive regulator in the process of seed germination.OsMYB3 may positively regulate the expression of the starch hydrolase gene through the GA signaling pathway to regulate seed germination.The kinase SnRK1 A can interact with OsMYB3.The interaction between OsMYB3 and kinase inhibits the transcription activation function of OsMYB3.It is speculated that the phosphorylation of this gene will inhibit the activity of its encoded protein.However,more issues need to be addressed,such as what are the downstream target genes that OsMYB3 directly binds to,the genetic position of OsMYB3 in GA pathway,how protein phosphorylation and sugar signals play roles in OsMYB3 mediated seed germination.And more efforts should be made to study the molecular mechanism of OsMYB3 involved in seed dormancy and germination.We believe that our results will provide a strong basis for control of viviparity in crops in the future.