Molecular Mechanism Of The LEC1-type Transcription Factor OsNF-YB7 Regulated Chlorophyll Biogenesis In The Embryo Of Rice

Studies have shown that chloroplasts in chloroembryos can carry out photosynthesis to provide oxygen and energy for early embryo development.On the other hand,chloroplast development in chloroembryos has important effects on physiological processes such as embryogenesis,seed germination,plant vegetative growth and so on.In addition,the development of chloroplast in embryo is also a key factor in normal embryo development and seed quality formation.The lack of chlorophyll in rape and other crop embryos will lead to a significant decrease in the content of unsaturated fatty acids and protein,which will seriously affect the seed quality[1].In-depth study of embryonic chlorophyll synthesis can not only explain the mechanism of chloroembryos formation,but also is of great significance for the improvement of green embryo crops.As a key transcription factor regulating seed development,LEAFYCOYLEDON1(LEC1)can promote the biosynthesis of chlorophyll in Arabidopsis thaliana cotyledons,but its mechanism is not clear.Unlike Arabidopsis thaliana,rice and other Gramineae plants lack the ability to synthesize chlorophyll.However,it is not clear what mechanism determines that chlorophyll cannot be accumulated in gramineous embryos.In this study,it was found that the loss of function of rice LEC1 homologous gene OsNF-YB7 led to abnormal chlorophyll accumulation in rice embryos,indicating that OsNFYB7 played an opposite role in regulating chlorophyll biosynthesis in rice embryos as in Arabidopsis LEC1.Further functional studies found that there was a physical interaction between OsNF-YB7 and OsGLK,a key regulator of chlorophyll biosynthesis.In this study,the molecular mechanism of green embryo formation regulated by the interaction between OsNF-YB7 and OsGLK1 was analyzed by means of genetics and molecular biology.The main results are as follows:1.Cytological analysis techniques such as transmission electron microscopy and fluorescence confocal microscopy were used to analyze the phenotype of the osnf-yb7 embryo.The results showed that the chloroembryo phenotype of the osnf-yb7 was caused by chlorophyll accumulation in the embryo.2.Through RNA-seq,qRT-PCR and other methods,it was found that the expression of genes related to chlorophyll synthesis and photosynthesis were up-regulated in osnf-yb7 embryos,indicating that OsNF-YB7 negatively regulates photosynthesis and chlorophyll biosynthesis.3.The results of transcriptional group and qRT-PCR experiment showed that OsNF-YB7 inhibited the expression of OsGLK1 in embryos.ChIP-qPCR and DLR experiments showed that OsNF-YB7 enriched and inhibited its activity in OsGLK1 promoter.4.Using CRISPR/Cas9 system,osnf-yb7osglk2 double mutant and osnf-yb7osglk1osglk2 triple mutant were successfully obtained.Phenotypic analysis showed that when all three genes were mutated,the chlorophyll accumulation in embryo decreased significantly.Furthermore,the OsGLK1 overexpression line(OsGLK1-OX)was constructed,and chlorophyll was significantly accumulated in the embryo.The above results suggest that the chlorophyll accumulation mediated by osnf-yb7 partly depends on the biological function of OsGLKs.5.Based on the analysis of OsGLK1-OX and osnf-yb7 embryo transcriptome data,it was found that OsGLK1 and OsNF-YB7 antagonized the regulation of chlorophyll synthesis and photosynthesis-related gene expression in embryos.Further analysis of their ChIP-seq data showed that OsNF-YB7 and OsGLK1 were bound to the same motif of the common target.6.The physical interaction between OsNF-YB7 and OsGLK1 was proved by biochemical experiments such as yeast two-hybrid and bimolecular fluorescence complementarity.Furthermore,DLR and EMSA methods were used to prove that OsNF-YB7 inhibited the biochemical activity of OsGLK1.Taken together,we believe that OsNF-YB7 plays a dual role in the regulation of chlorophyll biosynthesis in rice embryos.First,it functions as a transcription factor to repress downstream genes;second,it can interact with other transcription factors,such as OsGLKs,to interfere with its ability to activate chlorophyll biosynthesis and photosynthesis-related genes.