Transcription Factor OsNF-YC1 Regulates Grain Size By Coordinating The Transcriptional Activation Of OsMADS1 In Oryza Sativa L.

Rice is an important food crop in the world.Grain weight,grain number per panicle,and the number of panicles are the three factors that determine rice(Oryza sativa L.)yield.Of these,grain weight,which not only directly determines rice yield but also influences appearance and quality,is often considered the most important for rice production.Although many genes related to the control of rice grain size development have been studied and their molecular regulatory mechanisms have been initially clarified,their genetic regulatory networks still need to be elucidated.Here,we describe Os NF-YC1,a member of the NF-Y transcription factor family that positively regulates rice grain size.The Os NF-YC1 gene function was also analysed,with the following results.1.The knockout mutant osnf-yc1 was obtained by using CRISPR-Cas9 technology,and its grain width and grain thickness were reduced compared with the wild type,resulting in a lower thousand grain weight,but its grain length was not changed,the seeds showed a slight elongated grain shape.The number of primary branches remained unchanged,the number of secondary branches increased,the number of grains per panicle increased,and other traits such as plant height and yield per plant were not significantly different from the wild type.Using RNAi technology to down-regulate the expression of Os NF-YC1,the seeds in the Os NF-YC1-RNAi transgenic plants exhibited a phenotype of reduced grain width and grain thickness and reduced thousand grain weight,which was similar to the seed phenotype of the knockout mutant osnf-yc1.2.Cytological observation by scanning electron microscopy and paraffin sections revealed that the number of thin-walled cells in the outer layer of osnf-yc1 was significantly reduced and the cell area was significantly decreased.Meanwhile,detection of the expression of genes involved in cell cycle proteins revealed that the decrease in cell number might be related to the down-regulation of the expression of genes promoting cell proliferation.This suggests that Os NF-YC1 regulates seed development by affecting cell proliferation and cell expansion and thus seed development.3.Subcellular localization analysis in rice protoplasts,tobacco epidermal cells and transgenic strains of Os NF-YC1-GFP revealed that Os NF-YC1 protein was localized in the nucleus and cytoplasm.Meanwhile,transcriptional activation experiments in a yeast two-hybrid system showed that Os NF-YC1 is a transcription factor with transcriptional activation activity and the structural domain CBFD-NFYB-HFM has no transcriptional activation activity,while the transcriptionally active regions are located at its N and C terminus.4.Yeast two-hybrid assay,Bimolecular Fluorescence Complementation assay,and Pull-Down assay demonstrated that Os NF-YC1 interacts with Os MADS1 both in vitro and in vivo.Based on the deletion of the structural domain we constructed different vectors and further demonstrated by yeast two-hybrid assay that the conserved histone like structural domain CBFD-NFYB-HMF of Os NF-YC1 can directly interact with the conserved structural domain MADS-box of Os MADS1 transcription factor.To reveal the mechanism of the effect of Os NF-YC1 and Os MADS1 on seed development after interaction,we found that the expression of Os MADS1 at the transcriptional level and protein level was not significantly altered in the mutant osnf-yc1 by RT-q PCR,protein blotting,and in situ hybridization,and no significant ectopic expression occurred.The interaction of Os NF-YC1 with the transcription factor Os MADS1 was found to enhance the transcriptional activation activity of Os MADS1 by transient transcriptional activity assay.5.RT-q PCR analysis of Os MADS1 downstream target genes in mutant osnf-yc1 revealed that only Os MADS55 expression was highly significantly reduced to only34.8%/39.5% of WT,and to further support this conclusion,we showed by improved transient transcriptional activity assay that Os MADS55 promoter-driven LUC activity was induced by Os MADS1,and when Os NF-YC1 and Os MADS1 were co-expressed,Os MADS55 promoter-driven LUC activity was significantly enhanced.Meanwhile,we created Os MADS55 knockout mutants using CRISPR-Cas9 technology and found smaller seeds and reduced thousand grain weight by agronomic trait examination,which was similar to the phenotype of the mutant osnf-yc1.This indicates that Os NF-YC1 and Os MADS55 are not completely independent in terms of genetic relationship.