The Functional And Molecular Mechanism Analysis Of Rice Seed Specific Expressed Nuclear Factor-Y(NF-Y) Transcription Factor

Rice(Oryza sativa L.)is the foremost food source for nearly half of the global population.Improving rice yield and grain quality is the two crucial scientific issue in basic research and production practice.Rice grain size/shape directly influences rice yield,and is an important yield trait.Accumulation of seed storage substances is closely related to the quality of rice seed.In recent years,several factors that affect grain size and storage substance accumulation have been described in rice.However,the molecular mechanisms underlying the determination of grain size,and the transcriptional regulatory networks of synthesis and accumulation of storage substances,are still unclear.Nuclear factor-Y(NFY),a class of transcription factors,play important roles in multiple plant developmental processes.In this work,5 rice seed/endosperm-specific NF-Y genes(NF-YA8,NF-YB1,NFYB9,NF-YC10 and NF-YC12)were selected to identify their functions in seed/endosperm development by creating knockdown or knockout mutants and gene overexpression genetic materials,and their regulatory mechanisms were analyzed.The main results are as follows:1.A total of 5 rice/endosperm-specific expressed genes belonging to the NF-Y class of transcription factors were identified in rice genome.Expression profile analysis of 5 genes in different organs and tissues of ZH11 variety by qRT-PCR were performed.The results showed that their expression are endosperm-specific,indicating that rice seed/endosperm-specific NF-Y genes may involve in endosperm development.Here,we created some genetic materials of rice seed/endosperm-specific NF-Y genes by using reverse genetic methods,including gene knocking down(RNAi surpression),knocking out(CRISPR/Cas9 gene editing)or overpressing gene's expression.RNAi transgenic plants of 2 genes,CRISPR mutants of 5 rice seed/endosperm-specific NF-Y genes and gene overexpression materials of 2 genes have been obtained.2.Rice seed specific expressed NF-YC12 is a key multi-functional regulator in storage substance accumulation in Rice.NF-YC12,which encodes a putative nuclear factor-Y transcription factor subunit C,is predominantly expressed in aleurone layer and starchy endosperm,with the highest expression level between 7-14 days after pollination(DAP).Knockout of NF-YC12 significantly decreased grain weight and caused chalky endosperm.Scanning electron microscopy images of transverse sections of nf-yc12 grains indicated that starch granules were abnormal and irregular.Transmission electron microscopy analysis showed a growth arrest of starch granules in nf-yc12.The total starch and amylose contents were significantly lower than those in transgenic negative control(designated as WT).The prolamin content was decreased,while the glutelin content was higher than that in WT.The starch physicochemical properties(including the gelatinization properties of starch granules and the chain length distribution of amylopectin)were altered in nf-yc12 endosperm.Additionally,elevated expression of NF-YC12 significantly enhanced both grain size and weight.The storage substances of the seeds differed between NF-YC12-overexpression lines and the wild type.RNA sequencing analysis revealed that the genes related to starch biosynthesis and energy reserve metabolic process were enriched in the down-regulated category in nf-yc12 mutant,which is consistent with the key role of NFYC12 in rice endosperm development.We identified lower transcript levels of the selected genes associated with starch and protein synthesis in 7 DAP endosperm of nf-yc12 relative to WT.These data further indicated that the transcriptional regulatory function of NF-YC12 is associated with storage substance accumulation in rice seed.Targets of NF-YC12 in the developing endosperm were identified using ChIP-seq,in combination with RNA-seq analysis of the nf-yc12 mutant.ChIP-qPCR and yeast onehybrid assay further comfirmed that NF-YC12 was directly bound to the promoters of OsSUT1(Sucrose Transporter),OsGS1;3(Glutamine Synthetase1)and FLO6(FLOURY ENDOSPERM6).qRT-PCR results indicated that NF-YC12 positively regulates the expression of those 3 genes.These results indicated that OsSUT1,OsGS1;3 and FLO6 are the direct targets of NF-YC12 in rice endosperm.Furthermore,NF-YC12 was found to interact with NF-YB1 in vivo and in vitro.A model was proposed centre on NF-YC12-NFYB1 in the gene network.NF-YC12 may not only work in coordination with NF-YB1 to regulate the expression of SUTs in the aleurone layer,but also act as a direct activator of the downstream genes FLO6,OsGS1;3 and other undetermined targets to regulate the storage substance accumulation during endosperm development.Overall,this study demonstrates a transcriptional regulatory network of NF-YC12 and NF-YB1,which coordinates multiple pathways to regulate endosperm development and the storage substance accumulation in rice seed.3.Rice aleurone layer specific NF-YB1 regulates endosperm development and grain filling.Here,we report the function of a nuclear factor-Y transcription factor subunit B coding gene NF-YB1,which is expressed specifically in endosperm aleurone layer,using the CRISPR/Cas9 system and RNAi methods in rice.The NF-YB1-RNAi lines and NF-YB1 overexpression lines develops a chalky endosperm,indicating that the alter of NF-YB1 expression affects the quality of rice seed.However,the change of NF-YB1 expression did not affect the development of endosperm aleurone layer.Grains of nf-yb1 and NF-YB1-RNAi showned a lower 1000-grain weight and a higher grain chalkness rate,in comparion with that of WT.Meawhile,the total starch content and amylose content in nf-yb1 was significantly decreased and the chain length distribution of amylopectin in nf-yb1 was also altered.All of those results together indicate that NF-YB1 plays an important role in endosperm development and grain filling.4.Rice seed preferentially expressed factor NF-YC10 regulates rice grain size.NFYC10 show preferred expression patterns of endosperm.NF-YC10 is highly expressed in endosperm at 5-10 days after pollination.NF-YC10 protein is localized in the nucleus,and it has transcriptional activation activity in yeast.Knockout and suppression of NF-YC10 expression caused a dramatically reduction of grain weight and grain width.qRT-PCR results showwed that GW7 gene which is involved in the regulation of grain size was greatly upregulated in NF-YC10 RNAi lines and nf-yc10 mutants.NF-YC10 regulates grain size,at least in part,by influencing expression of several known genes related to grain size.Our findings reveal the function of NF-YC10 in grain size,suggesting that a novel important factor NF-YC10 has the potential to improve grain size and grain weight in rice.5.The expression pattern and functional analysis of rice NF-YA8 gene.GUS staining observation of transgenic plants of NF-YA8 promoter-reporter fusion vectors(pNFYA8::GUS)found that NF-YA8 only expressed in aleurone layer in rice endosperm,which are very similar to the expression pattern of NF-YB1 in rice.Result of qRT-PCR showed NF-YA8 is predominantly expressed in the middle and late stage of endosperm development,with the highest expression level between 7-10 days after pollination.It is consistent with the results of GUS staining,indicating that NF-YA8 expression is aleurone layer-specific.NF-YA8 protein is localized in the nucleus.yeast two-hybrid assay showed that NF-YA8 did not interact with NF-YB1 or NF-YC12.Knockout mutant of NF-YA8 was generated using the CRISPR/Cas9 approach.However,the nf-ya8 mutants did not show any obvious abnormalities in the endosperm appearance.