Functional Characterization Of NF-YB1,NF-YC12 And BHLH144 In Rice(Oryza Sativa L.)

Identification of seed development regulatory genes is the key for the genetic improvement of rice grain quality.Nuclear factor Y(NF-Y)heterotrimer complex containing NF-YA,B and C subunits are key transacription regulators in eukaryotes.Several seed-predominantly expressed NF-Ys have been known as master regulator in rice seed development,but the molecular mechanism remains unclear.Here,we report the functional characterization of a rice NF-Y heterotrimer complex NF-YB1-YC12-bHLH144,which is formed by the binding of NF-YB1 to NF-YC12 and then bHLH144 in a sequential order as indicated by yeast-three-hybrid andpull-down assays.CRISPR/Cas9-mediated knock-out of each of the complex genes resulted in alteration of grain qualities in all the mutants as well as reduced grain size in crnf-yb1 and crnf-yc12.RNA-seq analysis identified 1496 genes that were commonly-regulated by NF-YB1 and NF-YC12,including the key granule-bound starch synthase gene Wx.Degradation assay result showed that NF-YC12 and bHLH144 maintain NF-YB1 stability from the degradation mediated by ubiquitin126S proteasome,while NF-YB1 directly binds to the "G-box" domain of Wx promoter and activates its transcription as confirmed by EMSA,ChIP-PCR and transient luciferase activation assays,hence to regulate rice grain quality.We revealed a novel grain quality regulatory pathway controlled by NF-YB1-YC12-bHLH144 complex,in which NF-YB1 could attach to non-CCAAT cis element and activate target gene transcription.