OsbZIP76 interacts with OsNF-YBs and regulates endosperm cellularization in rice (Oryza sativa)

Cellularization is a key event during the development of the endosperm. Our understanding of the developmental regulation of cellularization has been limited for plants other than Arabidopsis. We found that the activation of OsbZIP76 coincided with the initiation of cellularization of rice. Either knockdown or knockout of OsbZIP76 led to precocious cellularization. Many genes involved in endosperm development or starch biosynthesis were prematurely activated in the caryopsis at two days after fertilization. The results implied that OsbZIP76 is involved in the regulation of cellularization in rice. As a putative transcription factor, OsbZIP76 alone lacked transcriptional activation activity. However, it was able to interact with OsNF-YB9 and OsNF-YB1, two nuclear factor Y (NF-Y) family transcription factors, both in yeast and in planta. OsbZIP76 and OsNF-YB9 showed similar endosperm-preferential expression patterns and the transiently expressed proteins were colocalized in the epidermal cells of tobacco. As with osnf-yb1 mutants, the osbzip76 mutants showed reduced seed size and reduced apparent amylose content of the seeds. We also confirmed that OsbZIP76 is an imprinted gene in rice, the expression of which depended on the genetic background. Our results suggested that OsbZIP76 is an endosperm-expressed imprinted gene to regulate development of the endosperm in rice. The process of cellularization in rice typically occurs from 3 through 5 DAF. OsbZIP76 coincided with the initiation of cellularization of rice. Either knockdown or knockout of OsbZIP76 led to precocious cellularization.Due to the high environmental temperature at the reproductive stage in the summer of 2018, the developmental process of rice caryopsis was promoted, which reflected by precocious cellularization at 2 DAF in WT as well as the mutant. We therefore used 2DAF samples to analyze the transcriptome changes in the caryopses of the osbzip76 mutants. RNA isolated from caryopses of the WT, osbzip76-1 and osbzip76-2 at 2 DAF was used for RNA-Seq. We set up three biological replicates for WT and two replicates for osbzip76-1 and osbzip76-2, two independent mutant alleles of OsbZIP76.