Genome-wide identification of grain filling genes regulated by the OsSMF1 transcription factor in rice

Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by gene promoters. Therefore, it is important to identify the binding motifs of transcription factors to better understand the networks associated with embryogenesis.

Here, we used a protein-binding microarray (PBM) to identify the binding motifs of OsSMF1, which is a basic leucine zipper transcription factor involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1 or OsbZIP58) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage protein synthesis, and it functions as a key regulator of starch synthesis. Quadruple 9-mer-based PBM analysis and electrophoretic mobility shift assay revealed that OsSMF1 bound to the GCN4 (TGA(G/C)TCA), ACGT (CCACGT(C/G)), and ATGA (GGATGAC) motifs with three different affinities. We predicted 44 putative OsSMF1 target genes using data obtained from both the PBM and RiceArrayNet. Among these putative target genes, 18, 21, and 13 genes contained GCN4, ACGT, and ATGA motifs within their 1-kb promoter regions, respectively. Among them, six genes encoding major grain filling proteins and transcription factors were chosen to confirm the activation of their expression in vivo. OsSMF1 was shown to bind directly to the promoters of Os03g0168500 (GCN4 motif), patatin-like gene (GCN4 motif), α-globulin (ACGT motif), rice prolamin box-binding factor (RPBF) (ATGA motif), and ONAC024 (GCN4 and ACGT motifs) and to regulate their expression.

The results of this study suggest that OsSMF1 is one of the key transcription factors that functions in a wide range of seed developmental processes with different specific binding affinities for the three DNA-binding motifs.