Spatial- and temporal-specific expression patterns are primarily regulated at the transcriptional level by the promoter. Therefore, it is important to determine the binding motifs of transcription factors to understand the networks associated with embryogenesis. Here, we used a protein-binding microarray (PBM) to determine the binding motif of OsSMF1, which is a basic leucine zipper transcription factor that is involved in the regulation of rice seed maturation. OsSMF1 (previously called RISBZ1) is known to interact with GCN4 motifs (TGA(G/C)TCA) to regulate seed storage proteins (SSPs). In addition, OsSMF1 (also known as OsbZIP58) functions as a key regulator of starch synthesis in the rice seed. Quadruple 9-mer-based PBM (Q9-PBM) and electrophoretic mobility shift assay (EMSA) experiments revealed that OsSMF1 binds to the ACGT (CCACGT(C/G)), GCN4 (TGA(G/C)TCA), and GCN4-like (GGATGAC) motifs with Kd values of 0.3353 μM, 0.6458 μM, and 1.117 μM, respectively. We also identified 60 putative OsSMF1 target genes using a combination of data from expression microarrays and RiceArrayNet (RAN) analysis. Of these OsSMF1 target genes, 20, 22, and 17 genes contained ACGT, GCN4, and GCN4-like motifs within the 2-kb promoter region, respectively. In addition to known target genes, we also identified 35 potential OsSMF1 target genes that have not been previously described in immature seeds. We also confirmed that OsSMF1 directly regulates Os03g0168500 (thioredoxin-related protein), RPBF, NAC6, and two hypothetical proteins (Os12g0621600 and Os11g0582400) in vivo. This study suggests that OsSMF1 functions in a wide range of seed development processes with specific binding affinities for three DNA binding motifs

The perturbation of the steady state of reactive oxygen species due to biotic and abiotic stresses in a plant could lead to protein denaturation through the modification of amino acid residues, including the oxidation of methionine residues. Methionine sulfoxide reductases (MSRs) catalyze the reduction of methionine sulfoxide back to the methionine residue. To assess the role of this enzyme, we generated transgenic rice using a pepper CaMSRB2 gene under the control of the rice Rab21 promoter with/without a selection marker, the bar gene. A drought resistance test on transgenic plants showed that CaMSRB2 confers drought tolerance to rice, as evidenced by less oxidative stress symptoms and a strengthened PSII quantum yield under stress conditions, and increased survival rate and chlorophyll index after the re-watering. The results from immunoblotting using a methionine sulfoxide antibody and nano-LC-MS/MS spectrometry suggest that porphobilinogen deaminase (PBGD), which is involved in chlorophyll synthesis, is a putative target of CaMSRB2. The oxidized methionine content of PBGD expressed in E. coli increased in the presence of H2O2, and the Met-95 and Met-227 residues of PBGD were reduced by CaMSRB2 in the presence of dithiothreitol. An expression profiling analysis of the overexpression lines also suggested that photosystems are less severely affected by drought stress. Our results indicate that CaMSRB2 might play an important functional role in chloroplasts for conferring drought stress tolerance in rice