Expression profiling was conducted with the Oryza sativa alternative splicing detecting microarray v.4 (OsASDM). Probe features are designed based on rice genome IRGSP_1.0 (http://rapdb.dna.affrc.go.jp/ ). The genome contains 37,868 genes. Among these 5,254 genes have alternative spliced sites, 11,938 transcripts. In the microarray, a total of 41,953 transcripts are covered from all the loci and 9112 alternative spliced transcripts. Four 60-nt long probes were designed from each transcript starting 60 bp ahead the end of stop codon and with shifting 30 bp so 4 probes cover 150 bp in the 3’ region of the gene. Genes from chloroplast (123) and mitochondria (74) and selection markers such as gfp, gus, hyg, bar, and kan are included. In total, he 125,956 probes were designed. To find organ specific transcripts RNA was prepared from leaf, root, panicle at 1 cm (P1cm). The signal intensity files were analyzed with limma package. Background correction and normalization were performed with libraries in the package. 13,486 genes are organ specific and 1,856 transcripts are alternatively spliced. Transcripts that specifically alternatively spliced in leaf are Os02t0197600-02_UE; Chlorophyll a-b binding protein 8, Os11t0707000-01_UE; Ribulose bisphosphate carboxylase/oxygenase, Os12t0291100-01_UE; ribulose 1,5-bisphosphate carboxylase small subunit. Transcripts that specifically alternatively spliced in root are Os03t0669100-02_UE; Deoxyuridine 5’-triphosphate nucleotidohydrolase, Transcripts that specifically alternatively spliced in tissues at P1cm are Os11t0210300-02_UE; Alcohol dehydrogenase 1, Os04t0631200-02_UE; Xyloglucan endotransglycosylase. Os03t0669100-02_UE ; Deoxyuridine 5’-triphosphate nucleotidohydrolase, Os11t0210300-02_UE ; Alcohol dehydrogenase 1, Os04t0631200-02_UE; Xyloglucan endotransglycosylase. These results show that OsASDM could be used to find alternatively spliced gene at ease.

Rice, as a model system of monocotyledon plants for genomic studies, is a main staple food for over half of the world population. A rice retrotransposon, Tos17, is active during tissue culture and its ability was wildly used in insertional mutagenesis. In this study we have produced 2,000 non-GM mutants induced by Tos17 in rice. We analyzed >2,000 flanking sequences of newly transposed Tos17 copies by the adaptor-ligation PCR method. The frequencies of Tos17 insertions in the genic and intergenic regions were 60.3% and 36.6%, respectively. We also selected four Tos17 insertion mutant lines for three TF genes which can be considered to be considered to be involved in rice seed development based on expression microarray data: osrem3, osta1, osbhlh1-1, and osbhlh1-2 mutant lines. According to Quadruple 9-mer-based protein binding microarray (Q9-UPBM) experiment, we found that the OsREM3, OsTA1, and OsbHLH1 bound to the ACACCAC, CACGTG, and GTAACA motifs, respectively. In combination of Q9-UPBM, RiceArrayNet analysis, and expression microarray data, we identified 8, 20, and 9 putative target genes of OsREM3, OsTA1, and OsbHLH1, respectively. We have been screening and characterizing the mutations by extensive phenotypic analysis as well as the functional analysis of genes.

Salt and drought stresses affect virtually every aspect of plant physiology and metabolism and thus limiting the productivity of crop plants worldwide. Salt and drought tolerance and adaptation in rice has been improved by engineering various genes related to transcription, signaling, accumulation of antioxidants and compatible solutes etc. Previously, we have produced 2,000 non-GM mutants induced by Tos17 in rice. We analyzed >2,000 flanking sequences of newly transposed Tos17 copies by the adaptor-ligation PCR method. We also identified significantly up- or down-regulated genes under drought, salt, or ABA stress in rice based on expression microarray data, which previously were performed from leaf at different developmental stages and conditions. For screening and characterizing the salt or drought tolerance mutations by extensive phenotypic analysis as well as the functional analysis of genes, we selected 133 mutant lines. To evaluate rice phenotypic traits under abiotic stress condition, we plan to investigate phenomics, which integrates technologies such as photonics, biology, computers, and robotics.

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

With the development of next generation sequencing (NGS) technology, the variation of sequences represented as SNP between cultivars becomes available at genome level. The major domestic cultivars with high yield have been developed by breeding of indica and japonica, it is important to localize the region of origin according to the genotype for further characterization of unique features of cultivars. For the localization of SNP at genome level, the paired end sequences of 6 major domestic rice cultivars, Ilmi, Ilpoom, Sulgaeng, Baekjinju1ho, Hwayoung and Woongwang were compared against Japonica and Indica Rice Genomes as reference genomes. The genomic DNAs were prepared from callus tissues and paired-end of the fragments were sequenced with NGS Sequencer, Illumina HISeq2000. About 50x coverage of paired-end sequences were trimmed according to the quality of the sequences, and errors were corrected with statistical analysis of kmers of 15. The trim-corrected sequences were mapped and variants were analyzed against reference genomes. The overall change rate of Ilmi against Nipponbare IRGSP 1.0 and Indica BGI 93-11 reference genomes were 0.92 base/1kb (1/1,079 base) and 8.09 base/1kb (1 base/123 bases), respectively. Among 6 cultivars, overall rate of Baekjinju1ho showed the lowest overall change rate of 0,53 base/1kb, and Hwayoung showed highest frequency of 0.92 base/1kb. Compared to high level in the range of change rate of 7.0-9.3 base/1kb against indica, domestic cultivars showed lower range of change rate 0.2-3.3 base/1kb with unique local high peak against japonica genome depend on the chromosomes. Compared to assembly of genome sequences, the variation of nucleotides compared to reference sequences is much faster and simple to characterize the genotype. The types of variation and the effect on functional categories will be presented.

Rice is an important model species and one of the most staple crops of the world. The use of rice appropriate promoters suitable for a specific target transgene is important for the control of spatial and temporal transgene expression. To isolate rice tissue-specific promoters, we exploited the potential of whole genome microarrays in 17 stages: callus, germinating seed, leaf, root, the size of the panicles before heading (1, 3, 5, 8, 10, 15, 20, and 22 cm), and the number of days after pollination (1, 3, 5, 11, 21 DAP) using a 300 K Rice Genome Microarray, covering 31,439 genes of the rice. Eight candidate genes for tissue-specific expression were selected in various organs and stage of reproductive development in rice: Histone H4 for constitutive expression, Dehydrin DHN1 for callus-specific expression, germinating seed-specific hypothetical protein, root-specific hypothetical protein, DNA topoisomerase and Retinoblastoma for expression at panicles before heading, heading-specific profiling, and invertase for expression at seed after pollination. Promoter regions of the selected genes were isolated and fused to the β-glucoronidase (GUS) reporter gene, and the constructs were introduced into rice plants. These promoters are highly active in the tissue-specific manner of rice and can be useful for the spatial and temporal enhancement of target gene(s).

The surveying of binding affinity between a particular transcription factor and DNA motifs is important in order to understand the developmental specific gene expression and regulatory networks of an organism. The microarray-based technologies (protein-binding microarrays; PBMs) provide useful predictions for understanding the transcriptional regulatory code in a genome-wide manner. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, named Q9-UPBM. Also, we developed rice promoter PBM (RPBM) using 19,480 rice promoter sequences containing 40 bp long probe with overlapping 20 bp (cover 1kb from 5’ upstream). We applied RISBZ1 protein, an endosperm specific basic leucine zipper transcription factor, to compare binding site specificities between Q9-UPBM and RPBM and find directly regulated promoter regions through the RPBM. Several cis-elements; Prolamin box (TGTAAAG), GCN4 motif (TGA(G/C)TCA), AACA motif (AACAAAA), and ACGT motif, are highly conserved in the promoters of cereal seed storage protein genes, and play a central role in controlling endosperm specific expression during seed maturation. Characterization of cis-elements and TFs has been performed on many storage protein genes of several crop plants, but the mechanisms are still poorly understood. Two chips provide RISBZ1 could bind to ACGT motif such as a CCACGTCA site and GGATGAC site as well as GCN4 motif known binding site. In RPBM binding affinity to CCACGTCA was highly significant, compared to GGATGAC site. The difference might be caused by the biased presence of specific promoter rather than Q9-UPBM. Also our results will provide direct insight into the importance of combinatorial interplay between cis-elements in regulating the expression of seed storage protein genes.

Gene expression profiles can serve as a valuable reference for deciphering gene functions. We exploited the potential of whole genome microarrays to measure the temporal expression profiles of rice genes in 13 stages of reproductive development. We could profile expression of 17,676 genes in at least one of the tissues. Differential expression analysis with compare to leaf and preceding stages of development revealed reproductive stage-preferential/-specific genes. we identified 35 genes expressing specifically during panicle and seed development. The metabolic/hormonal pathways and transcription factor families playing key role in reproductive development were elucidated after overlaying the expression data on the public databases and manually curated list of transcription factors, respectively. During floral meristem differentiation (P1cm) and male meiosis (P5cm), the genes involved in jasmonic acid and gebbellin biosynthesis were significantly upregulated. F11DAP stage of seed, containing enlargement organ, exhibited enrichment of transcripts involved in starch or sucrose biosynthesis. Genes regulating auxin biosynthesis were induced during early seed development. We validated the stage-specificity of regulatory regions of two panicle-specific genes, AK072471, Os08g0538700, and AK121412, an early seed-specific gene, in transgenic rice. The data generated here provides a snapshot of the underlying complexity of the gene networks regulating rice reproductive development.