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.

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).

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.