Circadian clock- and phytochrome-regulated Dof-like gene, Rdd1, is associated with grain size in rice.

We report here on the characterization of a putative Dof transcription factor gene in rice (Oryza sativa)--rice Dof daily fluctuations 1 (Rdd1). Daily oscillations in Rdd1 expression were retained after transferring to continuous dark (DD) or light (LL) conditions, indicating circadian regulation. However, Rdd1 showed arrhythmic expression in etiolated coleoptiles. Experiments revealed that the Rdd1 transcript accumulated up to 1 h after transferring from DD to LL conditions and decreased thereafter. We examined Rdd1 expression using phytochrome (phy)-deficient mutants, and the results showed that phyA and most likely phyB contributed to the regulation of Rdd1 expression. To further examine the role of Rdd1, transgenic rice plants were produced that carried Rdd1 in either a sense (RDD1-S) or antisense (RDD1-AS) orientation, driven by a constitutive promoter. The expression of endogenous Rdd1 in response to far-red light was found to be modified in RDD1-AS plants compared with wild-type (WT) or RDD1-S plants. In addition, RDD1-AS plants were smaller and flowered later than WT or RDD1-S plants; decreases in grain length, width and 1000-grain weight were also recorded. This study demonstrates that Rdd1 is a circadian clock and phy-regulated gene, which is associated with grain size in rice.

Accumulation of sense-antisense transcripts of the rice catalase gene CatB under dark conditions requires signals from shoots.

The amount of mRNA of the Oryza sativa L., cv. Nipponbare (rice) catalase gene, CatB, was decreased in the roots of intact seedlings kept in continuous darkness (DD). In contrast, sense and antisense unspliced CatB transcripts accumulated in the same tissue. Both strands cover the entire CatB-coding region, and form double-stranded RNA (dsRNA). The results of RNA dot-blot hybridization analysis using low molecular weight RNAs suggested that the sense and antisense CatB transcripts were more stable under DD conditions than under a light-dark regimen (LD). After shifting the lighting conditions from DD to LD, both the sense and antisense CatB transcripts were hardly detected, and the amount of CatB mRNA was restored. From these results, the antisense CatB transcripts might play a role in suppressing the normal processing of sense CatB transcript and also CatB protein synthesis by dsRNA formation, under conditions unsuitable for plant growth such as DD. This study indicates that signals transmitted from shoots are associated with the accumulation of sense and antisense CatB transcripts in roots under DD conditions, and that auxin is one of the possible signals.

Computational analysis suggests that alternative first exons are involved in tissue-specific transcription in rice (Oryza sativa).

MOTIVATION: Transcription start site selection and alternative splicing greatly contribute to diversifying gene expression. Recent studies have revealed the existence of alternative first exons, but most have involved mammalian genes, and as yet the regulation of usage of alternative first exons has not been clarified, especially in plants. RESULTS: We systematically identified putative alternative first exon transcripts in rice, verified the candidates using RT-PCR, and searched for the promoter elements that might regulate the alternative first exons. As a result, we detected a number of unreported alternative first exons, some of which are regulated in a tissue-specific manner. SUPPLEMENTARY INFORMATION: http://www.bioinfo.sfc.keio.ac.jp/research/intron.

Rice Annotation Database (RAD): a contig-oriented database for map-based rice genomics.

A contig-oriented database for annotation of the rice genome has been constructed to facilitate map-based rice genomics. The Rice Annotation Database has the following functional features: (i) extensive effort of manual annotations of P1-derived artificial chromosome/bacterial artificial chromosome clones can be merged at chromosome and contig-level; (ii) concise visualization of the annotation information such as the predicted genes, results of various prediction programs (RiceHMM, Genscan, Genscan+, Fgenesh, GeneMark, etc.), homology to expressed sequence tag, full-length cDNA and protein; (iii) user-friendly clone / gene query system; (iv) download functions for nucleotide, amino acid and coding sequences; (v) analysis of various features of the genome (GC-content, average value, etc.); and (vi) genome-wide homology search (BLAST) of contig- and chromosome-level genome sequence to allow comparative analysis with the genome sequence of other organisms. As of October 2004, the database contains a total of 215 Mb sequence with relevant annotation results including 30 000 manually curated genes. The database can provide the latest information on manual annotation as well as a comprehensive structural analysis of various features of the rice genome. The database can be accessed at http://rad.dna.affrc.go.jp/.

Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both a monocot and dicots.

The rice (Oryza sativa L.) catalase (EC 1.11.1.6) gene CatB is expressed in roots and cultured cells. We examined the promoter activity of its 5'-flanking region in a monocot and in two dicots. Transient expression assays in rice Oc and tobacco BY-2 suspension cell protoplasts showed that CatB's 5'-flanking DNA fragments (nucleotides -1066 to +298) had about 20 and 3-4 times as much promoter activity, respectively, as the CaMV 35S promoter. Serial deletion analyses of the CatB promoter region revealed that the shortest fragment (-56 to +298) still had about 10 times as much promoter activity as the CaMV 35S promoter in rice protoplasts. In tobacco protoplasts, the activity of the fragment (-56 to +298) was about half of the CaMV 35S promoter. Transgenic rice and Arabidopsis plants carrying GUS genes driven by the 5'-truncated CatB promoters were generated and their GUS activity was examined. The region ranging from -329 to +298 showed preferential expression in the roots of rice and Arabidopsis, and in the shoot apical meristems of Arabidopsis. In situ hybridization revealed that CatB was highly expressed in branch root primordia and root apices of rice. Fusion of the GUS gene to the region (-329 to +298) conferred strong expression in these same areas, indicating that the presence of this region was sufficient to express CatB specifically in the roots. There may be new regulatory element(s) in this region, because it contained no previously known cis-regulatory elements specific for gene expression in roots.

Rice Proteome Database based on two-dimensional polyacrylamide gel electrophoresis: its status in 2003.

The Rice Proteome Database is the first detailed database to describe the proteome of rice. The current release contains 21 reference maps based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of proteins from rice tissues and subcellular compartments. These reference maps comprise 11 941 identified proteins showing tissue and subcellular localization, corresponding to 4180 separate protein entries in the database. The Rice Proteome Database contains the calculated properties of each protein such as molecular weight, isoelectric point and expression; experimentally determined properties such as amino acid sequences obtained using protein sequencers and mass spectrometry; and the results of database searches such as sequence homologies. The database is searchable by keyword, accession number, protein name, isoelectric point, molecular weight and amino acid sequence, or by selection of a spot on one of the 2D-PAGE reference maps. Cross-references are provided to tools for proteomics and to other 2D-PAGE databases, which in turn provide many links to other molecular databases. The information in the Rice Proteome Database is updated weekly, and is available on the World Wide Web at http://gene64.dna.affrc.go.jp/RPD/.

A novel feature of microsatellites in plants: a distribution gradient along the direction of transcription.

A computer-based analysis was conducted to assess the characteristics of microsatellites in transcribed regions of rice and Arabidopsis. In addition, two mammals were simultaneously analyzed for a comparative analysis. Our analyses confirmed a novel plant-specific feature in which there is a gradient in microsatellite density along the direction of transcription. It was also confirmed that pyrimidine-rich microsatellites are found intensively near the transcription start sites, specifically in the two plants, but not in the mammals. Our results suggest that microsatellites located at high frequency in the 5'-flanking regions of plant genes can potentially act as factors in regulating gene expression.

RiceGAAS: an automated annotation system and database for rice genome sequence.

An extensive effort of the International Rice Genome Sequencing Project (IRGSP) has resulted in rapid accumulation of genome sequence, and >137 Mb has already been made available to the public domain as of August 2001. This requires a high-throughput annotation scheme to extract biologically useful and timely information from the sequence data on a regular basis. A new automated annotation system and database called Rice Genome Automated Annotation System (RiceGAAS) has been developed to execute a reliable and up-to-date analysis of the genome sequence as well as to store and retrieve the results of annotation. The system has the following functional features: (i) collection of rice genome sequences from GenBank; (ii) execution of gene prediction and homology search programs; (iii) integration of results from various analyses and automatic interpretation of coding regions; (iv) re-execution of analysis, integration and automatic interpretation with the latest entries in reference databases; (v) integrated visualization of the stored data using web-based graphical view. RiceGAAS also has a data submission mechanism that allows public users to perform fully automated annotation of their own sequences. The system can be accessed at http://RiceGAAS.dna.affrc.go.jp/.