Hiroyuki Ichida

Identification of plant promoter constituents by analysis of local distribution of short sequences

BackgroundPlant promoter architecture is important for understanding regulation and evolution of the promoters, but our current knowledge about plant promoter structure, especially with respect to the core promoter, is insufficient. Several promoter elements including TATA box, and several types of transcriptional regulatory elements have been found to show local distribution within promoters, and this feature has been successfully utilized for extraction of promoter constituents from human genome.ResultsLDSS (Local Distribution of Short Sequences) profiles of short sequences along the plant promoter have been analyzed in silico, and hundreds of hexamer and octamer sequences have been identified as having localized distributions within promoters of Arabidopsis thaliana and rice. Based on their localization patterns, the identified sequences could be classified into three groups, pyrimidine patch (Y Patch), TATA box, and REG (Regulatory Element Group). Sequences of the TATA box group are consistent with the ones reported in previous studies. The REG group includes more than 200 sequences, and half of them correspond to known cis-elements. The other REG subgroups, together with about a hundred uncategorized sequences, are suggested to be novel cis-regulatory elements. Comparison of LDSS-positive sequences between Arabidopsis and rice has revealed moderate conservation of elements and common promoter architecture. In addition, a dimer motif named the YR Rule (C/T A/G) has been identified at the transcription start site (-1/+1). This rule also fits both Arabidopsis and rice promoters.ConclusionLDSS was successfully applied to plant genomes and hundreds of putative promoter elements have been extracted as LDSS-positive octamers. Identified promoter architecture of monocot and dicot are well conserved, but there are moderate variations in the utilized sequences.

DNA adenine methylation changes dramatically during establishment of symbiosis

The DNA adenine methylation status on specific 5′‐GANTC‐3′ sites and its change during the establishment of plant–microbe interactions was demonstrated in several species of α‐proteobacteria. Restriction landmark genome scanning (RLGS), which is a high‐resolution two dimensional DNA electrophoresis method, was used to monitor the genomewide change in methylation. In the case of Mesorhizobium loti MAFF303099, real RLGS images obtained with the restriction enzyme MboI, which digests at GATC sites, almost perfectly matched the virtual RLGS images generated based on genome sequences. However, only a few spots were observed when the restriction enzyme HinfI was used, suggesting that most GANTC (HinfI) sites were tightly methylated and specific sites were unmethylated. DNA gel blot analysis with the cloned specifically unmethylated regions (SUMs) showed that some SUMs were methylated differentially in bacteroids compared to free‐living bacteria. SUMs have also been identified in other symbiotic and parasitic bacteria. These results suggest that DNA adenine methylation may contribute to the establishment and/or maintenance of symbiotic and parasitic relationships.

Heavy-ion beam mutagenesis identified an essential gene for chloroplast development under cold stress conditions during both early growth and tillering stages in rice

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice. Graphical Abstract We isolated and characterized the rice virescent mutant csv1. The mutant showed low temperature sensitivity not only early growth stages but also the tillering stage.

Morphological and physiological differences among cultivation lines of Undaria pinnatifida in a common garden experiment using a tank culture system

Undaria pinnatifida is grown for food and industrial materials worldwide; therefore, advanced breeding is needed to meet quality and productivity requirements. In this study, we examined regional lines of U. pinnatifida from five cultivation sites in Japan with different environmental conditions: Oga (OGA, the northern Sea of Japan coast), Hirota Bay (HRT, the northeastern Pacific coast), Matsushima Bay (MAT, the northeastern Pacific coast), Naruto (the Seto Inland Sea coast) and Shimonoseki (SIM, the southern Sea of Japan coast). The sporophytes of these lines were cultured in a tank culture system under controlled environmental conditions, and their morphological characteristics, nutrient uptake kinetics (Vmax, Ks and Vmax/Ks), and carbon, nitrogen and phosphorus contents were determined. Sporophytes from MAT grew faster, whereas those from SIM were smaller than those from the other sites. Although the blade thickness of sporophytes cultivated in the sea significantly differs among cultivation sites in the previous study, there was no significant difference in blade thickness among the regional lines cultivated in the tank. Sporophytes from OGA had the greatest Vmax/Ks values and significantly greater nitrogen contents than the other lines. Therefore, the morphological characteristics of MAT and SIM sporophytes, and the nutrient uptake kinetics of OGA sporophytes may have a genetic origin. This indicates that these lines may represent useful resources for selective breeding, with MAT sporophytes providing faster growth and OGA sporophytes being well-adapted to low-nutrient conditions.

Prediction of bipartite transcriptional regulatory elements using transcriptome data of Arabidopsis

Abstract In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of ‘4 bases—a spacer with a flexible length—4 bases’. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein–DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly.