Ryo Akashi

Sequencing and analysis of approximately 40,000 soybean cDNA clones from a full-length-enriched cDNA library.

A large collection of full-length cDNAs is essential for the correct annotation of genomic sequences and for the functional analysis of genes and their products. We obtained a total of 39 936 soybean cDNA clones (GMFL01 and GMFL02 clone sets) in a full-length-enriched cDNA library which was constructed from soybean plants that were grown under various developmental and environmental conditions. Sequencing from 5′ and 3′ ends of the clones generated 68 661 expressed sequence tags (ESTs). The EST sequences were clustered into 22 674 scaffolds involving 2580 full-length sequences. In addition, we sequenced 4712 full-length cDNAs. After removing overlaps, we obtained 6570 new full-length sequences of soybean cDNAs so far. Our data indicated that 87.7% of the soybean cDNA clones contain complete coding sequences in addition to 5′- and 3′-untranslated regions. All of the obtained data confirmed that our collection of soybean full-length cDNAs covers a wide variety of genes. Comparative analysis between the derived sequences from soybean and Arabidopsis, rice or other legumes data revealed that some specific genes were involved in our collection and a large part of them could be annotated to unknown functions. A large set of soybean full-length cDNA clones reported in this study will serve as a useful resource for gene discovery from soybean and will also aid a precise annotation of the soybean genome.

Discovery of natural Miscanthus (Poaceae) triploid plants in sympatric populations of Miscanthus sacchariflorus and Miscanthus sinensis in southern Japan

PREMISE OF THE STUDY Looming petroleum shortages and projected negative impacts of human-induced climate change may be partly alleviated by the development and use of bioenergy feedstock crops. Miscanthus ×giganteus, a highly productive sterile triploid hybrid grass that was discovered in Japan several decades ago, has considerable potential as an alternative source of energy. Given the risks, however, involved in the reliance upon production of one clone of this hybrid, which is a natural cross between Miscanthus sacchariflorus and Miscanthus sinensis, for lignocellulosic bioenergy production, natural occurrences of triploidy were investigated in sympatric populations of tetraploid M. sacchariflorus and diploid M. sinensis in Japan. METHODS Seeds were counted and DNA content was estimated by flow cytometry for plants of M. sacchariflorus and M. sinensis in several sympatric populations throughout Japan. Chromosomes were also counted for select plants. KEY RESULTS Based on seed-set data, M. sacchariflorus has significantly lower seed set than M. sinensis in Japan. Putative triploid seeds were found on M. sacchariflorus plants in southern Japan. CONCLUSIONS This is the first report of the natural occurrence of Miscanthus triploid plants in several decades. If found to be sterile and similar in productivity to the commonly cultivated clone of M. ×giganteus, these triploid plants might serve as additional sources of genetic variation for bioenergy production. Seed set data also indicates that other triploid plants might be found in more northern regions of Japan.

Lotus japonicus nodulation is photomorphogenetically controlled by sensing the red/far red (R/FR) ratio through jasmonic acid (JA) signaling

Light is critical for supplying carbon to the energetically expensive, nitrogen-fixing symbiosis between legumes and rhizobia. Here, we show that phytochrome B (phyB) is part of the monitoring system to detect suboptimal light conditions, which normally suppress Lotus japonicus nodule development after Mesorhizobium loti inoculation. We found that the number of nodules produced by L. japonicus phyB mutants is significantly reduced compared with the number produced of WT Miyakojima MG20. To explore causes other than photoassimilate production, the possibility that local control by the root genotype occurred was investigated by grafting experiments. The results showed that the shoot and not the root genotype is responsible for root nodule formation. To explore systemic control mechanisms exclusive of photoassimilation, we moved WT MG20 plants from white light to conditions that differed in their ratios of low or high red/far red (R/FR) light. In low R/FR light, the number of MG20 root nodules dramatically decreased compared with plants grown in high R/FR, although photoassimilate content was higher for plants grown under low R/FR. Also, the expression of jasmonic acid (JA) -responsive genes decreased in both low R/FR light-grown WT and white light-grown phyB mutant plants, and it correlated with decreased jasmonoyl-isoleucine content in the phyB mutant. Moreover, both infection thread formation and root nodule formation were positively influenced by JA treatment of WT plants grown in low R/FR light and white light-grown phyB mutants. Together, these results indicate that root nodule formation is photomorphogenetically controlled by sensing the R/FR ratio through JA signaling.

Enhanced Nodulation and Nitrogen Fixation in the Abscisic Acid Low-Sensitive Mutant enhanced nitrogen fixation1 of Lotus japonicus

The phytohormone abscisic acid (ABA) is known to be a negative regulator of legume root nodule formation. By screening Lotus japonicus seedlings for survival on an agar medium containing 70 μm ABA, we obtained mutants that not only showed increased root nodule number but also enhanced nitrogen fixation. The mutant was designated enhanced nitrogen fixation1 (enf1) and was confirmed to be monogenic and incompletely dominant. The low sensitivity to ABA phenotype was thought to result from either a decrease in the concentration of the plants endogenous ABA or from a disruption in ABA signaling. We determined that the endogenous ABA concentration of enf1 was lower than that of wild-type seedlings, and furthermore, when wild-type plants were treated with abamine, a specific inhibitor of 9-cis-epoxycarotenoid dioxygenase, which results in reduced ABA content, the nitrogen fixation activity of abamine-treated plants was elevated to the same levels as enf1. We also determined that production of nitric oxide in enf1 nodules was decreased. We conclude that endogenous ABA concentration not only regulates nodulation but also nitrogen fixation activity by decreasing nitric oxide production in nodules.

NBRP databases: databases of biological resources in Japan.

The National BioResource Project (NBRP) is a Japanese project that aims to establish a system for collecting, preserving and providing bioresources for use as experimental materials for life science research. It is promoted by 27 core resource facilities, each concerned with a particular group of organisms, and by one information center. The NBRP database is a product of this project. Thirty databases and an integrated database-retrieval system (BioResource World: BRW) have been created and made available through the NBRP home page (http://www.nbrp.jp). The 30 independent databases have individual features which directly reflect the data maintained by each resource facility. The BRW is designed for users who need to search across several resources without moving from one database to another. BRW provides access to a collection of 4.5-million records on bioresources including wild species, inbred lines, mutants, genetically engineered lines, DNA clones and so on. BRW supports summary browsing, keyword searching, and searching by DNA sequences or gene ontology. The results of searches provide links to online requests for distribution of research materials. A circulation system allows users to submit details of papers published on research conducted using NBRP resources.

Quantitative trait locus analysis of multiple agronomic traits in the model legume Lotus japonicus

The first quantitative trait locus (QTL) analysis of multiple agronomic traits in the model legume Lotus japonicus was performed with a population of recombinant inbred lines derived from Miyakojima MG-20 x Gifu B-129. Thirteen agronomic traits were evaluated in 2004 and 2005: traits of vegetative parts (plant height, stem thickness, leaf length, leaf width, plant regrowth, plant shape, and stem color), flowering traits (flowering time and degree), and pod and seed traits (pod length, pod width, seeds per pod, and seed mass). A total of 40 QTLs were detected that explained 5%-69% of total variation. The QTL that explained the most variation was that for stem color, which was detected in the same region of chromosome 2 in both years. Some QTLs were colocated, especially those for pod and seed traits. Seed mass QTLs were located at 5 locations that mapped to the corresponding genomic positions of equivalent QTLs in soybean, pea, chickpea, and mung bean. This study provides fundamental information for breeding of agronomically important legume crops.

Identification and Functional Analysis of Type III Effector Proteins in Mesorhizobium loti

Mesorhizobium loti MAFF303099, a microsymbiont of the model legume Lotus japonicus, possesses a cluster of genes (tts) that encode a type III secretion system (T3SS). In the presence of heterologous nodD from Rhizobium leguminosarum and a flavonoid naringenin, we observed elevated expression of the tts genes and secretion of several proteins into the culture medium. Inoculation experiments with wild-type and T3SS mutant strains revealed that the presence of the T3SS affected nodulation at a species level within the Lotus genus either positively (L. corniculatus subsp. frondosus and L. filicaulis) or negatively (L. halophilus and two other species). By inoculating L. halophilus with mutants of various type III effector candidate genes, we identified open reading frame mlr6361 as a major determinant of the nodulation restriction observed for L. halophilus. The predicted gene product of mlr6361 is a protein of 3,056 amino acids containing 15 repetitions of a sequence motif of 40 to 45 residues and a shikimate kinase-like domain at its carboxyl terminus. Homologues with similar repeat sequences are present in the hypersensitive-response and pathogenicity regions of several plant pathogens, including strains of Pseudomonas syringae, Ralstonia solanacearum, and Xanthomonas species. These results suggest that L. halophilus recognizes Mlr6361 as potentially pathogen derived and subsequently halts the infection process.

Somatic embryogenesis and plant regeneration from cultured immature inflorescences of apomictic dallisgrass (Paspalum dilatatum Poir.)

Abstract Plant regeneration from cultured immature inflorescences of Paspalum dilatatum was obtained by somatic embryogenesis. Embryonic callus was initiated from immature inflorescences (5–10 mm length) on Murashige and Skoog (MS) medium supplemented with 2.0–10.0 mg·l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and solidified with 0.2% (w/v) Gellan Gum. Somatic embryos could be induced at all concentrations of 2,4-D, but the maximal frequency of explants which produced somatic embryos occurred on medium containing 10.0 mg·l−1 2,4-D. Somatic embryos developed and germinated precociously when embryogenic calli were transferred to a medium containing 1.0 mg·l−1 each of kinetin and gibberellic acid (GA3). All regenerants were successfully grown to maturity. The embryogenic calli maintained their embryogenic capacity on Murashige and Skoog (MS) medium with 1.0 mg·l−1 2,4-D for more than 1 year.

Plant regeneration from seed-derived embryogenic callus and cell suspension cultures of bahiagrass (Paspalum notatum)

Abstract We have established a high-frequency plant regeneration system via somatic embryogenesis from seed-derived callus and cell suspension cultures in 6 genotypes of bahiagrass ( Paspalum notatum ). Embryogenic callus was initiated from mature seeds on Murashige and Skoog (MS) medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 3.0% sucrose and 0.3% Gellan Gum in the dark. Culture response was found to be correlated with genotype. ‘Pensacola’ had the best response in embryogenic callus formation, and 74% of the calli regenerated plants. Cell suspension cultures were established from embryogenic callus of ‘Pensacola’ in modified N 6 medium containing 1.0 mg/l 2,4-D. The suspension was composed of compact cell clusters. When smaller clusters (approx. 2.0–3.3 mm in diameter) were transferred to a solid MS medium with 3.0% sucrose but without hormones, plant regeneration was initiated at high frequency (28.6%). Morphological evidence is provided that regeneration of suspension cells occurred via embryogenesis. Regenerated plants were established to soil after culture in water at room temperature for 14 days for acclimatization.

Plant regeneration from suspension cultured-derived protoplasts of apomictic dallisgrass (Paspalum dilatatum Poir.)

Abstract Protoplasts were isolated from embryogenic suspension cells of apomictic dallisgrass (Paspalum dilatatum Poir.). The respective suspension cultures were initiated from immature inflorescence-derived embryogenic callus. Previous to protoplast isolation, suspension cells were treated with Murashige and Skoog (MS) liquid medium without sucrose and hormones. Due to this pretreatment protoplast yield and viability were dramatically increased. A maximum protoplast yield of 4–6 × 106· −1 fresh weight was obtained. Cell division and colony formation from pretreated protoplasts were found to be best in an agarose solidified KM8p medium at a density of 5–8 × 105· ml−1. The plating efficiency, based on colony formation after 2 weeks of culture, was 0.5–0.8%. Protoplast-derived colonies were transferred to a solidified MS medium containing 1.0 mg · 1−1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus proliferation. The calli formed embryonic structures which gave rise to green plants in 0.2% (w/v) Gellan Gum solidified MS medium with 1.0 mg·l−1 naphthaleneacetic acid (NAA) and 0.2 mg·l−1 benzylaminopurine (BAP). The regenerated plantlets were transferred to 1 2 MS hormone-free medium for further growth and root formation. Rooted plants could be transferred to soil.