Hisabumi Takase

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

Changes in the Bacterial Community of Soybean Rhizospheres during Growth in the Field

Highly diverse communities of bacteria inhabiting soybean rhizospheres play pivotal roles in plant growth and crop production; however, little is known about the changes that occur in these communities during growth. We used both culture-dependent physiological profiling and culture independent DNA-based approaches to characterize the bacterial communities of the soybean rhizosphere during growth in the field. The physiological properties of the bacterial communities were analyzed by a community-level substrate utilization assay with BioLog Eco plates, and the composition of the communities was assessed by gene pyrosequencing. Higher metabolic capabilities were found in rhizosphere soil than in bulk soil during all stages of the BioLog assay. Pyrosequencing analysis revealed that differences between the bacterial communities of rhizosphere and bulk soils at the phylum level; i.e., Proteobacteria were increased, while Acidobacteria and Firmicutes were decreased in rhizosphere soil during growth. Analysis of operational taxonomic units showed that the bacterial communities of the rhizosphere changed significantly during growth, with a higher abundance of potential plant growth promoting rhizobacteria, including Bacillus, Bradyrhizobium, and Rhizobium, in a stage-specific manner. These findings demonstrated that rhizosphere bacterial communities were changed during soybean growth in the field.

Multiplicity of the DNA-binding protein HBP-1 specific to the conserved hexameric sequence ACGTCA in various plant gene promoters

Multiplicity; DNA‐binding protein; Hexameric sequence; DNA‐protein interaction

DNA-binding protein(s) interacts with a conserved nonameric sequence in the upstream regions of wheat histone genes

A nuclear protein(s), HBP‐2, that binds to the upstream region of the wheat histone H4 gene was identified from a fractionated nuclear extract of wheat germ by DNase I footprinting. The DNase I‐protected region contained the conserved nonameric motif, CATCCAACG. Cross‐competition experiments that used the mobility shift assay showed that this nuclear protein(s) binds specifically to the upstream sequence that has been postulated to be a cis element of the wheat H3 gene. Our findings suggest that this DNA‐binding protein(s) may be a trans‐acting factor in the regulation of the transcription of wheat histone genes.

A Phosphofructokinase B-Type Carbohydrate Kinase Family Protein, NARA5, for Massive Expressions of Plastid-Encoded Photosynthetic Genes in Arabidopsis

To date, there have been no reports on screening for mutants defective in the massive accumulation of Rubisco in higher plants. Here, we describe a screening method based on the toxic accumulation of ammonia in the presence of methionine sulfoximine, a specific inhibitor of glutamine synthetase, during photorespiration initiated by the oxygenase reaction of Rubisco in Arabidopsis (Arabidopsis thaliana). Five recessive mutants with decreased amounts of Rubisco were identified and designated as nara mutants, as they contained a mutation in genes necessary for the achievement of Rubisco accumulation. The nara5-1 mutant showed markedly lower levels of plastid-encoded photosynthetic proteins, including Rubisco. Map-based cloning revealed that NARA5 encoded a chloroplast phosphofructokinase B-type carbohydrate kinase family protein of unknown function. The NARA5 protein fused to green fluorescent protein localized in chloroplasts. We conducted expression analyses of photosynthetic genes during light-induced greening of etiolated seedlings of nara5-1 and the T-DNA insertion mutant, nara5-2. Our results strongly suggest that NARA5 is indispensable for hyperexpression of photosynthetic genes encoded in the plastid genome, particularly rbcL.

Sequence-specific single-strand DNA-binding proteins that interact with the regulatory regions of wheat histone H3 and H4 genes

We identified two novel DNA-binding proteins, ssDBP-1 and ssDBP-2, in wheat germ nuclear extract that interact with the proximal sequences of the promoter regions of the wheat histone H3 and H4 genes. Mobility shift and methylation interference assays have demonstrated that these factors specifically bind to the single-strand DNA which partially overlaps the hexamer and octamer cis-elements of the H3 promoter. Both proteins are distinguishable from HBP-1a and HBP-1b which specifically bind to the H3 hexamer sequence. These ssDNA-binding proteins are supposed to regulate the transcription of the wheat histone genes.

Pyrosequencing assessment of rhizosphere fungal communities from a soybean field

Soil fungal communities play essential roles in soil ecosystems, affecting plant growth and health. Rhizosphere bacterial communities have been shown to undergo dynamic changes during plant growth. This study utilized 454 pyrosequencing to analyze rhizosphere fungal communities during soybean growth. Members of the Ascomycota and Basiodiomycota dominated in all soils. There were no statistically significant changes at the phylum level among growth stages or between bulk and rhizosphere soils. In contrast, the relative abundance of small numbers of operational taxonomic units, 4 during growth and 28 between bulk and rhizosphere soils, differed significantly. Clustering analysis revealed that rhizosphere fungal communities were different from bulk fungal communities during growth stages of soybeans. Taken together, these results suggest that in contrast to rhizosphere bacterial communities, most constituents of rhizosphere fungal communities remained stable during soybean growth.

Do soybeans select specific species of Bradyrhizobium during growth

Soybean is an important crop, with processed soybeans being the second largest source of vegetable oil and the largest source of animal protein feed in the world. Nodules on soybean roots are responsible for symbiotic nitrogen fixation, enabling soybean plants to obtain sufficient nitrogen for growth and seed production. Because nitrogen is an essential, but often limiting, nutrient for plant growth, improvements in nitrogen fixation are highly required in agriculture. We recently reported a comprehensive analysis of rhizosphere bacterial communities during soybean growth in a field in Kyoto prefecture, Japan. The bacterial communities of the rhizosphere changed significantly during growth, with potential plant growth-promoting rhizobacteria, including Bacillus, Bradyrhizobium, and Rhizobium, increasing in a stage-specific manner. In this addendum, we focus on changes in Bradyrhizobium during soybean growth, suggesting that soybean plants select for symbiotic partners.

Introduction of a 50 kbp DNA Fragment into the Plastid Genome

Plastid transformation technology has been used for the analysis and improvement of plastid metabolism. To create a transplastomic plant with a complicated and massive metabolic pathway, it is necessary to introduce a large amount of DNA into the plastid. However, to our knowledge, the largest DNA fragment introduced into a plastid genome was only 7 kbp long and consisted of just three genes. Here we report the introduction of foreign DNA of 23–50 kbp into the tobacco plastid genome with a bacterial artificial chromosome (BAC)-based plastid transformation vector. It was confirmed that the introduced DNA was passed on to the next generation. This is the first description of plastid transformation with a large amount of foreign DNA.

Dmc1 fluorescent foci in prophase I nuclei of diploid, triploid and hybrid lilies

Abstract. We examined the distribution of meiotic epitopes for the Dmc1 protein of lilies in a normal diploid, a triploid, and in a diploid species-hybrid. The triploid has an extra chromosome set; all three sets align, but only two of the three axes intimately pair at a given location. Our findings with the triploid support the idea that retention of the foci until the pachytene stage requires a successful homology check and synaptonemal complex (SC) initiation; the number of foci in the triploid diminishes by approximately 30% from early zygotene to pachytene, and the triploid pachytene values are similar to the pachytene values of the diploid. The species-hybrid lacks chromosome homology, has reduced SC formation and few reciprocal genetic exchanges. In this species-hybrid the number of foci at early zygotene is similar to that in the normal diploid but is dramatically reduced by mid-zygotene. The extent to which the number of Dmc1 foci is reduced is similar to the extent that SC formation is reduced. In contrast the extent of the reduction in reciprocal genetic exchange in the species-hybrid is much greater than the reduction in the number of foci. We conclude that Dmc1 protein is involved in homology checking, but the impact of failure to find homology affects SC formation and reciprocal genetic exchange differentially.