Biomedical subjects
Daisuke Shibata
Publications and source records attributed to Daisuke Shibata.
Analysis of gene expression in Arabidopsis thaliana by array hybridization with genomic DNA fragments aligned along chromosomal regions.
The availability of the entire genomic sequence of the higher plant Arabidopsis thaliana prompted an analysis of chromosomal regions for gene expression with the use of high-density DNA array filters spotted with genomic DNA fragments (genomic DNA array analysis). One TAC and nine P1 clones, each of which contains a genomic DNA insert of approximately 80 kb and was used for sequencing of chromosome 5, were arbitrarily selected for analysis. The total size of the genomic regions corresponding to these clones is 819 kb. A total of 339 DNA fragments (average size, 2.9 kb) that cover contiguously the 10 chromosomal regions was selected and spotted onto nylon filters. The filters were then subjected to hybridization with (33)P-labelled cDNA molecules that had been synthesized from polyadenylated RNA isolated from 3-week-old plants. Quantitative and reproducible measurement of hybridization signals allowed analysis of the transcription of all genes in the targeted regions that were expressed at a level above the limit of detection. The data revealed that the analysed chromosomal regions are rich in active genes, and that they also provided a basis for the identification of novel transcripts whose sequences are not represented in the expressed sequence tag (EST) database.
Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene.
Autophagy is an intracellular process for vacuolar bulk degradation of cytoplasmic components. The molecular machinery responsible for yeast and mammalian autophagy has recently begun to be elucidated at the cellular level, but the role that autophagy plays at the organismal level has yet to be determined. In this study, a genome-wide search revealed significant conservation between yeast and plant autophagy genes. Twenty-five plant genes that are homologous to 12 yeast genes essential for autophagy were discovered. We identified an Arabidopsis mutant carrying a T-DNA insertion within AtAPG9, which is the only ortholog of yeast Apg9 in Arabidopsis (atapg9-1). AtAPG9 is transcribed in every wild-type organ tested but not in the atapg9-1 mutant. Under nitrogen or carbon-starvation conditions, chlorosis was observed earlier in atapg9-1 cotyledons and rosette leaves compared with wild-type plants. Furthermore, atapg9-1 exhibited a reduction in seed set when nitrogen starved. Even under nutrient growth conditions, bolting and natural leaf senescence were accelerated in atapg9-1 plants. Senescence-associated genes SEN1 and YSL4 were up-regulated in atapg9-1 before induction of senescence, unlike in wild type. All of these phenotypes were complemented by the expression of wild-type AtAPG9 in atapg9-1 plants. These results imply that autophagy is required for maintenance of the cellular viability under nutrient-limited conditions and for efficient nutrient use as a whole plant.