Yukihisa Shimada

Uniconazole, a cytochrome P450 inhibitor, inhibits trans-zeatin biosynthesis in Arabidopsis.

Cytokinin (CK) is a plant hormone that plays important regulatory roles in many aspects of plant growth and development. Although functions of CK and its biosynthesis pathway have been studied extensively, there is still no efficient biosynthesis inhibitor, which would be useful for studying CK from a chemical genetic approach. Here, CK biosynthesis inhibitor candidates were searched for using a systematic approach. In silico screening of candidates were carried out using genome-wide gene expression profiles and prediction of target sites using global CK accumulation profile analysis. As a result of these screenings, it was found that uniconazole, a well known inhibitor of cytochrome P450 monooxygenase, prevents the biosynthesis of trans-zeatin, and that its target is CYP735As in Arabidopsis.

Arabidopsis Seedlings Over-Accumulated Indole-3-acetic Acid in Response to Aminooxyacetic Acid

Previously we identified aminooxy compounds as auxin biosynthesis inhibitors. One of the compounds, aminooxyacetic acid (AOA) inhibited indole-3-acetic acid (IAA) biosynthesis in rice and tomato. Here, we found that AOA induced auxin over-accumulation in Arabidopsis. The results suggest that auxin-related metabolic pathways are divergent among these plant species.

Screening of cDNA Clones for Plastid-targeted Proteins

We present a method to isolate cDNA clones that encode plastid-targeted proteins other than those encoding proteins of photosynthetic machinery. As an example, we have isolated clones for dark-inducible chloroplast proteins. Poly(A)+ RNA was isolated from 15-day-old radish cotyledons that had been kept in darkness for 24 h, and was used to prepare a library with the plasmid vector pBluescript. It was divided into groups of 8 or 12 clones, which were separately expressed in vitro in the presence of [35S]methionine. The labeled polypeptides were then incubated with isolated chloroplasts to see if they are imported by the chloroplasts. The group that showed positive signals after SDS-PAGE of the lysed chloroplasts were further analyzed for individual clones in the same manner. Thus, we were successful in isolating cDNA clones encoding chloroplast-targeted proteins that were expressed in radish cotyledons specifically in the dark. The method can be used to identify nuclear genes for nonphotosynthetic plastids, if an appropriate cDNA library is available.