Effects of exogenous phytohormones on plastid tRNA modifications in ragi coleoptiles

The effects of phytohormones on plastid tRNA modifications were investigated in ragi (Eleucine coracana) coleoptiles. Intact 7-day old dark-grown ragi seedlings were given phytohormone, indoleacetic acid (IAA) or isopentenyladenine (i6A) treatment and grown in the dark or under white fluorescent light; coleoptiles were harvested 24 hr following treatment, and plastid total tRNAs were isolated and analyzed for their content of modified nucleotides. A total of 14 modified nucleotides were identified in the total digests of ragi plastid total tRNA preparations; significant increases in the content of some modified nucleotides were observed following treatment of phytohormones in the dark and light. The relative amounts of pT, pm1G, pm7G and pm1A in IAA-treated dark-grown, pi6A, pm2G and pCm in IAA-treated light-grown, and pT and pm2G in i6A-treated light-grown ragi coleoptiles were 2 to 10 times higher than the untreated control coleoptile plastid total tRNA. In order to gain a better understanding of the effects of phytohormones on ragi plastid tRNA modifications, we purified plastid tRNA(Ile)(GAU) from coleoptiles of the aforementioned ragi seedlings and analyzed its modifed nucleotide content. We find that the content of pGm was 4 to 5 times higher in the tRNA(Ile)(GAU) purified from i6A- or IAA-treated dark-grown coleoptiles, and pm7G was 5 to 6 times higher in the tRNA(Ile)(GAU) of i6A-treated light-grown ragi coleoptiles. These results suggest that the synthesis or activity of some plastid-specific tRNA-modifying enzymes may be enhanced by i6A and IAA with two different modes of regulation, one operating in the light and the other operating in the dark.