Characterisation of a prokaryote-type tRNA-isopentenyltransferase gene from the moss Physcomitrella patens

Cytokinins are of critical importance to numerous developmental processes in plants. Two cytokinin biosynthetic pathways have been described; each one uses a different type of isopentenyltransferases (IPTs) as the key enzyme. In the first pathway, adenylate-IPTs (EC 2.5.1.27) prenylate adenylic nucleotides to cytokinin nucleotides, thus catalysing the direct de novo biosynthesis of free cytokinins. In the second pathway, tRNA-IPTs (EC 2.5.1.8) catalyse cytokinin formation by isopentenylation of tRNA, the degradation of which liberates cytokinin nucleotides. Seed plants have been shown to possess both forms of IPTs. Here, we report on the in-silico based identification and on the functional characterisation of an IPT encoding gene (PpIPT1) from the bryophyte Physcomitrellapatens. Analysis of the PpIPT1 amino acid sequence revealed high similarities to tRNA-IPTs of other plants. No adenylate-IPT genes were found in the Physcomitrella sequenced transcriptome/genome. PpIPT1 functionally complemented a defective tRNA-IPT gene of Saccharomyces cerevisiae (ScMOD5) in the strain MT-8. Dephosphorylated tRNA hydrolysates from PpIPT1-transformed MT-8 showed cytokinin activity in a moss bioassay and the presence of isopentenyladenosine in HPLC analysis, in contrast to those prepared from untransformed MT-8. A comparison of pro- and eukaryotic homologues revealed two classes of tRNA-IPTs; PpIPT1 belongs to a prokaryotic type with predicted chloroplast targeting. RT-PCR experiments revealed a stronger expression in the cytokinin overproducing mutant oveST25, thus indicating the potential role of PpIPT1 for cytokinin biosynthesis in the evolutionary old land plant Physcomitrella.