Abstract
To study cytokinin biosynthesis, we characterized a temperature-sensitive cytokinin-overproducing mutant, oveST25, of the moss Physcomitrella patens with respect to changes in cytokinin content during thermal induction in comparison to wild type. Our findings, based on combined liquid chromatography-mass spectrometry (LC-MS) analyses, show that thermoinduction caused a strong increase of extracellular N 6-(Δ2-isopentenyl)adenine (iP), N 6-(Δ2-isopentenyl)adenosine (iPR), cis-zeatin (cZ), cis-zeatin riboside (cZR) and its O-glucoside cZROG in oveST25. In contrast, no significant changes were measured in the wild type. To investigate the relevance of tRNA for cytokinin production in Physcomitrella, we determined cytokinins in tissue and culture medium as well as in tRNA hydrolysates. The analysis of cytokinins from whole-culture extracts of wild type revealed 56% of iP-type, 32% of cZ-type, and 11% of trans-zeatin (tZ)-type forms. In tRNA, 90% of cytokinins were represented by cZ-type and 8% by iP-type forms; tZ-type cytokinins were found only in trace amounts. The finding that the major free cytokinins are, albeit with altered proportions, also major forms in tRNA is compatible with the hypothesis of a strong tRNA-mediated biogenesis of cytokinins in this plant. Our RT-PCR-based studies on the expression of the tRNA-IPT gene, PpIPT1, revealed enhanced transcription levels in the cytokinin-overproducing oveST25 mutant at the inducing temperature of 25°C, but not at noninducing conditions (15°C). A wild-type transgenic line with cytokinin deficiency due to heterologous cytokinin oxidase/dehydrogenase overexpression (AtCKX2) also exhibited enhanced PpIPT1 expression levels, indicating that cytokinin deficiency might upregulate tRNA-mediated cytokinin biosynthesis. The evidence that the tRNA-mediated pathway might be mainly responsible for biosynthesis of isoprenoid cytokinins in Physcomitrella is strongly supported by the recent release of the Physcomitrella genomic sequence where only tRNA-IPTs but no adenylate-IPTs are present.
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