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Molecular and biochemical evidence for tRNA-dependent cytokinin biosynthesis in the ancient land plant Physcomitrella patens (Hedw.) B.S.G.
Molekulare und biochemische Beweise für die tRNA-abhängige Biosynthese von Cytokininen in der frühen Landpflanze Physcomitrella patens (Hedw.) B.S.G.
Yevdakova, Natalya A.
(2007) Natalya A. Yevdakova, Klaus von Schwartzenberg (2007) CHARACTERIISATION OF A PROKARYOTE-TYPE tRNA-ISOPENTENYLTRANSFERASE GENE FROM THE MOSS PYSCOMITRELLA PATENS. Planta 226: 683-695.
Dokument 1.pdf (2.857 KB)
Freie Schlagwörter (Deutsch):
Biosynthese von Cytokininen , tRNA , Physcomitrella patens
Freie Schlagwörter (Englisch):
cytokinin biosynthesis , tRNA , Physcomitrella patens
42.41 , 42.13 , 42.21 , 35.70 , 42.52
Lorbiecke, Rene (PD Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
Cytokinins are of critical importance to numerous developmental processes in plants. There have been described two cytokinin biosynthetic pathways, each one using a different type of isopentenyltransferases (IPTs) as a key enzyme. In the first pathway, adenylate-IPTs (EC 188.8.131.52) prenylate adenylic nucleotides to cytokinin nucleotides thus catalysing the direct de novo biosynthesis of free cytokinins. In the second pathway, tRNA-IPTs (EC 184.108.40.206) 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.
In the thesis I report on the in silico based identification and on the functional characterisation of an IPT encoding gene (PpIPT1) from the bryophyte Physcomitrella patens (Hedw.) B.S.G.. Analysis of the PpIPT1 amino acid sequence revealed high similarities to tRNA-IPTs of other plants. No adenylate-IPT genes were found in the sequenced Physcomitrella 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.
Physcomitrella is known as an especially useful model system for the research of cytokinin biosynthesis due to its cytokinin overproducing mutants. I have characterised a temperature sensitive ove mutant, oveST25, for changes in its cytokinin content during the thermal induction. Cytokinins were determined in tissue and culture medium as well as in tRNA-hydrolysates by combined liquid chromatography-mass spectrometry (LC-MS).
Not depending on temperature conditions cis-zeatin riboside-O-glucoside (cZROG) was found to be the predominant form in the tissue of oveST25 and wild type; in culture medium cis-zeatin-O-glucoside (cZOG) predominated in both genotypes. Thermoinduction in oveST25 caused a drastic increase of extracellular N6-isopentenyladenine (iP) and cis-zeatin-riboside-O-glucoside (cZROG), 10fold and 4fold respectively. In the wild type no significant changes were measured.
In a comparative analysis of cytokinin contents in whole cultures and in tRNA-hydrolysates cZ-type cytokinins were found to be predominant in both fractions (99% and 91%, respectively, in wild type of Physcomitrella). The iP-type represented the second dominant group with ~1% in whole culture- and >8% in tRNA-cytokinins. The resemblance of cytokinin distribution in tRNA and whole cultures of Physcomitrella suggests a tRNA origin of cytokinins in Physcomitrella.
RT-PCR-based expression studies with the tRNA-IPT gene PpIPT1 in the oveST25 mutant revealed enhanced transcription levels at the inducing temperature of 25ºC compared to non-inducing conditions (15ºC). A transgenic wild type line with cytokinin deficiency due to cytokinin oxidase/dehydrogenase overexpression (tCKX7) exhibited also high PpIPT1 expression levels indicating that cytokinin deficiency might upregulate tRNA-mediated cytokinin biosynthesis.
Presented results indicate a potential role of PpIPT1 for cytokinin biosynthesis in Physcomitrella and point out the relevance of the tRNA-mediated pathway for cytokinin production in moss.