Dataset Information

Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis.

ABSTRACT: The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the question of whether cytokinins in moss derive from tRNA exclusively. Targeted gene knockout of ipt1 (d|ipt1) along with localization studies revealed that the chloroplast-bound IPT1 was almost exclusively responsible for the A37 prenylation of tRNA in Physcomitrella. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based cytokinin profiling demonstrated that the total amount of all free cytokinins in tissue was almost unaffected. However, the knockout plants showed increased levels of the N (6) -isopentenyladenine (iP)- and trans-zeatin (tZ)-type cytokinins, considered to provide active forms, while cis-zeatin (cZ)-type cytokinins were reduced. The data provide evidence for an additional and unexpected tRNA-independent cytokinin biosynthetic pathway in moss. Comprehensive phylogenetic analysis indicates a diversification of tRNA-IPT-like genes in bryophytes probably related to additional functions.

SUBMITTER: Lindner AC

PROVIDER: S-EPMC4036517 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis.

Lindner Ann-Cathrin AC Lang Daniel D Seifert Maike M Podlešáková Kateřina K Novák Ondřej O Strnad Miroslav M Reski Ralf R von Schwartzenberg Klaus K

Journal of experimental botany 20140401 9

The moss Physcomitrella patens is part of an early divergent clade of land plants utilizing the plant hormone cytokinin for growth control. The rate-limiting step of cytokinin biosynthesis is mediated by isopentenyltransferases (IPTs), found in land plants either as adenylate-IPTs or as tRNA-IPTs. Although a dominant part of cytokinins in flowering plants are synthesized by adenylate-IPTs, the Physcomitrella genome only encodes homologues of tRNA-IPTs. This study therefore looked into the questi ...[more]

PMID: 24692654

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Project description:BackgroundThe moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2.ResultsOur results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis.ConclusionsOur study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.

Dataset Information

Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis.

Publications

Isopentenyltransferase-1 (IPT1) knockout in Physcomitrella together with phylogenetic analyses of IPTs provide insights into evolution of plant cytokinin biosynthesis.

Similar Datasets

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