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Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

ABSTRACT: Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- and rehydration-responsive proteins, respectively. The functional categories of the most differentially-expressed proteins were seed maturation, defence, protein synthesis and quality control, and energy production. Strikingly, most of the late embryogenesis abundant (LEA) proteins were expressed at a basal level under control conditions and their synthesis was strongly enhanced by dehydration, a pattern that was confirmed by RT-PCR. Actinoporins, phosphatidylethanolamine-binding protein, arabinogalactan protein, and phospholipase are the likely dominant players in the defence system. In addition, 24 proteins of unknown function were identified as novel dehydration- or rehydration-responsive proteins. Our data indicate that Physcomitrella adopts a rapid protein response mechanism to cope with dehydration in its leafy-shoot and basal expression levels of desiccation-tolerant proteins are rapidly upgraded at high levels under stress. This mechanism appears similar to that seen in angiosperm seeds.

SUBMITTER: Cui S

PROVIDER: S-EPMC3254677 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature

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Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

Cui Suxia S Hu Jia J Guo Shilei S Wang Jie J Cheng Yali Y Dang Xinxing X Wu Lili L He Yikun Y

Journal of experimental botany 20111011 2

Physcomitrella patens is an extremely dehydration-tolerant moss. However, the molecular basis of its responses to loss of cellular water remains unclear. A comprehensive proteomic analysis of dehydration- and rehydration-responsive proteins has been conducted using quantitative two-dimensional difference in-gel electrophoresis (2D-DIGE), and traditional 2-D gel electrophoresis (2-DE) combined with MALDI TOF/TOF MS. Of the 216 differentially-expressed protein spots, 112 and 104 were dehydration- ...[more]

PMID: 21994173

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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

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

Publications

Proteome analysis of Physcomitrella patens exposed to progressive dehydration and rehydration.

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