Dataset Information

Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

ABSTRACT: Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in developing seeds of flowering plants. The moss Physcomitrella patens contains the highly conserved DEK1 gene. Using efficient gene targeting, we generated a precise PpDEK1 deletion (?dek1), which resulted in normal filamentous growth of protonema. Two distinct mutant phenotypes were observed: an excess of buds on the protonema, and abnormal cell divisions in the emerging buds resulting in developmental arrest and the absence of three-dimensional growth. Overexpression of a complete PpDEK1 cDNA, or the calpain domain of PpDEK1 alone, successfully complements both phenotypes. These results in P. patens demonstrate the morphogenetic importance of the DEK1 protein in the control of oriented cell divisions. As it is not for protonema, it will allow dissection of the structure/function relationships of the different domains of DEK1 using gene targeting in null mutant background.

SUBMITTER: Perroud PF

PROVIDER: S-EPMC4285852 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

Perroud Pierre-François PF Demko Viktor V Johansen Wenche W Wilson Robert C RC Olsen Odd-Arne OA Quatrano Ralph S RS

The New phytologist 20140521 3

Orientation of cell division is critical for plant morphogenesis. This is evident in the formation and function of meristems and for morphogenetic transitions. Mosses undergo such transitions: from two-dimensional tip-growing filaments (protonema) to the generation of three-dimensional leaf-like structures (gametophores). The Defective Kernel 1 (DEK1) protein plays a key role in the perception of and/or response to positional cues that specify the formation and function of the epidermal layer in ...[more]

PMID: 24844771

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Project description:COR413 genes belong to a poorly characterized group of plant-specific cold-regulated genes initially identified as part of the transcriptional activation machinery of plants during cold acclimation. They encode multispanning transmembrane proteins predicted to target the plasma membrane or the chloroplast inner membrane. Despite being ubiquitous throughout the plant kingdom, little is known about their biological function. In this study, we used reverse genetics to investigate the relevance of a predicted chloroplast localized COR413 protein (PpCOR413im) from the moss Physcomitrella patens in developmental and abiotic stress responses. Expression of PpCOR413im was strongly induced by abscisic acid (ABA) and by various environmental stimuli, including low temperature, hyperosmosis, salinity and high light. In vivo subcellular localization of PpCOR413im-GFP fusion protein revealed that this protein is localized in chloroplasts, confirming the in silico predictions. Loss-of-function mutants of PpCOR413im exhibited growth and developmental alterations such as growth retardation, reduced caulonema formation and hypersensitivity to ABA. Mutants also displayed altered photochemistry under various abiotic stresses, including dehydration and low temperature, and exhibited a dramatic growth inhibition upon exposure to high light. Disruption of PpCOR413im also caused altered chloroplast ultrastructure, increased ROS accumulation, and enhanced starch and sucrose levels under high light or after ABA treatment. In addition, loss of PpCOR413im affected both nuclear and chloroplast gene expression in response to ABA and high light, suggesting a role for this gene downstream of ABA in the regulation of growth and environmental stress responses. Developmental alterations exhibited by PpCOR413im knockout mutants had remarkable similarities to those exhibited by hxk1, a mutant lacking a major chloroplastic hexokinase, an enzyme involved in energy homeostasis. Based on these findings, we propose that PpCOR413im is involved in coordinating energy metabolism with ABA-mediated growth and developmental responses.

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

Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

Publications

Defective Kernel 1 (DEK1) is required for three-dimensional growth in Physcomitrella patens.

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