RESUMO
The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, ß, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Ácidos Graxos/metabolismo , Fosfolipases A/metabolismo , Fosfolipídeos/metabolismo , Óleos de Plantas/metabolismo , Sementes/enzimologia , Acil Coenzima A/análise , Acil Coenzima A/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Ácidos Graxos/análise , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Mutação , Especificidade de Órgãos , Fosfatidilcolinas/metabolismo , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Óleos de Plantas/análise , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA de Plantas/genética , Sementes/citologia , Sementes/genética , Triglicerídeos/análise , Triglicerídeos/metabolismo , Regulação para CimaRESUMO
The phospholipase A(2) (PLA(2)) superfamily of lipolytic enzymes is involved in a number of essential biological processes, such as inflammation, development, host defense, and signal transduction. Despite the proven involvement of plant PLA(2)s in many biological functions, including senescence, wounding, elicitor and stress responses, and pathogen defense, relatively little is known about plant PLA(2)s, and their genes essentially remain uncharacterized. We characterized three of four Arabidopsis thaliana PLA(2) paralogs (PLA(2)-ß, -γ, and -δ) and found that they (1) are expressed during pollen development, (2) localize to the endoplasmic reticulum and/or Golgi, and (3) play critical roles in pollen development and germination and tube growth. The suppression of PLA(2) using the RNA interference approach resulted in pollen lethality. The inhibition of pollen germination by pharmacological PLA(2) inhibitors was rescued by a lipid signal molecule, lysophosphatidyl ethanolamine. Based on these results, we propose that plant reproduction, in particular, male gametophyte development, requires the activities of the lipid-modifying PLA(2)s that are conserved in other organisms.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Germinação , Fosfolipases A2/metabolismo , Pólen/crescimento & desenvolvimento , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/enzimologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Complexo de Golgi/enzimologia , Lisofosfolipídeos/metabolismo , Mutação , Fosfolipases A2/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Pólen/genética , Pólen/ultraestrutura , Interferência de RNA , RNA de Plantas/genéticaRESUMO
Plant secretory phospholipases A(2) (sPLA(2)s) probably play important roles in phospholipid signaling based on the data reported from other organisms, but their functions are poorly understood because of the lack of cloned sPLA(2) genes. In this study, we cloned and characterized an Arabidopsis secretory phospholipase A(2)-gamma (AtsPLA(2)-gamma) cDNA, and examined its enzymatic properties. The recombinant protein of AtsPLA(2)-gamma showed maximal enzyme activity at pH 8.0, and required Ca(2+) for activity. Moreover, AtsPLA(2)-gamma showed sn-2 position specificity but no prominent acyl preference, though it showed head group specificity to phosphatidylethanolamine rather than to phosphatidylcholine. AtsPLA(2)-gamma was found to predominate in the mature flower rather than in other tissues, and subcellular localization analysis confirmed that AtsPLA(2)-gamma is secreted into the intercellular space.