RESUMEN
* Reductive catabolism of pyrimidine nucleotides occurs via a three-step pathway in which uracil is degraded to beta-alanine, CO(2) and NH(3) through sequential activities of dihydropyrimidine dehydrogenase (EC 1.3.1.2, PYD1), dihydropyrimidinase (EC 3.5.2.2, PYD2) and beta-ureidopropionase (EC 3.5.1.6, PYD3). * A proposed function of this pathway, in addition to the maintenance of pyrimidine homeostasis, is the recycling of pyrimidine nitrogen to general nitrogen metabolism. PYD expression and catabolism of [2-(14)C]-uracil are markedly elevated in response to nitrogen limitation in plants, which can utilize uracil as a nitrogen source. * PYD1, PYD2 and PYD3 knockout mutants were used for functional analysis of this pathway in Arabidopsis. pyd mutants exhibited no obvious phenotype under optimal growing conditions. pyd2 and pyd3 mutants were unable to catabolize [2-(14)C]-uracil or to grow on uracil as the sole nitrogen source. By contrast, catabolism of uracil was reduced by only 40% in pyd1 mutants, and pyd1 seedlings grew nearly as well as wild-type seedlings with a uracil nitrogen source. These results confirm PYD1 function and suggest the possible existence of another, as yet unknown, activity for uracil degradation to dihydrouracil in this plant. * The localization of PYD-green fluorescent protein fusions in the plastid (PYD1), secretory system (PYD2) and cytosol (PYD3) suggests potentially complex metabolic regulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitrógeno/metabolismo , Nucleótidos/metabolismo , Pirimidinas/metabolismo , Uracilo/metabolismo , Amidohidrolasas/metabolismo , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Expresión Génica , Técnicas de Inactivación de Genes , Genes de Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Redes y Vías Metabólicas , Mutación , Plantas Modificadas GenéticamenteRESUMEN
Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) catalyzes the committed step in the de novo synthesis of uridine-5'-monophosphate (UMP), from which all other pyrimidine nucleotides are made. In Arabidopsis, ATCase is encoded by a single PYRB gene, whose expression was regulated by tissue pyrimidine availability. RT-PCR and PYRB:GUS expression profiles showed markedly increased expression of PYRB in root tissues during the first 5days after germination, as seed pyrimidine reserves were exhausted and de novo synthesis was required to support new growth. Growth of seedlings in the presence of the ATCase inhibitor N-(phosphonacetyl)-l-aspartate (PALA) resulted in complete developmental arrest at the day 5 stage, which was reversible upon addition of exogenous uracil. Arabidopsis RNAi lines exhibiting 70-95% reductions in PYRB transcript and ATCase protein levels had delayed growth and development, produced smaller plants with reduced root to shoot biomass ratios, few flowers, and siliques that produced smaller seeds with greatly reduced viability, compared with wild type plants. The severity of the phenotype was correlated with the extent of PYRB silencing and was reversible by pyrimidine addition. These results suggest that de novo synthesis is required, although minimal activities, supplemented by efficient salvaging pathway activities, are able to meet metabolic demands for pyrimdines during growth and development. Coordinate changes in expression of salvage and catabolic pathway genes in RNAi plants indicate that pyrimidine metabolism responds dynamically to changes in tissue pyrimidine availability.