Expression and functional analysis of aspartate transcarbamoylase and role of de novo pyrimidine synthesis in regulation of growth and development in Arabidopsis

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 5 days 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.