Functional and Developmental Impact of Cytosolic Protein N-Terminal Methionine Excision in Arabidopsis

Abstract

Protein N-terminal methionine (Met) excision (NME) is carried out by two types of Met aminopeptidases (MAPs), MAP1 and MAP2, in eukaryotes. Three enzymes, MAP1A, MAP2A, and MAP2B, have been identified in the cytoplasm of Arabidopsis (Arabidopsis thaliana). MAP transcript quantification revealed a predominance of MAP2B and developmental and organ-specific regulation of both MAP1A and MAP2s. By combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic NME in Arabidopsis and of the respective contributions of the two types of cytoplasmic MAPs throughout development. dsRNA interference and knockout (KO) plant lines targeting either MAP1A alone or both MAP2s simultaneously were constructed and shown to display wild-type phenotypes. In the MAP1A KO context, modulating MAP2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development, with particularly strong effects on the root system. Reciprocally, complete MAP2 inhibition in various MAP1A knocked-down genetic backgrounds also generated a gradient of developmentally abnormal plants, but the effects on the root system were milder than in the KO context. In the absence of MAP2 activity, the severity of the phenotype in the MAP1A knocked-down lines was correlated to the extent of MAP1A mRNA accumulation. Complete cytoplasmic NME inactivation blocked development after plant germination. Thus, in plants, (1) cytoplasmic NME is essential; (2) MAP1A and MAP2s are functionally interchangeable, which is not the case in fungi and animals, as a complete block of either MAP-type activity does not cause any visible molecular or phenotypic effect; and (3) a minimal level of cytoplasmic MAP is required for normal development.