NADP-malic enzyme family from Arabidopsis thaliana

GERRARD WHEELER, M. C.; TRONCONI, M. A.; ANDREO, C. S.; DRINCOVICH, M. F.; MAURINO, V. G.

Iguazú, Argentina

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2004

SAIB

The A. thaliana genome contains four genes encoding putative NADP-malic enzymes (NADP-ME). One of the genes encodes a putative plastidic protein, while the other three do not possess any organellar targeting sequence. In order to characterize the AtNADP-ME gene family, the full length cDNAs of each isoform were isolated by RT-PCR. The amplified products were subcloned into pET-32 expression vector. The recombinant proteins were purified and used for kinetic and structural characterizations, as well as for the production of polyclonal antibodies. In order to analyze the NADP-ME pattern of expression in A. thaliana, promoter regions of all four members of the NADP-ME family were amplified by PCR and cloned into the binary vector pGPTV-BAR, which carries the GUS gene. The plasmids containing the chimeric MEA::GUS genes were introduced into A. thaliana by A. tumefaciens mediated transformation. Transgenic lines were analyzed for GUS activity. On the other hand, different SALK lines with T-DNA insertions in the different A. thaliana NADP-ME genes were obtained. Loss-of-function mutants not exhibit major phenotypic differences respect to wild type at non-stressed conditions. However, comparative enzymatic activity assays in several tissues indicated specific patterns of expression of the different isoforms, as obtained by GUS activity. In summary, we present the characterization of a complete NADP-ME gene family, which is the basis to unreveal the biological function of each NADP-ME isoform.