NADP-malic enzyme family from A. thaliana: functional characterization of T-DNA insertion mutants

GERRARD WHEELER M; ZANOR, M.; DRINCOVICH MF,; CARLOS SANTIAGO ANDREO; MAURINO VG,

Rosario

Congreso; XLII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular, SAIB.; 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

The A. thaliana genome contains four genes encoding NADP-malic enzymes (me1-4). ME4 is localized to plastids, whereas the other three isoforms (ME1-3) are cytosolic. ME2 y ME4 are the only proteins showing a constitutive pattern of expression. Furthermore, ME2 is responsible for the major part of NADP-ME activity in mature plant tissues. In order to elucidate the particular role of each family member in Arabidopsis metabolism, several T-DNA mutants defective in the me genes were analyzed. In addition, homozygous double and triple mutants were generated by crossing. The distribution of NADP-ME activity obtained in different organs was in agreement with expected results. However, none of the single, double or triple mutant combinations showed an obvious phenotype under normal growth conditions. Thus, different stresses were imposed to insertion lines. After high light treatment, the knock-out mutants without ME2 are more sensitive than the wild-type and exhibited severe photo oxidative damage. In view of this result, the me2 gene under control of CaMV35S promoter was introduced into these lines by A. tumefaciens mediated transformation. Transgenic lines were analyzed for NADP-ME activity, native PAGE electrophoresis and western blot. It could be envisioned that ME2 is required in cases where the export of malate to the cytosol is increased, probably contributing to the malate valve.