PRODUCTION AND CHARACTERIZATION OF FOUR ARABIDOPSIS LINES OVEREXPRESSING THYLAKOIDAL ASCORBATE PEROXIDASE (tAPX)

Ascorbate peroxidases (APX) are present in plant cells in the cytoplasm, in the peroxisomes and in the chloroplasts (stromatic sAPX and thylakoidal tAPX ) and catalyse the reduction of H2O2 to water by using ascorbate as electron donor. We produced Arabidopsis lines overexpressing tAPX by transforming Arabidopsis with a construct in which the Arabidopsis tAPX cDNA has been cloned in the pBE2113-GUS transformation vector. Tranformants have been selected in Kanamycin and 9 independent T2 single insertion homozygous lines have been isolated. Northern blots analysis, RT-PCR and then western blot analysis with a monoclonal antibody against spinach APX confirmed overexpression of the tAPX cDNA in 4 lines, out of the 9 tested. We assayed, in these 4 lines, tAPX activity both electrophoretically (by native PAGE) and spectrophotometrically: tAPX activity, in the overexpressing lines ranges from 1.4 to 4 times with respect to control. The four overexpressing lines are phenotipically identical to control in terms of growth parameters. During light photoinhibition their photochemical efficiency Fv/Fm declines like that of control, thus confirming data found in the recent literature that chloroplastic APX are the primary target of photoinhibition . Most interestingly, the overexpressing lines are more resistant than control line to various oxidative treatments (Paraquat, Nitric oxide, Cupper) thus confirming in vivo the role played by tAPX in the protection against oxidative stress