Isolation, characterization, and overexpression of pea cytosolic ascorbate peroxidase
Webb, Robert Paul
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The damage to cellular proteins, DNA and lipids caused by the accumulation of reduced oxygen intermediates is collectively referred to as oxidative stress. Plants have evolved a number of antioxidative enzymes that ameliorate oxidative stress by scavenging toxic oxygen species. Ascorbate peroxidase (APX) is a hemoperoxidase that catalyzes the reduction of hydrogen peroxide to water by utilizing ascorbate as an electron donor. We isolated a pea cytosolic APX cDNA which was used to make chimeric gene constructs that express cytosolic APX in either the cytosol (cytAPX) or chloroplast (chlAPX). The gene constructs were developed in binary vectors and mobilized into Agrobacterium for leaf disk transformation into Nicotianana tabacum cv. Xanthi. The cytAPX transgenic plants showed a 3.8-fold increase in APX activity compared to the nonexpressing control plants. The chlAPX transgenic plants showed a 13-fold increase over the control plants. In order to determine if the increased levels of APX activity conferred protection against oxidative stress, leaf disks from two independent lines of both cytAPX and chlAPX transgenic plants were exposed to the superoxide-generating herbicide methyl viologen. The cytAPX transgenic plants showed significant protection from methyl viologen at concentrations of 1.2 and 2.4 µM. However, the chlAPX transgenic plants showed no protection at any of the concentrations tested.