Browsing by Subject "Effects of stress on"
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Item Isolation, characterization, and overexpression of pea cytosolic ascorbate peroxidase(Texas Tech University, 1995-12) Webb, Robert PaulThe 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.Item Molecular analysis and expression of glutathione S-transferase-coding genes in tobacco(Texas Tech University, 1996-12) Roxas, Virginia P.Glutathione S-transferases (GSTs: EC 2.5.1.18) are enzymes that detoxify xenobiotic compounds by covalently linking glutathione to a substrate, forming a glutathione conjugate. Two auxin-responsive cDNAs (Nt 107 and parB) that code for GSTs were Isolated from 2,4-D (2,4-dichlorophenoxyacetic acid)-treated tobacco (Nicotiana tabacum cv. Xanthi) suspension culture cells using reverse transcription polymerase chain reaction. Expression of Nt 107 in Escherischia coli resulted in >300-fold increase in GST specific activity, demonstrating that Nt 107 encodes an active GST. E. coli cultures that express Nt 707 also exhibited glutathione peroxidase (GPX: EC 1.11.1.9) activity against the substrates cumene hydroperoxide, hydrogen peroxide and tert-butyl hydroperoxide. These findings indicate that Nt 107 is likely to be actively involved in the protection against cell stress. To determine the factors that affect the expression of Nt 107, we have analyzed its response to a wide variety of factors in the wild type tobacco. Stress-signaling compounds that include abscisic acid, hydrogen peroxide, methyl jasmonate and salicylic acid; heavy metals CuSO^ and FeS04; and salt were found to induce Nt 707 expression in a concentration-dependent manner, indicating that Nt 107 participates in the responses of plants to stress conditions.