Responses of antioxidative systems to acute ozone stress in transgenic poplar (Populus tremula × P. alba) over-expressing glutathione synthetase or glutathione reductase
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Wild-type hybrid poplar (Populus tremula × P. alba) plants and transgenic lines over-expressing glutathione reductase (GR) either in the cytosol (ca. 5-fold) or in the chloroplast (150- to 200-fold) or glutathione synthetase (GSS) in the cytosol (ca. 200-fold) were exposed for 3 days to ambient air or air containing 300 nl l–1 ozone for 7 h day–1. The contents and oxidation state of antioxidants (glutathione; ascorbate), the in vitro activities of target enzymes (GR, GSS), as well as the in vitro activities of ascorbate consuming (ascorbate peroxidase, APX) and regenerating (monodehydroascorbate reductase, MDAR; dehydroascorbate reductase, DHAR) enzymes were determined in the developing 2nd and the mature 5th leaves from the apex of the plants. The changes in the activities of the target enzymes GR and GSS also affected other components of the leaves' antioxidative system. Over-expression of GR in the chloroplast enhanced total glutathione contents of the leaves 2- to 3-fold. Foliar GSSG levels were enhanced in all transgenic poplar lines compared to wild-type plants. In developing leaves of transgenic plants, over-expressing GR in the cytosol or chloroplasts, APX and DHAR activities were higher, in mature leaves similar or lower than in wild-type plants. These differences between poplar lines did not mediate different sensitivities of the leaves to acute ozone exposure (Strohm et al., J Exp Bot 50:365–374). Still, components of the antioxidative system of the leaves showed both similar or specific reactions to acute ozone exposure in different poplar lines. Irrespective of leaf age total glutathione contents increased 2- to 3-fold in ozone-exposed leaves of all poplar lines. Also GSSG contents of the leaves increased, with the exception of plants over-expressing GR in the chloroplasts. Still a highly reduced state of glutathione was maintained in wild-type (>90%) and transgenic lines (ca. 80%) irrespective of ozone exposure. Apparently, GR activity was sufficient in all lines to compensate for ozone-mediated glutathione oxidation. Foliar GR activity increased up to 100% as a consequence of ozone treatment in wild-type and transgenic plants over-expressing GR in the cytosol, but not in plants over-expressing GR in the chloroplasts. The combined increase in total glutathione contents and GR activity in response to acute ozone exposure did not prevent injury and, therefore, cannot be considered a useful mechanism of defence, but rather an indicator of ozone-mediated oxidative stress. Total ascorbate contents and the activities of ascorbate consuming (APX) or regenerating enzymes (MDAR, DHAR) were not significantly affected by acute ozone exposure in all poplar lines. Ozone exposure strongly oxidised the foliar ascorbate pool except in leaves over-expressing GR in the chloroplast. Apparently, these leaves possess an enhanced capacity for ascorbate regeneration as a consequence of the strongly enhanced chloroplast GR activity and the simultaneously increased APX activity and glutathione levels. The enhanced antioxidative capacity did not prevent ozone-mediated injury to the leaves, most likely due to different sites of primary ozone reactions. Despite higher total ascorbate contents and higher APX activities in developing than in mature leaves, these and other components of the antioxidative system analysed in this study were not responsible for the higher sensitivity to acute ozone exposure of mature compared to developing poplar leaves.
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