Factors Affecting the Enhancement of Oxidative Stress Tolerance in Transgenic Tobacco Overexpressing Manganese Superoxide Dismutase in the Chloroplasts

Abstract

Two varieties of tobacco (Nicofiana fabacum var PBD6 and var SR1) were used to generate transgenic lines overexpressing Mnsuperoxide dismutase (MnSOD) in the chloroplasts. The overexpressed MnSOD suppresses the activity of those SODs (endogenous MnSOD and chloroplastic and cytosolic Cu/ZnSOD) that are prominent in young leaves but disappear largely or completely during aging of the leaves. The transgenic and control plants were grown at different light intensities and were then assayed for oxygen radical stress tolerance i n leaf disc assays and for abundance of antioxidant enzymes and substrates in leaves. Transgenic plants had an enhanced resistance to methylviologen (MV), compared with control plants, only after growth at high light intensities. I n both varieties the activities of FeSOD, ascorbate peroxidase, dehydroascorbate reductase, and monodehydroascorbate reductase and the concentrations of glutathione and ascorbate (all expressed on a chlorophyll basis) increased with increasing light intensity during growth. Most of these components were correlated with M V tolerance. It is argued that SOD overexpression leads to enhancement of the tolerance to MV-dependent oxidative stress only if one or more of these components is also present at high levels. Furthermore, the results suggest that i n var SR1 the overexpressed MnSOD enhances primarily the stromal antioxidant system.The photosynthetic electron transport chain in higher plant chloroplasts contains, at the acceptor side of PSI, a number of auto-oxidizable enzymes. For example, Fd in the reduced state can react with oxygen, yielding the superoxide anion radical (OJ (Furbank and Badger, 1982;Asada and Takahashi, 1987), and it has been shown that superoxide is also generated in the aprotic membrane interior (Elstner and Frommeyer, 1978;Takahashi and Asada, 1988). Superoxide does not seem to be particularly toxic, although it does inactivate a number of metal-containing enzymes such as Fd-linked nitrate reductase, catalase, and peroxidases

Modification of reactive oxygen species scavenging capacity of chloroplasts through plastid transformation

“…This finding runs counter to some previous observations (e.g. Slooten et al 1995, McKersie et al 1999, Kwon et al 2002), on nuclear transformants, which suggest that over-expression of chloroplast-targeted SODs suppresses the activity of the native enzymes, although this finding is far from universal among the various SOD over-expressing lines. In fact there are two publications which support our observation.…”

“…Expression of MnSOD (normally located in the mitochondria, but targeted to the chloroplast in most of these studies) resulted in improved frost tolerance in alfalfa (McKersie et al 1993), cold and oxidative stress tolerance in maize (Van Breusegem et al 1999), drought (Wang et al 2005) and salt tolerance in rice (Tanaka et al 1999), and salt tolerance in poplar (Wang et al 2010). Levels of the Fe or Cu/Zn SODs, normally associated with chloroplasts, have also been enhanced, resulting in improved cold tolerance in alfalfa (McKersie et al 1999(McKersie et al , 2000, salt and drought tolerance (Badawi et al 2004b) in tobacco, and methyl viologen (MV-a superoxide-generating herbicide) tolerance in tobacco (Sen Gupta et al 1993;Slooten et al 1995) and poplar (Arisi et al 1998). Other ROS scavenging enzymes have also attracted attention.…”

Light and Excess Manganese

“…Leaf antioxidant content varies with growing conditions, and the effect of light intensity has been addressed recently (Slooten, et al, 1995;Grace and Logan, 1996). Plants growing under high light are more frequently exposed to reactive oxygen species (the superoxide radical, H 2 O 2 , and singlet oxygen) generated in the plastids than plants growing at low light intensity, and consequently might respond by accumulating higher amounts of antioxidants in leaf tissue.…”

“…Enzymes such as SOD, GR, ASPX, and DAS reductase, and com-pounds such as ascorbate and glutathione are actively involved in the ascorbate-glutathione scavenging cycle, controlling reactive oxygen species not only in the chloroplast but also in the cytosol (for reviews, see Alscher and Hess, 1993;Foyer and Mullineaux, 1994). The involvement and role of antioxidants in protection against oxidative stress have been demonstrated using transgenic plants (Foyer et al, 1994;Allen, 1995;Slooten et al, 1995), and genetic variability in the content of antioxidant elements has been reported in several species (Sevilla et al, 1988;Daza et al, 1993;Guzy and Heath, 1993). Tolerance of oxidative stresses such as the herbicide paraquat and NaCl were partially associated with a better antioxidant response in tolerant genotypes (Gossett et al, 1994;Hernández et al, 1995).…”

The role of antioxidant enzymes in photoprotection

“…For example, a decrease in the activity of the key antioxidant enzymes caused by shading resulted in the disappearance of the beneficial effect of MnSOD overproduction on the methyl viologen resistance of transgenic tobacco plants (Slooten et al 1995). The overproduction of SOD itself can cause an increase in the activity of other antioxidant enzymes (Sen Gupta et al 1993a, b;Kingston-Smith and Foyer 2000); however, this effect is not always observed (Payton et al 2001).…”