Transgenic Plants With Modified Antioxidant Enzyme Levels

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Because of the involvement of AOS in a wide variety of environmental stresses, antioxidant enzymes are interesting molecular targets for the production of new plant varieties that can cope with these stresses. Several antioxidative stress enzymes have been genetically engineered into plants to assess their potential capacity for enhancing oxidative stress tolerance. The beneficial effects observed in some of these transgenic plants can lead to interesting agronomic applications. The following section gives an overview of the state of the art concerning transgenic plants with modified levels of antioxidant enzyme levels.

A. Transgenic Plants with Elevated Superoxide Dismutase Levels

The first report on transgenic plants overproducing SOD was, however, not promising. Transgenic tobacco plants with 30- to 50-fold increased SOD activity levels due to the production of a petunia chloroplastic Cu/ZnSOD were not more resistant against methyl viologen (MV) (49). The tolerance toward oxidative stress is easily tested in vitro with MV (also known as paraquat), which is a light-activated herbicide. In the light, MV becomes an electron acceptor from photosystem I (PSI), subsequently reducing dioxygen to 02 • In this way, the herbicide strongly enhances the formation of superoxide radicals and is a fairly good mimic of the superoxide-forming process that occurs in vivo in illuminated chloroplasts. The MV also accepts electrons from the respiratory electron transport chain in the mitochondria and forms superoxide radicals in the dark too. Transgenic tomato plants with two- to fourfold increased SOD levels by producing the same chloroplastic Cu/ZnSOD were not better protected against photoinhibitory conditions known to increase oxidative stress (high light, low temperatures, and low C02 concentrations).

Pitcher et al. (50) showed that the same transgenic tobacco plants were not protected against ozone stress. Although the activity levels of enzymes involved in H202 scavenging (APX, GR, etc.) were not measured in both cases, the lack of induced tolerance is plausibly explained by the inability of the plants to cope with the elevated levels of H202 that were produced by the enhanced dismutation of superoxide radicals. Transgenic potato plants overproducing tomato cytosolic or chloroplastic Cu/ZnSOD lacked a chlo-rotic and wilting phenotype that was seen in wild-type plants after dipping their shoots in 100 |xM MV. In culture medium with 10 (jlM MV, root cultures from the same transgenic potatoes grew at rates similar to those in control medium (51). In contrast to the very high overproduction of SOD

reported in the transgenic tobacco plants (49), these transgenic potato plants had only a modest increase in Cu/ZnSOD activity. In this way, too high accumulation of H202 is avoided, preventing promotion of hydroxyl formation by H202 and Oj interaction. A correlation between MV tolerance and chilling tolerance was found in transgenic tobacco plants that overproduce a chloroplast-localized Cu/ZnSOD from pea at twofold higher levels than the endogenous FeSOD (52,53). In agreement, moderate cytosolic overproduction of a pea Cu/ZnSOD in transgenic tobacco confers partial resistance to ozone-induced foliar necrosis (54). Up to sixfold enhancement of SOD activity led to 30 to 50% less visible foliar damage. The beneficial effect of the transgene could, however, be detected only in older leaves (leaves 3 to 5). In the youngest leaves (5 and 6), no difference between transgenic and wild-type plants could be observed. Because Cu/ZnSODs are sensitive to H202, it is also possible that some of the introduced Cu/ZnSOD activity is inhibited by its own end product. In this way, MnSOD, which is insensitive to H202, was introduced as a better candidate for engineering oxidative stress tolerance.

The MnSOD from Nicotiana plumbaginifolia has indeed been more consistently reported to confer resistance to oxidative stress. Transgenic tobacco plants overproducing the nuclear-encoded mitochondrial MnSOD in either mitochondria or chloroplasts (by replacing the mitochondrial transit sequence with a chloroplast transit peptide) were less sensitive to MV. Overproduction of MnSOD in the mitochondria rendered the plants more tolerant to MV in dark but not in light incubations with MV. By contrast, MnSOD overproduction in the chloroplasts made the plants more tolerant to MV in both dark and light incubations (55). The SOD overproduction in the chloroplasts also led to the induction of other antioxidant enzymes (FeSOD, APX, DHAR peroxidase) and higher glutathione and ascorbate levels (56). Enhanced SOD activity in the mitochondria had only a minor effect on ozone tolerance in transgenic tobacco. However, overproduction of SOD in the chloroplasts resulted in a three- to fourfold reduction of visible ozone injury (Fig. 1) (57). In addition to the fact that MnSOD is resistant against H202, the subcellular location may probably play an important role in determining whether positive or negative effects arise from SOD overproduction. Targeting SODs to organelles with a higher risk of AOS production seems more efficient than cytosolic overproduction. Because during most abiotic stresses the chloroplast is the main site of AOS production, it is considered the target organelle for protection against oxidative stress.

Because of their original presence in plant chloroplasts, FeSOD proteins are theoretically better candidates for overproduction in the chloroplasts. Under conditions in which H202 accumulation is not the limiting factor, the biochemical properties of the FeSOD proteins should be better

PBD6 W.T. MitSODI UitSOD2 ChlSODI ChlSOD2

Figure 1 Foliar injury of leaf 6 from wild-type (a) and transgenic tobacco overproducing superoxide dismutase (b) after 7 days of ozone exposure, (c) Leaf injury in transgenic versus wild-type tobacco plants after 1 week of ozone fumigation. The values given show the mean ± standard error of the mean for wild-type plants (PBD6 W.T.), transgenic plants overproducing SOD targeted to the mitochondria (MitSODI and MitSOD2), and transgenic plants overproducing SOD targeted to the chloroplasts (ChlSODI and ChlSOD2). Means with the same letter are not significantly (P < 0.05) different. (©Mac-millan Publishers, reprinted with permission.)

PBD6 W.T. MitSODI UitSOD2 ChlSODI ChlSOD2

Figure 1 Foliar injury of leaf 6 from wild-type (a) and transgenic tobacco overproducing superoxide dismutase (b) after 7 days of ozone exposure, (c) Leaf injury in transgenic versus wild-type tobacco plants after 1 week of ozone fumigation. The values given show the mean ± standard error of the mean for wild-type plants (PBD6 W.T.), transgenic plants overproducing SOD targeted to the mitochondria (MitSODI and MitSOD2), and transgenic plants overproducing SOD targeted to the chloroplasts (ChlSODI and ChlSOD2). Means with the same letter are not significantly (P < 0.05) different. (©Mac-millan Publishers, reprinted with permission.)

adapted to function optimally under the specific conditions within the chlo-roplast. This characteristic was indeed shown by overproduction in transgenic tobacco of the mature FeSOD from Arabidopsis thaliana coupled to a chloroplast transit peptide (58). Transgenic FeSOD protected both the plasmalemma and PSII against superoxide generated during illumination of leaf discs impregnated with MV. Overproduction of a mitochondrial MnSOD in the chloroplasts protected only the plasmalemma, but not PSII, against MV. This difference was attributed to the higher membrane affinity of FeSOD, allowing this enzyme to scavenge superoxide radicals at the site of their formation, i.e., near PSI (58).

The potential of SOD overproduction in stress engineering strategies has been reported for three crops, cotton, alfalfa, and maize. Transgenic cotton plants that produce chloroplast-localized MnSOD are more tolerant against chilling-induced oxidative stress (59). Transgenic alfalfa plants with twice the amount of total SOD-overproducing N. plumbaginifolia MnSOD in both the mitochondria and the chloroplast were tested for increased tolerance against oxidative stress by incubating leaves with the herbicide aci-fluorfen. Acifluorfen is a photobleaching, p-nitrodiphenyl ether herbicide that promotes accumulation of the chlorophyll precursor protoporphyrin; in the light it generates singlet oxygen that causes peroxidation in the tonoplast, plasmalemma, and chloroplast envelope. Increased resistance against acifluorfen correlated with increased freezing stress tolerance. One transgenic plant, propagated by cuttings, had increased regrowth capacities compared with nontransgenic plants when subjected to sublethal freezing temperatures from — 8°C to — 16°C (60). When viability was measured by electrolyte leakage or by tetrazolium staining, differences between transgenic and wildtype plants were minor or nonexistent, despite differences in winter survival among the plants (61). Two other experiments with two transgenic alfalfa plants producing MnSOD indicated the value of engineering oxidative stress tolerance to improve environmental stress tolerance in crop plants. For the first time, transgenic plants overproducing antioxidant enzymes were also tested in a natural field environment. The transgenic plants tended to show reduced injury from water deficit stress as determined by chlorophyll fluorescence, electrolyte leakage, and regrowth from crowns. Over a 3-year field trial, vigor and survival of transgenic plants were significantly improved (62).

It should be noted that the preceding experiments were done by using only a few transgenic plants obtained by transformation of an alfalfa cultivar with poor agronomic performance and originally not well adapted to winter conditions. McKersie et al. (62) expanded their original observations by transforming two elite alfalfa plants that are adapted to the field environment, and they examined many more independent transgenics under both laboratory and field conditions. This detailed assessment confirmed the earlier observation that transgenics had a greater survival and yield (total shoot dry matter production) in the field.

Increased winter survival was also correlated with increased SOD levels in transgenic alfalfa plants overproducing FeSOD but not with any beneficial effects on photosynthesis, growth, or oxidative stress tolerance in fro

One leaves. This correlation can be explaine d enging capacity in the root, thereby enhai ing injury (63). Another more attractive els of H202 produced by SOD overpro toxicity, H202 is well recognized to ai defense responses (10,64,65). Altered 1 mation process that induces a general de: adverse environmental conditions during We evaluated transgenic maize pi baginifolia MnSOD or an A. thaliana Fe fused to a chloroplast transit peptide flower mosaic virus 35S (CaMV35S) p was correctly targeted to chloroplasts distinguished on SOD activity gels, tolerance to MV. The growth characteris followed during growth at ambient (15-17°C) in growth cabinets. Although ments had a growth advantage compared tically significant increase in growth coi

To extrapolate the in vitro oxid^i growth effects during environmental str are currently evaluating the initiated fie] rope to scale up the experiments and to plants under natural environmental stress periments revealed that in transgenic mainly (although not exclusively) locate1 sheath cells (67). The low levels of Mn be attributed to a different expression c but posttranscriptional or posttranslatic MnSOD cannot be excluded. Wilson et cular-specific activity of /3-glucuronidas transformed with a CaMV35S-GUS fusi Because endogenous SOD and AK die sheath cells, whereas GR and DHA in the mesophyll tissue of maize, a cle system between the mesophyll and bund This differential localization correlates respective enzymes. Because NADPH GR and DHAR activities are rate limite ential distribution of antioxidants is, of deal with oxidative stress. The overpro each cell type could restore this natu:

i!i by the increased superoxide-scav-ncing recovery from primary freez-explanation relates to enhanced lev-iuction. In addition to its potential :t as a signal molecule in several 202 levels might induce an accli-fense response to tolerate better the winter survival (63). ants that overproduce an N. plum-SOD complementary DNA (cDNA) m pea under control of the cauli-romoter. The recombinant MnSOD nd its enzymatic activity could be transgenic line showed enhanced tics of transgenic maize lines were -25°C) and chilling temperatures the transgenic lines in all experi-with the wild-type lines, no statis-ulild be observed (66). tive stress tolerance to improved ;ss conditions, such as chilling, we d trials at different locations in Eu-<f;heck the behavior of the transgenic conditions. Immunolocalization ex-maize the recombinant MnSOD is d in the chloroplasts of the bundle SOD in the mesophyll cells could ipacity of the CaMV35S promoter, nal regulation of the recombinant al. (68) have also shown the vase (GUS) in leaves of maize plants on.

i activities are restricted to the bun-R activities could be detected only :ir partitioning of the AOS defense le sheath cells must be present (69). with the need for NADPH of the limited in the bundle sheath cells, d in this compartment. This differ-course, crucial for maize plants to duction of AOS scavengers within ral imbalance and, hence, confer a higher tolerance to maize plants against chilling-associated oxidative stress. The exclusive presence of the transgenic MnSOD in the bundle sheath cells probably influenced the expected protective effect of MnSOD in the transgenic maize plants during chilling stress.

Transgenic maize lines overproducing an A. thaliana FeSOD in the chloroplasts also suffered less from paraquat damage than controls as indicated by decreased membrane leakiness and by higher photosynthetic activity. In contrast to the MnSOD lines, the transgenic FeSOD maize plants also exhibited a significantly increased growth rate at low temperatures (as estimated from fresh weight and summed leaf length determinations) (70). These and previous results in tobacco suggest that FeSOD is a better candidate enzyme to protect plants against oxidative stress. The reason could be the difference in suborganellar location between the overproduced MnSOD and FeSOD. Because of their chloroplastic and mitochondrial origin, respectively, FeSOD and MnSOD might have different properties. Van Camp et al. (58) showed that in tobacco the transgenic FeSOD is at least partially bound to the chloroplast membrane, whereas transgenic MnSOD behaves more like a stromal enzyme. This differential subcellular location might provoke different protective effects against oxygen radicals. Transgenic FeSOD might be able to bind electrostatically to the chloroplast membrane in the vicinity of the site of radical production, resulting in increased protective properties.

B. Transgenic Plants with Modulated Ascorbate Peroxidase or Catalase Levels

Overproduction of an A. thaliana APX in tobacco chloroplasts provided almost complete protection of the PSII reaction center against aminotriazole. Aminotriazole inhibits catalase and in this way provokes accumulation of H202. Tolerance to MV was slightly better, but tolerance to eosin (a singlet oxygen generator) or chilling-induced photoinhibition was not enhanced (L. Slooten, personal communication). Transgenic tobacco plants overproducing an A. thaliana peroxisomal APX were protected against aminotriazole but not against MV (causing mainly AOS formation in the chloroplast) (71). These data show that H202 formed in the peroxisomes can diffuse and affect photosynthetic activities in the chloroplast. Protecting plant cells against oxidative stress during photorespiratory conditions can hence be done by scavenging H202 at the place of production in the peroxisomes or by providing the chloroplast with extra H202-scavenging capacity. In an ozone-sensitive transgenic tobacco, a 10-fold increase in chloroplastic APX activity was not effective against cellular injury caused by ozone stress (72). Overproduction in tobacco chloroplasts of the other major H202 scavenger, catalase, led to tolerance against MV and drought stress conditions (73,74).

By combining superoxide scavengers and H202 scavengers, even better results can be envisaged, the most spectacular results having been reported in insects. In Drosophila melanogaster, the overproduction of SOD and cat-alase resulted in a delay of aging and greater longevity, whereas overproduction of either of the two enzymes alone had only minor effects (75,76). Transgenic tobacco overproducing an Escherichia coli GR or a rice Cu/ ZnSOD were fivefold more resistant against ion leakage caused by MV treatments. Crossings between both transgenic lines were highly tolerant to MV concentration of 50 jjlM, whereas control plants were sensitive to MV concentrations as low as 1 fxM (77).

Underproduction of antioxidative stress enzymes often increases sensitivity to the experienced stress. Tobacco plants with decreased APX or GR activity, obtained by antisense technology, are more susceptible to ozone stress and MV, respectively (78,79). Transgenic plants deficient in catalase (class I) can be grown only under low light conditions (<100 |xmol m"~2 sec-1 photosynthetic photon fluence rate). When exposed to higher light intensities, these plants developed white necrotic lesions on the leaves after 1 to 2 days. Lesion formation was induced by photorespiration because damage was prevented under elevated C02. Stress analysis revealed that Cat 1-deficient plants were more sensitive to paraquat, salt, and ozone stress, indicating that Catl is a key component of several stress defenses (10,65,80,81).

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