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. 1998 Jan;116(1):107–116.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

Hans-Peter Mock 1, Ulrich Keetman 1,2, Elisabeth Kruse 1, Barbara Rank 2, Bernhard Grimm 1,*
PMCID: PMC35148

Abstract

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Tetrapyrroles function as prosthetic groups of various proteins for the transfer of electrons and for the sensing of redox states. In plants the dominant tetrapyrrolic end product is chlorophyll, which harvests and converts light into chemical energy. Unbound chlorophyll and accumulating precursors can easily be photo-oxidized and are known photosensitizers in diphenyl ether herbicide action and in photodynamic therapy (Rebeiz et al., 1984; Menon et al., 1989). They absorb radiant energy and produce mainly singlet oxygen, although they can also generate other oxygen radicals (Arakane et al., 1996).

Reactive oxygen species are produced in all organisms in response to various environmental conditions. When plants are exposed to high-light intensities, high or low temperatures, or ozone or air pollution, more reactive oxygen species are generated than the scavenging mechanisms can detoxify (Alscher et al., 1997). Moreover, reactive oxygen species can also be formed in metabolic pathways. Oxidative stress can result in necrosis, programmed cell death (apoptosis), or the induction of protective mechanisms. The reactive oxygen species themselves are proposed to trigger these cellular responses. Plants are particularly subjected to oxidative stress when oxygen is generated during photosynthesis.

The chloroplasts possess an elaborate system for scavenging reactive oxygen species, which comprises both enzymatic and nonenzymatic compounds (Foyer et al., 1994; Asada, 1996). APX scavenges the hydrogen peroxide generated by the action of SOD and thereby prevents the chemical formation of other toxic oxygen species (Asada, 1994). The MDA radical formed is an indicator of oxidative stress in leaves (Heber et al., 1996). The regeneration of ascorbate is accomplished by the action of MDAR, DHAR, and GR. The latter enzymes, along with APX, belong to the Halliwell-Asada pathway (Creissen et al., 1994; Foyer et al., 1994).

In the vicinity of the thylakoid membranes, MDA can also be photoreduced through the mediation of Fd (Miyake and Asada, 1992; Foyer and Lelandais, 1993). If not scavenged, the MDA radical can spontaneously disproportionate into ascorbate and DHA, which in turn can be reduced by DHAR to ascorbate using GSH as an electron donor. The microcompartmentation of antioxidative enzymes with respect to photosynthetic processes producing radicals was summarized recently by Asada (1996). The generation of reactive oxygen species and consequently the localization of the enzymes involved in detoxification are not, however, restricted to the plastids (Scandalios, 1993; Alscher et al., 1997).

Under normal growth conditions the risk of photo-oxidative damage from intermediates in chlorophyll biosynthesis is low. Only protochlorophyllide bound to NADP-protochlorophyllide oxidoreductase accumulates in etiolated tissue. A regulatory feedback mechanism prevents the steady increase of protochlorophyllide. In light-adapted plants the synthesis of chlorophyll is tightly controlled. Feeding ALA to plants can circumvent the regulatory feedback control of ALA synthesis and induce an excess accumulation of protoporphyrin IX and Mg-porphyrins. In the presence of light the accumulated nonphototransformable protochlorophyllide generates singlet oxygen through a type II photosensitization reaction, which photodynamically damages the plants (Dodge, 1994). Herbicides of the diphenyl ether type inhibit protoporphyrinogen oxidase, leading to accumulation of protoporphyrin IX and subsequently to photodynamic damage to leaves. The paradoxical accumulation of the inhibited enzyme's product was resolved when it was discovered that protoporphyrinogen was oxidized to protoporphyrin IX outside of the plastid, most likely by peroxidases (Jacobs et al., 1996).

We were initially interested in studying the consequences of deregulated tetrapyrrole synthesis resulting from the expression of antisense RNA for UROD (Mock and Grimm, 1997) or CPO (Kruse et al., 1995a). The transgenic plants that we produced developed leaf lesions. Uro(gen) or copro(gen) accumulated up to 500-fold compared with the wild type, and the amount of excessive tetrapyrroles was correlated with the intensity of leaf damage. Leaf necrosis was almost absent when plants were grown under dim light or with short light periods (Mock and Grimm, 1997), indicating that the destructive processes induced by accumulating porphyrins were dependent on light intensity. Leaf lesions usually appeared on fully developed leaves of the transgenic lines, although the accumulation of uro(gen) or copro(gen) was highest in young leaves.

Apparently, the cellular mechanisms that protect plant cells against the phototoxic effects of tetrapyrroles are most active in young, developing leaves. To understand how the accumulation of porphyrins in different cell compartments leads to the production of necrotic lesions, we decided to characterize the plant's defense mechanisms to oxidative stress by measuring antioxidant enzyme activities and mRNA levels. An induction of the protective enzymes as well as decreases in the levels of several low-molecular-mass antioxidants were found.

MATERIALS AND METHODS

Tobacco (Nicotiana tabacum var Samsun NN) plants expressing antisense genes for UROD (Mock and Grimm, 1997) or CPO (Kruse et al., 1995a) and control plants were raised in growth chambers (25°C, 16 h of light, 300 μmol quanta PAR m−2 s−1). Eight- to 10-week-old transgenic and control plants with the same number of leaves were harvested 2 h after the onset of light. Leaves were pooled from several plants (n ≥ 6 for each harvest and each line), frozen in liquid nitrogen, and stored at −80°C.

The primary transformants and the progenies of line 2 with UROD antisense RNA expression (Mock and Grimm, 1997) and of line PL 1/3 with CPO antisense RNA expression (Kruse et al., 1995a) were used for the analysis. UROD antisense line 12 (Mock and Grimm, 1997) and CPO antisense line PL 1/41 (Kruse et al., 1995a) were also used in preliminary experiments and yielded results consistent with the present data.

RNA Isolation and Analysis

Isolation of RNA and northern analysis were performed as described by Kruse et al. (1995a). The cDNA clones for the SOD and CAT isoforms, GPX, and APX were generously provided by D. Inzé (University of Gent, Belgium). Specific probes were prepared according to the method of Willekens et al. (1994b) and radiolabeled by nick translation in the presence of [32P]dCTP according to the manufacturer's protocol (GIBCO-BRL). Ten micrograms per lane of total RNA was separated on formaldehyde-containing agarose gels (Sambrook et al., 1989). Equal loading of RNA was confirmed by ethidium bromide staining. Transfer to nylon membranes (Hybond N, Amersham) was performed by vacuum blotting. Membranes were hybridized at 55°C overnight and then washed twice for 15 min at 55°C with 2× SSC containing 0.1% SDS.

Determination of Enzyme Activities

Protein extracts for determining GR and APX activity were prepared as described by Aono et al. (1995). GR and APX were assayed according to the method of Aono et al. (1991) and Nakano and Asada (1981), respectively. For the determination of MDAR and DHAR, leaf material was homogenized in the buffer system of Moran et al. (1994). MDAR and DHAR assays were conducted as described by Foyer et al. (1989) and Hossain and Asada (1984), respectively. Total SOD activity was determined as described by Kruse et al. (1995a). CAT was extracted as described by Moran et al. (1994). Extracts were applied to gel filtration on NAP 10 columns (Pharmacia) equilibrated with extraction buffer, and then enzyme activity was determined according to the method of Aebi (1984).

For the analysis of SOD isoforms, extracts were prepared and subjected to nondenaturing gel electrophoresis as described by Van Camp et al. (1994). Gels were stained according to the method of Beauchamp and Fridovich (1971) and documented with an imaging system (Vilber Lourmat, Marne La Vallée, France). Individual SOD isoforms were identified as described by Van Camp et al. (1994) and quantitated with the software package (Bio 1D) of the imaging system (Vilber Lourmat). All spectrophotometric assays were run on a diode array spectrophotometer (model DU 7400, Beckman) at 25°C.

Miscellaneous

Ascorbate was determined according to the procedures given by Law et al. (1983) and glutathione was determined as described by Smith et al. (1984). Protein content was determined according to the method of Bradford (1976) using BSA as the standard. Western analysis was performed as described by Kruse et al. (1995a). Monoclonal antibodies against spinach cytosolic APX (Saji et al., 1990) and an antiserum against rye CAT (Hertwig et al., 1992) were kindly provided by Dr. Saji (National Institute for Environmental Studies, Onagawa, Tsukuba, Japan) and Prof. Feierabend (Botanical Institute, University of Frankfurt, Frankfurt/Main, Germany), respectively.

Statistical Treatment

Plant material from three independent harvests was used for biochemical analysis. For each independent sample, analysis of enzyme activities and determination of low-molecular-weight antioxidants were performed at least in triplicate. Results from different harvests were combined by normalizing values for each wild-type leaf to 100%. Western and northern analyses were performed for each harvest; typical results are shown in the figures.

RESULTS

Tetrapyrrole Accumulation Is Accompanied by Changes in the Activity of Enzymes Involved in Oxidative Stress Defense

We have already reported the initial characterization of transgenic tobacco plants expressing antisense RNA for UROD (Mock and Grimm, 1997) and CPO (Kruse et al., 1995a). Lower activities of UROD or CPO led to the accumulation of large amounts of potentially phototoxic porphyrin(ogen)s. Compared with wild-type plants, the leaves of the transformants were smaller and contained wilted areas with necrotic lesions of whitish, desiccated tissue (Fig. 1). The lesions were not uniformly distributed over the leaf surface (Fig. 1, B and C).

Figure 1.

Figure 1

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Leaf 6 (counted from the top) of wild-type (SNN; left), UROD antisense (URODAS; middle), and CPO antisense (CPOAS; right) plants. Plants were grown for 10 weeks in soil at a light intensity of 300 μmol quanta PAR m−2 s−1. All photographs are shown at the same magnification.

In view of the well-established ability of porphyrins to trigger the light-dependent formation of reactive oxygen species, we anticipated an antioxidative stress response. Increases in total SOD activity in CPO (Kruse et al., 1995a) and UROD (data not shown) antisense plants were determined and compared with controls. The higher SOD activity could have resulted from the enhanced production of superoxide anions and would consequently demand sufficient capacity for detoxifying hydrogen peroxide to prevent the subsequent formation of more reactive oxygen species such as the hydroxyl radical (Asada, 1994). This prompted us to verify whether the activities of other enzymes of the oxidative stress defense system were stimulated in the porphyric UROD and CPO antisense plants (Fig. 2).

Figure 2.

Figure 2

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Activity of enzymes participating in the Halliwell-Asada pathway in the leaves of wild-type (white bars), UROD antisense (gray bars), and CPO antisense (black bars) plants. For purposes of comparison, the activities of the control leaves were always set at 100% and the data were averaged from three independent experiments. Leaves were counted from the top to the bottom. The following specific enzyme activities, given in nanokatals per milligram of protein, were determined in wild-type leaves: APX, 16.3 (minimum value in leaf 5) to 66.5 (maximum value in leaf 11); MDAR, 3.52 (minimum value in leaf 5) to 7.0 (maximum value in leaf 11); DHAR, 6.07 (minimum value in leaf 11) to 10.58 (maximum value in leaf 5); and GR, 0.58 (minimum value in leaf 5) to 1.25 (maximum value in leaf 11).

In the leaves of the two lines expressing UROD or CPO antisense RNA, the total activity of soluble APX was higher than in those of control plants in all of the developmental stages investigated (Fig. 2). Older leaves of the transformants especially showed up to 3-fold higher APX activity compared with the wild type (Fig. 2, see leaf 9 for UROD and leaf 11 for CPO). Increases in soluble APX activity could be attributed to plastidal or cytosolic isoforms (Asada, 1996). A severalfold increase in APX activity would necessitate the additional regeneration of ascorbate from MDA by MDAR. The activity of MDAR in wild-type leaves increased with leaf age. In all of the leaves of UROD and CPO antisense plants the MDAR activity was slightly higher than in corresponding leaves of wild-type plants (Fig. 2).

A second, ascorbate-regenerating system was also analyzed. DHAR activity in the leaves of wild-type plants was shown to either remain constant or decrease with leaf development in several independent experiments. The specific DHAR activities in leaf extracts of UROD or CPO antisense plants were lower than in the corresponding leaves of wild-type plants (Fig. 2). The regeneration of GSH was finally achieved by GR using reducing equivalents. Cytosolic, plastidal, and mitochondrial isoforms have been described (Edwards et al., 1990; Creissen et al., 1994). Total GR activities in wild-type tobacco leaves were always slightly higher in older than in younger leaves. GR activity was higher (6–54%) in all leaves of both UROD and CPO antisense plants (Fig. 2) than in wild-type plants.

Can Compartment-Specific Responses of the Antioxidative Protection System Be Distinguished in the Two Transgenic Lines with UROD or CPO Antisense Genes?

That the subcellular localization of UROD and CPO in plants is confined to the plastids has been proven by cell-fractionation studies (Smith et al., 1993) and by plastid translocation experiments with in vitro-synthesized precursor proteins of UROD or CPO (Kruse et al., 1995b; Mock et al., 1995). Therefore, uro(gen) or copro(gen) will initially accumulate in the plastids.

We were interested in distinguishing between the intensity and developmental course of the antioxidative response in the plastids and that in other cellular compartments. The various SOD isoforms in leaves of different ages from UROD antisense line 2 and control plants were assayed to compare temporal coordination between plastidal SOD and the activity of SOD isoforms in other compartments (Fig. 3). Total SOD activity for each extract calculated by summing the values of all isoforms was 5 to 57% higher in extracts of UROD antisense plants than in extracts of wild-type plants. Higher total SOD activities in UROD antisense plants were attributed to increased activities of mitochondrial Mn-SOD and cytosolic Cu/Zn-SOD. The levels of plastidal Cu/Zn-SOD isoforms were significantly higher only in leaf 5. Plastidal Fe-SOD activity was nearly doubled in leaf 5 but only slightly increased in older leaves of UROD antisense plants compared with the wild type. Similar results were obtained for CPO antisense plants (data not shown).

Figure 3.

Figure 3

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Analysis of SOD isoform activities in leaves of wild-type (white bars) and UROD antisense (gray bars) plants. Protein extracts were separated by native gel electrophoresis and stained for SOD activity. Quantitation of spots representing individual SOD isoforms was performed for n = 9 gels by image analysis with extracts from three independent series of experiments. SOD activity is given in relative units. Leaves were counted from the top to the base. cyt, Cytosolic form; pl, plastidal form.

We subsequently investigated the activity of CATs, which contribute to the cellular oxidative stress defense system by dismutating hydrogen peroxide to water and oxygen (Scandalios, 1994; Willekens et al., 1995). Most plant CATs have been localized in peroxisomes, with the exception of the CAT-3 isoform of maize (Scandalios, 1994). Using an antisense approach Chamnongpol et al. (1996) recently showed that a deficiency in CAT may lead to photosensitive plants with severe necrotic lesions. Total CAT activity increased with leaf age in wild-type plants (Fig. 4). Compared with wild-type controls, the total CAT activity was similar in younger leaves of the two transformants and decreased slightly in older leaves (Fig. 4). Western analysis with an antiserum against CAT revealed no differences in protein content between transgenic plants and controls (data not shown).

Figure 4.

Figure 4

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CAT activity in leaves of wild-type (white bars), UROD antisense (gray bars), and CPO antisense (black bars) plants. CAT activity was assayed spectrophotometrically by monitoring the initial decrease in hydrogen peroxide upon the addition of protein extract.

We concluded that an accumulation of photosensitive porphyrins led to a general stimulation of the enzymatic activities involved in the oxidative stress defense. This analysis indicates the generation of additional reactive oxygen species in UROD and CPO antisense plants.

Porphyrin-Induced Oxidative Stress Enhances Levels of RNA Encoding Detoxifying Enzymes

Higher activities of enzymes involved in oxidative stress defense in porphyrin-accumulating plants could result from changes in their gene expression. Transcript levels of antioxidative enzymes were rapidly modified upon exposure of the plants to various stress conditions (Tsang et al., 1991; Willekens et al., 1994b).

In wild-type leaves the transcripts of different SOD isoforms were most abundant in the youngest leaves and steadily declined during leaf development (Fig. 5A). With the exception of the plastidal Cu/Zn-SOD, higher levels of SOD mRNAs accumulated in the corresponding leaves of UROD or CPO antisense plants. The elevated gene expression found in the porphyric plants corresponded with higher activities of individual SOD isoenzymes (Fig. 3).

Figure 5.

Figure 5

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Northern analysis of antioxidant mRNA levels in leaves of wild-type (SNN), UROD antisense (UROD AS), and CPO antisense (CPO AS) plants. Equal amounts of RNA (10 μg) were separated on formaldehyde-containing agarose gels. Equal loading of RNA was checked by ethidium bromide staining. After RNA was blotted onto nylon membranes, hybridization was performed with specific probes for cytosolic (cyt. Cu/ZnSOD), mitochondrial (MnSOD), plastidal FeSOD (FeSOD), and plastidal Cu/ZnSOD (pl. Cu/ZnSOD; A); CAT (CAT 1–3) isoforms (B); and GPX (C). A representative blot from three independent experiments is shown.

Three classes of CAT isoforms with differential expression have been described for Nicotiana plumbaginifolia (Willekens et al., 1994a): CAT1 displayed high levels of expression in photosynthetically active tissues, CAT2 was preferentially expressed in vascular tissues, and CAT3 was preferentially expressed in seeds and young seedlings (Willekens et al., 1994a). Transcripts encoding these individual CATs exhibited differential responses to various stresses in N. plumbaginifolia (Willekens et al., 1994b). They were also increased in UROD and CPO antisense plants compared with controls (Fig. 5B). Differences in CAT mRNA abundance between transgenic and control plants were most pronounced in fully developed leaves. Increased mRNA levels for all of the CAT isoforms, coupled with unaltered or even reduced enzyme activity, could be best explained by the higher transcriptional activities required to balance higher protein turnover rates (Willekens et al., 1995). The sensitivity of CAT toward photoinactivation has been observed in vivo and in vitro (Feierabend and Engel, 1986; Gantchev and Vanlier, 1995). Salt-stress-suppressed translation strongly affected levels of CAT because of its elevated photosensitivity (Streb and Feierabend, 1996).

GPX mRNA levels were drastically increased in the two transgenic lines (Fig. 5C). The probe used in our experiments most likely encodes the phospholipid hydroperoxide GPX (Willekens et al., 1994b), and the increased transcript levels could reflect enhanced lipid peroxidation in our transformants.

Steady-state mRNA levels of cytosolic APX were not significantly altered in UROD or CPO antisense plants relative to wild-type plants (data not shown), despite the higher enzyme activities observed (Fig. 2). This observation resembles the salt-stress-induced increase in ascorbate activity in radish that also was not paralleled by differences in mRNA levels (Lopez et al., 1996). It was recently proposed that, in the presence of 3,4-dihydroxyphenolic compounds, guaiacol-type peroxidases could function as ascorbate-dependent, hydrogen-peroxide-detoxifying enzymes (Mehlhorn et al., 1996). Western analysis with a monoclonal antibody against cytosolic APX (Saji et al., 1990) demonstrated an increased protein content that was correlated with the increased APX activity (Fig. 6). Further analysis of APX isoforms by native gel electrophoresis would provide evidence for the contribution of each cellular APX isoform to the observed increase in total activity.

Figure 6.

Figure 6

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Western analysis of leaf extracts from wild-type (SNN), UROD antisense (URODAS), and CPO antisense (CPOAS) plants. Equal amounts of protein were separated on SDS-PAGE and transferred to membranes by semidry blotting. Membranes were immunostained using a monoclonal antibody against spinach cytosolic APX.

In summary, the strong increase in mRNA levels for most of the enzymes analyzed reflects increased transcriptional activity of genes for the antioxidative defense system in our transformants. Increased transcript levels for nonplastidal SOD isoforms and for CATs indicate that the stimulation of the cellular response was not confined to the plastids, as already indicated by increased activities of cytosolic and mitochondrial SOD isoforms (Fig. 3).

Levels of Low-Molecular-Weight Antioxidants

The cellular arsenal for scavenging reactive oxygen species and toxic organic radicals includes a number of small molecules such as ascorbate, GSH, tocopherol, and the carotenoids (Foyer, 1993; Hausladen and Alscher, 1993; Hess, 1993; Pallet and Young, 1993). Increased activities of SOD and APX in UROD and CPO antisense plants were accompanied by higher levels of the enzymes involved in the recycling of ascorbate. We analyzed total and reduced ascorbate and glutathione pools in wild-type and transgenic plants (Fig. 7). The total amount of ascorbate and glutathione varied depending on the plant and on leaf age. Ascorbate contents in the wild-type plants ranged from 2.77 to 5.95 μmol/g fresh weight in leaf 5 and declined steadily as the leaves grew older, declining to 1.17 to 3.19 μmol/g fresh weight in leaf 11. The two photosensitized transformants contained less ascorbate (Fig. 7). The ascorbate content was 13 to 25% lower in UROD antisense plants and 30 to 50% lower in CPO antisense plants.

Figure 7.

Figure 7

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Total ascorbate and glutathione contents and percentage of reduced ascorbate and GSH in leaves of wild-type (white bars), UROD antisense (URODAS; gray bars), and CPO antisense (CPOAS; black bars) plants. For purposes of comparison, the total ascorbate or glutathione content of the wild type was set at 100% for all leaves investigated. Data were averaged from three independent sets of experiments.

The decreased total ascorbate content in the leaves of the antisense plants was accompanied by a lower percentage of the reduced form (Fig. 7). The percentage of reduced ascorbate was even lower in CPO antisense than in UROD antisense plants. In parallel, a decrease in the ascorbate content was apparent in plants after wounding and was accompanied by increased activities of cytosolic MDAR (Grantz et al., 1995). This result is consistent with our findings of simultaneously increased MDAR activities and lowered ascorbate content in the porphyric plants (Fig. 3). Parallel to the decrease in ascorbate content, the two antisense lines showed diminished amounts of total glutathione and GSH (Fig. 7). In wild-type leaves the glutathione content varied from 400 to 500 nmol/g fresh weight in leaf 5 and declined steadily during leaf development, reaching 122 to 206 nmol/g fresh weight in leaf 11.

DISCUSSION

Transgenic Tobacco Plants with Reduced UROD or CPO Activity Display Similar Antioxidative Defense Responses

Transgenic tobacco plants with reduced UROD or CPO activity were characterized by severe leaf necrosis (Fig. 1; Kruse et al., 1995a; Mock and Grimm, 1997). Biochemical analysis revealed high levels of the porphyrinogen substrates or their photosensitizing oxidized forms (Kruse et al., 1995a; Mock and Grimm, 1997). As presented in this manuscript the selected transgenic lines showed an induction of a broad range of antioxidative stress defense reactions. The changes in the individual antioxidative components are remarkably similar in both UROD and CPO antisense plants, which is consistent with a general response mechanism against tetrapyrrole-induced oxidative stress. Both transgenic lines exhibited increases in mRNA levels for most of the enzymes involved in the detoxification of reactive oxygen (Fig. 5). Elevated levels of transcripts resulted in increased enzyme activities for the mitochondrial and cytosolic SOD isoforms (Fig. 3) but not for CAT, which in older leaves exhibits a decrease in total activity compared with the wild type (Fig. 4).

The stimulation of the enzymatic antioxidative defense system also led to increased activities of GR, MDAR, and APX (Fig. 2). Overexpression of GR in tobacco or poplar was accompanied by increased tolerance to oxidative stress conditions (Aono et al., 1991; Foyer et al., 1995). Therefore, we assume that the activation of genes and increased activities of protective enzymes were induced to counteract reactive oxygen species formed because of the photo-oxidation of accumulating tetrapyrroles.

Reduced Levels of Low-Molecular-Weight Antioxidants May Limit the Capacity of the Antioxidative Defense System in UROD and CPO Transformants

Previously, we demonstrated that CPO antisense plants contained lower levels of tocopherol (Kruse et al., 1995a); however, the level of protective carotenoids was not significantly changed in UROD (Mock and Grimm, 1997) and CPO (Kruse et al., 1995a) antisense plants. By analyzing low-molecular-weight antioxidants in the current study, we were able to demonstrate that the ascorbate and GSH levels are usually reduced in both transgenic lines (Fig. 7).

Several functions in the oxidative stress defense system have been ascribed to ascorbate (Foyer, 1993). We suggest that ascorbate is also important in keeping tetrapyrrolic intermediates such as uro(gen) and copro(gen) in their reduced form, thus preventing the formation of photodynamically active porphyrins. This is consistent with the observation that a diet supplemented with ascorbate can cure chemically induced uroporphyria in a rat mutant lacking the final enzyme for ascorbate synthesis (Sinclair et al., 1995). Moreover, it has been shown that the oxidation of uro(gen) to uro can be inhibited by ascorbate in vitro (Jacobs et al., 1996).

Our data indicate that the transformants have only a limited capacity to maintain the pool of low-molecular-weight antioxidants. This might be due to the limited capacity for de novo synthesis of the antioxidants and/or limitations in the supply of reducing equivalents. Investigations of chlorophyll fluorescence parameters, photosynthesis rates, and carbohydrate metabolism will show the capacity of the transformants to provide reducing equivalents necessary in the response against oxidative stress (Kehrer and Lund, 1994).

Is the Antioxidative Stress Response in UROD or CPO Antisense Plants Restricted to the Plastids?

We asked whether the accumulation of photodynamic tetrapyrroles would induce a local or a general stress response. At present we assume that the porphyrin(ogens) initially accumulate in the plastids. As long as the tetrapyrroles remain in this compartment, reactive oxygen species generated are probably scavenged by the plastidal protective system. However, accumulated tetrapyrroles may subsequently leak out of the plastids and then be distributed into and photosensitize other cellular compartments. In analogy, the application of diphenyl-ether-type herbicides caused the accumulation of photodestructive protoporphyrin IX in the cytoplasm (Lehnen et al., 1990).

The increased levels of transcripts encoding SOD and CAT isoforms and the higher content of cytosolic APX protein in the transformants reflected an induction of a general antioxidative response rather than of a plastid-specific one. To be consistent with the hypothesis of a local stress response (Alscher et al., 1997), the actual increase of CAT RNA or of several nonplastidal SOD isoforms would require the distribution of porphyrins and/or reactive oxygen species throughout the cell. Hydrogen peroxide, for example, is able to cross the membranes of several compartments and is thought to be involved in signaling processes, e.g. during plant-pathogen interaction (Mehdy, 1996). Alternatively, changes in the pro-oxidant/antioxidant state in the plastids might trigger an antioxidative response in other compartments. Transcript levels of cytosolic Cu/Zn-SOD of pine (Pinus sylvestris; Wingsle and Karpinski, 1996) and Nicotiana (Hérouart et al., 1993) were shown to be regulated by the cellular redox state. At present the question of how photosensitzation of tetrapyrroles mediates the signals for the cellular antioxidative response remains open. Future studies must aim to localize the subcellular distribution of accumulated tetrapyrroles in UROD or CPO antisense plants.

Oxidative Stress Responses in UROD and CPO Antisense Plants: A Comparison with Other Systems Accumulating Photosensitive Tetrapyrroles

The deleterious effects of accumulated tetrapyrroles are well documented in porphyric diseases caused by inherited defects (Elder and Roberts, 1995) or induced by chemicals such as hexachlorbenzene (Ockner and Schmidt, 1961). Deficiency mutations of UROD have been described in humans and are similar to our antisense plants (Elder and Roberts, 1995, and refs. therein), Escherichia coli (Sasarman et al., 1975), and yeast (Kurlandzka et al., 1988). Patients with porphyria disease may suffer from severe light sensitivity as a consequence of accumulated tetrapyrroles. To our knowledge, mutants defective in UROD have not yet been presented in higher plants. One of the first detectable alterations of cesium-chloride-treated barley seedlings was the accumulation of uro(gen) as a result of UROD inhibition and subsequent photodynamic damage to the leaves (Shalygo et al., 1997).

The mode of action of cesium on UROD is not fully understood, but its inhibitory effect can be reversed by high doses of potassium salts, indicating the involvement of a potassium channel (N.V. Shalygo and H.-P. Mock, unpublished data). The accumulation of photosensitive tetrapyrrole intermediates was also provoked in plants by the application of diphenyl-ether-type herbicides affecting protoporphyrinogen oxidase (Witkowski and Halling, 1988) or by feeding ALA (Rebeiz et al., 1984). However, increased tolerance to the photodynamic herbicides has been attributed to an improved detoxification of reactive oxygen species.

Analysis of several tobacco lines for susceptibility toward acifluorfen revealed that the more resistant lines were characterized by a higher a priori ascorbate content but not by increased activities of antioxidative enzymes (Gullner et al., 1991). This indicates that a constitutive level of ascorbate rather than enzymatic antioxidants may be more important in protecting plants from such a stress. However, after treatment with the herbicide the tolerant plants also showed stronger induction of GR activity (Gullner et al., 1991). In contrast, cucumber (Cucumis sativus) cotyledons treated with acifluorfen showed strong and immediate decreases in the levels of ascorbate and GSH and, simultaneously, in the activity of antioxidative enzymes (Kenyon and Duke, 1985). The differences in the response toward acifluorfen observed between tobacco and cucumber may be explained by a species-specific toxicity of the applied herbicide. Acifluorfen treatment resulted in very rapid damage to the cucumber seedlings, whereas tobacco showed visible injury only after several days.

The addition of acifluorfen to photoauxotrophic soybean (Glycine max) cell cultures prompted a dose-dependent response of the antioxidative defense system (Knörzer et al., 1996). The ascorbate content was increased at low concentrations of herbicide but was lower than in controls when high doses were applied (Knörzer et al., 1996). The activities of APX, GR, and MDAR increased with the acifluorfen concentration. DHAR activity was increased at low doses of the herbicide but decreased at higher doses. This enzyme could be more sensitive to the oxidative stress imposed by tetrapyrroles. The results obtained with soybean cell cultures are more similar to the response pattern of the antioxidative defense of UROD and CPO antisense plants. Differences in the antioxidative response between herbicide-treated plants and UROD or CPO antisense transformants could be explained by different exposure times and photodynamic effects of the accumulated porphyrins. The phototoxicity of protoporphyrin IX might be higher than that of uro and copro (Menon et al., 1989). Also, UROD and CPO antisense plants are faced with accumulating photosensitive tetrapyrroles throughout their entire development, whereas the herbicide was applied only at a certain developmental stage of the plants.

The gradual inhibition of target enzyme expression in the tetrapyrrole pathway by different intensities of antisense RNA synthesis facilitates the selection of appropriate transgenic lines for our studies. UROD and CPO antisense plants are characterized by their differential sensitivity to light intensities. The transgenic lines can be propagated under low-light conditions that do not lead to extensive lesion formation (Mock and Grimm, 1997), whereas the transfer to high light induces cell death (H.-P. Mock, unpublished observations).

Transformants that induce the synthesis and stimulate the activity of protective enzymes against reactive oxygen species provide a model system for investigating tetrapyrrole-induced oxidative stress. The antioxidative stress response resembles that of plants treated with herbicides that provoke the accumulation of photosensitizing tetrapyrroles (Dodge, 1994). These systems rely on the application of xenobiotic agents, and the plants' responses include detoxification of the herbicides (Dodge, 1994). Analysis of transgenic plants that induce necrosis formation after transfer from low- to high-light growth conditions will allow us to analyze the kinetics of cellular responses caused by photodynamically active tetrapyrroles.

ACKNOWLEDGMENTS

We thank Elena Barthel for excellent technical assistance. Grants from the Deutsche Forschungsgemeinschaft to B. Grimm are gratefully acknowledged. Prof. Dr. J. Feierabend (Frankfurt/Main, Germany) and Dr. H. Saji (Onagawa, Tsukuba, Japan) are thanked for their generous gifts of antisera. We also thank Prof. Dr. D. Inzé (Gent, Belgium) for providing cDNA clones and Dr. Christian Langebartels (München, Germany) and Dr. H. Härtel (Gatersleben, Germany) for critical reading of the manuscript.

Abbreviations:

ALA

δ-aminolevulinate

APX

ascorbate peroxidase

CAT

catalase

CPO

coproporphyrinogen oxidase

copro(gen)

coproporphyrin(ogen)

DHAR

dehydroascorbate reductase

GPX

glutathione peroxidase

GR

glutathione reductase

MDA

monodehydroascorbate

MDAR

monodehydroascorbate reductase

SOD

superoxide dismutase

UROD

uroporphyrinogen decarboxylase

uro(gen)

uroporphyrin(ogen)

Footnotes

1

This work was partially supported by grants from the Deutsche Forschungsgemeinschaft (nos. 936/3-1 and 936/4-1).

LITERATURE  CITED

  1. Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–126. doi: 10.1016/s0076-6879(84)05016-3. [DOI] [PubMed] [Google Scholar]
  2. Alscher RG, Donahue JL, Cramer CL. Reactive oxygen species and antioxidants: relationships in green cells. Physiol Plant. 1997;100:224–233. [Google Scholar]
  3. Aono M, Kubo A, Saji H, Aatori T, Tanaka K, Kondo N. Resistance to active oxygen toxicity of transgenic Nicotiana tabacum that expresses the gene for glutathione reductase from Escherichia coli. Plant Cell Physiol. 1991;32:691–697. [Google Scholar]
  4. Aono M, Saji H, Fujiyama K, Sugita M, Kondo N, Tanaka K. Decrease in activity of glutathione reductase enhances paraquat sensitivity in transgenic Nicotiana tabacum. Plant Physiol. 1995;107:645–648. doi: 10.1104/pp.107.2.645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Arakane K, Ryu A, Hayashi C, Masunaga T, Shinmoto K, Mashiko S, Nagao T, Hirobe M. Singlet oxygen (1Δg) generation from coproporphyrin in Propionibacterium acnes on irradiation. Biochem Biophys Res Commun. 1996;223:578–582. doi: 10.1006/bbrc.1996.0937. [DOI] [PubMed] [Google Scholar]
  6. Asada K. Induction and action of active oxygen species in photosynthetic tissues. In: Foyer C, Mullineaux P, editors. Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. Boca Raton, FL: CRC Press; 1994. pp. 77–104. [Google Scholar]
  7. Asada K. Radical production and scavenging in the chloroplasts. In: Baker NR, editor. Photosynthesis and the Environment. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1996. pp. 123–150. [Google Scholar]
  8. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971;44:276–287. doi: 10.1016/0003-2697(71)90370-8. [DOI] [PubMed] [Google Scholar]
  9. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  10. Chamnongpol S, Willekens H, Langebartels C, Van Montagu M, Inzé D, Van Camp W. Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light. Plant J. 1996;10:491–503. [Google Scholar]
  11. Creissen GP, Edwards EA, Mullineaux PM. Glutathione reductase and ascorbate peroxidase. In: Foyer CH, Mullineaux PM, editors. Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. Boca Raton, FL: CRC Press; 1994. pp. 343–364. [Google Scholar]
  12. Dodge AD. Herbicide action and effects on detoxification processes. In: Foyer C, Mullineaux P, editors. Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. Boca Raton, FL: CRC Press; 1994. pp. 219–236. [Google Scholar]
  13. Edwards EA, Rawsthorne S, Mullineaux PM. Subcellular distribution of multiple forms of glutathione reductase in leaves of pea (Pisum sativum L.) Planta. 1990;180:278–284. doi: 10.1007/BF00194008. [DOI] [PubMed] [Google Scholar]
  14. Elder GH, Roberts AG. Uroporphyrinogen decarboxylase. J Bioenerg Biomembr. 1995;27:207–214. doi: 10.1007/BF02110035. [DOI] [PubMed] [Google Scholar]
  15. Feierabend J, Engel S. Photoinactivation of catalase in vitro and in leaves. Arch Biochem Biophys. 1986;251:567–576. doi: 10.1016/0003-9861(86)90365-6. [DOI] [PubMed] [Google Scholar]
  16. Foyer CH. Ascorbic acid. In: Alscher RG, Hess JL, editors. Antioxidants in Higher Plants. Boca Raton, FL: CRC Press; 1993. pp. 31–58. [Google Scholar]
  17. Foyer CH, Dujardyn M, Lemoine Y. Responses of photosynthesis and the xanthophyll and ascorbate-glutathione cycles to changes in irradiance, photoinhibition and recovery. Plant Physiol Biochem. 1989;27:751–760. [Google Scholar]
  18. Foyer CH, Lelandais M (1993) The roles of ascorbate in the regulation of photosynthesis. In HY Yamamoto, ed, Photosynthetic Responses to the Environment. American Society of Plant Physiologists, Rockville, MD, pp 88–101
  19. Foyer CH, Lelandais M, Kunert KJ. Photooxidative stress in plants. Physiol Plant. 1994;92:696–717. [Google Scholar]
  20. Foyer CH, Sourjau N, Perret S, Lelandais M, Kunert KJ, Pruvost C, Jouanin L. Overexpression of glutathione reductase but not glutathione synthetase leads to increases in antioxidant capacity and resistance to photoinhibition in poplar trees. Plant Physiol. 1995;109:1047–1057. doi: 10.1104/pp.109.3.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gantchev TG, Vanlier JE. Catalase inactivation following photosensitization with tetrasulfonated metallophthalocyanines. Photochem Photobiol. 1995;62:123–134. doi: 10.1111/j.1751-1097.1995.tb05248.x. [DOI] [PubMed] [Google Scholar]
  22. Grantz AA, Brummell DA, Bennett AB. Ascorbate free radical reductase mRNA levels are induced by wounding. Plant Physiol. 1995;108:411–418. doi: 10.1104/pp.108.1.411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gullner G, Komivés T, Király L. Enhanced inducibility of antioxidant systems in a Nicotiana tabacum L. biotype results in acifluorfen resistance. Z Naturforsch. 1991;46c:875–881. [Google Scholar]
  24. Hausladen A, Alscher RG. Glutathione. In: Alscher RG, Hess JL, editors. Antioxidants in Higher Plants. Boca Raton, FL: CRC Press; 1993. pp. 1–30. [Google Scholar]
  25. Heber U, Miyake C, Mano J, Ohno C, Asada K. Monodehydroascorbate radical detected by electron paramagnetic resonance spectrometry is a sensitive probe of oxidative stress in intact leaves. Plant Cell Physiol. 1996;37:1066–1072. [Google Scholar]
  26. Hérouart D, Van Montagu M, Inzé D. Redox-activated expression of the cytosolic copper/zinc superoxide dismutase gene in Nicotiana. Proc Natl Acad Sci USA. 1993;90:3108–3112. doi: 10.1073/pnas.90.7.3108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hertwig B, Streb P, Feierabend J. Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions. Plant Physiol. 1992;100:1547–1553. doi: 10.1104/pp.100.3.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hess JL. Vitamin E, α-Tocopherol. In: Alscher RG, Hess JL, editors. Antioxidants in Higher Plants. Boca Raton, FL: CRC Press; 1993. pp. 111–134. [Google Scholar]
  29. Hossain MA, Asada K. Purification of dehydroascorbate reductase from spinach and its characterization as a thiol enzyme. Plant Cell Physiol. 1984;25:85–92. [Google Scholar]
  30. Jacobs JM, Jacobs NJ, Kuhn CB, Gorman N, Dayan FE, Duke SO, Sinclair JF, Sinclair PR. Oxidation of prophyrinogens by horseradish peroxidase and formation of a green pyrrole pigment. Biochem Biophys Res Commun. 1996;227:195–199. doi: 10.1006/bbrc.1996.1488. [DOI] [PubMed] [Google Scholar]
  31. Kehrer JP, Lund LG. Cellular reducing equivalents and oxidative stress. Free Radical Biol Med. 1994;17:65–75. doi: 10.1016/0891-5849(94)90008-6. [DOI] [PubMed] [Google Scholar]
  32. Kenyon WH, Duke SO. Effects of acifluorfen on endogenous antioxidants and protective enzymes in cucumber (Cucumissativus L.) cotyledons. Plant Physiol. 1985;79:862–866. doi: 10.1104/pp.79.3.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Knörzer OC, Durner J, Böger P. Alterations in the antioxidant system of suspension-cultured soybean cells (Glycinemax) induced by oxidative stress. Physiol Plant. 1996;97:388–396. [Google Scholar]
  34. Kruse E, Mock H-P, Grimm B. Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative stress defense system. EMBO J. 1995a;14:3712–3720. doi: 10.1002/j.1460-2075.1995.tb00041.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kruse E, Mock H-P, Grimm B. Coproporphyrinogen III oxidase from barley and tobacco—sequence analysis and initial expression studies. Planta. 1995b;196:796–803. [PubMed] [Google Scholar]
  36. Kurlandzka A, Zoladek T, Rytka J, Labbe-Bois R, Urban-Grimal D. Biochem J. 1988;253:109–116. doi: 10.1042/bj2530109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Law MY, Charles SA, Halliwell B. Glutathione and ascorbic acid in spinach (Spinacia oleracea) chloroplast. The effect of hydrogen peroxide and of paraquat. Biochem J. 1983;210:899–903. doi: 10.1042/bj2100899. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lehnen LP, Sherman TD, Becerril JM, Duke SO. Tissue and cellular localization of acifluorfen-induced prophyrins in cucumber cotyledons. Pestic Biochem Physiol. 1990;37:329–248. [Google Scholar]
  39. Lopez F, Vansuyt G, Casse-Delbart F, Fourcroy P. Ascorbate peroxidase activity, not the mRNA level, is enhanced in salt-stressed Raphanus sativus plants. Physiol Plant. 1996;97:13–20. [Google Scholar]
  40. Mehdy MC, Sharma YK, Sathasivan K, Bays NW. The role of activated oxygen species in plant disease resistance. Physiol Plant. 1996;98:365–374. [Google Scholar]
  41. Mehlhorn H, Lelandais M, Korth HG, Foyer CH. Ascorbate is the natural substrate for plant peroxidases. FEBS Lett. 1996;378:203–206. doi: 10.1016/0014-5793(95)01448-9. [DOI] [PubMed] [Google Scholar]
  42. Menon IA, Persad SD, Haberman HF. A comparison of the phototoxicity of protoporphyrin, coproporphyrin and uroporphyrin using a cellular system in vitro. Clin Biochem. 1989;22:197–200. doi: 10.1016/s0009-9120(89)80077-3. [DOI] [PubMed] [Google Scholar]
  43. Miyake C, Asada K. Thylakoid-bound ascorbate peroxidase in spinach chloroplasts and photoreduction of its primary oxidation product the monodehydroascorbate radicals in thylakoids. Plant Cell Physiol. 1992;33:541–553. [Google Scholar]
  44. Mock H-P, Grimm B. Reduction of uroporphyrinogen decarboxylase by antisense RNA expression affects activities of other enzymes involved in tetrapyrrole biosynthesis and leads to light-dependent necrosis. Plant Physiol. 1997;113:1101–1112. doi: 10.1104/pp.113.4.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mock H-P, Trainotti L, Kruse E, Grimm B. Isolation, sequencing and expression of cDNA sequences encoding uroporphyrinogen decarboxylase from tobacco and barley. Plant Mol Biol. 1995;28:245–256. doi: 10.1007/BF00020244. [DOI] [PubMed] [Google Scholar]
  46. Moran JF, Becana M, Iturbe-Ormatexe I, Fechilla S, Klucas RV, Aparicio-Tejo P. Drought induces oxidative stress in pea plants. Planta. 1994;194:346–352. [Google Scholar]
  47. Nakano Y, Asada K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 1981;22:867–880. [Google Scholar]
  48. Ockner RK, Schmid R. Acquired porphyria in man and rat due to hexachlorbenzene intoxication. Nature. 1961;189:499. doi: 10.1038/189499a0. [DOI] [PubMed] [Google Scholar]
  49. Pallet KE, Young AJ. Carotenoids. In: Alscher RG, Hess JL, editors. Antioxidants in Higher Plants. Boca Raton, FL: CRC Press; 1993. pp. 59–89. [Google Scholar]
  50. Rebeiz CA, Montazer-zouhoor A, Hopen HJ, Wu SM. Photodynamic herbicides. I. Concept and phenomenology. Enzyme Microbiol Technol. 1984;6:390–401. [Google Scholar]
  51. Saji H, Tanaka K, Kondo N. Monoclonal antibodies to spinach ascorbate peroxidase and immunochemical detection of the enzyme in eight different plant species. Plant Sci. 1990;69:1–9. [Google Scholar]
  52. Sambrook J, Frisch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989. [Google Scholar]
  53. Sasarman A, Chartrand P, Proschek R, Desrochers M, Tardif D, Lapointe C. Uroporphyrin-accumulating mutants of Escherichiacoli K-12. J Bacteriol. 1975;124:1205–1212. doi: 10.1128/jb.124.3.1205-1212.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Scandalios JG. Oxygen stress and superoxide dismutases. Plant Physiol. 1993;101:7–12. doi: 10.1104/pp.101.1.7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Scandalios JG. Regulation and properties of plant catalases. In: Foyer CH, Mullineaux PM, editors. Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants. Boca Raton, FL: CRC Press; 1994. pp. 275–315. [Google Scholar]
  56. Shalygo NV, Averina NG, Grimm B, Mock H-P. Influence of cesium on tetrapyrrole biosynthesis in etiolated and greening barley leaves. Physiol Plant. 1997;99:160–168. [Google Scholar]
  57. Sinclair PR, Gorman N, Sinclair JF, Walton HS, Bement WJ, Lambrecht RW. Ascorbic acid inhibits chemically induced uroporphyria in ascorbate-requiring rats. Hepatology. 1995;22:565–572. [PubMed] [Google Scholar]
  58. Smith AG, Marsh O, Elder GH. Investigation of the subcellular location of the tetrapyrrole-biosynthesis enzyme coproporphyrinogen oxidase in higher plants. Biochem J. 1993;292:503–508. doi: 10.1042/bj2920503. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Smith IK, Kendall AC, Keys AJ, Turner JC, Lea PJ. Increased levels of glutathione in a catalase-deficient mutant of barley (Hordeum vulgare L.) Plant Sci Lett. 1984;37:29–33. [Google Scholar]
  60. Streb P, Feierabend J. Oxidative responses accompanying photoincactivation of catalase in NaCl-treated rye leaves. Bot Acta. 1996;109:125–132. [Google Scholar]
  61. Tsang EWT, Bowler C, Hérouart D, Van Camp W, Villaroel R, Genetello C, Van Montagu M, Inzé D. Differential regulation of superoxide dismutases in plants exposed to environmental stress. Plant Cell. 1991;3:783–792. doi: 10.1105/tpc.3.8.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Van Camp W, Willekens H, Bowler C, Van Montagu M, Inzé D, Reupold-Popp P, Sanderman H, Langebartels C. Elevated levels of superoxide dismutase protect transgenic plants against ozone damage. Biotechnology. 1994;12:165–168. [Google Scholar]
  63. Willekens H, Inzé D, Van Montagu M, Van Camp W. Catalases in plants. Mol Breed. 1995;1:207–228. [Google Scholar]
  64. Willekens H, Langebartels C, Tiré C, Van Montagu M, Inzé D, Van Camp W. Differential expression of catalase genes in Nicotiana plumbaginifolia (L.) Proc Natl Acad Sci USA. 1994a;91:10450–10454. doi: 10.1073/pnas.91.22.10450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Willekens H, Van Camp W, Van Montagu M, Inzé D, Langebartels C, Sandermann H. Ozone, sulfur dioxide, and ultraviolet B have similar effects on mRNA accumulation of antioxidant genes in Nicotiana plumbaginifolia L. Plant Physiol. 1994b;106:1007–1014. doi: 10.1104/pp.106.3.1007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Wingsle G, Karpinski S. Differential redox regulation by glutathione of glutathione reductase and CuZn-superoxide dismutase gene expression in Pinus sylvestris L. needles. Planta. 1996;198:151–157. doi: 10.1007/BF00197598. [DOI] [PubMed] [Google Scholar]
  67. Witkowski DA, Halling BP. Accumulation of photodynamic tetrapyrroles induced by acifluorfen-methyl. Plant Physiol. 1988;87:632–637. doi: 10.1104/pp.87.3.632. [DOI] [PMC free article] [PubMed] [Google Scholar]

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