Plant Physiol. (1998) 116: 107-116
Defense Responses to Tetrapyrrole-Induced Oxidative Stress in
Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or
Coproporphyrinogen Oxidase Activity1
Hans-Peter Mock,
Ulrich Keetman,
Elisabeth Kruse,
Barbara Rank, and
Bernhard Grimm*
Institut für Pflanzengenetik und Kulturpflanzenforschung,
Corrensstrasse 3, D-06466 Gatersleben, Germany (H.-P.M.,
U.K., E.K., B.G.); and Institut für Biologie,
Humboldt-Universität zu Berlin, Unter den Linden 6 (Sitz:Philippstrasse 13), D-10099 Berlin, Germany (U.K.,
B.R.)
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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.
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INTRODUCTION |
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.
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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 s1). 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).
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| Figure 1.
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 m2 s1.
All photographs are shown at the same magnification.
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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).
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| Figure 2.
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).
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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).
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| Figure 3.
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.
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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).
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| Figure 4.
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.
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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).
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| Figure 5.
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.
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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.
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| Figure 6.
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.
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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.
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| Figure 7.
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.
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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.
| |
FOOTNOTES |
1
This work was partially supported by grants from
the Deutsche Forschungsgemeinschaft (nos. 936/3-1 and 936/4-1).
*
Corresponding author; e-mail grimm{at}ipk-gatersleben.de; fax
49-39-482-5136.
Received June 5, 1997;
accepted October 6, 1997.
| |
ABBREVIATIONS |
Abbreviations:
ALA, 
-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).
| |
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.
| |
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