Plant Physiology 132:1335-1343 (2003)
© 2003 American Society of Plant Biologists
ENVIRONMENTAL STRESS AND ADAPTATION
Potato Plants Lacking the CDSP32 Plastidic Thioredoxin Exhibit Overoxidation of the BAS1 2-Cysteine Peroxiredoxin and Increased Lipid Peroxidation in Thylakoids under Photooxidative Stress
Mélanie Broin and
Pascal Rey*
Commissariat à l'Energie Atomique (CEA)/Cadarache, Direction des
Sciences du Vivant, Département d'Ecophysiologie Végétale
et de Microbiologie, Laboratoire d'Ecophysiologie de la Photosynthèse,
13108 Saint-Paul-lez-Durance cedex, France
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ABSTRACT
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The CDSP32 protein (chloroplastic drought-induced stress protein of 32 kD)
is a thioredoxin participating in the defense against oxidative damage. We
recently have identified in vitro the BAS1 2-Cys peroxiredoxin, a
peroxide-detoxifying enzyme, as a target for CDSP32. Here, we report the
characterization under stress conditions of transgenic potato (Solanum
tuberosum) plants lacking CDSP32 with regard to the BAS1 redox state and
the level of lipid peroxidation. Under control conditions, BAS1 is present at
similar levels both in wild-type (WT) and transgenic plants. Under drought and
methyl viologen treatment, CDSP32-lacking plants display, compared with WT, an
increased proportion of BAS1 monomer corresponding to an overoxidized form of
the protein. Leaf discs from transgenic plants treated with methyl viologen
exhibit earlier degradation of BAS1 than WT plants do. Using several
approaches, i.e. a probe emitting fluorescence when reacting with peroxides,
high-performance liquid chromatography determination of lipid hydroxy fatty
acid content, and measurement of chlorophyll thermoluminescence, we show a
higher lipid peroxidation level under methyl viologen treatment in thylakoids
from CDSP32-lacking plants compared with WT. These data show that CDSP32 is a
critical component in the defense system against lipid peroxidation in
photosynthetic membranes, likely as a physiological electron donor to the BAS1
peroxiredoxin.
Thioredoxins are proteins present in all organisms from bacteria to human
(Homo sapiens). Despite different amino acid sequences, they display
a typical folding and are characterized by a conserved redox active center,
Cys-Gly-Pro-Cys, able to reduce disulfide bridges of target proteins
(Eklund et al., 1991 ).
Initially described as electron carriers in ribonucleotide reduction in
Escherichia coli, thioredoxins serve as redox carriers in a wide
variety of physiological processes
(Arnér and Holmgren,
2000). In bacteria, yeast (Saccharomyces cerevisiae), and
animal cells, most thioredoxin genes are induced by oxidative conditions
(Kuge and Jones, 1994;
Paget et al., 1998;
Ritz et al., 2000), and
numerous reports have shown the participation of these proteins in oxidative
stress responses (Muller,
1991; Takemoto et al.,
1998). In various organisms, thioredoxins are involved
particularly in the protection against oxidative stress as electron donors for
thioredoxin peroxidases (or peroxiredoxins), which detoxify hydrogen peroxide
(H2O2) and alkyl hydroperoxides (Chae et al.,
1994a,
1994b;
Poole et al., 2000;
Goyer et al., 2002).
Three main types of thioredoxins with multiple isoforms have been described
in plants. The h-type is located in cytosol and likely participates
in different processes such as seed germination, plant reproduction, cell
communication, and cell division (Mouaheb
et al., 1998; Meyer et al.,
1999). The plastidic f- and m-types are involved
in the control of the enzyme activities of CO2 fixation cycle in
relation to light intensity
(Schürmann and Jacquot,
2000). In contrast to other organisms, the participation of plant
thioredoxins in oxidative stress responses has only emerged recently. Verdoucq
et al. (1999) showed that
Arabidopsis h3 thioredoxin was able to interact
with a yeast peroxiredoxin. Mouhaeb et al. (1998) and Issakidis-Bourguet et
al. (2001) reported that
thioredoxins h3 and m from Arabidopsis
could complement the H2O2 hypersensitivity of a yeast
thioredoxin-deficient mutant. In the past few years, we identified another
type of plastidic thioredoxin, induced by drought and oxidative stress
conditions (Pruvot et al.,
1996; Rey et al.,
1998; Broin et al.,
2000). The protein, designated CDSP32 (chloroplastic
drought-induced stress protein of 32 kD), is composed of two thioredoxin
domains with only one active redox disulfide center in the C-terminal part
(Rey et al., 1998). Transgenic
potato (Solanum tuberosum) plants lacking the CDSP32 protein due to a
cosuppression phenomenon displayed an increased susceptibility to
photooxidative stress conditions (Broin et
al., 2002). In vitro incubation assays and affinity chromatography
indicated that the 2-Cys peroxiredoxin BAS1 is likely a main target for CDSP32
(Broin et al., 2002). In plant
cells, the 2-Cys peroxiredoxin type is localized in chloroplasts where it
participates in the antioxidant defense system (Baier and Dietz,
1997,
1999;
Baier et al., 2000). Based on
in vitro assays, the protein, which is functional as a homodimer, has been
shown to reduce H2O2 and alkyl hydroperoxides
(König et al., 2002).
In the present study, we further characterized transgenic potato plants
lacking the CDSP32 thioredoxin to gain insight about its function in the
protection against oxidative damage. We report that, compared with wild type
(WT), transgenic plants displayed under stress conditions substantial changes
with regards to the redox state of the BAS1 peroxiredoxin and to the level of
lipid peroxidation. The participation of CDSP32 in the detoxification of lipid
hydroperoxides is discussed in relation to a function of electron donor to the
BAS1 peroxiredoxin.
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RESULTS
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BAS1 Abundance in the Different Organs of WT and CDSP32-Lacking
Plants
In WT plants grown under control conditions, the CDSP32 thioredoxin was
present in leaves and not detected in other organs (flowers, stems, tubers,
and roots; Fig. 1A). In the
transgenic lines previously shown to lack the thioredoxin in leaves
(Broin et al., 2002), the
protein was also found to be absent in other organs
(Fig. 1A), even when loading
larger amounts of protein (data not shown). The BAS1 peroxiredoxin abundance
was compared in proteins of WT and transgenic lines separated under reducing
conditions. A high abundance of the BAS1 23-kD monomer was observed in all
organs of both plant types (Fig.
1B). Note that WT and transgenic plants displayed a similar
pattern with regard to BAS1 abundance in the different organs, the highest
level being observed in leaves.
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Figure 1. Immunoblot analysis of CDSP32 and BAS1 abundances in the different organs
of WT and CDSP32-lacking potato lines. Soluble proteins were separated by
SDS-PAGE under reducing conditions. Proteins (10 µg) from WT potato and a
CDSP32-lacking (CDSP32–) plant (D4 line) were analyzed using the CDSP32
(A) and BAS1 (B) antisera diluted 1:1,000 (v/v) and 1:10,000 (v/v),
respectively. The bands corresponding to CDSP32 and BAS1 were revealed at 32
and 23 kD, respectively. WF, Whole flowers; L, leaves; S, stems; T, tubers; R,
roots.
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Abundance and Redox State of BAS1 in Leaves under Drought Stress
We investigated the BAS1 abundance and redox state in whole potato plants
subjected to water deficit, down to a leaf relative water content of around
65%. Under these conditions, the CDSP32 amount increased in leaves of WT
compared with control conditions as previously reported
(Rey et al., 1998), but the
protein was not detected in transgenic lines (data not shown). In leaf
proteins from well-watered plants separated under reducing conditions, a
similar abundance of BAS1 peroxiredoxin was noticed both in WT and in
CDSP32-lacking lines (Fig. 2B).
Under drought conditions, no change in BAS1 abundance was noticed in WT,
whereas a level reduced by around one-third (image analysis performed using
Genetools) was observed in the two lines deprived of CDSP32
(Fig. 2B). Proteins were
separated in the absence of reducing agent to quantify the proportion of BAS1
dimer, which corresponds to the functional form of the protein
(Dietz et al., 2002). Under
well-watered conditions, BAS1 was found mainly as a dimer at around 43 to 45
kD (around 87% and 80% in WT and transgenic lines, respectively;
Fig. 2, C and D). Due to the
presence of one or two disulfide bridges, two dimer forms with distinct
electrophoretic mobility were revealed as reported by Dietz et al.
(2002). Although a slight
decrease in the proportion of BAS1 dimer (down to 77%) was noticed in WT under
drought, the proportion of this form was strongly reduced (around 53%) in
water-stressed transgenic plants, particularly in the D4 line
(Fig. 2, C and D). Note that in
our experiments, no BAS1 oligomerization was observed under stress, conversely
to the data reported by König et al.
(2002) in barley (Hordeum
vulgare). In thylakoids of wilted potato plants, BAS1 was found only as a
dimer, and its relative abundance was much lower compared with that observed
in soluble proteins (data not shown).
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Figure 2. Immunoblot analysis of the BAS1 abundance and redox state in leaves from WT
and CDSP32-lacking potato lines under drought stress. Leaf soluble proteins
from whole plants (A–C) were separated by SDS-PAGE under reducing (A and
B) or nonreducing (C) conditions. Proteins (5 and 15 µg in reducing and
nonreducing conditions, respectively) were analyzed using the Rubisco (A) and
BAS1 (B and C) antisera diluted 1:4,000 (v/v) and 1:10,000 (v/v),
respectively. The Rubisco immunoblot is presented to show homogeneous loading
of lanes. The bands corresponding to large Rubisco subunit and BAS1 dimer and
monomer were revealed at around 50, 43 to 45, and 23 kD, respectively. Two WT
plants and two CDSP32-lacking (D4 and D18) plants were either grown under
control conditions (H2O+) or subjected to water shortage for 10 d
(H2O–). D, Percentage of BAS1 protein present as a dimer in
leaf-soluble proteins. The data were obtained by image analysis, using
Genetools (Syngene, Cambridge, UK), of immunoblot of proteins separated under
nonreducing conditions (as in C). The values presented were calculated from
data originating from five independent plants per treatment. Tr, D4 and D18
plants.
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Abundance and Redox State of BAS1 in Leaf Discs Subjected to
Photooxidative Stress
We reported previously that leaf discs from CDSP32-lacking plants exposed
to light in the presence of the photooxidizing PSI acceptor, methyl viologen
(Babbs et al., 1989), exhibit
compared with WT increased damage within the photosynthetic apparatus
(Broin et al., 2002). Western
analyses were carried out to investigate the abundance and redox state of the
BAS1 peroxiredoxin after a 4-h period of light exposure in the presence of
methyl viologen. At this stage, leaf discs from transgenic plants started to
exhibit photooxidative damage, whereas WT discs displayed almost no stress
symptoms (data not shown). When proteins were separated under reducing
conditions, a similar BAS1 abundance was revealed both in WT and in transgenic
discs incubated on water (Fig.
3A). Under nonreducing conditions, much lower percentages of BAS1
dimer (less than 45%) were revealed in discs incubated on water
(Fig. 3, B and C) compared with
those of control whole plants (more than 80%,
Fig. 2D). Note that WT discs
were found to display a significantly higher percentage of BAS1 dimer than
discs from transgenic plants (41% and 25%, respectively). In methyl
viologen-treated discs, blots from gels run in the presence of dithiothreitol
revealed a lower BAS1 level in both plant types (around 25% less in comparison
with control discs incubated on water, Fig.
3A). A band with a lower size compared with that of the BAS1
monomer was revealed using the BAS1 antiserum only in the two CDSP32-lacking
lines. In the D4 line, the relative abundance of this band was estimated,
using Genetools, to be around 30% of that of the BAS1 monomer. Under
nonreducing conditions during migration, the proportion of BAS1 dimer fell
down to less than 20% in both disc types treated with methyl viologen
(Fig. 3, B and C). Note also
that only one dimer form was detected in the presence of the photooxidizing
compound (Fig. 3B), likely
indicating substantial changes in the redox state of the BAS1 dimer under
stress.
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Figure 3. Immunoblot analysis of the BAS1 abundance and redox state in leaf discs
from WT and CDSP32-lacking potato lines under photooxidative stress
conditions. Soluble proteins from leaf discs (A and B) were separated by
SDS-PAGE under reducing (A) or nonreducing (B) conditions. Proteins were
analyzed using the BAS1 antiserum as described in
Figure 2. Leaf discs from two
independent WT plants or two CDSP32-lacking (D4 and D18) plants were incubated
either on water (MV–) or on 1 µM methyl viologen (MV+) for
4 h in the light with a 12-h dark break. C, Percentage of BAS1 protein present
as a dimer in leaf discs. The data were obtained by image analysis, using
Genetools, of immunoblot of proteins separated under nonreducing conditions
(as in B). The values presented were calculated from data originating from six
independent plants per treatment. Tr, D4 and D18 plants.
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Analysis of the Production of Peroxides in Methyl Viologen-Treated
Protoplasts Using a Fluorescent Probe
We further characterized CDSP32-lacking plants by investigating the
localization of peroxides under photooxidative stress generated using methyl
viologen. For this purpose, we used 2', 7'-dichlorofluorescein
diacetate (H2DCF-DA), a chemical probe emitting fluorescence when
reacting with organic peroxides and H2O2 but not with
the superoxide anion (Zhu et al.,
1994). In plants, H2DCF-DA has been used to monitor the
H2O2 release in soybean (Glycine max) after a
pathogen attack (Levine et al.,
1994) and in mitochondria of tobacco (Nicotiana tabacum)
plants modified in alternative oxidase level
(Maxwell et al., 1999).
Investigations were carried out on protoplasts using laser-scanning confocal
microscopy. In non-treated protoplasts both from WT and from CDSP32-deprived
lines, the dichlorofluorescein (DCF) fluorescence was hardly detectable (data
not shown). Note that no DCF fluorescence was observed in non-fully digested
cells, characterized by a noncircular shape, due to H2DCF-DA
inability to cross the cell wall. In methyl viologen-treated protoplasts, a
much higher DCF green fluorescence signal was recorded in CDSP32-lacking lines
than in WT (Fig. 4, right). The
DCF fluorescence pattern was found to be close to that of the natural
chlorophyll fluorescence (Fig.
4, left), strongly suggesting the occurrence of a higher
production of peroxides in plastids of transgenic plants.
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Figure 4. Analysis of the production of peroxides using H2DCF-DA in
protoplasts from WT and CDSP32-lacking potato lines under methyl viologen
treatment. Laser-scanning confocal microscope images of protoplasts from WT
and CDSP32-lacking (CDSP32–) potato lines labeled with
H2DCF-DA and treated with 10 µM methyl viologen under
light for 5 min. Left, Natural chlorophyll (Chl) fluorescence. Right, DCF
fluorescence. The same magnification (x350) was used for all images. The
experiment was repeated three times with similar fluorescence intensity
signals in D4 and D18 CDSP32-lacking lines.
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Analysis of the Production of Lipid Hydroperoxydes in Methyl
Viologen-Treated Leaf Discs
The amount of lipid hydroperoxides in control and methyl viologen-treated
leaf discs was measured after reduction using HPLC analysis of hydroxy fatty
acids. The level of most hydroxy fatty acids was found to be higher in discs
subjected to methyl viologen for 4 h 30 min under light than in discs
incubated on water under the same conditions
(Fig. 5A; data not shown).
However, the 13-HOTE amount, which was very high both in WT and in transgenic
discs under control conditions, was found to be much lower in the presence of
methyl viologen (Fig. 5A; data
not shown). We investigated 13-HOTE chirality to verify whether the high
content of the fatty acid in control discs was due to chemical oxidation or to
the activity of a specific lipoxygenase induced during the preparation of leaf
discs. Royo et al. (1996)
reported the activation of a specific lipoxygenase by wounding in potato
plants, leading to the generation of 13-HOTE, a precursor of jasmonic acid, a
molecule involved in defense reactions
(Blechert et al., 1995). After
the first HPLC, the peak of 13-HOTE was taken and analyzed by chiral
chromatography, as described in "Materials and Methods," to
determine its enantiomer composition. As shown in
Figure 5C (upper), 95% 13-HOTE
was found chiral (13S-HOTE) in control discs, in contrast to 9-HOTE, which
appeared to be racemic. These data indicate that the high 13-HOTE level in
discs incubated on water very likely originates from the activity of a
specific lipoxygenase induced by wounding. In discs treated with methyl
viologen, both 13-HOTE and 9-HOTE were found to be racemic, thus reflecting a
membrane autoxidization process (Fig.
5C, lower). The much lower 13-HOTE content during methyl viologen
treatment indicates that the lipoxygenase is very likely inactivated under
severe photooxidative stress.
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Figure 5. HPLC analysis of the hydroxy fatty acid content in leaf discs from WT and
CDSP32-lacking potato lines treated with methyl viologen. Leaf discs from WT
and CDSP32-lacking (CDSP32–) lines were floated on water (CT) or 1
µM methyl viologen (MV) and exposed to light for 2 h, then to
dark for 16 h, and to light for 2 h 30 min, before analysis. Hydroperoxy fatty
acids were extracted and analyzed by HPLC after reduction. A, Chromatograms of
hydroxy fatty acids from CDSP32– leaf discs, CT (—), and MV (---).
B, Amount of hydroxy fatty acids (9-, 12-, and 16-hydroxy-octadecatrienoic
acid [HOTE] and 9- and 13-hydroxy-octadecadienoic acid) in WT and
CDSP32– leaf discs incubated on water ( ) or on 1 µM
methyl viologen ( ). Values are means (±SD) of three
determinations on 1 g of leaf discs from two plants per type (two WT, one D4,
and one D18). The experiment was repeated twice with similar results. C,
Chromatograms of chiral HPLC analysis of 13-HOTE (left) and 9-HOTE (right)
from CDSP32– leaf discs under control conditions (CT, upper) and methyl
viologen treatment (MV, lower).
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In discs floated on water, the content of all hydroxy fatty acids (9-, 12-,
and 16-HOTE and 9- and 13-hydroxy-octadecadienoic acid, except 13-HOTE) was
around 1,000 nmol g–1 dry weight both in WT and
CDSP32-lacking lines (Fig. 5B).
The content was significantly higher under methyl viologen treatment, reaching
1,500 nmol g–1 dry weight in WT and around 2,700
nmol g–1 dry weight in transgenic lines
(Fig. 5B). These data clearly
reveal a higher production of lipid hydroperoxydes due to membrane
autoxidization in leaf discs from transgenic lines under photooxidative
stress.
Analysis of Lipid Peroxidation in Thylakoids of Methyl
Viologen-Treated Leaf Discs
The level of chlorophyll thermoluminescence (TL) signal, which has been
correlated to the level of lipid peroxidation in thylakoids
(Vavilin and Ducruet, 1999),
was measured in discs subjected either to control conditions or to
photooxidative stress generated by 1 µM methyl viologen for 270
min under light. In non-treated discs from WT and CDSP32-lacking lines, the
peak of chlorophyll TL at 130°C was not detected (data not shown). In
methyl viologen-treated discs, a high TL signal at 130°C was recorded
(Fig. 6), indicating the
production of lipid hydroperoxides during the treatment. The signal was 2-fold
higher in transgenic lines than in WT, showing increased lipid peroxidation in
the thylakoids of plants lacking CDSP32.
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Figure 6. Analysis of the thylakoid lipid peroxidation level by measuring the
chlorophyll TL signal in leaf discs from WT and CDSP32-lacking potato lines
treated with methyl viologen. Leaf discs from WT and CDSP32-lacking
(CDSP32–) lines were floated on water containing 1 µM
methyl viologen and exposed to light for 2 h, then to dark for 16 h, and to
light for 2 h 30 min, before analysis. The level of chlorophyll TL signal at
130°C was recorded. Values are means (±SD) from 24 discs
originating from three independent experiments with two plants per type (two
WT, one D4, and one D18) in each experiment. AU, Arbitrary unit.
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DISCUSSION
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Modifications in the Abundance and Redox State of the BAS1
Peroxiredoxin in Plants Lacking the CDSP32 Thioredoxin
This paper first documents the characteristics of WT and CDSP32-deprived
plants with regard to the abundance and redox state of BAS1, a plastidic
peroxide-detoxifying enzyme, which has been presumed to be a target in planta
for the CDSP32 thioredoxin (Broin et al.,
2002). A substantially decreased proportion of BAS1 dimer, the
functional form of the protein, was revealed under drought stress in
transgenic plants and in discs of both plant types under methyl viologen
treatment. In in vitro assays, oxidizing stress conditions have been reported
to provoke overoxidation and monomerization of 2-Cys and type II
peroxiredoxins such as plant BAS1
(König et al., 2002) or
yeast Ahp1 (Prouzet-Mauléon et al.,
2002), respectively. Based on these reports and on the fact that a
very high percentage of BAS1 monomer was found in leaf discs subjected to a
severe photooxidizing treatment generated by methyl viologen, we conclude that
the stress-induced BAS1 monomer observed in our experiments corresponds to an
overoxidized form of the protein. These findings corroborate those of
Rabilloud et al. (2002), who
concluded that mammal 2-Cys peroxiredoxins constitute targets for peroxides,
and indicate that overoxidation of such peroxiredoxins also occurs in plants
under stress conditions. It is worth mentioning that, in comparison with
well-watered whole plants, leaf discs incubated on water for a few hours in
the light displayed substantial changes with regard to the BAS1 redox state.
We conclude from this observation that, even in the absence of methyl
viologen, discs are subjected to rather stressful conditions leading to
changes in the plastidic redox state and in the photosynthetic activity, as
reported by Kato et al.
(2002). Altogether, these
data, showing a significantly higher percentage of BAS1 monomer in transgenic
plants than in WT under drought and in leaf discs floated on water, indicate
that CDSP32-lacking lines display increased overoxidation of BAS1 under
stress. Further, despite no variation in the BAS1 level under control
conditions, transgenic plants have a lower 2-Cys peroxiredoxin abundance than
WT under water deficit, likely indicating accelerated protein degradation. In
other respects, under methyl viologen treatment, up to 30% of BAS1 monomer was
found with a lower molecular mass in discs from CDSP32-deprived plants. It is
noteworthy that BAS1 monomer has a higher propensity to partial proteolysis
(König et al., 2002) and
that overoxidation of a 2-Cys mammal peroxiredoxin leads to inactivation and
accelerated degradation of the protein
(Rabilloud et al., 2002).
Following on from this, we propose that the BAS1 form displaying a lower
molecular mass in transgenic lines under methyl viologen treatment originates
from a degradation process, which would be accelerated as a result of earlier
overoxidation of the protein.
Participation of the CDSP32 Thioredoxin in Detoxification of Lipid
Hydroperoxides
In comparison with WT, CDSP32-lacking plants exhibited a higher level of
DCF fluorescence signal in protoplasts subjected to methyl viologen treatment.
Because close fluorescence patterns were observed for DCF and chlorophyll, we
concluded that a more elevated production of H2O2 and/or
organic peroxides occurs in plastids in the absence of the thioredoxin. In
other respects, we noticed approximately 2-fold higher levels of hydroxy fatty
acid content and chlorophyll TL signal in leaf discs from transgenic lines
treated with methyl viologen than in WT discs. Taken collectively, these data
show that plants without CDSP32 are characterized by an increased production
of lipid hydroperoxides in thylakoids under photooxidative stress. Based on
the results obtained using DCF, we propose that the higher level of lipid
peroxidation in transgenic plants under stress originates either from direct
oxidation of fatty acids or from an increase in H2O2
content. These findings are a substantial contribution concerning the function
of the thioredoxin, which appears to be an essential component for limiting
lipid peroxidation in the photosynthetic membranes.
CDSP32. A Physiological Electron Donor to the BAS1
Peroxiredoxin?
In chloroplasts, thylakoid membranes are particularly rich in
polyunsaturated fatty acids prone to peroxidation
(Browse et al., 1994).
Detoxification of lipid hydroperoxides is of peculiar importance because these
compounds drive autocatalytic chain reactions and can provoke severe damage in
the photosynthetic membranes (Asada,
1996; Havaux and Niyogi,
1999). Besides low-Mr compounds, two main
enzymes, the phospholipid hydroperoxide glutathione peroxidase
(Mullineaux et al., 1998) and
the 2-Cys peroxiredoxin (Dietz et al.,
2002), are likely to participate in reduction of plastidic lipid
hydroperoxides. On the basis of its abundance and localization, BAS1 is
presumed to be the essential enzymatic component for detoxifying
hydroperoxides within thylakoid membranes and for maintaining integrity of the
photosynthetic apparatus (Dietz et al.,
2002). Accordingly, antisense down-regulation of BAS1 expression
has been reported to lead to impairment in PSII efficiency in young
Arabidopsis leaves (Baier and Dietz,
1999), but the effect was slight probably due to a still
substantial protein amount. Similarly, Klughammer et al.
(1998) reported that
disruption of the 2-Cys peroxiredoxin gene in Synechocystis sp. PCC
6803 results in increased stress sensitivity of the photosynthetic metabolism.
In other respects, yeast strains lacking or overexpressing the Ahp1p 2-Cys
peroxiredoxin exhibit increased or decreased susceptibility, respectively, to
an alkyl hydroperoxide, tertbutyl hydroperoxide
(Lee et al., 1999). Lee et al.
(1999) also reported that the
Ahp1p peroxiredoxin requires the presence of a thioredoxin to perform its
antioxidant function because yeast mutants deprived of thioredoxin were found
to be highly sensitive to tert-butyl hydroperoxide, even when Ahp1p was
overexpressed. These data indicate that, in various organisms, the 2-Cys
peroxiredoxin activity level is correlated with the level of stress
sensitivity. Previously, we showed that CDSP32-lacking lines display, compared
with WT, a lower photosynthetic activity and a reduced chlorophyll content
under photooxidative stress conditions
(Broin et al., 2002). The data
presented in this study indicate that under stress, these lines concomitantly
exhibit an increased proportion of inactivated BAS1 protein due to
overoxidation and a higher level of lipid peroxidation. As a consequence, we
conclude that the increased damage in the photosynthetic membranes of
CDSP32-lacking lines originates from a reduced detoxification capacity of
H2O2 and/or of hydroperoxides due to accelerated
inactivation of BAS1. We propose that this inactivation results from the
absence of the CDSP32 thioredoxin, which would normally reduce the
peroxiredoxin. To unambiguously show that BAS1 constitutes a target for CDSP32
in planta, it would be worth investigating the presence of the heterodimeric
complex formed of the two proteins in plants overexpressing the CDSP32
thioredoxin mutated at its active site.
In contrast to CDSP32, BAS1 is present at high levels in all organs of
control WT plants. However, in Chinese cabbage (Brassica campestris
L. subsp. pekinensis), Cheong et al.
(1999) reported substantially
lower levels of the 2-Cys peroxiredoxin in roots and flowers compared with
that observed in leaves. These data raise the question, at least in potato, of
the role of BAS1 in roots and flowers where plastids are not photosynthetic
and not as developed as in leaves. Further, they suggest that electron donors
to BAS1 different from CDSP32 exist in these organs and also in leaves in the
absence of stress. König et al.
(2002) showed that spinach
(Spinacia oleracea) thioredoxins f and m reduce
oxidized BAS1 in in vitro assays. Motohashi et al.
(2001) identified more than 14
targets for spinach thioredoxin m, including the 2-Cys BAS1
peroxiredoxin. However, in these experiments, BAS1 was found to interact with
thioredoxin m in a much less specific manner compared with the
results published for CDSP32 (Broin et al.,
2002). CDSP32 could constitute an electron donor to BAS1 more
particularly in leaves under stress conditions when other antioxidant enzymes
such as ascorbate peroxidase are inactivated
(Dietz et al., 2002). Note
also that very recent data reveal that CDSP32 is more abundant in young
developing leaves in potato, likely indicating that the protein plays a
protective function also during leaf development
(Broin et al., 2003). In
Arabidopsis plants antisense for the BAS1 gene, an enhancement of the
enzyme activities associated with ascorbate metabolism was noticed, but no
change in glutathione metabolism was observed
(Baier et al., 2000). Further
work is needed to characterize plants either lacking or overexpressing CDSP32
to investigate how the thioredoxin is integrated in the antioxidant network
during stress and development.
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MATERIALS AND METHODS
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Plant Material, Drought, and Photooxidative Treatments
Potato (Solanum tuberosum cv Désirée) WT and
transgenic plants were propagated in vitro and transferred for growth in vivo
in a phytotron (photosynthetic photon flux density = 200 µmol
m–2 s–1, 12-h night,
23°C/19°C day/night). Two independent lines, designated D4 and D18
(Broin et al., 2002) and
cosuppressed for CDSP32 expression, were used. A gradually increasing
water deficit was applied on 3-week-old plants by withholding watering for
around 10 d. Relative water content was determined on leaf pieces as
previously described (Rey et al.,
1998). For incubation experiments, 1.4-cm diameter discs were
excised from young well-expanded leaflets of 3-week-old plants. Discs were
incubated in the phytotron on water containing 0.1% (v/v) Tween 20 with or
without 1 µM methyl viologen (Sigma, St. Louis) in the light
(375 µmol photons m–2
s–1) for 2 h, then in the dark for 12 h and in the
light for 2 to 2 h 30.
Protein Extraction, SDS-PAGE, and Western Analysis
For preparing leaf-soluble proteins, leaflet or disc samples were blended
in liquid N2, and the powder was resuspended in 50 mM
Tris-HCl (pH 8.0) and 1 mM phenylmethylsulfonyl fluoride, with or
without reducing agent (50 mM  -mercaptoethanol), and
centrifuged (10000g, 4°C, 10 min). The soluble proteins were
precipitated at –20°C by the addition of 1 volume of acetone to the
supernatant. The soluble proteins from other organs (whole flowers, stems,
tubers, and roots from 6-to 8-week-old plants) were prepared similarly using
50 mM -mercaptoethanol in the extraction buffer.
Protein content was determined using a modified Lowry method (Sigma).
One-dimensional electrophoresis (Laemmli,
1970) was performed with 13% (w/v) acrylamide gels with or without
dithiothreitol (0.1 M) in the solubilization buffer and using an
Mr electrophoresis calibration kit (BioRad Laboratories,
Hercules, CA). After SDS-PAGE, proteins were electroblotted onto 0.45
µM nitrocellulose (Pall Gelman Sciences, Pall Corporation, Ann
Arbor, MI) for western analysis. The sera raised against the potato CDSP32
N-terminal region (Rey et al.,
1998) and the Arabidopsis BAS1 protein
(Broin et al., 2002) were used
diluted 1:1,000 (v/v) and 1:10,000 (v/v), respectively. Bound antibodies were
detected using an anti-rabbit IgG alkaline phosphatase conjugate diluted
1:10,000 (v/v; Sigma). Image analysis and estimation of the intensity of bands
were carried out using Genetools.
Preparation of Protoplasts
Protoplasts were prepared from young fully expanded leaves from 3-week-old
plants, cut into slices, and incubated for 16 h (at 28°C in the dark) on a
maceration fluid containing 0.45 M mannitol, the macro- and
micro-elements of Murashige and Skoog
(1962), 1 mM
CaCl2 (pH 5.8; washing medium) supplemented with 0.5% (w/v)
cellulase O-R-10 (Yakult Honsha Co., Tokyo) and 0.5% (w/v) macerozyme R-10
(Yakult Honsha Co.). Protoplasts were purified using a Percoll gradient
procedure (Mills and Joy,
1980) and resuspended to 500 µg chlorophyll
mL–1 in washing medium.
Confocal Microscopy
The intracellular production of H2O2 and organic
peroxides was investigated using H2DCF-DA (Molecular Probes,
Eugene, OR), a compound converted, when taken up by cells, to the
membrane-impermeant derivative H2DCF by esterases. H2DCF
is nonfluorescent but rapidly oxidized to the highly fluorescent DCF by
reaction with the intracellular peroxides
(Zhu et al., 1994).
Fresh protoplasts were diluted to 200 µg chlorophyll
mL–1 in 50 µL of washing medium containing 100
mM Tricine (pH 7.5). After simultaneous addition of methyl viologen
(10 µM final concentration) and H2DCF-DA (5
µM final concentration), the reaction mix was illuminated for 5
min (photosynthetic photon flux density = 300 µmol
m–2 s–1) and analyzed
using a Fluoview krypton-argon laser scanning confocal microscope (Olympus,
Tokyo). DCF and chlorophyll were excited at 488 nm, and the emitted
fluorescence was detected through 530-/20-nm bandpass and >660-nm longpass
filters, respectively. The laser intensity was identical in all experiments.
Data were computerized and imported into Adobe Photoshop 5.0 (Adobe Systems,
Mountain View, CA) for the preparation of figures.
Hydroxy Fatty Acid and Hydroperoxy Fatty Acid Analysis
Free and bonded hydroxy and hydroperoxy fatty acids were analyzed using
HPLC, as free hydroxy fatty acids, after NaBH4 reduction and
hydrolysis. Control and methyl viologen-treated leaf discs (1 g fresh weight)
were frozen in liquid nitrogen and homogenized in 0.2 M NaOH and 5%
(w/v) NaBH4 in the presence of an internal reference,
15-hydroxy-11, 13(Z,E)-eicosadienoic acid (40 nmol
g–1 fresh weight). Extraction and HPLC analysis
were performed as described by Degousée et al.
(1994) and Rustérucci
et al. (1999), using a Zorbax
RX-SIL silica column (Interchim, Montluçon, France). Hydroxy fatty
acids isomers were detected at 234 nm and identified using standards
(Degousée et al.,
1995). Quantification was performed with reference to
15-hydroxy-11, 13(Z,E)-eicosadienoic acid, assuming a similar extinction
coefficient at 234 nm for all hydroxy fatty acids.
For chirality analysis of 13-HOTE and 9-HOTE, the corresponding hydroxy
fatty acid was taken after HPLC analysis through a Zorbax RX-SIL silica column
(Interchim). The hydroxy fatty acids were then analyzed by HPLC using a
Chiralcel OD-H silica column (Interchim), and the isomers were eluted with a
hexane:isopropanol:acetic acid (95:5:0.1 [v/v]) isocratic flux (1 mL
min–1) and detected at 234 nm.
Chlorophyll TL
The excited forms of lipid peroxides are able to transfer their energy to
chlorophyll, and heating a leaf sample results in the desexcitation of
chlorophyll through photon emission. This process, termed chlorophyll TL, is
used to estimate the level of lipid peroxidation within thylakoids
(Stallaert et al., 1995).
Measurements were carried out on 1.4-cm-diameter leaf discs, treated (or not)
with 1 µM methyl viologen as described above, according to
Ducruet and Miranda (1992) and
Broin et al. (2000).
|
ACKNOWLEDGMENTS
|
|---|
We are very grateful to Dr. Jean-Luc Montillet (Laboratoire de
Radiobiologie Végétale, DEVM, CEA/Cadarache,
Saint-Paul-lez-Durance, France) for valuable advice on HPLC analysis of
hydroxy fatty acids and for critical reading of the manuscript. We are also
grateful to Dr. Michel Havaux (Laboratoire d'Ecophysiologie de la
Photosynthèse, DEVM, CEA/Cadarache) for helpful advice in chlorophyll
TL experiments. We wish to thank Jean-Pierre Agnel (Laboratoire de
Radiobiologie Végétale, Département d'Ecophysiologie
Végétale et de Microbiologie, CEA/Cadarache) for help in
analysis of hydroxy fatty acids and Jacqueline Massimino, Françoise
Eymery, and Véronique Cardettini (Laboratoire d'Ecophysiologie de la
Photosynthèse, Département d'Ecophysiologie
Végétale et de Microbiologie, CEA/Cadarache) for assistance in
growing plant material.
Received February 3, 2003;
returned for revision March 2, 2003;
accepted March 31, 2003.
|
FOOTNOTES
|
|---|
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.103.021626.
*
Corresponding author; e-mail
pascal.rey{at}cea.fr;
fax 33–4–42–25–62–65.
|
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ABSTRACT
RESULTS