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Plant Physiol. (1998) 118: 493-504
Light and Excess Manganese1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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The effect of light intensity on antioxidants, antioxidant enzymes, and chlorophyll content was studied in common bean (Phaseolus vulgaris L.) exposed to excess Mn. Leaves of bean genotypes contrasting in Mn tolerance were exposed to two different light intensities and to excess Mn; light was controlled by shading a leaflet with filter paper. After 5 d of Mn treatment ascorbate was depleted by 45% in leaves of the Mn-sensitive genotype ZPV-292 and by 20% in the Mn-tolerant genotype CALIMA. Nonprotein sulfhydryl groups and glutathione reductase were not affected by Mn or light treatment. Ten days of Mn-toxicity stress increased leaf ascorbate peroxidase activity of cv ZPV-292 by 78% in low light and by 235% in high light, and superoxide dismutase activity followed a similar trend. Increases of ascorbate peroxidase and superoxide dismutase activity observed in cv CALIMA were lower than those observed in the susceptible cv ZPV-292. The cv CALIMA had less ascorbate oxidation under excess Mn-toxicity stress. Depletion of ascorbate occurred before the onset of chlorosis in Mn-stressed plants, especially in cv ZPV-292. Lipid peroxidation was not detected in floating leaf discs of mature leaves exposed to excess Mn. Our results suggest that Mn toxicity may be mediated by oxidative stress, and that the tolerant genotype may maintain higher ascorbate levels under stress than the sensitive genotype.
Mn is an essential micronutrient for most living organisms. It is
involved in redox reactions as a cofactor for different enzymes (for a
detailed list of enzymes, see Burnell, 1988 In Mn-sensitive plant species, the deleterious effect of Mn toxicity is
often observed in the shoots as stunted growth, chlorosis, crinkled
leaves, and brown lesions or "speckles." However, the response of
plants to excess Mn is affected by leaf age (Horst, 1988 Although the physiological mechanisms of Mn toxicity and tolerance are
still unknown, several reports suggest a role for excess Mn in the
induction of oxidative stress. Gerretsen (1950) The toxic effects of heavy metals, both essential and nonessential
elements, have been linked to the production of free radicals (De Vos
and Schat, 1991 Leaf antioxidant capacity is modified by long-term light acclimation
(Grace and Logan, 1996 Plant Material
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
) such as the Mn-containing
isozyme of Mn-SOD, which is one of the essential mechanisms in
protection against oxidative stress in plants (for review, see Bowler
et al., 1994). Mn is also important in the water-splitting system that
provides electrons to PSII. Various soil conditions often present in
acid and volcanic soils can lead to Mn reduction and create Mn toxicity
in many natural and agricultural systems (Foy et al., 1978; Carver and
Ownby, 1995).
), temperature
(Heenan and Carter, 1977; Rufty et al., 1979), soil nutrient balance,
soil pH, genotype, and light intensity. The effect of light intensity
on Mn-toxicity symptoms was first reported in 1935, when McCool (1935)
found that plants grown in low light displayed fewer symptoms of Mn
toxicity than those grown in high light. Subsequent reports with
different crops found a similar effect of low light (Sirkar and Amin,
1974; Elamin and Wilcox, 1986; Horiguchi, 1988; Nable et al., 1988).
Wissemeier and Horst (1992) reported that symptoms of Mn toxicity
(localized brown spots and callose formation) in mature leaves of
cowpea were enhanced under low-light conditions, but did not rule out that chlorosis in immature leaves might be enhanced under high-light conditions. However, a factor that complicates the interpretation of
most previous studies of the effect of light intensity on Mn-toxicity symptoms is the fact that plants grown in low light usually accumulate less foliar Mn that those grown at a higher light intensity, as found
in maple trees (McCain and Markley, 1989).
proposed that
Mn-induced chlorosis was not caused by inhibition of chlorophyll synthesis but rather by photooxidation of chlorophyll. Campbell and
Nable (1988) suggested that low light intensity may also decrease the
photodestruction of chlorophyll in plants suffering Mn toxicity. In pea
del Rio et al. (1985) reported the induction of Mn-SOD in plants
exposed to high levels of Zn and Mn. Also, Leidi et al. (1987a, 1987b)
found high Mn-SOD activity in soybean genotypes grown under excess Mn.
Panda et al. (1986) reported lipid peroxidation in aging, isolated
chloroplasts treated with excess Mn.
). Free radicals are usually formed as by-products of
normal biological reactions, but their lifespan and diffusion into the
cell space are closely controlled by the cell antioxidant system.
Enzymes such as SOD, GR, ASPX, and DAS reductase, and compounds such as
ascorbate and glutathione are actively involved in the
ascorbate-glutathione scavenging cycle, controlling reactive oxygen
species not only in the chloroplast but also in the cytosol (for
reviews, see Alscher and Hess, 1993; Foyer and Mullineaux, 1994). The
involvement and role of antioxidants in protection against oxidative
stress have been demonstrated using transgenic plants (Foyer et al.,
1994; Allen, 1995; Slooten et al., 1995), and genetic variability in
the content of antioxidant elements has been reported in several
species (Sevilla et al., 1988; Daza et al., 1993; Guzy and Heath,
1993). Tolerance of oxidative stresses such as the herbicide paraquat
and NaCl were partially associated with a better antioxidant response
in tolerant genotypes (Gossett et al., 1994; Hernández
et al., 1995).
) and might confer tolerance to oxidative
stresses. Variability in antioxidant capacity within common bean
genotypes exists (Guzy and Heath, 1993), but it is not known if
tolerance to Mn toxicity is correlated with better antioxidant capacity
of tolerant genotypes. Because Mn might promote oxidative stress, it is
important to characterize the response and levels of antioxidants in
leaves accumulating excess Mn. It is also important to explore how that
response is modified by light intensity within and among contrasting
genotypes suffering Mn-toxicity stress. In this study we used a system
that permitted equivalent Mn accumulation in tissue at different
irradiance levels to examine the combined effect of light intensity and
Mn-toxicity stress on antioxidants and chlorophyll in leaves of two
bean genotypes with contrasting sensitivity to Mn.
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
).
Also, in a study of the effect of excess Mn on
CO2 assimilation of these two genotypes it was
found that excess Mn decreased CO2 assimilation mainly in immature leaves by causing rapid degradation of chlorophyll. Mature leaves were less affected (González and Lynch, 1997), although both leaf types accumulated excess Mn at comparable rates.
2
s1 (high light = 100% incident PAR) and
the other received 220 µmol m2
s1 (low light = 25% of maximum PAR).
Plants were harvested at 5 and 10 d after Mn treatment was
initiated, and leaf tissue was split into three subsamples: (a) two
leaf discs of 1.24 cm2, which were used for
chlorophyll analysis; (b) a subsample, which was designated for
nutrient analysis; and (c) the remaining tissue, which was frozen in
liquid nitrogen and stored at 
70°C for biochemical analysis.
Mn Analysis
Tissue was dried at 70°C, weighed, ashed at 500°C for 12 h, dissolved in 0.1 N HCl, and analyzed for Mn content by atomic absorption spectrophotometry.Chlorophyll
Chlorophyll was extracted overnight in a dark, cold room by placing two leaf discs of 1.24 cm2 into 2 mL of N,N-dimethylformamide (Sigma). Aliquots were taken to measure the A664.5 and A667 in a spectrophotometer, and chlorophyll content was calculated according to the method of Inskeep and Bloom (1985).
Enzyme Assays
For enzyme activity measurements all operations were carried out at 4°C by keeping the samples in an ice bath until assays were completed.). Two leaf discs (1.24 cm2) were
ground with 600 µL of a cold buffer containing Bicine (50 mM, pH 8.0), NaHCO3 (20 mM), MgCl2 (20 mM),
Na-EDTA (1 mM), PVP (1%), and leupeptin sulfate (5 µM). Leaf extract was spun at 14,000g for 15 min and diluted 1:1 with a solution containing 62.5 mM Tris-HCl (pH 6.8) and 10% glycerol. Protein in the supernatant was
determined using Bradford's reagent (Bio-Rad).
.
,N-tetramethylethylenediamine (28 mM) for 25 min; the previous buffer was replaced with
100 mL of a solution containing nitroblue tetrazolium (2 mM, Sigma) in buffer phosphate (36 mM, pH 7.8)
and the gel was shaken for 30 min. Gels were illuminated for 15 min or
until white bands appeared, the solution was replaced with phosphate
buffer (36 mM, pH 7.8), and gels were placed in a light box
to continue the reaction very slowly. Gels were fixed in a solution
containing 30% methanol, 7.5% glycerol for 1 h, and dried in a
gel-drying membrane (Bio-Rad). Dried gels were scanned and the
integrated density calculated. To determine if peroxidase interfered
with the SOD assay in the activity gel, samples were run and a gel was
vertically split into two sections. One section was stained for SOD
activity, and the other section was stained for peroxidase with
3,3,5,5-tetramethylbenzidine (Guikema and Sherman, 1980; as described
by Vallejos, 1983).
). pCMB inhibits the activity of ASPX, and by
running the assay with and without pCMB one may determine the activity
of ASPX in the leaf extract. The effect of pCMB on ASPX was
confirmed by assaying ASPX in the activity gel (Mitler and Zilinskas,
1993) and in the spectrophotometer on randomly selected samples.
1
cm1; Nakano and Asada, 1981) at 25°C in a
reaction mixture containing KPO4 (50 mM, pH 7.0), EDTA (5 mM), ascorbate (0.5 mM), and H2O2 (0.1 mM) in a final volume of 1 mL. Each assay was run
three times. ASPX activity was calculated by subtracting the change in
absorbance measured in the presence of the inhibitor from the
absorbance measured without the inhibitor.
where GSTNB is glutathione-nitrobenzoic acid.
with
slight modifications. The reaction mixture contained phosphate buffer
(100 mM, pH 7.5), EDTA (2.5 mM), DTNB (0.75 mM), NADPH (0.1 mM), GSSG (1 mM),
and leaf extract in a volume of 1 mL. The reaction was run at 30°C,
activated by adding NADPH, and formation of TNB was followed at 412 nm
for 1 min (Smith and Vierheller, 1988). Changes in absorbance were
translated to nanomoles of GSSG from a standard curve prepared with
commercial GR (Sigma) and known concentrations of GSSG.
Water-Soluble Compounds
Nonprotein SH groups were determined by a modification of the protocol of Tanaka et al. (1985), and in common bean are assumed to
represent mainly GSH (Guzy and Heath, 1993). Four leaf discs were
homogenized with a mortar and pestle in 2 mL of 10% (w/v) TCA
containing 1.5 mM EDTA, and centrifuged for 15 min at
14,000g. The supernatant was split into two aliquots and
stored at 70°C. For SH determination one aliquot was thawed and
kept cold. From each sample 100 µL of supernatant was added to 790 µL of 150 mM K2HPO4 with 6.3 mM EDTA, added while vortex mixing, followed by 10 µL of
5 N NaOH to bring the pH close to 7.3, which is appropriate for color development. Then, 100 µL of 6 mM DTNB in 0.15 mM phosphate buffer was added and mixed vigorously. Tubes
were incubated at room temperature for 10 min before
A412 determination because of the formation
of TNB. Absorbances were converted to SH concentration using a
standard curve prepared in a similar way but including known amounts of
GSH instead of leaf supernatant.
. The assay
is based on the reduction of Fe3+ to
Fe2+ by ascorbic acid in acidic solution. The
Fe2+ then forms complexes with bipyridyl,
producing a pink color that absorbs at 525 nm. Total ascorbate (AS plus
DAS) is determined through a reduction of DAS to AS by DTT. For the
concentrations cited, 100 µL of the frozen aliquot was assayed for
total ascorbate content, and the other half was assayed for AS only.
DAS concentrations were then deduced from the difference. To determine
if the Mn that was present in high-Mn tissue interfered with the
development of color, we added several concentrations of
MnSO4 to known amounts of AS and read the
A525.
Lipid Peroxidation
Lipid peroxidation was assayed by the reaction of MDA with thiobarbituric acid, which produces a pinkish chromagen (Du and Bramlage, 1992). The thiobarbituric acid reagent consisted of 20%
(w/v) TCA, 0.65% (w/v) thiobarbituric acid, and 0.01% (w/v) butylated
hydroxy toluene. From an independent set of plants grown in the
greenhouse, leaf discs (23 mm in diameter) from fully expanded leaves
were floated in nutrient solution containing 0.01, 0.8 and 3.2 mM MnSO4 in Petri dishes in the
growth chamber for a 12-h photoperiod and with high light intensity
(670 µmol m2 s1).
Leaf discs were placed in the Petri dishes at the end of the photoperiod and kept there for at least 120 h. Each day four discs from each genotype and each Mn treatment were sampled. For Mn determination we harvested discs at 0, 3, and 6 h after
they were placed in the nutrient solution. Discs were vigorously
stirred in cold CaCl2 (10 mM) for 10 min to remove excess Mn. Two discs were used for Mn content and two
discs were frozen in liquid nitrogen and assayed for chlorophyll
determination and lipid-peroxidation products. Discs were extracted
with 1 mL of anhydrous ethanol, mixed with the same volume of
thiobarbituric acid reagent, and heated for 25 min at 90°C. Samples
were centrifuged for 15 min at 14,000g and supernatant was
measured spectrophotometrically at 532 and 600 nm. Data were calculated
as nanomoles of MDA per milligram of dry tissue using the extinction
coefficient of 156,000 M1
cm1.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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All of the variables measured in this report were affected by light intensity regardless of Mn treatment or genotype. Consequently, the effect of Mn-toxicity stress on a given variable within a light treatment is expressed as a percentage by comparing the value observed in Mn control samples with the value in samples treated with high Mn. Changes observed in a response variable are compared across light treatments.
Chlorophyll and Mn Content
High-light leaves had less chlorophyll content than low-light leaves regardless of Mn treatment. Excess foliar Mn caused chlorosis at both light treatments and in both genotypes; however, chlorosis was significantly greater in high-light leaves of the susceptible genotype ZPV-292. Chlorosis among light treatments was not significantly different at 5 d, but at 10 d high-light leaves suffered more chlorosis than low-light leaves. In low light we observed a 12% loss of chlorophyll in the tolerant cv CALIMA and a 24.2% loss in the susceptible cv ZPV-292. In high light, cv CALIMA suffered a 26.7% decrease in chlorophyll compared with 34.4% in cv ZPV-292 (Fig. 1B). In the susceptible genotype ZPV-292, loss of chlorophyll among light treatments occurred at similar foliar Mn concentrations; however, leaflets of the genotype CALIMA accumulated slightly less Mn at low light (Fig. 2B).
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Antioxidant Enzymes
The effect of pCMB allowed us to discriminate the reduction of ascorbate attributable to ASPX from that attributable to the less-specific GPX. When leaf extracts were incubated with pCMB, the white bands indicating the activity of ASPX in the activity gel disappeared almost completely. When a leaf extract had high ASPX activity in the spectrophotometric assay, it also displayed wide bands in the activity gel (data not shown). After 5 d of Mn treatment (Fig. 2A), the activity of ASPX increased by 87% and 64% in leaflets of the susceptible genotype ZPV-292 receiving high and low light, respectively; in cv CALIMA, the activity of ASPX in high-Mn plants was not significantly different from that in the control plants at any light treatment (Fig. 2A). A similar response pattern was observed after 10 d (Fig. 2B), but the effect of light intensity was more evident: in leaflets receiving high light the activity of ASPX increased by 270% in the susceptible cv ZPV-292 and by 130% in the tolerant cv CALIMA. At low light, even though there was an increase in the activity of ASPX, the magnitude was substantially lower; nonetheless, genotypic differences were conserved (Fig. 2B). It is important to note that at least in the susceptible genotype ZPV-292, the differential increase in activity of ASPX between high and low light occurred at identical foliar Mn.). At
5 d GPX activity increased by almost 250% in the susceptible
genotype ZPV-292, whereas in the tolerant genotype CALIMA the activity
was not significantly different from that in the control plants with
corresponding light treatments (Fig. 2A). At 10 d the effect of
light intensity and the differences between genotypes were more
striking. GPX activity in cv CALIMA with low light and high Mn was 87%
higher than in the control, but in high light it was 160% higher than
in the corresponding control. Although in the susceptible genotype
ZPV-292 light intensity had less effect, GPX activity was considerably
higher than in the tolerant cv CALIMA with similar light and Mn
treatments (Fig. 2B).
Water-Soluble Compounds
Lipid Peroxidation
In this report we examined the combined effect of light intensity
and excess Mn on both chlorophyll content and level and activity of
antioxidants in leaves of two common bean genotypes. Although previous
studies have suggested that low illumination delayed (McCool, 1935 Received March 6, 1998;
accepted June 24, 1998.
Abbreviations:
AS, reduced ascorbate.
ASPX, ascorbate
peroxidase.
DAS, dehydroascorbate.
DTNB, 5,5
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Vierheller TLTCA
(1988)
Assay of glutathione reductase in crude tissues homogenates using 5,5, 1994). Occasionally,
a faint band appeared between Mn-SOD and Cu/Zn that might represent
Fe-SOD, because it was tolerant of KCN but inhibited by
H2O2.
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Figure 3.
Typical pattern of SOD isozymes observed in bean
leaves. A, SOD activity gel loaded with different amounts of protein
required to produce an equivalent change in integrated density (B) of
SOD bands. C, SOD activity observed in 1, ZPV-292 Control Mn; 2, ZPV-292 High Mn; 3, CALIMA Control Mn; 4, CALIMA High Mn. A
nondenaturing gel was used to measure the activity of SOD. Gels were
soaked in 100 mL of a solution containing riboflavin 28 µM, KPO4 (36 mM, pH 7.8) and
temed (28 mM) for 25 min. This buffer was replaced with 100 mL of NBT (nitroblue tetrazolium) (2 mM) in
KPO4 (36 mM, pH 7.8) and shaken for 30 min.
Gels were illuminated for 15 min until white bands were detected. The
top band was H2O2- and KCN-tolerant which is
typical of Mn-SOD. The middle and lower bands disappeared when gels
were soaked in KCN, a typical reaction of Cu/Zn-SOD isozymes.
reported that excess Zn and Mn induced a
Mn-containing SOD in pea. In our study the presence of this occasional
band was not associated with a given treatment or genotype, and was not
quantified. Peroxidase stained as a blue band at the upper part of the
gel and did not migrate to the same distance as SOD (data not shown).
Thus, we ruled out the interference of peroxidase with the bands of SOD
in the activity gels. The pattern of SOD isozymes observed here has
been reported for bean (Corpas et al., 1991) and pea (Burke and Oliver,
1992). For instance, the Mn-SOD, which is resistant to
H2O2, appears to be found
in the mitochondrial matrix (Bowler et al., 1994, and refs. therein). The Cu/Zn-SODs, which are KCN sensitive, have been studied extensively in many species and exist in more than one cell compartment, but at
least one isozyme is always present in the cytosol and is referred to
as Cu/Zn-SOD I (Bowler et al., 1994). Another isoform, the Cu/Zn-SOD
II, has been found associated with the chloroplast in bean (Corpas et
al., 1991), as well as in other species (Bowler et al., 1992, 1994, and
refs. therein).
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Figure 4.
Relative activity of SOD isozymes in mature leaves
of two different bean genotypes grown in silica sand irrigated with
nutrient solution containing different doses of Mn (black bars = 5 µM; gray bars = 200 µM Mn) for 10 d (experiment 3). Light intensity was controlled by shading one leaflet
with Whatman No. 1 filter paper (Low Light = 220 µmol
m 2 s1; High Light = 870 µmol
m2 s1). Asterisks represent means
(n = 3; three leaflets assayed once each) that were
significantly different (LSD = 5%) from control plants
within a light treatment. See ``Materials and Methods'' for details
about SOD activity and scanning. Plants were harvested at 5 (A) and 10 (B) d.
1 dry weight by that time (Fig. 5).
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Figure 5.
Mn concentration (A), chlorophyll content, and MDA
concentration in leaf discs of mature leaves of two different genotypes
(CALIMA [tolerant, B] and ZPV-292 [susceptible, C]). Leaf discs
were floated in Petri dishes holding a nutrient solution containing
different levels of Mn (0.01, 0.8, and 3.2 mM Mn as
MnSO4). Leaf discs received 670 µmol photons PAR
m 2 s1 for 12 h daily. The composition
of the nutrient solution is described in ``Materials and Methods''.
Each point represents the average of two observations. In A, closed
symbols represent Mn concentration in the discs of the tolerant
genotype CALIMA, and open symbols represent Mn concentration in the
discs of the susceptible genotype ZPV-292.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
;
Sirkar and Amin, 1974; Horiguchi, 1988; Nable et al., 1988) or even
increased (Wissemeier and Horst, 1992) the symptoms of Mn toxicity,
only one study focused specifically on the effect of light intensity on
chlorophyll content of Mn-stressed plants, and to our knowledge no
previous reports have addressed the combined effect of light intensity
and Mn toxicity on leaf antioxidants. Horiguchi (1988) grew common bean
and maize under four different light regimes (100%, 80%, 40%, and
5% of the total radiation) and several Mn levels. Leaf chlorophyll
content for all three light levels were compared in plants grown at 32 ppm Mn and control plants (0.32 ppm Mn). At high light there was a reduction in leaf chlorophyll content of 48%, followed by 40% at
medium light and 0% at low-light treatment; however, there was a
4.6-fold difference in foliar Mn between leaves grown in high light
(1820 ppm Mn) and low light (396 ppm Mn), which confounds the effect of
light intensity with the effect of foliar Mn concentration. Nevertheless, although quantitative data were not presented, the author
claimed that bean leaves having 340 ppm Mn and grown at high light
displayed severe chlorosis.
),
suggesting that different mechanisms of Mn toxicity might occur at
different leaf developmental stages.
;
Grace and Logan, 1996). Plants growing under high light are more
frequently exposed to reactive oxygen species (the superoxide radical,
H2O2, and singlet oxygen)
generated in the plastids than plants growing at low light intensity,
and consequently might respond by accumulating higher amounts of
antioxidants in leaf tissue. The higher accumulation of antioxidants
might protect the tissue against other events that result in oxidative
stress. For instance, the extra accumulation of antioxidants in pea
plants grown at a relatively high light intensity, compared with plants grown in low light seemed to confer more tolerance to paraquat (Gillham
and Dodge, 1987). In our experiments control leaflets of both genotypes
receiving high light contained greater amounts of antioxidants than
leaflets receiving low light, except for GR and GPX activity. This
scenario changed when Mn toxicity was imposed, and the antioxidant
systems of the two genotypes responded differently.
). Aono et al. (1991) showed that transformed plants
expressing bacterial GR in the cytosol were more resistant to paraquat
than untransformed plants. More recently, Donahue et al. (1997)
described an increment in cytosolic ASPX transcript in pea leaves
treated with paraquat, and no responses of any isoform of SOD were
detected, but a new Fe-SOD isoform was induced by paraquat that was not associated with tolerance.
). The authors found that the susceptible genotype Bragg showed
elevated total SOD under conditions of Mn toxicity and low Fe. However,
it is difficult to discern a pattern among genotypes because Lee, a
genotype regarded as tolerant of excess Mn (Heenan and Carter, 1976),
also displayed elevated SOD activity. Leidi et al. (1987b) tested the
effect of various Fe and Mn concentrations on soybean. These authors
determined the activity of Mn-SOD and Cu/Zn-SOD by assaying the leaf
extracts in the presence of 1 mM NaCN. Their results
indicated that most of the increased activity in response to the Fe-
and Mn-toxicity stress corresponded to Mn-SOD, with very little
associated with Cu/Zn-SOD. This function is contrary to the findings of
most reports, which indicate that Cu/Zn is the most abundant isozyme in
soybean and in most of the species assayed (Bowler et al., 1994). Leidi
et al. (1987b) pointed out that these values were unexpectedly low
compared with the values observed when SOD was electrophoretically
determined. The effect of Mn toxicity, according to the authors,
resulted in the elevated activity of Mn-SOD regardless of the Fe level
in the growing medium (Leidi et al., 1987b). In our studies we detected a slight increase in Mn-SOD activity, mainly in the susceptible cv
ZPV-292, but the most significant response was observed in the
cytosolic SOD (Cu/Zn-SOD I) in high-light and excess-Mn treatments.
). Of the two isozymes in the chloroplast, one is
bound to the thylakoid membranes (Miyake and Asada, 1992) and the other
is in the stroma (Chen and Asada, 1989). From our results it is not
possible to tell which isozyme of ASPX is responding to the Mn
treatment. However, we could speculate that perhaps it is the cytosolic
fraction that is being activated under Mn toxicity. This speculation is
based on the observed increment of the activity of the cytosolic
isozyme of SOD and the consequent production of
H2O2. It is more likely
that the ASPX activity observed is responding to the accumulation of
the substrate (H2O2) in the putative site where it is being produced. After 5 d of Mn
treatment, SOD and ASPX increased their activity by 68% and 86%,
respectively, and after 10 d this increment was 275% in SOD
activity and 235% in ASPX activity, indicating a close coordination in
their response to control oxygen reactive species, perhaps within the
same cell compartment. Also, Mitler and Zilinskas (1993) reported that
the activity of the ASPX detected in native gels co-migrated with the
pure cytosolic ASPX enzyme, and that their attempts to detect chloroplastic ASPX were successful only when fresh, intact chloroplasts were ruptured and subjected to native electrophoresis. No detection of
chloroplastic ASPX was possible when frozen chloroplasts or leaf
extracts were used for native electrophoresis (Mitler and Zilinskas,
1993). When we tested the effect of pCMB by assaying the activity of
ASPX in the activity gels, we used conditions similar to those used by
Mitler and Zilinskas (1993), and the samples with higher ASPX activity
in the spectrophotometric assay also displayed wide bands in the gels,
presumably representing the activity of cytosolic ASPX.
), was observed only at 10 d and mainly at high
light. This result is quite interesting because glutathione is involved
in the regeneration of reduced ascorbic acid, a reaction catalyzed by
DAS reductase. If ascorbate is oxidized to monodehydroascorbate and
then to DAS, one would expect a depletion of the GSH pool and increased
activity of GR, to keep the GSSG-to-GSH ratio in balance. However,
ascorbate could also be regenerated by monodehydroascorbate reductase
(Foyer, 1993). Measurement of total ascorbate showed that a depletion
of AS was not followed by an accumulation of DAS, refuting the
possibility that GR or DAS reductase is inhibited under Mn toxicity
stress and is the cause of the decreased pool of AS.
) is one of the essential
water-soluble antioxidants present in millimolar concentrations in the
chloroplast, and is the substrate used by ASPX to control the level of
photosynthetically generated
H2O2 (Nakano and Asada, 1981) as part of the ascorbate-glutathione cycle. Also, ascorbate is
involved in the regeneration of the membrane-bound antioxidants 
-tocopherol and zeaxanthin, affording protection against lipid peroxides and singlet oxygen, respectively. The pool of ascorbate in
the cell is kept at a fairly constant level, and the loss of ascorbate
might reflect the degree of stress imposed (Stegmann et al., 1991).
Because ascorbate functions in leaves mainly as a reductant, its redox
state is pivotal for its function as an antioxidant (Foyer, 1993). A
higher increase in the chloroplast ascorbate content of NaCl-treated
leaves was found in a tolerant pea genotype (Hernández et al.,
1995). Bean genotypes tolerant of ozone stress had a higher ascorbate
content than the susceptible genotypes (Guzi and Heath, 1993). Change
in the ascorbate redox state from reduced to oxidized ascorbate was
found in tobacco leaves exposed to Fe stress (Kampfenkel et al., 1995)
without net changes in the amount of ascorbate. Sirkar and Amin (1974) found a slight increase in total ascorbate in cotton plants suffering Mn toxicity.
), and that Mn was bound to oxalate in Mn hyperaccumulator plants
(Memon and Yatazawa, 1984). Thus, if susceptible genotypes respond to
excess Mn by increasing the synthesis of oxalate at the expense of
ascorbic acid, the deleterious effect of Mn toxicity might be
indirectly enhanced because of a depletion of this important
antioxidant. In nonaqueous fractionation studies (González, 1996)
oxalate-like crystals were found to accumulate high levels of Mn in
leaves of both genotypes, although no quantitative comparison among
genotypes was attempted. Tolerant genotypes might have developed a
different strategy to deal with excess Mn in leaf tissue, such as
organelle or tissue compartmentation.
) may differ from ours because of the experimental
system used.
found that the
Mn-tolerant soybean genotype Lee was more tolerant to ozone stress than
the Mn-susceptible genotype Bragg. The tolerance of soybean genotypes
to ozone stress was associated with the endogenous presence of
kaempferol glycosides, particularly K3 through K6 and K9. Flavonoids
can absorb short wavelengths and protect the plants against the
damaging effect of UV light. Thus, because light is a determining
element in both the onset of chlorosis and the increment of antioxidant
enzyme activities in Mn-treated plants, the relationship between
light-absorbing substances and tolerance to Mn toxicity deserves
further study.
), where ascorbic acid is synthesized (Loewus and Loewus, 1987), events happening outside of the chloroplast leave this organelle with lower
concentrations of antioxidant, a situation that promotes chlorosis when
the tissue is exposed to higher irradiance. This chain of events would
not require excess Mn in chloroplasts of mature leaves.
1
This research was partially supported by the
Centro Internacional de Agricultura Tropical.
FOOTNOTES
2
Present address: Department of Agronomy and
Range Science, University of California, One Shields Avenue, Davis, CA
95616.
*
Corresponding author; e-mail jpl4{at}psu.edu; fax
1-814-863-6139.
ABBREVIATIONS
-dithiobis(2-nitrobenzoic
acid).
GPX, guaiacol peroxidase.
GR, glutathione reductase.
GSSG, oxidized glutathione.
MDA, malondialdehyde.
pCMB, p-chloromercuribenzoate.
SOD, superoxide dismutase.
LITERATURE CITED
Top
Abstract
Introduction
Methods
Results
Discussion
References
-dithiobis(2-nitrobenzoic acid).
Anal Biochem
175:
408-413
[CrossRef][ISI][Medline]
