to Environmental Stress [T. A. Mansfield, L. Fowden and J. S. Stoddard, eds.]. Chapman and Hall, London. pp. 263-273. 6. Slooten, L., Capian, K., Van Camp, W.
環境毒性学会誌（Jpn. J. Environ. Toxicol.），11（1），1-9，2008
Glutathione-Ascorbate Cycle for Phytoremediation of Mercury by Eichhornia crassipes (Mart.) Solms Upma Narang*, A.K. Thukral*, Renu Bhardwaj*, S.K. Garg** *
Department of Botanical & Environmental Sciences, ** Department of Molecular Biology & Biochemistry, Guru Nanak Dev University, Amritsar − 143005, India.
ABSTRACT The activities of ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, monodehydroascorbate reductase, and ascorbic acid and glutathione contents increased in response to mercury accumulation in E. crassipes. This enhancement in the glutathione-ascorbate Cycle components was observed in response to mercury in solution up to a concentration of 100 μg l-1, whereas, at a concentration of 1000 μg l-1 the enzyme activities decreased. Roots accumulated maximum amount of Hg, and there was a significant positive correlation between Hg accumulated and components of the glutathione-ascorbate cycle in E. crassipes, during phytoremediation of mercury. Key words: E. crassipes, mercury, antioxidants, antioxidative enzymes.
. hydroxyl radicals（ OH）in plants. An increase in
1. Introduction Release of heavy metals such as Cd, Cu, Pb, Cr
ROS levels in cells is extremely dangerous because
and Hg into the environment is highly detrimental
these highly reactive molecules damage the main
to human health as these are non-degradable and
classes of cell components such as proteins, polysac-
1）
get bio-accumulated in the food chain . When
charides, lipids and nucleic acids. Free radical scav-
placed in effluents containing heavy metals, water
enging system operates in the cytoplasm and
hyacinth has been shown to readily absorb Cu, Pb, . The formation of strong
chloroplasts in a series of reactions, in which the . reduction of activated oxygen（ O2- and H2O2）is
complexes of metals like lead and mercury with the
achieved eventually at the expense of photosyntheti-
thiol group and the pluridentate ligands（glycine,
cally or enzymatically produced reductant, NAD（P）
histidine and cysteine）results in the modification
5） H . NADPH reduces the reaction products of
（inhibition or enhancement）of the activities of cer-
ascorbate peroxidase i.e., dehydroascorbate or mon-
tain enzymes and proteins. A common feature of
odehydroascorbate either directly or via
different stress factors is their potential to increase
glutathione6） DHAR and MDHAR are the enzymat-
Zn, Hg, Cd and Ni
2 - 4）
ic components of the glutathione-ascorbate cycle the production of reactive oxygen species such as . 1 singlet oxygen（ O2），superoxide radical（ O2 ）， （also known as ascorbate-glutathione cycle） hydrogen peroxide（H2O2）and the most toxic
involved in the regeneration of reduced ascorbate7）.
−1−
Glutathione-Ascorbate Cycle for Phytoremediation
Ascorbate is the most important antioxidant in
2. MATERIALS AND METHODS The plants of E. crassipes were cultured in 10%
plants, exercising a fundamental role in the removal 8）
of hydrogen peroxide . Oxidation of ascorbate
Hoagland’ s nutrient medium containing 0, 1, 10,
occurs in two sequential steps, first producing mon-
100 and 1000 μg l-1 of Hg as HgCl2. A volume of 5
odehydroascorbate, and if not rapidly re-reduced to
litres of each treatment solution was maintained in
ascorbate, the monodehydroascorbate disproportion-
10-litre troughs in replicates of three for each treat-
ates to ascorbate and dehydroascorbate. The two
ment at a temperature of 27±2℃, with three E.
enzymes involved in the regeneration of reduced
crassipes plants in each trough. The accumulation of
ascorbate are dehydroascorbate reductase and mon-
Hg in the plants was studied over a period of three
9）
odehydroascorbate reductase . Glutathione（GSH）
weeks and it was observed that after two weeks the
is the major low molecular weight thiol compound
uptake of mercury by the plants was negligible.
in plants. It acts as disulphide reductant to protect
Therefore, different plant parts（roots, petioles and
thiol groups on enzymes, reacts with singlet oxygen
leaf laminae）were harvested after 7 and 14 days
and hydroxyl radicals and also participates in regen-
of Hg treatment. E. crassipes plants were cultured
eration of ascorbate. GSH is regenerated by the
under static conditions to simulate the growth of
10）
enzyme glutathione reductase . The phytoremedia-
the plant in the area in static waters. Hg was deter-
tors use their internal defence system during phy-
mined using cold vapor atomic absorption spec-
toremediation leading to environmental restoration.
trophotometery（Mercury Analyzer, ECIL, Model
The components of antioxidative system mentioned
5800/E） ．For the estimation of glutathione reduc-
above constitute the ascorbate-glutathione cycle
tase, 1 g of plant tissue was homogenized in pre-
（Halliwell-Asada pathway） ，and are given in Fig. 1.
chilled mortar and pestle with 3 ml of 100 mM
E. crassipes is an efficient remediator of heavy met-
potassium phosphate buffer（pH 7.0）under ice-
10）
especial-
cold conditions. For estimation of ascorbate peroxi-
ly in tropical climates. The hypothesis tested is
dase, dehydroascorbate reductase and monodehy-
that accumulation of Hg in the plant would cause
droascorbate reductase, 5 mM ascorbate was added
the oxidative stress and would stimulate the compo-
to the extraction buffer. The homogenate was cen-
nents of ascorbate-glutathione cycle of the plant to
trifuged at 15,000 g at 5℃ for 20 minutes and the
manage this stress.
supernatant was used for enzyme analysis. The
als and grows luxuriantly in wastewaters
plant extracts for ascorbic acid and glutathione content estimation were prepared by homogenizing 1 g of fresh plant tissue in pre-chilled mortar and pestle under ice cold conditions in 3 ml of Tris buffer（50 mM, pH 10.0）containing 1 mM EDTA. The homogenate was centrifuged at 12,000 g for 15 minutes and the supernatant was used for the antioxiFig.1 Halliwell-Asada Pathway（Ascorbate-Glutathione cycle）．（APX, ascorbate-peroxidase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase; GR, glutathione reductase. Modified from May et al. 1998 11）．）
dant assays. The activities of ascorbate peroxidase （APX）（EC 1.11.1.11）12）, glutathione reductase （GR） （EC 1.6.4.2）13）, dehydroascorbate reductase （DHAR） （EC 1.8.5.1）14）, and monodehydroascor-
−2−
Narang et al
Fig.2 Initial contents of mercury (μg g-1 dry wt.), antioxidative enzymes activities (units mg-1 protein) and antioxidants contents (mg g-1 FW) in E. crassipes (n=3).
Fig.3 Mercury accumulation (μg g-1 dry wt) by E. crassipes on treatment with HgCl2 (μg/ L, n=3).
Fig.4 APX activity (units mg-1 protein) in E. crassipes on treatment with HgCl2 (μg/ L, n=3).
Fig.5 GR activity (units mg-1 protein) in E. crassipes on treatment with HgCl2 (μg/ L, n=3).
bate reductase（MDHAR） （EC 1.1.5.4）15）, and the
tured in solutions containing HgCl2 are given in
16）
contents of ascorbic acid（AA） , and glutathione 17）
Figs. 2 and 3. It was observed that roots accumulat-
（GSH） were determined after 7 and 14 days of
ed maximum amount of mercury, followed by leaf
culturing. Statistical analysis was done for descrip-
laminae and petioles. The APX activities increased
tive statistics, regression, ANOVA and Tukey’ s multiple comparison test.
th -1 on the 14 day of treatment with 100 μg l in the
order, roots > petioles > leaf laminae（Fig. 4） ．The GR activities attained the maximum values in all the plant parts for 100 μg l-1 concentration on 14th
3. RESULTS The initial enzyme activities and antioxidant contents in E. crassipes and distribution of mercury
day of treatment, the order being, roots > laminae > petioles（Fig. 5） ．
content in different plant parts of E. crassipes cul-
−3−
DHAR activity was also found to increase with
Glutathione-Ascorbate Cycle for Phytoremediation
Fig.6 DHAR activity (units mg-1 protein) in E. crassipes on treatment with HgCl2 (μg/ L, n=3).
Fig.7 MDHAR activity (units mg-1 protein) in E. crassipes on treatment with HgCl2 (μg/ L, n=3).
Fig.8 AA content (mg g-1 FW) in E. crassipes on treatment with HgCl2 (μg/ L, n=3).
Fig.9 GSH content (mg g-1 FW) in E. crassipes on treatment with HgCl2 (μg/ L, n=3).
the increase in concentration of mercury in solution
respectively（Fig. 8） ．
in the order, roots > petioles > leaf laminae（Fig.
New leaves and roots sprouted in both controls
6） ．MDHAR activities increased in the order peti-
and treatments in the second, which led to increase
th
oles = leaf laminae > roots on 14 day for 100μg l
-1
treatment（Fig. 7） ．
in ascorbic acid content. On the 7 th day of treatment, the ascorbic acid content in roots, petioles
The zero day ascorbic acid content was -1
and leaf laminae increased from 1 μg l-1 to 1000 μ
FW in roots, 0.058 mg
-1 th g l concentration. The 14 day observations
g-1 FW in petioles and 0.129 mg g-1 FW in leaf lam-
showed the ascorbic acid content to be maximum
observed to be 0.03 mg g
th
th
for 1000μg l-1 treatment as 0.580 mg g-1 FW in
days were observed to be 0.035 and 0.447 mg g-1
-1 -1 roots, 0.799 mg g- FW in petioles and 1.295 mg g
FW（roots） ，0.143 and 0.612 mg g-1 FW（petioles）
FW in leaf laminae. The glutathione content on
inae. The ascorbic acid contents on 7
-1
and 14
and 0.307 and 0.967 mg g FW（leaf laminae）
treatment with mercuric chloride（Fig. 9）was
−4−
Narang et al
TABLE 1. Treatment x Dose interactions using two-way ANOVA.
TABLE 2. Regression between mercury content (X), and enzyme activities and antioxidant contents (Y) in roots of E. crassipes after 14 days of treatment.
observed to be maximum for 1μg l-1 concentration th
plant parts（roots, petioles and leaf laminae）and
on 7 day. For the rest of treatments, the GSH con-
mercury concentration. The two-way ANOVA for the
tent remained lower than these values but higher
effect of days x concentration revealed significant
-1
than the initial values. At 1000 μg l concentration
differences among all treatments except for APX
the plants became brittle.
activity in leaf laminae and MDHAR activity in
On the basis of one way ANOVA, variations in
roots and petioles. The significance of interactions
mercury content, antioxidative enzyme activities
between concentration on mercury and treatment
and antioxidant contents were found to be signifi-
period for implies that the effect of Hg concentra-
th
th
cant for all plant parts both on the 7 and 14
tion is affected by duration of treatment and vice
days. Table 1 describes the interaction between
versa. Similarly, significant differences were
days of treatment（7 and 14 days）and mercury
observed for all parameters on 7th and 14th days in
concentration（0, 1, 10, 100 and 1000 μg l-1 ）and
different plant parts（plant parts x concentration）
−5−
Glutathione-Ascorbate Cycle for Phytoremediation
in all the treatments except for MDHAR activity on th
APX activity in Sesbania drummondii seedlings
7 day. The treatment x dose interactions in the
exposed to mercury. Ascorbate peroxidase stimula-
two-way ANOVA’ s clearly indicated that the effect of
tion due to As accumulation in Pteris vittata
21）
，
22）
Hg concentration on antioxidative components
and Cu intoxication in Zea mays
depends on the plant parts. In order to find rela-
reported. The reduction of H2O2 by APX results in
tion between the mercury accumulated by the
the production of dehydroascorbate or monodehy-
plants and changes in the enzyme activities regres-
droascorbate. The monodehydroascorbate is con-
sion analyses were done for the data after 14 days
verted back to ascorbate with the help of MDHAR,
of treatment. It was observed that antioxidative
whereas DHAR catalyses the recovery of ascorbic
enzymes and antioxidants are positively correlated
acid from dehydroascorbate involving the oxidation
with the Hg content in the roots of plants cultured
of GSH to GSSG. So, an enhancement in the activi-
-1
in concentrations up to 100 μg l （Table 2） ．
has also been
ty of ascorbate peroxidase must in turn induce DHAR and MDHAR as also observed in the present
4. DISCUSSION
study. Glutathione reductase converts the oxidized
In plant cells, ascorbic acid is a major antioxi-
glutathione（GSSG）to reduced glutathione（GSH） ，
dant involved in the ascorbate-glutathione cycle and
which is involved in the quenching of free radicals
has been shown to play an important role in pollu-
either directly or by getting oxidized in the recovery
tion tolerance. Enhanced ascorbic acid content was
of ascorbate. The enhancement of enzyme activities
also observed in roots and leaves of Brassica juncea
being observed in the present course of study is in
plants that were found to be effective accumulators
accordance with the earlier reports. Increase in dif-
of Cr, Fe, Zn and Mn when given tannery sludge
ferent enzyme activities in B. juncea shoots under
treatment
18）
．On exposure to Cd, the ascorbate
level was observed to be comparatively higher in E. 19）
Zn treatment Vicia faba
23）
，Cd treatment in sunflower
25）
24）
and Cu stress in Phaseolus vulgaris
， 8）
．During the
has also been reported to increase the antioxidative
present investigation, the ascorbic acid content
enzymes. The enhanced activity of glutathione
increased to 7.9 fold in roots, 3.4 fold in petioles
reductase in the
and 2.3 fold in leaf laminae of plants treated with
enhanced production of GSH. An increase in GSH
crassipes than in Pistia stratiotes
-1
1000 μg l concentration on 7th day. A further th
present study suggests the
content of 58% in roots, 43% in petioles and 29% in
stimulation of 27 to 34% was observed on 14 day.
leaf laminae was observed in plants treated with 1
The enhanced level of ascorbic acid can be attrib-
μg l-1 mercuric chloride after 7 days of treatment.
uted to its role as a free redical quencher. In the
For the rest of the treatments, GSH content was
ascorbate − glutathione cycle, ascorbate peroxidase
maintained at a comparatively lower level despite of
is the primary enzyme involved in scavenging of
the fact that it showed a concentration dependent
H2O2 using ascorbate as a substrate. The APX activ-
increase on 14th day in all plant parts. This signifies
ity during the present study was found to increase
the active stimulation of GSH content in response
in response to mercury stress level showing the
to mercury. However, the maintenance of lowered
maximum increase of 314% in roots, 104% in peti-
level of GSH at higher concentration and prolonged
oles and 351% in leaf laminae. The study finds sup-
exposure could be attributed to its being simultane-
20）
port from Israr et al. who reported increase in
ously utilized either in production of phytochelatins
−6−
Narang et al
food crops. J Zhejiang Univ Sci B. 8: 1-13.
or in the recovery of ascorbate. But the stimulated activity of GR did not allow the abrupt fall in the
2.
Riddle, S. G., Tran, H. H., Dewitt, J. G. and
GSH level. In literature also, GSH content has been
Andrews, J. C.（2002） ．Field, laboratory and
reported to increase initially followed by decrease
X-ray absorption spectroscopic studies of mer-
with increase in metal concentration and passage of
cury accumulation by water hyacinth. Environ
time in wheat leaves in response to Zn and Cr
Sci Technol. 36: 1965-1970.
stress
26）
．Initial increase in GSH content due to
3.
Lu, X., Kruatrachue, M., Pokethitiyook, P. and
Cd exposure（50 μM）has also been reported in
Homyok, K.（2004）．Removal of cadmium
Phragmites australis, which subsequently dropped at
and zinc by water hyacinth, Eichhornia cras-
high Cd（100 μM）treatment
27）
sipes. Science Asia. 30: 93-103.
． 4.
5. CONCLUSIONS
Jayaweera, M. W., Kasturiarachchi, J. C., Kularatne, R. K. and Wijeyekoon, S. L.（2008） ．
Exposure to heavy metals provokes pronounced
Contribution of water hyacinth（Eichhornia
responses of antioxidative systems, but the quan-
crassipes（Mart.）Solms）grown under differ-
tum of response is dependent on the tissue ana-
ent nutrient conditions to Fe-removal mecha-
lyzed and the intensity of stress. There was an
nisms in constructed wetlands. Journal of
increase in the mercury content in different plant
Environmental Management. 87: 450-460.
parts of E. crassipes cultured in solutions HgCl2.
5.
Polle, A. and Rennenberg, H.（ 1993）．
Roots accumulated maximum amount of mercury
Significance of antioxidants in plant adaptation
in roots, to the extent of 71.68 times with respect
to environmental stress. In: Plant Adaptation
to the concentration in water after 14 days. This
to Environmental Stress [T. A. Mansfield, L.
was accompanied with an increase in the activities/
Fowden and J. S. Stoddard, eds.]. Chapman
contents of glutathione ascorbate pathway in all the
and Hall, London. pp. 263-273.
plant parts. There was a significant positive correla-
6.
Slooten, L., Capian, K., Van Camp, W., Van
tion between the amount of mercury accumulated
Montagu, M., Symbesma, C. and Inze, D.
by roots and components of antioxidative defence
（1995） ．Factors affecting the enhancement of
system. Generally the interactions between days of
oxidative stress tolerance in transgenic tobacco
treatment x mercury and plant parts x mercury
overexpressing manganese superoxide dismu-
were significant. Thus the components of ascorbate
tase in the chloroplasts. Plant Physiol. 107:
− glutathione pathway in E. crassipes interact in a
737-750.
coordinated manner for the removal of reactive oxy-
7.
Leterrier, M., Corpas, F. J., Barroso, J. B.,
gen species generated as a result of exposure to
Sandalio, L. M. and del Rio, L. A.（2005） ．
mercury making it an efficient phytoremediator.
Peroxisomal monodehydroascorbate reductase. Genomic clone characterization and functional
REFERENCES
analysis under environmental stress conditions.
1.
Plant Physiol. 138: 2111-2123.
Islam, E., Yang, X. E., He, Z. L. and Mahmood, Q.（2007） ．Assessing potential dietary toxici-
8.
ty of heavy metals in selected vegetables and
−7−
Gupta, M., Cuypers, A., Vangronsveld, J. and Clijsters, H.（1999）．Copper affects the
Glutathione-Ascorbate Cycle for Phytoremediation
enzymes of the ascorbate-glutathione cycle and
9.
17. Sedlak, J. and Lindsay, R. H.（ 1968）．
its related metabolites in the roots of Phaseolus
Estimation of total, protein bound and non-pro-
vulgaris. Physiol Plant. 106: 262-267.
tein sulphydryl groups in tissue with Ellman’ s
Arora, A., Sairam, R. K. and Srivastava, G. C.
reagent. Anal Biochem. 25: 192-205.
（2002） ．Oxidative stress and antioxidative sys-
18. Singh, S. and Sinha, S.（2005） ．Accumulation of
tem in plants. Curr Sci. 82: 1227-1238.
metals and its effects in Brassica juncea（L.）
10. Prasad, M. N. V. and Strazalka, K.（2002） ．
Czern.（cv Rohini）grown on various amend-
Physiology and Biochemistry of Metal Tolerance
ments of tannery waste. Ecotoxicol Environ Saf.
in Plants. Kluwer Academic Publishers,
62: 118-127. 19. Sanita di Toppi, L., Vurro, E., Rossi, L.,
Dordrecht. 11. May, M., Vernoux, T., Leaver, C., Van Montagu,
Marabottini, R., Musetti, R., Careri, M., Maffini,
M. and Inze, D.（1998） ．Glutathione home-
M., Mucchino, C., Corradini, C. and Badiani, M.
ostasis in plants: implications for environmen-
（2007） ．Different compensatory mechanisms in
tal sensing and plant development. J Exp Bot.
two metal-accumulating aquatic macrophytes
49: 649-667.
exposed to acute cadmium stress in outdoor artificial lakes. Chemosphere. 68: 769-780.
12. Nakano, Y. and Asada, K.（1981） ．Hydrogen peroxide is scavenged by ascorbate specific-per-
20. Israr, M., Sahi, S., Datta, R. and Sarkar, D.
oxidase in spinach chloroplasts. Plant Cell
（2006）．Bioaccumulation and physiological
Physiol. 22: 867.
effects of mercury in Sesbania drummondii. Chemosphere. 65: 591-598.
13. Carlberg, I. and Mannervik, B.（1975）． Purification of the flavoenzyme glutathione
21. Srivastava, M., Ma, L. Q., Singh, N. and Singh, S.
reductase from rat liver. J Biol Chem. 250:
（2005） ．Antioxidant responses of hyperaccumu-
5475-5480.
lator and sensitive fern species to arsenic. J Exp Bot. 56: 1335-1342.
14. Dalton, D. A, Russel, S. A., Hanus, F. J., Pascoe, G. A. and Evans, H. J.（1986）．
22. Tanyolac, D., Ekmekci, Y. and Unalan, S.（2007） ．
Enzymatic reactions of ascorbate and glu-
Changes in photochemical and antioxidant
tathione that prevent peroxide damage in soy-
enzyme activities in maize（Zea mays L.）leaves
abean root nodules. Proc Natl Acad Sci., USA.
exposed to excess copper. Chemosphere. 67: 89-98. 23. Prasad, K. V. S. K., Paradha Saradhi, P. and
83: 3811-3815. 15. Hossain, M. A., Nakano, Y. and Asada, K.
Sharmila, P.（1999）．Concerted action of
（1984） ．Monodehydroascorbate reductase in
antioxidant enzymes and curtailed growth under
spinach chloroplasts and its participation in
zinc toxicity in Brassica juncea. Environ Exper
regeneration of ascorbate for scavenging hydro-
Bot. 42: 1-10. 24. Gallego, S. M., Benavides, M. P. and Tomaro, M.
gen peroxide. Plant Cell Physiol. 25: 385-395. 16. Roe, J. H. and Kuether, C. A.（1943） ．The
L.（1999） ．Effect of cadmium ions on oxidative
determination of ascorbic acid in whole blood
defense system in sunflower cotyledons. Biol
and urine through the 2,4-dinitrophenyl
Plant. 42: 49-55.
hydrazine derivative of dehydroascorbic acid. J
25. Cordova Rosa, E. V., Valgas, C., Souza-Sierra, M. M., Correa, A. X. R. and Radetski, C. M.（2003） ．
Biol Chem. 147: 399-407.
−8−
Narang et al
Biomass growth, micronucleus induction and antioxidant stress enzyme responses in Vicia faba exposed to cadmium in solution. Environ Toxicol Chem. 22: 645-649. 26. Panda, S. K., Chaudhary, I. and Khan, M. H. （2003） ．Heavy metals induce lipid peroxidation and affect antioxidants in wheat leaves. Biol Plant. 46: 289-294. 27. Pietrini, F., Iannelli, M. A., Pasqualini, S. and Massacci, A.（2003） ．Interaction of cadmium with glutathione and photosynthesis in developing leaves and chloroplasts of Phragmites australis （Cav.）Trin. ex Steudel. Plant Physiol. 133: 829837. （受付：2007年8月4日；受理：2008年5月11日）
−9−
