Glutathione S-transferase: an enzyme for chemical defense in plants

Abstract

Chemical defenses in plants can be classified in two distinct groups: 
constitutive and induced defenses. Glutathione (GSH) a tripeptide (-L-glutamyl-
L-cisteinyl-glycine, first reported in 1988 as a philothion, distributed in the 
intracellular space of plants, animals, and microorganisms has two general 
functions: to remove toxic metabolites from the cell and to maintain cellular 
sulfhydril groups in their reduced form. Glutathione (GSTs: EC 2.5.1.18) are 
enzymes that detoxify endobiotic and xenobiotic compounds by covalent linking of 
glutathione to hydrophobic substrate. GST enzymes have been identified and 
characterized in insects, bacteria, and in many plant species. Plant glutathione 
s-transferase shows three groups based on the phylogenetic tree and genetic 
distance. There is evidence that maize GST has a small region of high amino acid 
sequence identify with mammalian alpha, mu, and pi class GST subunits. These 
classes are present only in animals and yeasts and not present in bacteria and 
plants. Herbicide detoxification by glutathione s-transferase has been widely 
studied. Maize seedling GST activities was classified in the order alachlor ( 
fluorodifen ( atrazine ( metolachlor. GST activity sequence observed for 
atrazine was Zea mays ( P. miliaceum (( S. bicolor = S. faberi = D. sanguinalis 
= Abutilon theophrasti ( E. crus-galli. Although little is known about other 
biological GST functions, there is some evidences that GST also may be induced 
by heavy pathogen attack and heavy metals. Gst1, induced by pathogen attack, 
presents in wheat showed similarity to with maize GST. Maize glutathione s-
transferase Bronze2 (Bz2) is induced by cadmium treatment. A review of the 
glutathione s-transferase importance for detoxification in plants is presented.


Introduction

Plants and other organisms have developed mechanisms to defend themselves 
against herbivores, insects, pathogens, or chemical compounds that are harmful 
to their survival. These strategies have been classified in two distinct groups: 
constitutive and induced defenses. Constitutive defenses also referred to as 
"natural" or "innate" resistance provide natural protection while induced 
defenses are created by an external agent such as an insect, pathogen, or 
xenobiotic. One induced mechanism is activation of specific enzymes that 
inactivate chemical compounds or detoxify heavy metals in plants.
	Glutathione ((-L-glutamyl-L-cisteinyl-glycine), a submajor constituent of 
all cells and almost always the major nonprotein thiol compound present in 
cells, was first reported in 1888 by De Rey-Pailhade as a philothion (RH2) and 
shown to be responsible for formation of hydrogen sulfide when yeast cells were 
crushed with elemental sulfur. He also reported that philothion were present in 
beef muscle, beef liver, sheep brain, lamb small intestine, fish muscle, egg 
white, fresh sheep blood, and in freshly picked asparagus tips. Hopkins renamed 
philothion in 1921 as glutathione (Boyland and Chasseaud 1969; Meister 1989). 
Glutathione (GSH) shows two peptide bounds, two carboxylic acid groups, one 
amino group, and one thiol group (Fig. 1).

Fig. 1. Chemical structure of glutathione.
The high number of hydrophilic functional groups in glutathione combined with 
its low molecular weight leads to high water solubility (Kosower 1976). 
Glutathione that is distributed in the intracellular space of plants, animals, 
and microorganisms has two general functions: to remove toxic metabolites from 
the cell and to maintain cellular sulfhydril groups in their reduced form 
(Liebman and Greenberg 1988). Glutathione metabolism involves many reactions 
(Fig. 2) where glutathione is synthesized, degraded, conjugated or oxidized. In 
one of these pathways, the glutathione S-transferase (GSTs, E.C.2.5.1.18), a 
family of multifunctional isozymes in vertebrates, plants, insects and aerobic 
microorganisms (Armstrong 1993), catalyzes both GSH-dependent conjugation and 
reduction (Ketterer et al. 1993).

Fig. 2. Description of the metabolism of glutathione with all pathways possible. 
1 (-glutamylcysteine synthetase; 2 GSH synthetase; 3 (-glutamyltranspeptidase; 4 
dipeptiddases; 5 (-glutamylcyclotransferase; 6 5-oxoprolinase; 7 GSH s-
transferase; 8 N-acethyltransferase; 9 GSH peroxidases; 10 GSH thiol 
transferases; 11 reaction of free radicals with GSH; 12 gluthatione disulfide 
(GSSG) reductase; 13 transport of (-Glu-(Cys)2 (Meister 1989).

Glutathione s-transferase detoxifies endobiotic and xenobiotic compounds by 
covalently linking glutathione to a hydrophobic substrate, forming less reactive 
and more polar gluatathione s-conjugate (Neuefeind et al. 1997). Glutathione S-
transferases are dimeric enzymes that show five independent classes, alpha, mu, 
pi, sigma (Buetler and Eaton 1992; Drogg et al. 1995; Ketterer et al. 1993; 
Neuefiend 1997), and theta (Buetler and Eaton 1992; Drogg et al. 1995; Neuefiend 
1997). Classes alpha, mu and pi initially proposed by Mannervick et al. in 1988 
(Buetler and Eaton 1992), are presents only in animals and yeasts but absent in 
bacteria and plants (Pemble and Taylor 1992). The very heterogeneous theta 
class, reported by Meyer et al 1991 (Buetler and Eaton 1992) are presents in 
yeasts, plants, bacteria, rats, humans, chickens, salmon, and non veterbrates 
such as flies and apparently absent in lower animals such as molluscs, 
nematodes, and platyhelminthes (Taylor et al 1993). Buetler and Eaton in 1992 
assembled a database of 71 full length and near full length amino acid sequences 
of glutathione s-transferases from various vertebrates, eukaryotes and 
prokaryotes. With this database they established a dendrogram identifying 
classes alpha, mu, pi, theta, and sigma.


Detoxification of chemical compounds

Detoxification in plants is a process (Fig. 3) tat metabolizes foreign 
compounds. It can be separated into two sequential processes: chemical 
(modification) transformation, and compartmentation. These two processes are 
divided into three phases: phase I (activation) reactions, phase II 
(conjugation), and phase III (internal compartmentation and storage processes) 
(Coleman 1997; Marrs 1996; Sabnderman 1992).
	Phase I usually involves hydrolysis catalyzed by esterases and amidases or 
oxidation catalyzed by the cytochrome P-450 system. Phase II involves the 
deactivation synthesis of xenobiotic or a phase I-activated metabolite by 
covalent linkage to an endogenous hydrophilic molecule, such as glucose, 
malonate or glutathione resulting in a nontoxic or less toxic compound. Phase II 
is catalyzed by glucosyl-, malonyl, or glutathione transferases. In phase III, 
the inactive water soluble conjugates formed in phase II, are exported from the 
cytosol by membrane-located proteins, which initiate the compartmentalization 
and storage in the vacuole (soluble conjugates) or in the cell wall (insoluble 
conjugates) (Coleman 1997; Marrs 1996; Sabnderman, 1992).

Fig. 3. Detoxification process of xenobiotics in plants. Broken arrows represent 
a proposed patway for the glucosylation of xenobiotics in the Golgi, followed by 
release of the metabolites into the apoplast. CT, glutathione-conjugate 
transporte; AT, ATP-dependent xenobiotic anion (taurocholate) transporter; GT, 
ATP-dependent glucoside-conjugate transporter; VP, vacuolar peptidase (Coleman 
et al., 1997).


Glutathione s-transferase in plants

	Glutathione s-transferases (GST), first discovered because of their 
ability to metabolize toxic exogenous compounds, have been identified and 
characterized in insects (Harold and Ottea 1997), in bacteria (Zablotowich et 
al. 1995), and in many plants such as maize (Edwards and Owen 1986; Rossini et 
al. 1996; Jablonkai and Hatzios 1991; Scarponi et al. 1992; Jepson et al. 1994; 
Holt et al. 1995; Marrs et al. 1995; Hatton et al. 1996; Dixon et al. 1997; 
Marrs and Walbot 1997), wheat (Jablonkai and Hatzios 1991; Mauch and Dudler 
1993, Romano et al. 1993; Edwards and Cole 1996; Riechers et al. 1996; Riechers 
et al. 1997), tobacco (Droog et al. 1995), dwarf pine (Schroder and Rennenberg 
1992), soybean (Ulmasov et al. 1995; Andrews et al. 1997), Arabidopsis thaliana 
(Reinemer et al. 1996), barley (Romano et al. 1993; Wolf et al. 1996), Setaria 
spp. (Wang and Dekker 1995), carnation (Meyer et al. 1991), potato (Hahn and 
Strittmatter 1994), chickpea (Hunatti and Ali 1990, 1991), sorghum (Gronwald et 
al. 1987; Dean et al. 1990), velvetleaf (Anderson and Gronwald 1991) and 
sugarcane (Singhal et al. 1991). According to Lamoureux and Rusness (1993) cited 
by Marrs (1996) there are over 33 plant species with GST activity, although in 
many cases the GSTs have been not purified. Drogg et al. (1995) proposed three 
categories of glutathione s-transferase in plants, type I, II, and III (Fig. 4), 
based on the phylogenetic tree and genetic distance obtained in the evolutionary 
relationship between 16 plant GST protein sequences available. Type I contains 
three exons and two introns, type II contains ten exons and nine introns, and 
type III contains two exons and one intron (Marrs 1996). Plant glutathione s-
transferases are comparable to theta class, although type II also corresponds in 
sequence and intron position to alpha class GST in mammalian. Taylor et al 
(1987) cited by Taylor et al (1993) show evidence that maize GST has a small 
region of high amino acid sequence same as with mammalian alpha, mu, and pi 
class GST subunits.




Fig. 4. Phylogenetic tree of plant type I, II, and III glutathione s-transferase 
obtained from Genbank or PIR (Marrs, 1996).


		Type I GST includes GST I, GST II, GST III, and GST IV present in 
maize that show substrate specificity toward herbicides such as alachlor, 
atrazine, or metolachlor. Other GSTs classified in this group are GstA (gene) 
cDNA WIR56 in wheat, parB (gene) in tobacco, PMA239X14, AW124, gst2/Atpm24, 
ERD11, and ERD13 in Arabidopsis thaliana. Type I GST was also cloned from 
broccoli, sugarcane, Silene cucubalis, and Hyoscyamus muticus. Type II is 
represented only by GST1 (pSR8) and GST2 from carnation whose substrate 
specificity is unknown although there is speculation that they participate in 
lipid peroxidation. Type III consist of GmHsp26A or GHT2 in soybeans, prp1-1 
(gene) also called Gst1 from potato, parA/Nt114, parC/Nt107, Nt103, and Nicotina 
plumbaginfolia msr1 (pLS216) from tobacco, bronze -2 from maize, and GST5 from 
Arabidopsis thaliana, first identified as a set of homologous genes, is induced 
toward auxin, ethylene, pathogen infection, heavy metals, and heat shock. 
Another glutathione s-transferases such as Sorghum GSTs 1-6, GST I, II, III, IV 
from chickpea, soluble (37kD), soluble (47 kD), and microsomal from pea, GST25 
and GST26 from wheat, GST1 from Arabidopsis thaliana, GST I and GST II from 
Picea abies, Zea mays (function as 30 kD monomer) from maize, and Phaseolus 
vulgaris from French bean have been characterized, but because of the amino acid 
sequence still unavailable impeding grouping in classes I, II, and III (Marrs, 
1996).


Herbicide detoxification in plants

	Mortimer (1997) discussed the possibility of phenological adaptation in 
weeds in terms of an evolved response to herbicides, however this phenological 
adaptation remains largely unexplored. On the other hands one basis for 
selectivity is the fact that plants can detoxify some herbicides fast enough to 
avoid accumulation to phytotoxic levels. Herbiicides, usually highly lipophilic, 
metabolized by enzymatic reactions change reactivity and polarity. These 
compounds are eliminated by conjugation, detoxification, deposition, and so on, 
more rapidly than they are replenished (Devine et al. 1993). The basic reaction 
of herbicide detoxification is oxidation, reduction, hydrolysis, and conjugation 
(Duke 1985). A much less phytotoxic product is created or in many cases the 
product is inactive (Cole 1994). Glutathione s-transferase is one enzyme that 
participates in herbicide detoxification. A very important conjugation in plant 
herbicide metabolism is the thiol reaction attack of the GSH to an eletrophilic 
substrate with a displacement of a nucleophile (Fig. 5) (Armstrong 1993; Kreuz 
et al. 1996). These reactions usually are catalyzed by glutathione s-transferase 
as first reported by Lamourex et al. (1970) in maize with conjugation of 
glutathione and atrazine (Kreuz et al. 1996).

R-X  +  GST  (r) R-SG  +  HX

Fig. 5. Generic reaction, atrazine, tridiphane, EPTC, fluorodifen, and 
aciflurofen reaction catalyzed by glutathione s-transferase.


	Several GSTs have been characterized in maize such as GST I, a homodimer 
of 29kDa subunits, GST II, a heterodimer of 27 kDa and 29 kDa subunits, GST III, 
a homodimer of 26kDa subunits, and GST IV, a homodimer of 27kDa subunits. GST I 
and GST II comprise 1 to 2% of the soluble protein in maize. GST 27 present in 
both GST II and IV were present in RNA isolated from maize root but expression 
was not detected in RNA from aerial parts of the plant. GST 29 was expressed in 
the stem and leaves and to lower levels in pollen and endosperm. GST 27 besides 
play a role in isoforms which are not only involved in detoxification of 
xenobiotics but also catalyze the conjugation of glutathione to endogenous 
substrates (Jepson et al. 1994)
	GST activity present in cultured cells to the enzyme extracted from maize 
cultivars resistant to s-triazine herbicides was compared. Precipitates of crude 
enzyme extracts from maize showed activity 0.04, 0.08, 0.09, and 0.002 mol 
product/mg protein min, respectively for cv. DeKalb leaves, cv. Fronica leaves, 
cv. LG11 leaves, and Black Mexican Sweet Corn suspension culture harvested 7 
days after incubation (DAI). When atrazine was used as a substrate, GSH 
conjugation rate catalyzed by glutathione s-transferase was very low at all 
suspension culture harvests. GST activity varied from 1.3 to 3.2 mol product/mg 
protein min. when metolachlor was used as a substrate. Leaf GST activity in 
atrazine substrate was 0.022 (7 DAI), 0.022 (10 DAI), and 0.015 (14 DAI) mol 
product/g fresh weight cells min, and 9.3 (4 DAI), 17.1 (7 DAI), 10.6 (10 DAI), 
and 11.4 (14 DAI) mol product/g fresh weight cells min when metolachlor was used 
as a substrate (Edwards and Owen 1986).
	Hatton et al. (1996) characterized the glutathione dependent herbicide-
detoxification system in maize and in associated weeds, by determining GST 
activities. In saturating substrate concentrations, maize seedlings contained 
GST activities toward differing classes of substrates was classified in the 
order alachlor ( fluorodifen ( atrazine ( metolachlor. The same activity 
sequence was obtained by Digitaria sanguinalis, Echinochloa crus-galli, Panicum 
miliaceum, Setaria faberi, and Sorghum bicolor. The GST activity sequence 
observed for atrazine was Zea mays ( P. miliaceum (( S. bicolor = S. faberi = D. 
sanguinalis = Abutilon theophrasti ( E. crus-galli. Their results establish a 
good correlation between the relative GSH specific activity toward alachlor and 
an excellent correlation with metolachlor.
	All GST activity of maize cv. Pioneer 3394 and Artus were higher in roots 
than shoots toward alachlor, metolachlor, and fluorodifen while shoots showed 
higher GST activity toward atrazine. In shoots without the herbicide-safener 
dichlormid, GST activity observed was atrazine = alachlor = metolachlor ( 
fluorodifen. When shoots where treated with dichlormid, GST activity increased 
for all herbicides. The highest GST root activity was observed toward the 
chloroacetanilides increased in Pioneer 3394 when safener was used (Dixon et al 
1997).
	Jablonkai and Hatzios (1991) studied the role of shoot and root glutahione 
and glutathione s-transferase activity in response of two maize hybrids and one 
wheat cultivar to acetochlor, a chloroacetanilide herbicide. GST activity showed 
a strong dependence on acetochlor concentration. GSH/acetochlor ratio at lower 
acetochlor concentrations was high, corresponding to higher acetochlor conjugate 
formeatiion. Root GST activity in both hybrids was greater and more inducible by 
acetochlor pretreatment than in susceptible wheat.
	Analysis for characterization of glutathione s-transferase isoforms in 
maize showed that susceptible and intermediate lines exhibited impaired 
functions of GST-27 and GST-29 subunits, respectively. GST IV appears to be the 
principal detoxifying enzyme for alachlor, although GST I and II are required to 
achieve tolerance to high rates of alachlor (Rossini et al. 1996).
	Glutathione s-transferase was purified from maize roots treated with 
safeners, GST II and GST I. GST II, a heterodimer of 29 and 27-kDa subunits, was 
present in untreated seedling roots and absent in other maize organs. GST II 
seedling root activity varied from 20 to 40% in untreated seedling roots but in 
roots treated with safeners, GST II enzymatic activity increased to 45% of the 
total seedling root GST activity. GST II was about seven times more active 
against alachlor than GST I . GST 29, common in GST I and GST II, was present in 
all treated and untreated maize organs with safeners while GST 27 was found only 
in untreated maize organs at low levels. GST 27 was induced in all the major 
aerial organs (Holt et al. 1995).
	Although piperonyl butoxide (PBO) was reported to increase the herbicidal 
activity of triazines in various plants, Varsano et al. (1992) observed that 
atrazine conjugation with GSH, catalyzed by GST, in maize was not inhibited by 
PBO. This was assumed to be due to cellular level action. PBO inhibition of GST 
was rather limited and did not explain the observed synergistic effect of PBO 
and atrazine. 
	Ezra and Stephenson (1985) found that proso millet (Panicum miliaceum) 
roots had about 50% of the GSH of corn roots. Proso millet shoot showed less 
than 10% the GST activity of proso millet roots equivalent to 33% of the corn 
root enzyme. Shoot enzymatic activity in proso millet is equivalent as 10% of 
the corn shoot enzyme. Glutathione concentration was 35.9 and 65.4 (g GSH/ g 
fresh weight for proso millet root and corn root, respectively. Safener R-25788 
elevated GSH activity to 62.7 but not increased proso millet GST activity. The 
difference observed in tolerances to atrazine and EPTC between corn and proso 
millet was related to the different rate of metabolism of the herbicide by proso 
millet.
	The effect of herbicide antidotes on the glutathione s-transferase of 
etiolated Sorghum bicolor shoot was investigated by Dean et al. (1990). 
Untreated seedlings showed two distinct GSTs, a major (GST-1) with activity 
toward CDNB (1-chloro-2,4-dinitrobenzene) and a minor (GST-7) toward 
metolachlor. Treated with various antidotes, sorghum seedlings show four to five 
additional GST with activity toward metolachlor and little or no activity toward 
CDNB. The GST (metolachlor) activity in etiolated shoots was increased 1.8 fold 
and 6.5 fold by 24 hour treatment with 1(M and 160(M metolachlor respectively. 
According to the author these results are consistent with the finding that 
antidotes protect sorghum against metolachlor inducing de novo synthesis of 
glutathione s-transferase.
	Glutathione s-transferase activity in sorghum was increased less than two 
fold when CDNB (1-chloro-2,4-dinitrobenzene) was used as a substrate. In 
contrast, when metolachlor was the substrate, GST activity was increased in 
average 18 fold in response to antidote treatment. When metolachlor was used as 
a substrate, the enzymatic activity in untreated sorghum, was 0.07 nmol/mg 
protein hr while the enzymatic activity in treated sorghum with antidotes varied 
from 0.36 to 2.08 nmol/mg protein hr. The relative ability of a particular 
antidote to enhance GST activity was highly correlated with protection against 
metolachlor. The GST (metolachlor) activity in moles GSH-metolachlor conjugated/ 
mg protein hr relation was established as a function of shoot length (expressed 
in % of antidote-treated control) * 0.032 - 0.963 (Gronwald et al. 1987).
	Glutathione s-transferase's contribution to herbicide detoxification and 
selectivity suspension in soybean, Abutilon theophrasti, Amaranthus retroflexus, 
Digitaria sanguinalis, Echinochloa crus-galli, Ipomoea hederacea, Setaria 
faberi, and Sorghum halepense was determined. For the leguminosae GST activity 
was determined using homoglutathione (hGSH, (-L glutamyl-L-cysteinyl-(-alanine) 
while for the other species GSH was used. In cultured cells of soybean, GST 
activity was fomesafen ( metolachlor = acifluorfen ( chlorimuron ethyl. Soybean 
cultured cell and 14 day old seedling showed the highest specific activity with 
metolachlor and fomesafen. When fomesafen was used as a substrate the order of 
GST activity was soybean (( E. crus-galli ( D. sanguinalis ( S. halepense = S. 
faberi while A. theophrasti,  A. retroflexus, and  I. hederacea  did not show 
any GST activity. When metolachlor was used as a substrate the order was soybean 
( A. theophrasti (( S. halepense (  A.  retroflexus ( I. hederacea while E. 
crus-galli, D. sanguinalis , and S. faberi  did not show any GST activity. The 
results showed correlation between the selectivity of fomesafen and the GST 
activity for all species studied while for metolachlor the correlation between 
selectivity and the GST activity was observed only toward broadleaved weeds 
(Andrews et al. 1997). Metolachlor accumulation in roots and shoots was greater 
in soybean than in corn seedlings due to a higher herbicide-induced activity of 
glutathione s-transferase in corn (Scarponi et al. 1992).

	The most GST (atrazine) activity in Abutilon theophrasti was observed in 
leaf tissue while low levels were detected in stems and no activity was observed 
in roots. GST concentration was 5.6 fold greater in leaf tissue than in stems. 
In this study, there was evidence that enhanced GST activity may result in the 
development of herbicide resistance in weeds (Anderson and Gronwald 1991).
	Bread wheat and other Triticum species contain GST activity toward 
fenoxaprop in roots, shoots, and unbleached plain flour. GST activity was 
increased with treatment of safeners. GST activity measured in 5, 7, 10, and 12 
day old seedlings were 86, 80, 60, and 71 nkats/g protein for shoot and 196, 
152, 113, and 100 nkats/g protein for roots respectively. When safener was used, 
shoot and root GST activities increased. GST activity obtained with safener 
treatment varied from 182 to 235 nkats/g protein for shoot and from 280 to 468 
nkats/g protein for roots. GST detoxification of fenoxaprop in wheat is in 
contrast to the metabolic fate of the diclofop that is hydroxylated by the 
cytochrome P450. The GST activity responsible for fenoxaprop detoxification was 
10 fold lower toward fenoxaprop-ethyl suggesting that the herbicide requires 
ester hydrolysis to create rapid detoxification by glutathione conjugation 
(Edwards and Cole 1996).
	Glutathione s-transferase levels in wheat shoots and wheat shoot relatives 
with and without safeners were quantified (Riechers et al. 1996). Maize GST 
antibody indicated presence of GST activity in untreated wheat and wheat 
relatives with safener enzymatic levels increased to a similar extent. On the 
other hand GST dimethenamid assay determined little or no constitutive GST 
dimethenamid activity. The GST dimethenamid activity also was increased with 
safener treatment. GST dimethenamid activity varied from ND (not detectable) to 
107 pmol/mg protein*min in approximately 58 wheat line (Fig. 6).

Fig 6. Glutathione s-transferase dimethenamid activity in crude extracts of 
wheat and wheat relatives (Riechers et al. 1996).


Glutathione s-transferase in pathogen defenses

Although plant glutathione s-transferase has been largely studied with regard to 
its role in herbicide detoxification, little is known about other biological 
functions of GST in plants. Evidence that pathogen attack results in an 
effective increase of mRNa coding a protein homologous to glutathione s-
transferase was presented by Dubler et al. (1991) for wheat. Mauch and Dubler 
(1993) characterized a pathogen-induced gene from wheat that was called GstA1 
based on sequence similarity showed with maize glutathione s-transferase. GstA1 
was expressed at a low basal level in healthy control plants. GstA1 level 
increased drastically and remained elevated for two days after plant inoculation 
with Erysiphe graminis f. sp. hordei and E. g. f. sp. tritici. The result 
observed indicated that the GstA1 expression is generally in response to 
infection. This was supported by GstA1 induction by Puccinia recondita f. sp. 
tritici, another fungal pathogen. One member of the prp1 gene family responsive 
to pathogen, ppr1-1 that encodes a cytosolic glutathione s-transferase from 
potato increased upon infection with Phytophthora infestans (Hahn and 
Strittmater 1994).


Glutathione s-transferase induced by heavy metals

	Plants have mechanisms that maintain essential metal concentrations 
between deficient and toxic limits as well as mechanisms to keep nonessential 
metals at low toxicity thresholds (Rauser 1995). Plant glutathione s-transferase 
also may be induced by heavy metals, wounding, ethylene, and ozone to generate 
an active oxygen species (Fig 7) during oxidative stress.  (Marrs 1996). 
	Maize seedlings were incubated in solutions of plant hormones, sodium 
chloride, cobalt chloride, cadmium chloride, and sodium arsenite or placed under 
environmental stress conditions (cold, hypoxia - flooding roots with water, and 
heat shock). Cadmium induced strongly maize glutathione s-transferase Bronze2 
(Bz2) and GST III. Salt stress, hypoxia, and heat shock had no effect on Bz2, 
whereas plant hormone, cold, and arsenite induced Bz2 transcript accumulation. 
As a result of cadmium treatment spliced Bz2 increase 20 fold and unspliced Bz2 
increased more than 50 fold over the levels of unspliced Bz2 Rna in control 
protoplasts. B37, a maize inbred line was the only one that showed a cadmium-
responsive GST activity (Marrs and Walbot 1997). Mauch and Dudler 1993 observed 
that wheat GST25 and GST26 activities also increased in the presence of cadmium. 

Fig. 7. Active oxygen species inducing GST levels that metabolize the toxic 
produts of lipid peroxidation and DNA damage (Marrs 1996). 

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