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Plant glutathione S -transferases:
enzymes with multiple functions in
sickness and in health
Robert Edwards, David P. Dixon and Virginia Walbot
Glutathione S -transferases (GSTs) are abundant proteins encoded by a highly divergent,
ancient gene family. Soluble GSTs form dimers, each subunit of which contains active sites
that bind glutathione and hydrophobic ligands. Plant GSTs attach glutathione to electrophilic
xenobiotics, which tags them for vacuolar sequestration. The role of GSTs in metabolism is
unclear, although their complex regulation by environmental stimuli implies that they have
important protective functions. Recent studies show that GSTs catalyse glutathione-dependent isomerizations and the reduction of toxic organic hydroperoxides. GSTs might also have
non-catalytic roles as carriers for phytochemicals.
C
ellular survival requires the management of reactive oxygen
species, endogenous phytochemicals and exogenous toxins,
including diverse xenobiotics added to the environment by
humans. Furthermore, many cellular processes function optimally
only within a narrow redox range. Glutathione (GSH), the tripeptide
g-Glu–Cys–Gly, plays a central role in the crucial processes of
detoxification and redox buffering1. GSH is abundant in plants, typically exceeding 1 mM in the cytoplasm1. Plants also contain several
GSH-dependent detoxifying enzymes2, most notably glutathione
S-transferases (GSTs), which collectively constitute .1% of the
soluble protein in maize leaves3. Individual gene analysis and genomics projects indicate that plants have 25 or more genes coding for
GSTs, and that the proteins share as little as 10% amino acid identity.
In both crops and weeds, GSTs detoxify electrophilic herbicides
by catalysing their conjugation with GSH (Ref. 4; Fig. 1). The Sglutathionylated metabolites are tagged for vacuolar import by
ATP binding cassette (ABC) transporters, which selectively transport GSH conjugates5. GSTs are composed of two subunits with
molecular masses in the range of 25–27 kDa and are either homodimers of a single gene product or heterodimers of subunits
encoded by different genes. Individual GST isoenzymes can selectively detoxify specific xenobiotics, with species differences in
GST specificity and capacity determining herbicide selectivity4.
Paradoxically, in spite of the specific and crucial roles of individual GST enzymes in xenobiotic metabolism, their roles in endogenous plant metabolism have remained an enigma. There are only a
few reports of natural products [such as the phytoalexin medicarpin
(Fig. 1)] being conjugated with GSH (Ref. 6) in spite of a burgeoning literature showing that the expression of specific GSTs vary
markedly during plant development (cell division, senescence) and
after exposure to pathogens, changes in environmental conditions
and chemical treatments3. GSTs with high affinity for auxins3 and
cytokinin7 have been suggested to contribute to hormone homeostasis. Indeed, the enhancement of GST expression has become a
marker for plant response to stress, although the functional significance of selective GST expression is only just emerging.
One role of this article is to introduce the diversity of GSTs,
highlighting novel features of the plant GST family compared
with other eukaryotes. The complexity and antiquity of GST
genes and their functioning as homo- and heterodimeric proteins
present challenges in both classification and nomenclature. However, the core of the article is a consideration of the functions of
GSTs in endogenous plant metabolism. In particular that GSTs:
(1)
(2)
(3)
Catalyse conjugation reactions with natural products
similar to those observed with xenobiotics.
Function as binding and carrier proteins for phytochemicals
between cellular compartments.
Catalyse alternative GSH-dependent biotransformation
reactions.
GST-gene diversity: who counts as a family member?
GSTs are an ancient and diverse protein family, existing as multigene families in bacteria, fungi, animals and plants (Fig. 2). The
‘classic’ definition of a GST requires the catalytic formation of
GSH conjugates with xenobiotic substrates. The compound 1chloro-2,4-dinitrobenzene (CDNB) is used as a model substrate
(Fig. 1) but some GSTs show little activity with this compound4.
Furthermore, highly electrophilic substrates spontaneously conjugate with GSH under physiological conditions, with GSTs accelerating the reaction only modestly8. Whatever their substrate,
GSTs should selectively bind GSH or natural homologues, such
as homoglutathione (g-Glu–Cys–b-Ala) and hydroxymethyl
glutathione (g-Glu–Cys–Ser), which are commonly found in
legumes and grasses, respectively9. Affinity chromatography
using GSH as a ligand is widely used to extract GSTs, although
resins with GSH conjugates can be useful in the recovery of specific GSTs from cellular extracts10.
Members of the GST family show the greatest sequence similarity in their GSH-binding domain; four highly conserved amino
acids in this domain are a ‘signature motif’ for GSTs, but this
motif is not sufficient to identify a GST. By applying the dual
definition of GSH binding and catalysis of GSH conjugation with
electrophiles, we can distinguish GSTs from proteins with related
sequences that have evolved alternative functions. Proteins
related to the GST superfamily, such as the crystallin protein
in the squid lens11 and elongation factor 1-gamma proteins
(Ref. 12), have lost their capacity either to bind GSH or to use
the thiol in conjugation reactions. Some offshoots of the GST
family currently have no assignable functions. For example, several stress-induced proteins in bacteria resemble GSTs (Ref. 13),
as does the herbicide safener inducible 27-kDa In2-1 protein
from maize14.
Classification of plant GSTs based on sequence
An accurate GST classification scheme would require knowledge
of the proteins’ in vivo role(s) to name individual GST enzymes
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genes have three exons. The other large
group, type III, consisted mainly of auxinOH SG
OH
induced GSTs, with the genes containing
HO
HO
OH
two exons. Types I and III GSTs show
OH
.50% sequence divergence and have now
been placed in separate classes15,16. Type-II
+ GSH
GSTs
have ten exons and are much closer
O
O
to the mammalian zeta GSTs (Ref. 2).
Isoliquirtigenin
Recently, a Type-IV grouping was proposed
for several Arabidopsis genes that are simi(b)
lar to classical mammalian theta enzymes2.
HO
O
HO
O
During 1999, dozens of plant GST
sequences were reported from diverse
angiosperm sequencing projects (e.g.
GSH
maize, tomato, soybean and cotton), with
SG
O
.25 genes for GSTs identified to date in
OCH3
OCH3
HO
Medicarpin
the Arabidopsis genome alone. This diversity makes the catch-all theta classification
(c)
inappropriate. Some plant GSTs clearly
GS
group with specific mammalian types but
R
R
O
O
+ GSH
there are two distinct plant-specific types2
(Fig. 2). Because the principle of GreekOH
OH
letter designations is widely used for nonR = H − cinnamic acid
plant GSTs (Ref. 15), we suggest that a
R = OH − coumaric acid
new nomenclature system be adopted for
(d)
plant GST genes (Box 1; Table 1). ThereNO2
NO2
fore, the classification of plant GSTs
should be amended to include the followO2N
O2N
Cl + GSH
SG + HCl
ing new classes:
• Phi (F) – a plant-specific class replacing
1-Chloro-2,4-dinitrobenzene (CDNB)
Type I.
• Zeta (Z) – replacing Type II.
(e)
• Tau (U) – a plant-specific class replacing
O
O
SG
Type III.
Cl
• Theta (T) – replacing Type IV.
This nomenclature is applicable to SyneN
N
+ GSH
+ HCl
chocystis spp., whose genome contains
O
O
genes for four GST-like proteins without
the intron–exon gene structure of higher
Alachlor
plants. At the predicted-amino-acid level,
Trends in Plant Science
the gene designated gst1 (SwissProt
P74665) is a zeta GST; ORF sll1902
Fig. 1. Glutathione conjugation reactions. Glutathione (GSH) forms conjugates with the chal(P73835) shows the greatest similarity to
cone isoliquirtigenin (a) and the pterocarpan phytoalexin medicarpin (b). These addition reacthe tau-class gene, maize In2-1 (P49248).
tions occur spontaneously under basic conditions and can be readily reversed by reducing the
The gst gene (Q55139) is a phi-class mempH. (c) The phenylpropanoids cinnamic acid and coumaric acid undergo addition reactions
with GSH to form the respective conjugates. This reaction is radical driven and is catalysed
ber, and ORF slr0236 (P72690) has similarby an ascorbate peroxidase. (d and e) The classic detoxification reactions catalysed by GSTs
ity to the tau GSTs. We can conclude that
with the model substrate 1-chloro-2,4-dinitrobenzene and the herbicide alachlor, respectively.
the two plant-specific classes, tau and phi,
In both cases, the glutathione substitution products are stable and the reaction irreversible.
are characteristic of both simple and complex photosynthetic organisms and might
be required to accommodate the metabolic
properly; current schemes use amino acid sequence similarity and consequence of active oxygen generated by photosynthesis.
gene organization. Although this understates the functional hetAnother interesting feature of the GST family is the genes’
erogeneity of this family, we can ask whether gene similarity fore- arrangement in repeating units on plant chromosomes, which
casts functional capabilities. Using a classification system based strongly suggests that there have been numerous gene duplion immunological cross-reactivity and sequence relatedness, sol- cations. Chromosome II of Arabidopsis contains seven adjacent
uble mammalian GSTs have been divided into the alpha, mu, pi, tau-class genes, and all three of the known theta-class genes in the
sigma, theta and zeta classes2,15 (Fig. 2). Most nonmammalian Arabidopsis genome occur as a cluster and the two known
GSTs were lumped into the highly heterogeneous theta class, zeta-class genes are arranged in tandem.
which was viewed as closest to the original progenitor of all
eukaryotic GSTs. However, the view that plant GSTs are a primi- Structure of plant GSTs
tive subgroup is far from the truth.
X-ray crystallography has shown a similar three-dimensional toThree distinct types of plant GSTs were recognized initially3. pology for three phi-class GSTs from Arabidopsis17 and maize18,19,
Type I included GSTs with herbicide-detoxifying activity; these which share only 20% sequence identity. Each subunit has an active
(a)
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GST detoxification
Box 1. Enzyme and gene nomenclature
Sigma
Alpha
Isomerase
MAAI
Pi
Zeta
Mu
Theta
GPOX detoxification
Tau
Phi
Ligandin, isomerase,
GST detoxification
GPOX detoxification
Hormone responsive
Ligandin
GST detoxification
GPOX detoxification
Trends in Plant Science
Fig. 2. A deduced glutathione S-transferase (GST) phylogenetic
tree showing the relationships between the major GST classes and
the extent of sequence diversity within each class. The relationships between the classes are adapted from Ref. 2. For each class,
the triangle height is proportional to the number of sequences in the
SwissProt database, and the triangle width is proportional to the
sequence divergence (100% minus the average % identity between
each pair of amino acid sequences). The lengths of the connecting
bars represent the approximate extent of divergence between the
classes. To illustrate the scale, the tau class shows 59% divergence
(41% average identity) and includes 44 sequences. Established
functions for family members are listed next to each triangle. The
theta class sequences have been limited to those closely related to
the mammalian theta sequences to avoid the inclusion of highly
divergent sequences placed into this class by default.
site that includes a conserved GSH-binding site (G-site) located in
the N-terminal domain and a C-terminal cosubstrate-binding
domain (H-site), which will accommodate a diverse range of
hydrophobic compounds. The G-site is specific for GSH and facilitates the formation of the catalytically active thiolate anion of GSH.
In all three phi GSTs, the active sites are situated on either side of a
large, open cleft formed between the subunits, allowing access to
large planar and spherical molecules (Fig. 3). However, each active
site interacts minimally with the adjacent subunit17–19. Crystallography has also shown that certain GSTs can bind an additional GSH
molecule adjacent to the active site17, although the functional significance of this secondary binding site has received little attention.
In addition to the diversity of GST genes in each species, heterodimer formation could give rise to up to (1 1 2 1 3 1…1 n)
distinct heterodimers, where n is the number of GST genes of a
particular class. The importance of GST dimerization in xenobiotic metabolism was shown by determining the cause of sensitivity to the herbicide alachlor in an inbred maize line20: ZmGST II
(Table 1) subunits were unable to dimerize with one another to
form the ZmGST II–II homodimer, which is highly active in conjugating and detoxifying alachlor (Fig. 1).
Studies with coexpressed GST subunits in recombinant bacteria
suggest that dimerization is spontaneous but restricted to subunits of
the same class. In the case of the maize tau GSTs ZmGST V and
ZmGST VI, heterodimerization was as likely as homodimerization21,
We propose a new gene and enzyme nomenclature for glutathione Stransferases (GSTs) to simplify future assignments and to align designations more closely to evolutionary history. The current naming
of GSTs is confusing because enzymes are named in their order of
purification and genes by their order of sequencing. This produces
situations in which enzyme I is encoded by gene 3. Heterodimeric
proteins are given a specific name, which further obscures which
genes encode the subunits.
We propose that plant genes be named using a species designation, a gene-class identifier, and a number within that class. For
example, XyGstZ1 indicates that the gene was isolated from species
Xy (such as At for Arabidopsis thaliana, Zm for Zea mays), Z denotes
zeta class and the numeral 1 indicates that it is the first gene of the
zeta class from that species.
At the protein level, XyGSTZ1-1 would indicate a homodimer
encoded by XyGstZ1; XyGSTZ1-2 would indicate a heterodimer
encoded by the XyGstZ1 and XyGstZ2 genes. For example, the maize
enzyme GST V–VI (Ref. 21) would become ZmGSTU1-2. This system is compatible with the current practice for animal GSTs as well
as with the plant-genetics conventions of using italics for genes and
standard type for proteins. Table 1 shows the application of this
nomenclature to maize and Arabidopsis GSTs.
Table 1. Suggested new nomenclature applied to
maize and Arabidopsis GSTs
Species
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Zea mays
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Arabidopsis
Old name
Proposed name
ZmGST I
ZmGST II
ZmGST III
ZmGST V
ZmGST VI
ZmGST VII
Bronze2
AtGST10
GST1, PM239x14
GST2, PM24.1
GST3, ERD11
GST4, ERD13
GST6
GST7
GST8
GST11
GST5, 103-1a
ZmGSTF1
ZmGSTF2
ZmGSTF3
ZmGSTU1
ZmGSTU2
ZmGSTU3
ZmGSTU4
AtGSTT1
AtGSTF1
AtGSTF2
AtGSTF3
AtGSTF4
AtGSTF5
AtGSTF6
AtGSTF7
AtGSTF8
AtGSTU1
whereas heterodimer formation was favoured with the maize phi
GSTs ZmGST I and ZmGST II (Ref. 22).
Do heterodimers perform unique roles? The available kinetic
measurements, performed with xenobiotic substrates in vitro,
have detected only ‘additive’ properties in heterodimers21,23. We
hypothesize that, in vivo, heterodimers might uniquely recognize
specific phytochemicals or exhibit unique kinetic properties
essential for metabolite management. Alternatively, heterodimer
formation might determine the subcellular localization or control
the rates of protein turnover.
GSTs and endogenous metabolism: vexing problems
identifying in vivo substrates
GSTs evolved long before plants were challenged to detoxify pesticides and synthetic pollutants, therefore what are the physiological
roles of GSTs that were shaped by natural selection? Ironically, the
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detoxified by phi GSTs from sorghum23,
and tau GSTs from wheat have similar
activities10. Pathogen-inducible GSTs probably also detoxify exogenous natural products, though of exogenous origin, such as
phytotoxins produced by competing plants
or by invading microbial pathogens. A
good example of this is the detoxification
of isothiocyanates, reactive compounds
released from glucosinolate precursors
formed in Brassica species. These allelochemicals can suppress the growth of competing plants but are excellent substrates for
tau and phi GSTs in wheat and maize2,10.
Although inducible GSTs might use
stress metabolites as substrates, their role
Fig. 3. Two views of a space-filled model of the crystal structure of the Arabidopsis phi
in GSH conjugation of natural products in
class glutathione S-transferase (GST) dimer AtGSTF2-2. (a) A side-on view to illustrate the
healthy plants is less clear. Until recently,
large active-site cleft. (b) A view into the active sites. For both views, both subunits are visthe best candidates for endogenous phytoible; the lower subunit is shaded from red at the N-terminus to yellow at the C-terminus and
chemicals detoxified by GSTs were the
the upper subunit is shaded from blue at the N-terminus to green at the C-terminus. Each of
phenylpropanoids cinnamic acid and
the two active sites is occupied by a molecule of S-hexylglutathione (white). The glucoumaric acid (Fig. 1). However, recent
tathione moiety is close to the dimer interface and interacts exclusively with the N-terminal
analysis has demonstrated that these conjudomain of the GST, whereas the S-hexyl moiety lies further from the dimer interface and
interacts with both N-terminal and C-terminal domains of the protein.
gates are formed by a radical-mediated
process catalysed by an ascorbate peroxidase rather than by a GST (Ref. 28).
GSTs of herbivorous animals have long been accorded the role of Anthocyanins were also proposed as likely substrates for conjugametabolizing dietary toxins, most of which are plant natural prod- tion because a GST is required for the last cytoplasmic step of
ucts. Interestingly, GSTs are frequently highly abundant in crops, a biosynthesis in both maize (Bz2 gene) and petunia29 (An9 gene). In
characteristic exploited in selective herbicides, where the GSTs the absence of the GST-mediated step, anthocyanin accumulates
which detoxify them can be 20-fold more abundant in the crop than in the cytoplasm, suggesting that conjugate formation was a prein the competing weeds24. In an evolutionary context, the evidence requisite for vacuolar sequestration. However, to date, conjugates
available in wheat and its relatives suggests that enhanced GST of non-electrophilic flavonoids have not been detected by
expression is not the result of modern breeding but is instead char- high-pressure liquid chromatography (HPLC) of plant extracts
acteristic of all the progenitor Triticum species used in its domesti- or enzymatic reactions30. Both the AN9 and BZ2 GSTs are
cation25,26. It is possible that, because they reduce stress, a high GST flavonoid-binding proteins (L.A. Mueller and V. Walbot, uncapacity is a prerequisite for successful artificial selection for high published), and the current model proposes that these gene prodyield under diverse and adverse conditions.
ucts prevent anthocyanin oxidation and cross-linking in the
Do the four distinct classes of plant GSTs recognize different cytoplasm, and hence indirectly aid vacuolar sequestration.
substrates? As discussed below, emerging evidence suggests that
While screening other flavonoids for their ability to undergo
zeta and theta GSTs in plants and animals have conserved GSH- glutathione conjugation, we have recently identified the beta-Sdependent activities that have little to do with conventional conju- glutathionyl derivative of the chalcone isoliquirtigenin (4,29,
gating activity. By contrast, when assayed for GSH-conjugating 49-trihydroxychalcone) as being formed in vitro (Fig. 1). Signifiactivity towards xenobiotics, the phi and tau GSTs of maize2 and cantly, this compound was formed at physiological pH in
wheat10 have a surprisingly similar spectrum of activities. If the the absence of plant proteins and would readily deconjugate to
phi and tau GSTs have distinct functions, these are likely to be isoliquirtigenin and GSH under mildly acidic conditions. The
evident only with endogenous substrates.
GSH conjugate of the pterocarpan medicarpin can also breakdown to re-release the parent phytoalexin at acidic pH (Fig. 1).
GSTs conjugating natural products
This conjugate, but not the parent phytoalexin, is readily transAfter glutathionation, xenobiotic conjugates are rapidly imported ported into resealed minivacuoles by an ABC transporter in vitro6.
into the vacuole before being further metabolized into a range of Intriguingly, these results suggest that GSH conjugation of elecknown sulphur-containing metabolites4. By contrast, the GSH con- trophilic natural plant products could be more widespread than
jugate of caftaric acid identified in wine must and a sulphur- previously thought; a role in vacuolar targeting might have been
containing metabolite of gibberellic acid, termed gibberthione, are missed because the conjugates are degraded in the acidic condiamong the few candidates for GSH conjugation of endogenous tions of most vacuoles or in the course of phytochemical analysis.
plant products27. This suggests either that GSH conjugation is not
an important route of detoxification for endogenous metabolites or GSTs functioning as binding proteins
that the elusive conjugates are unstable and have escaped detection. A hypothesis to explain why a specific GST is needed for anthoStress-inducible GSTs might act to conjugate metabolites aris- cyanin sequestration is that these proteins act as carriers. The coning from oxidative damage. For example, cytotoxic alkenals cept of GSTs as carrier proteins or ‘ligandins’ was first proposed
derived from the peroxidation of natural products, such as fatty in the early 1970s, based on the finding that a similar protein was
acids, are well-defined substrates of GSTs in oxidatively stressed identified as the cellular binding factor for diverse steroids, drugs
animal cells2,3. In plants, 4-hydroxynonenal, a toxic alkenal and mammalian metabolites3. Later, cloning and identification of
released following oxidative damage of membranes, is actively ‘ligandin’ as a GST resulted in the same quandary currently faced
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by those studying plant GSTs: an enzyme in search of substrates
for conjugation. The fact that GSTs have been identified while
searching for auxin- and cytokinin-binding proteins when using
photoaffinity labels3,7 suggests that these proteins might have
functions in the binding and transport of plant hormones. Similarly, both phi and tau GSTs have high affinities for tetrapyrroles
and porphyrin metabolites21,27. With both tetrapyrroles and plant
hormones, binding inhibits GST activity toward xenobiotics, but
the inhibitory ligands do not undergo conjugation. Similarly, the
CDNB conjugating activity of petunia AN9, a phi GST, is inhibited by flavonols, flavones and anthocyanins29. There is ample
evidence that specific plant GSTs that bind defined plant metabolites fit the definition of ligandins.
GSTs catalysing GSH-dependent peroxidase and isomerase
reactions
By catalysing the nucleophilic attack of GSH on hydroperoxides,
GSTs can catalyse the reduction of organic hydroperoxides to the
less-toxic monohydroxy alcohols, the resulting sulfenic acid
derivative of GSH then spontaneously forming a disulfide with
another GSH molecule (Fig. 4). Tobacco seedlings overexpressing a tobacco tau GST with a high glutathione peroxidase activity
are more tolerant of chilling and osmotic dehydration than wildtype plants31.
A further link between GSTs functioning as glutathione peroxidases and oxidative-stress tolerance was discovered in black
grass32 (Alopecurus myosuroides). Herbicide-resistant weeds that
are cross-resistant to multiple classes of herbicides express a phi
GST (AmGSTF1) that is a highly active glutathione peroxidase;
this GST is barely detectable in herbicide-sensitive black grass.
AmGSTF1 appears to contribute to herbicide resistance by preventing the accumulation of cytotoxic hydroperoxides of natural
products, which can be formed either directly or indirectly as a
result of injury by herbicides with diverse modes of action. Significantly, AmGSTF1 is related to cereal phi GSTs that are
induced in crops following treatment with herbicide safeners, providing a connection between safener-mediated protection of crops
and oxidative stress tolerance32.
GSTs are also essential for the isomerization of specific
metabolites (Fig. 4). The proposed mechanism involves the transient formation of a GSH adduct, spontaneous isomerization of
the compound and finally the release of the isomer and GSH. An
excellent example is the human zeta GST, which functions as a
maleylacetoacetate isomerase (MAAI), a key enzyme in phenylalanine catabolism. MAAI catalyses the cis–trans isomerization
of maleylacetoacetate to fumarylacetoacetate (Fig. 4). This reaction has been known for some time to be GSH dependent and
associated with a GST; the recent complementation of an MAAIdeficient strain of Aspergillus nidulans with the human zeta
GST confirming its identity as the MAAI enzyme33. This GSTmediated cis–trans isomerization reaction involves the reversible
addition of GSH to the cis double bond; after rotation, GSH is
eliminated and the trans isomer is formed. In view of their
sequence similarity, plant zeta GSTs probably have a similar
activity. The carnation (Dianthus caryophyllus) zeta genes are
induced during senescence2,3, which is consistent with a role in the
degradation of aromatic amino acids.
GST-mediated isomerase reactions recently identified in animals include prostaglandin-H E-isomerase activity34 and the isomerization of 13-cis retinoic acid to all-trans retinoic acid35. In the
latter case, the enzyme catalysing the reaction is clearly a GST, yet
the reaction does not require GSH and these enzymes appear to
have recruited other proteinaceous thiols to catalyse the reaction.
In plants, the isomerase activity of GSTs has been demonstrated
(a)
R− O − OH + GSH
R−OH + GS − OH
GSH
GS–SG
O–
(b)
O–
O
O–
O
O–
OH O O
Maleylacetoacetate
OH O O
Fumarylacetoacetate
(c)
N
N
N
N
N
S
O
S–
Br
GSH
N
SG
Br
O
S
N
N
GSH
N
Br
O
Trends in Plant Science
Fig. 4. Examples of glutathione-S-transferase (GST)-mediated and
glutathione (GSH)-dependent reactions that do not result in the
formation of GSH conjugates as end products. (a) GST functioning
as a glutathione peroxidase, using GSH to reduce an organic
hydroperoxide (ROOH) to the monohydroxy alcohol (ROH). (b) A
zeta GST catalyzing a cis–trans isomerization of maleylacetoacetate to fumarylacetoacetate. (c) GST catalyzing the isomerization of a thiadiazolidine proherbicide to the phytotoxic
triazolidine, showing the proposed reaction mechanism and the
formation of the GSH-conjugated intermediate.
using thiadiazolidine herbicides; these are bioactivated by GSTmediated isomerization to triazolidines, which are potent inhibitors
of protoporphyrinogen oxidase36. A likely mechanism for isomerization involves nucleophilic attack at the carbonyl group by GSH,
a process that is activated by the GST (Fig. 4). Ring opening then
allows rotation around the N–C bond, with the C5N double bond
transferred to the C–S group; the nitrogen atom attacks the carbonyl group to reform the ring and eliminate the glutathione36.
Given the flexible architecture of the active site of plant and mammalian GSTs, it now appears likely that other isomerase activities
carried out by undefined enzymes might be catalysed by GSTs.
Conclusions
Recent advances in molecular genetics and biochemistry have
unveiled a surprising complexity in the variety of GST genes and
enzymes in plants. We have tried to highlight some tantalizing reasons for this diversity: members of an ancient gene family have
been recruited to perform diverse physiological functions based
around their ability to activate a readily available cellular thiol
while coordinately binding a diverse range of hydrophobic ligands.
GST functions have direct cytoprotective activities and they might
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be essential to preserve plants during environmental stress and disease, as well as supporting normal development. We consider the
frontier of research to involve matching individual GSTs with their
in vivo ligands and showing which interactions result in catalysis.
Because of the extreme divergence among GSTs, sequence analysis alone cannot uncover function. Perhaps even more intriguing are
the hints that some GSTs might be carrier proteins for reactive plant
metabolites, controlling their activity and cellular distribution. In the
coming years, our ability to manipulate the expression of GSTs in
plants through gene disruption and transgenic approaches will allow
us to test these models of GST function in vivo.
Acknowledgements
Support for our research on plant GSTs is supported by grants
from the US National Science Foundation to V.W. (IBN 9603927)
and from the Biotechnology and Biological Sciences Research
Council, Zeneca Agrochemicals and Aventis Crop Science to R.E.
D.D. was supported by Aventis Crop Science.
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Robert Edwards* and David P. Dixon are at the Dept of Biological
Sciences, University of Durham, Durham, UK DH1 3LE;
Virginia Walbot is at the Dept of Biological Sciences, Stanford
University, Stanford, CA 94305-5020, USA.
*Author for correspondence (tel 144 191 374 2428;
fax 144 191 374 2417; e-mail [email protected]).