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, typ-ically exceeding 1 mMin 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 gen-omics 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 S-glutathionylated metabolites are tagged for vacuolar import by ATP binding cassette (ABC) transporters, which selectively trans-port GSH conjugates5. GSTs are composed of two subunits with molecular masses in the range of 25–27 kDa and are either homo-dimers of a single gene product or heterohomo-dimers of subunits encoded by different genes. Individual GST isoenzymes can selec-tively detoxify specific xenobiotics, with species differences in GST specificity and capacity determining herbicide selectivity4.Paradoxically, in spite of the specific and crucial roles of individ-ual GST enzymes in xenobiotic metabolism, their roles in endogen-ous 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 burgeon-ing literature showburgeon-ing 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 auxins3and cytokinin7have been suggested to contribute to hormone homeosta-sis. Indeed, the enhancement of GST expression has become a marker for plant response to stress, although the functional signifi-cance 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. How-ever, the core of the article is a consideration of the functions of GSTs in endogenous plant metabolism. In particular that GSTs:
(1) Catalyse conjugation reactions with natural products similar to those observed with xenobiotics.
(2) Function as binding and carrier proteins for phytochemicals between cellular compartments.
(3) 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 multi-gene 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 1-chloro-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 conju-gate with GSH under physiological conditions, with GSTs accel-erating 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 spe-cific GSTs from cellular extracts10.
Members of the GST family show the greatest sequence simi-larity 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, sever-al 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 vivorole(s) to name individual GST enzymes
Plant glutathione S -transferases:
enzymes with multiple functions in
sickness and in health
Robert Edwards, David P. Dixon and Virginia Walbot
properly; current schemes use amino acid sequence similarity and gene organization. Although this understates the functional het-erogeneity of this family, we can ask whether gene similarity fore-casts functional capabilities. Using a classification system based on immunological cross-reactivity and sequence relatedness, sol-uble mammalian GSTs have been divided into the alpha, mu, pi, sigma, theta and zeta classes2,15 (Fig. 2). Most nonmammalian
GSTs were lumped into the highly heterogeneous theta class,
which was viewed as closest to the original progenitor of all eukaryotic GSTs. However, the view that plant GSTs are a primi-tive subgroup is far from the truth.
Three distinct types of plant GSTs were recognized initially3. Type I included GSTs with herbicide-detoxifying activity; these
genes have three exons. The other large group, type III, consisted mainly of auxin-induced GSTs, with the genes containing two exons. Types I and III GSTs show .50% sequence divergence and have now been placed in separate classes15,16. Type-II GSTs have ten exons and are much closer to the mammalian zeta GSTs (Ref. 2).
Recently, a Type-IV grouping was proposed for several Arabidopsisgenes that are simi-lar to classical mammalian thetaenzymes2.
During 1999, dozens of plant GST sequences were reported from diverse angiosperm sequencing projects (e.g. maize, tomato, soybean and cotton), with .25 genes for GSTs identified to date in the Arabidopsisgenome alone. This diver-sity makes the catch-all thetaclassification
inappropriate. Some plant GSTs clearly group with specific mammalian types but there are two distinct plant-specific types2 (Fig. 2). Because the principle of Greek-letter designations is widely used for non-plant GSTs (Ref. 15), we suggest that a new nomenclature system be adopted for plant GST genes (Box 1; Table 1). There-fore, the classification of plant GSTs should be amended to include the follow-ing new classes:
• Phi (F) – a plant-specific class replacing Type I.
• Zeta (Z) – replacing Type II.
• Tau (U) – a plant-specific class replacing Type III.
• Theta (T) – replacingType IV.
This nomenclature is applicable to Syne-chocystis spp., whose genome contains genes for four GST-like proteins without the intron–exon gene structure of higher plants. At the predicted-amino-acid level, the gene designated gst1 (SwissProt P74665) is a zeta GST; ORF sll1902
(P73835) shows the greatest similarity to the tau-class gene, maize In2-1 (P49248).
The gstgene (Q55139) is a phi-class
mem-ber, and ORF slr0236(P72690) has similar-ity to the tauGSTs. We can conclude that
the two plant-specific classes, tauand phi,
are characteristic of both simple and com-plex photosynthetic organisms and might be required to accommodate the metabolic consequence of active oxygen generated by photosynthesis.
Another interesting feature of the GST family is the genes’ arrangement in repeating units on plant chromosomes, which strongly suggests that there have been numerous gene dupli-cations. Chromosome II of Arabidopsiscontains seven adjacent
tau-class genes, and all three of the known theta-class genes in the
Arabidopsis genome occur as a cluster and the two known
zeta-class genes are arranged in tandem.
Structure of plant GSTs
X-ray crystallography has shown a similar three-dimensional to-pology for three phi-classGSTs from Arabidopsis17and maize18,19,
which share only 20% sequence identity. Each subunit has an active
Fig. 1.Glutathione conjugation reactions. Glutathione (GSH) forms conjugates with the
chal-cone isoliquirtigenin (a) and the pterocarpan phytoalexin medicarpin (b). These addition reac-tions occur spontaneously under basic condireac-tions and can be readily reversed by reducing the pH. (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 by an ascorbate peroxidase. (d and e) The classic detoxification reactions catalysed by GSTs with the model substrate 1-chloro-2,4-dinitrobenzene and the herbicide alachlor, respectively. In both cases, the glutathione substitution products are stable and the reaction irreversible.
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 facili-tates the formation of the catalytically active thiolate anion of GSH. In all three phiGSTs, 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. Crystallogra-phy has also shown that certain GSTs can bind an additional GSH molecule adjacent to the active site17, although the functional sig-nificance of this secondary binding site has received little attention. In addition to the diversity of GST genes in each species, het-erodimer formation could give rise to up to (1 1 2 1 3 1…1 n) distinct heterodimers, where nis the number of GST genes of a particular class. The importance of GST dimerization in xenobi-otic metabolism was shown by determining the cause of sensitiv-ity 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 con-jugating 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 tauGSTs ZmGST V and
ZmGST VI, heterodimerization was as likely as homodimerization21,
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 pes-ticides and synthetic pollutants, therefore what are the physiological roles of GSTs that were shaped by natural selection? Ironically, the
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 relation-ships 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.
Trends in Plant Science Alpha
Pi
Theta
Zeta GST detoxification
Isomerase MAAI
Hormone responsive Ligandin
GST detoxification GPOX detoxification Ligandin, isomerase,
GST detoxification GPOX detoxification GPOX detoxification
Sigma
Mu
Tau
Phi
Table 1. Suggested new nomenclature applied to maize and Arabidopsis GSTs
Species Old name Proposed name
Zea mays ZmGST I ZmGSTF1
Zea mays ZmGST II ZmGSTF2
Zea mays ZmGST III ZmGSTF3
Zea mays ZmGST V ZmGSTU1
Zea mays ZmGST VI ZmGSTU2
Zea mays ZmGST VII ZmGSTU3
Zea mays Bronze2 ZmGSTU4
Arabidopsis AtGST10 AtGSTT1
Arabidopsis GST1, PM239x14 AtGSTF1
Arabidopsis GST2, PM24.1 AtGSTF2
Arabidopsis GST3, ERD11 AtGSTF3
Arabidopsis GST4, ERD13 AtGSTF4
Arabidopsis GST6 AtGSTF5
Arabidopsis GST7 AtGSTF6
Arabidopsis GST8 AtGSTF7
Arabidopsis GST11 AtGSTF8
Arabidopsis GST5, 103-1a AtGSTU1
Box 1. Enzyme and gene nomenclature
We propose a new gene and enzyme nomenclature for glutathione S
-transferases (GSTs) to simplify future assignments and to align des-ignations 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 gene3. 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 desig-nation, a gene-class identifier, and a number within that class. For example, XyGstZ1indicates that the gene was isolated from species Xy(such as Atfor Arabidopsis thaliana, Zmfor Zea mays), Z denotes zetaclass and the numeral 1 indicates that it is the first gene of the zetaclass 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 XyGstZ1and XyGstZ2genes. For example, the maize
enzyme GST V–VI (Ref. 21) would become ZmGSTU1-2. This
GSTs of herbivorous animals have long been accorded the role of metabolizing dietary toxins, most of which are plant natural prod-ucts. Interestingly, GSTs are frequently highly abundant in crops, a characteristic exploited in selective herbicides, where the GSTs which detoxify them can be 20-fold more abundant in the crop than in the competing weeds24. In an evolutionary context, the evidence available in wheat and its relatives suggests that enhanced GST expression is not the result of modern breeding but is instead char-acteristic of all the progenitor Triticumspecies used in its domesti-cation25,26. It is possible that, because they reduce stress, a high GST capacity is a prerequisite for successful artificial selection for high yield under diverse and adverse conditions.
Do the four distinct classes of plant GSTs recognize different substrates? As discussed below, emerging evidence suggests that
zetaand thetaGSTs in plants and animals have conserved
GSH-dependent activities that have little to do with conventional conju-gating activity. By contrast, when assayed for GSH-conjuconju-gating activity towards xenobiotics, the phiand tauGSTs of maize2and
wheat10have a surprisingly similar spectrum of activities. If the
phi and tau GSTs have distinct functions, these are likely to be
evident only with endogenous substrates. GSTs conjugating natural products
After glutathionation, xenobiotic conjugates are rapidly imported into the vacuole before being further metabolized into a range of known sulphur-containing metabolites4. By contrast, the GSH con-jugate of caftaric acid identified in wine must and a sulphur-containing metabolite of gibberellic acid, termed gibberthione, are among the few candidates for GSH conjugation of endogenous plant products27. This suggests either that GSH conjugation is not an important route of detoxification for endogenous metabolites or that the elusive conjugates are unstable and have escaped detection. Stress-inducible GSTs might act to conjugate metabolites aris-ing from oxidative damage. For example, cytotoxic alkenals derived from the peroxidation of natural products, such as fatty acids, are well-defined substrates of GSTs in oxidatively stressed animal cells2,3. In plants, 4-hydroxynonenal, a toxic alkenal released following oxidative damage of membranes, is actively
detoxified by phi GSTs from sorghum23,
and tau GSTs from wheat have similar
activities10. Pathogen-inducible GSTs prob-ably also detoxify exogenous natural prod-ucts, 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 allelo-chemicals can suppress the growth of com-peting plants but are excellent substrates for
tauand phiGSTs in wheat and maize2,10.
Although inducible GSTs might use stress metabolites as substrates, their role in GSH conjugation of natural products in healthy plants is less clear. Until recently, the best candidates for endogenous phyto-chemicals detoxified by GSTs were the phenylpropanoids cinnamic acid and coumaric acid (Fig. 1). However, recent analysis has demonstrated that these conju-gates are formed by a radical-mediated process catalysed by an ascorbate peroxi-dase rather than by a GST (Ref. 28). Anthocyanins were also proposed as likely substrates for conjuga-tion because a GST is required for the last cytoplasmic step of biosynthesis in both maize (Bz2gene) and petunia29(An9gene). In the absence of the GST-mediated step, anthocyanin accumulates in the cytoplasm, suggesting that conjugate formation was a pre-requisite for vacuolar sequestration. However, to date, conjugates of non-electrophilic flavonoids have not been detected by high-pressure liquid chromatography (HPLC) of plant extracts or enzymatic reactions30. Both the AN9 and BZ2 GSTs are flavonoid-binding proteins (L.A. Mueller and V. Walbot, un-published), and the current model proposes that these gene prod-ucts prevent anthocyanin oxidation and cross-linking in the cytoplasm, and hence indirectly aid vacuolar sequestration.
While screening other flavonoids for their ability to undergo glutathione conjugation, we have recently identified the beta
-S-glutathionyl derivative of the chalcone isoliquirtigenin (4,29, 49-trihydroxychalcone) as being formed in vitro(Fig. 1). Signifi-cantly, this compound was formed at physiological pH in the absence of plant proteins and would readily deconjugate to isoliquirtigenin and GSH under mildly acidic conditions. The GSH conjugate of the pterocarpan medicarpin can also break-down to re-release the parent phytoalexin at acidic pH (Fig. 1). This conjugate, but not the parent phytoalexin, is readily trans-ported into resealed minivacuoles by an ABC transporter in vitro6. Intriguingly, these results suggest that GSH conjugation of elec-trophilic natural plant products could be more widespread than previously thought; a role in vacuolar targeting might have been missed because the conjugates are degraded in the acidic condi-tions of most vacuoles or in the course of phytochemical analysis. GSTs functioning as binding proteins
A hypothesis to explain why a specific GST is needed for antho-cyanin sequestration is that these proteins act as carriers. The con-cept of GSTs as carrier proteins or ‘ligandins’ was first proposed in the early 1970s, based on the finding that a similar protein was identified as the cellular binding factor for diverse steroids, drugs and mammalian metabolites3. Later, cloning and identification of ‘ligandin’ as a GST resulted in the same quandary currently faced
Fig. 3.Two views of a space-filled model of the crystal structure of the Arabidopsisphi
class glutathione S-transferase (GST) dimer AtGSTF2-2. (a) A side-on view to illustrate the
large active-site cleft. (b) A view into the active sites. For both views, both subunits are vis-ible; the lower subunit is shaded from red at the N-terminus to yellow at the C-terminus and the upper subunit is shaded from blue at the N-terminus to green at the C-terminus. Each of the two active sites is occupied by a molecule of S-hexylglutathione (white). The
glu-tathione moiety is close to the dimer interface and interacts exclusively with the N-terminal domain of the GST, whereas the S-hexyl moiety lies further from the dimer interface and
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. Simi-larly, both phiand tauGSTs 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 phiGST, is
inhib-ited by flavonols, flavones and anthocyanins29. There is ample evidence that specific plant GSTs that bind defined plant metab-olites 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 overexpress-ing a tobacco tauGST with a high glutathione peroxidase activity
are more tolerant of chilling and osmotic dehydration than wild-type plants31.
A further link between GSTs functioning as glutathione perox-idases 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 pre-venting 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. Sig-nificantly, AmGSTF1 is related to cereal phi GSTs that are
induced in crops following treatment with herbicide safeners, pro-viding 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 tran-sient 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 zetaGST, which functions as a
maleylacetoacetate isomerase (MAAI), a key enzyme in phenyl-alanine catabolism. MAAI catalyses the cis–transisomerization of maleylacetoacetate to fumarylacetoacetate (Fig. 4). This reac-tion has been known for some time to be GSH dependent and associated with a GST; the recent complementation of an MAAI-deficient strain of Aspergillus nidulans with the human zeta
GST confirming its identity as the MAAI enzyme33. This GST-mediated cis–transisomerization reaction involves the reversible addition of GSH to the cisdouble 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 ani-mals include prostaglandin-H E-isomerase activity34and the isom-erization of 13-cisretinoic acid to all-transretinoic 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
using thiadiazolidine herbicides; these are bioactivated by GST-mediated isomerization to triazolidines, which are potent inhibitors of protoporphyrinogen oxidase36. A likely mechanism for isomer-ization 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 car-bonyl group to reform the ring and eliminate the glutathione36. Given the flexible architecture of the active site of plant and mam-malian 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 rea-sons 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
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
maleylaceto-acetate to fumarylacetomaleylaceto-acetate. (c) GST catalyzing the isomeri-zation of a thiadiazolidine proherbicide to the phytotoxic triazolidine, showing the proposed reaction mechanism and the formation of the GSH-conjugated intermediate.
Trends in Plant Science R− O − OH + GSH R−OH + GS − OH
GSH
GS –SG
OH OH
O–
O O O
O–
O–
O O O
O–
Maleylacetoacetate Fumarylacetoacetate (a)
(b)
(c)
N
N S
N
O
Br
N Br
N N
O S GSH
N Br
N N
S–
O SG
be essential to preserve plants during environmental stress and dis-ease, as well as supporting normal development. We consider the frontier of research to involve matching individual GSTs with their in vivoligands and showing which interactions result in catalysis.
Because of the extreme divergence among GSTs, sequence analy-sis 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.
References
1Noctor, G. and Foyer, C.H. (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Phys. Plant Mol. Biol.49, 249–279 2Dixon, D.P. et al.(1998) Glutathione-mediated detoxification systems in
plants. Curr. Opin. Plant Biol.1, 258–266
3Marrs, K.A. (1996) The functions and regulation of glutathione S-transferases in plants. Annu. Rev. Plant Phys. Plant Mol. Biol.47, 127–158
4Cole, D.J. and Edwards, R. (2000) Secondary metabolism of agrochemicals in plants. In Metabolism of Agrochemicals in Plants(Roberts, T.R., ed.), pp. 107–154, John Wiley & Sons
5Rea, P.A. (1999) MRP subfamily ABC transporters from plants and yeast. J. Exp. Bot. 50, 895–913
6Li, Z-S. et al.(1997) Vacuolar uptake of the phytoalexin medicarpin by the glutathione conjugate pump. Phytochemistry45, 689–693
7Gonneau, J. et al.(1998) A Nicotiana plumbaginifoliaprotein labeled with an azido cytokinin agonist is a glutathione S-transferase. Physiol. Plant.103, 114–124 8Coleman, J.O.D. et al.(1997) Detoxification of xenobiotics by plants: chemical
modification and vacuolar compartmentation.Trends Plant Sci.2, 144–151 9Noctor, G. et al.(1998) Glutathione: biosynthesis, metabolism and relationship
to stress tolerance explored in transformed plants.J. Exp. Bot.49, 623–647 10 Cummins, I. et al.(1997) Purification of multiple glutathione transferases
involved in herbicide detoxification from wheat (Triticum aestivumL.) treated with the safener fenchlorazole-ethyl. Pestic. Biochem. Physiol. 59, 35–49
11 Tomarev, S.I. and Piatigorsky, J. (1996) Lens crystallins of invertebrates: diversity and recruitment from detoxification enzymes and novel proteins. Eur. J. Biochem.235, 449–465
12 Koonin, E.V. et al.(1994) Eukaryotic translation elongation factor 1-gamma contains a glutathione transferase domain – study of a diverse, ancient protein superfamily using motif search and structural modeling. Protein Sci.11, 2045–2054
13 Serizawa, H. and Fukuda, R. (1987) Structure of the gene for the stringent starvation protein of Escherichia coli. Nucleic Acids Res.15, 1153–1163 14 Hershey, H.P. and Stoner, T.D. (1991) Isolation and characterization of cDNA
clones for RNA species induced by substituted benzenesulfonamides in corn. Plant Mol. Biol.17, 679–690
15 Hayes, J.D. and McLellan, L.I. (1999) Glutathione and glutathione-dependent enzymes represent a co-ordinately regulated defence against oxidative stress. Free Radic. Res.31, 273–300
16 Droog, F. (1997) Plant glutathione S-transferases, a tale of theta and tau. J. Plant Growth Regul.16, 95–107
17 Reinemer, P. et al.(1996) Three-dimensional structure of glutathione S-transferase from Arabidopsis thalianaat 2.2 Å resolution: structural characterization of herbicide-conjugating plant glutathione S-transferases and a novel active site architecture.J. Mol. Biol.255, 289–309
18Neuefeind, T. et al.(1997) Crystal structure of herbicide-detoxifying maize glutathione S-transferase-I in complex with lactoylglutathione: evidence for an induced-fit mechanism.J. Mol. Biol. 274, 446–453
19Neuefeind, T. et al.(1997) Cloning, sequencing, crystallization and X-ray structure of glutathione S-transferase III from Zea maysvar. mutin: a leading enzyme in detoxification of maize herbicides.J. Mol. Biol.274, 577–587 20Rossini, L. et al.(1996) Characterization of glutathione S-transferase isoforms
in three maize inbred lines exhibiting differential sensitivity to alachlor.Plant Physiol.112, 1595–1600
21Dixon, D.P. et al.(1999) Dimerization of maize glutathione transferases in recombinant bacteria. Plant Mol. Biol.40, 997–1008
22Sommer, A. and Böger, P. (1999) Characterization of recombinant corn glutathione S-transferase isoforms I, II, III and IV.Pestic. Biochem. Physiol. 63, 127–138
23Gronwald, J.W. and Plaisance, K.L. (1998) Isolation and characterization of glutathione S-transferase isozymes from sorghum.Plant Physiol.117, 877–892 24Hatton, P.J. et al.(1999) Glutathione transferases involved in herbicide
detoxification in the leaves of Setaria faberi(giant foxtail).Physiol. Plant. 105, 9–16
25Edwards, R. and Cole, D.J. (1996) Glutathione transferases in wheat (Triticum) species with activity towards fenoxaprop-ethyl and other herbicides.Pestic. Biochem. Physiol. 54, 96–104
26Riechers, D.E. et al.(1996) Variability of glutathione S-transferase levels and dimethenamid tolerance in safener-treated wheat and wheat relatives.Pestic. Biochem. Physiol.56, 88–101
27Lamoureux, G.L. and Rusness, D.G. (1993) Glutathione in the metabolism and detoxification of xenobiotics in plants. In Sulfur Nutrition and Assimilation in Higher Plants(De Kok, L.J. et al., eds), pp. 221–237, SPB Academic Publishing 28Dean, J.V. and Devarenne, T.P. (1997) Peroxidase-mediated conjugation of
glutathione to unsaturated phenylpropanoids: evidence against glutathione S-transferase involvement.Physiol. Plant.99, 271–278
29Alfenito, M.R. et al.(1998) Functional complementation of anthocyanin sequestration in the vacuole by widely divergent glutathione S-transferases. Plant Cell10, 1135–1149
30Walbot, V. et al.Do glutathione S-transferases acts as enzymes or carrier proteins for their natural substrates. In Sulphur Metabolism in Plants(Brunold, C. et al., eds), Verlag Paul Haupt, Berr, Switzerland (in press)
31Roxas, V.P. et al.(1997) Overexpression of glutathione S
-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during stress. Nat. Biotechnol.15, 988–991
32Cummins, I. et al.(1999) A role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple herbicides in black-grass. Plant J.18, 285–292
33Fernández-Cañón, J.M. and Peñalva, M.A. (1998) Characterization of a fungal maleylacetoacetate isomerase gene and identification of its human homologue. J. Biol. Chem.273, 329–337
34Meyer, D.J. et al.(1996) Purification and characterization of prostaglandin-H E-isomerase, a sigma-class glutathione S-transferase, from Ascaridia galli. Biochem. J.13, 223–227
35Chen, H. and Juchau, M.R. (1998) Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cisretinoic acid to all-transretinoic acid in vitro. Biochem. J.336, 223–226
36Jablonkai, I. et al.(1997) Chemical catalysis of the isomerization of a peroxidising herbicidal thiadiazolidine. In Proceedings of the Brighton Crop Protection Conference – Weeds, pp. 771–776, British Crop Protection Council, Farnham, UK
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;
