Download Engineering and genetic approaches to modulating the

Copyright ß Physiologia Plantarum 2006, ISSN 0031-9317
Physiologia Plantarum 126: 382–397. 2006
REVIEW
Engineering and genetic approaches to modulating the
glutathione network in plants
Spencer Maughana and Christine H. Foyerb,*
a
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK
Crop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK
b
Correspondence
*Corresponding author,
e-mail: [email protected]
Received 10 October 2005; revised 26
November 2005
doi: 10.1111/j.1399-3054.2006.00684.x
Reduced glutathione (GSH) is the most abundant low-molecular weight thiol
in plant cells. It accumulates to high concentrations, particularly in stress
situations. Because the pathway of GSH synthesis consists of only two
enzymes, manipulation of cellular glutathione contents by genetic intervention has proved to be relatively straightforward. The discovery of a new
bacterial bifunctional enzyme catalysing GSH synthesis but lacking feedback
inhibition characteristics offers new prospects of enhancing GSH production
and accumulation by plant cells, while the identification of g-glutamyl
cysteine and glutathione transporters provides additional possibilities for
selective compartment-specific targeting. Such manipulations might also be
used to affect plant biology in disparate ways, because GSH and glutathione
disulphide (GSSG) have crucial roles in processes as diverse as the regulation
of the cell cycle, systemic acquired resistance and xenobiotic detoxification.
For example, depletion of the total glutathione pool can be used to
manipulate the shoot : root ratio, because GSH is required specifically for
the growth of the root meristem. Similarly, chloroplast g-glutamyl cysteine
synthetase overexpression could be used to increase the abundance of
specific amino acids such as leucine, lysine and tyrosine that are synthesized
in the chloroplasts. Here we review the aspects of glutathione biology related
to synthesis, compartmentation and transport and related signalling functions
that modulate plant growth and development and underpin any assessment
of manipulation of GSH homeostasis from the viewpoint of nutritional
genomics.
Abbreviations – g-EC, g-glutamyl cysteine; g-ECS, g-glutamyl cysteine synthetase; APR, adenosine 50 phosphosulphate reductase; BS, bundle sheath; BSO, 1-buthionine-SR-sulfoximine; CAT, catalase; CDK, cyclin-dependent kinase; ER, endoplasmic
reticulum; FW, fresh weight; GGT, g-glutamyl transpeptidase; GPX, glutathione peroxidase; GR, glutathione reductase; GRX,
glutaredoxin; GS, glutathione-S; GSH, reduced glutathione; GSH-S, glutathione synthetase; GSNO, S-nitrosoglutathione;
GSSG, glutathione disulphide; GST, glutathione S-transferase; GS-X, glutathione conjugates; Hgt1p, high-affinity glutathione
transporter; JA, jasmonic acid; MAPK, mitogen-activated protein kinase; mBBr, monobromobimane; MRP, multidrug-resistant
transporter; NPR1, non-expressor of PR proteins 1; OPT, oligopeptide transport proteins; PC, phytocheletin; PCD, programmed
cell death; PMF, proton motive force; PR, pathogenesis related; QTL, quantitative trait loci; RNS, reactive nitrogen species;
ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired resistance; TGA, a group of bZIP (basic leucine
repeat)-type transcription factors; TRX, thioredoxin.
382
Physiol. Plant. 126, 2006
Introduction
The thiol tripepide glutathione [g-Glu-Cys-Gly; reduced
glutathione (GSH)] does not immediately come to mind
in considerations of human nutrition. However, this
ubiquitous tripeptide thiol is a vital intracellular and
extracellular protective antioxidant against oxidative/
nitrosative stresses, which play a key role in the control
of many human diseases. GSH is also important in
immunity modulation in animals as well as in remodelling of the extracellular matrix, apoptosis and mitochondrial respiration in disease. Mammalian cancer cells
often accumulate GSH, where there is also a metabolic
switch to glycolysis rather than TCA cycle as a major
energy source (Pozuelo Rubio et al. 2004). Interactions
of GSH with antiapoptotic factors such as Bcl-2 in cancer cells have been linked to radiation and multidrug
resistance (Ortega et al. 2003). However, because high
GSH concentrations are essential for both antioxidant
and immune defence systems in mammals, tissue GSH
levels can be regulated, particularly in malnourished
patients through diet and nutrition before therapeutic
treatments (Bray and Taylor 1993).
Plants make abundant amounts of glutathione, and its
synthesis and accumulation in either the chloroplast or
cytosolic compartments of the plant cell can be
enhanced or decreased relatively easily by genetic engineering approaches (Strohm et al. 1995, Creissen et al.
1996, Noctor et al. 1998a, b, Zhu et al. 1999, Xiang
et al. 2001). Ectopic expression of g-glutamyl cysteine
synthetase (g-ECS, also called glutamate cysteine ligase)
(Noctor et al. 1996) and certain enzymes of sulphur
assimilation pathway (Harms et al. 2000) or glutathione
reductase (GR) (Foyer et al. 1991) in transgenic plants
results in substantial increases in leaf glutathione.
Glutathione contents have been increased by at least
five-fold the values of wild-type plants without negative
effects (Noctor et al. 1998b, 2002a). Only one report to
date has indicated that an enhanced capacity to generate and accumulate the intermediate g-glutamyl
cysteine (g-EC) can produce negative effects (Creissen
et al. 1999). The increased capacity to generate glutathione and enhance cellular glutathione pools leads
to higher rates of sulphur assimilation, modified amino
acid metabolism and enhanced stress tolerance (Noctor
et al. 1998a, b). Transformed poplar trees with an
increased capacity to synthesize and accumulate glutathione are currently being used in the field for bioremediation purposes to purify and restore soils polluted
by human activities (Peuke and Rennenberg, 2005a, b).
Genetic engineering approaches have led to an
improved understanding of how compartment-specific
alterations in glutathione synthesis affect metabolism
Physiol. Plant. 126, 2006
and defence. Similarly, the analysis of mutants deficient
in glutathione has greatly advanced current concepts of
how GSH and glutathione disulphide (GSSG) regulate
cell signalling and development. Chemical inhibition of
GSH synthesis using 1-buthionine-S, R-sulfoximine
(BSO), a specific inhibitor of g-ECS (Griffith and
Meister 1979, Griffith 1982, Maughan and Cobbett
2003), has been widely used to study the effects of
GSH depletion in plants (Hell and Bergman 1990,
Vernoux et al. 2000). Analysis of Arabidopsis thaliana
T–DNA insertion and EMS mutants selected by their
ability to grow on BSO concentrations that are inhibitory to the wild-type has indicated that different
mechanisms can confer BSO resistance (Maughan
2003).
Since its initial discovery in yeast over 125 years ago,
glutathione has been shown to have a prodigious number
of critical functions (Fig. 1). GSH is a powerful reducing
agent and is a key player in the plant redox-state (Noctor
and Foyer 1998, Noctor et al. 2002a). Here we will
focus on how manipulation of GSH synthesis and metabolism has advanced current concepts of glutathione
homeostasis and function, concentrating on aspects
related to cell signalling, development and defence
that may be of importance in any consideration of nutritional genomics involving glutathione. The characteristics and functional importance of putative thiol-based
reactive oxygen species (ROS) sensors and associated
redox-signalling cascades have recently been discussed
Abiotic
Heavy metal detoxification
Salinity protection
UV protection
Systemic Acquired
Resistance
Protein
thiolation
Xenobiotic detoxification
Drought protection
Biotic
ROS
scavenging
Calcium
signaling
PCD
MAPK
cascades
Oxidative brust
Signaling
Cell cycle progression
Root development
Sulfur storage
Sulfur sensing
Carbon - Sulfur - Nitrogen
Metabolism
Fig. 1. The functions of glutathione in plants linked by essential signalling functions that integrate plant growth, development and defence
processes.
383
(Foyer and Noctor 2005). In this context, we will concentrate here on the implications for the plant of modulating glutathione homeostasis.
Like ascorbate, glutathione limits the lifetime of ROS
and lipid peroxide signals. Moreover, GSH alters calcium signalling in plants (Gomez et al. 2004a) and
participates in the calcium-dependent pathways of
ROS-signal transduction (Rentel and Knight 2004).
Through oxidation to GSSG, other signalling cascades
can be initiated through thiol-disulphide exchange and
thiolation. Moreover, the interaction between GSH and
nitric oxide, catalysed by the enzyme formaldehyde
dehydrogenase (also known as class III alcohol dehydrogenase), is also important in signal transduction as
S-nitrosoglutathione (GSNO) is thought to be a stable
NO transport form (Liu et al. 2001). The formaldehyde
dehydrogenases, which also catalyse the NAD-dependent
formation of S-formylglutathione and S-hydroxymethyl
glutathione, are regulated by wounding and salicylic
acid (SA) (Diaz et al. 2003). Hence, glutathione is
directly implicated in the coordinated control of signalling by ROS and reactive nitrogen species (RNS)
that determines cell fate in situations such as pathogen
attack.
It is important to note that thioredoxins (TRXs), glutathione and lipid peroxides all target the same protein
residues, allowing us to envisage a dynamic signalling
network where proteins are modulated by these three
signalling systems according to their respective concentrations and the associated enzymes such as glutaredoxins (GRXs).
Control of plant growth and development
Glutathione regulation is important in a number of key
processes associated with plant growth and development. These include the following:
The cell cycle and plant development
Current knowledge of how cell division is controlled
and how it interacts with different aspects of plant
development such as morphogenesis, architecture and
growth rate has greatly increased over the last 5 years.
Cell-cycle regulation involves components that respond
to signals from the external environment as well as intrinsic developmental programmes, and it ensures that DNA
is replicated with high fidelity within the constraints of
prevailing environmental conditions (Dewitte et al. 2003,
de Jager et al. 2005). Cyclin-dependent kinases (CDKs)
play a central role in cell-cycle regulation, with negative
(KRP proteins) and positive (D-type cyclins) regulators
acting downstream of environmental inputs at the G1
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checkpoint (Dewitte and Murray 2003, de Jager et al.
2005). The activity of the CDK/cyclin complexes is
regulated by a network of regulatory mechanisms
including transcription, proteolysis, phosphorylation/
dephosphorylation, interaction with regulatory proteins
and intracellular trafficking.
In Arabidopsis, the embryo cells of the dry seed are
arrested in the G1 phase of the cell cycle. Germination
is initiated by water uptake and the resumption of metabolism and cell division. Nitric oxide is a potent regulator of germination in dormant seeds such as those of
Arabidopsis (Bethke et al. 2004, 2006). Germination is
stimulated by the presence of environmental nitrate
from which NO can be synthesized via nitrate reductase. The relatively low abundance of GSH within the
dry seed means that GSH has either to be regenerated
from oxidized or bound forms upon water uptake or that
it has to be synthesized de novo during germination to
achieve the high levels present in seedlings. Low GSH
levels in the dry seed and during germination may prevent excessive formation of GSNO, allowing sufficient
free NO to be present to stimulate the germination
process. However, it is also possible that NO accumulation serves as a trigger for GSH synthesis during
germination.
While it is well established that the G1 checkpoint is
very sensitive to environmental conditions, it has only
recently become clear that this control might be exerted
through oxidation–reduction (redox) regulation and
associated components, particularly glutathione and
ascorbate, whose concentrations and redox state influences G1 progression. Glutathione and ascorbate exert
independent controls on the plant cell cycle (Potters
et al. 2004).
Numerous interactions between the plant and environment take place at the root/soil interface.
Monobromobimane (mBBr) has been widely used to
detect GSH in vivo. Application of this GSH-localization system to Arabidopsis roots shows that GSH accumulates in the vascular system and root tip (Fig. 2A).
The relatively high concentration of GSH in root
tips, where actively dividing cells are found (Fig. 2A),
suggests that growing tissues have a requirement
for GSH. Indeed, studies using the GSH-deficient
ROOTMERISTEMLESS1 mutant (rml1) which has less
than 2% of the wild-type GSH levels have shown that
without GSH root meristem cells arrest in G1 (Cheng
et al. 1995, Vernoux et al. 2000). This mutation only
affects post-embryonic root development, and while
overall the roots are shorter, the cell files and types are
not aberrant. It is important to note that in contrast to the
root meristem, the shoot meristem is able to produce all
the aboveground organs with development and timing
Physiol. Plant. 126, 2006
A
F
B
D
C
E
G
Fig. 2. The effects of 1-buthionine-S, R-sulfoximine (BSO) on tissue
glutathione and root growth in Arabidopsis thaliana. The root of a 7-dayold seedling shown in (A) highlights the fact that reduced glutathione
(GSH) is most abundant in the root tip and in the vasculature as
indicated by the blue fluorescence of mBBr. Untreated Arabidopsis
cells (B) were stained for GSH which accumulates in the vacuole (C).
Equivalent BSO-treated (1 mM) cells (D, E) lack any significant staining
indicating the depletion of GSH. In (F), the root apical meristem is
demarcated by the staining of dividing cells by using a fusion of the
GUS enzyme to a mitotic cyclin (CYCB1;1; Colon-Carmona et al. 1999).
Inhibition of GSH synthesis by BSO (1 mM) cell division causes cessation
of root growth (G).
that are comparable with the wild-type despite very low
GSH levels.
The rml1 phenotype can also be induced chemically
by treating cells or seedlings with BSO to lower GSH
levels (Fig. 2B–G). This causes the cells in the root
meristem to stop dividing, and, like the root meristem
cells of rml1, they arrest in the G1 phase (Fig. 2F,G).
These results suggest that GSH plays a key role in
potentiating signals permitting cell-cycle progression
and hence root growth. Similarly, root nodule formation
is prevented when GSH synthesis is blocked by addition
of BSO, suggesting that like the root meristem, the
nodule meristem is unable to develop in the absence
Physiol. Plant. 126, 2006
of GSH (Frendo et al. 2005). While the absolute content
of GSH and the GSH : GSSG ratio clearly influence
cell-cycle progression, the targets of glutathione action
are unknown at present. In mammalian systems, this
regulation includes transcription of the retinoblastoma
gene product as well as its activity, which function
directly downstream of G1 activators (Yamauchi and
Bloom 1997).
Given the importance of glutathione redox status in
regulating cell division, it is perhaps not surprising that
the GSH : GSSG ratio also regulates embryogenesis in
plants (Belmonte and Yeung 2004, Belmonte et al.
2005) as it does in animals. Application of GSH in the
second half of embryo development results in precocious germination, while application of GSSG improved
somatic embryogenesis (Belmonte and Yeung 2004,
Belmonte et al. 2005). The redox status of the embryo
glutathione pool delineates specific stages of embryo
development both in vivo and in vitro. A high GSH/
GSSG ratio is essential for cell division and proliferation
during the initial stages of embryo development, but
later in development, the redox status of the glutathione
pool decreases (Belmonte et al. 2005). This metabolic
shift in cellular glutathione redox status appears to be
required for continual embryonic development, and
proper acclimation of storage product deposition is possibly facilitated through interactions with hormones
such as abscisic acid. Zygotic embryos are held in G1
until germination-specific D-type cyclins promote cell
division (Mausubelel et al. 2006). It is thus tempting to
suggest that redox controls may act upstream of
germination-specific cell-cycle regulation in a fashion
similar to the regulation of division in the root apical
meristem.
Because GSH is a modulator of general plant development, it is not surprising that it has also been implicated in senescence processes (Ogawa et al. 2002). One
specific mode of action affects the activity of the ubiquitin-dependent 26S proteasome. The activity of this
protein degradation pathway may be influenced by cellular redox status in plants as it is in animals, where high
GSH/GSSG ratios impede the binding of target proteins
(Hochstrasser 1998, del Pozo et al. 1998, Theriault et al.
2000, Sangerman et al. 2001).
Plant defence and systemic acquired resistance
GSH is a transcriptional regulator. It enhances the DNAbinding activity of NFkB, a transcription factor that is
central to the regulation of the oxidative stress response
in animal systems. Few homologues of major animal or
microbial redox-sensitive elements and factors have
been reported in plants to date apart from the activator
385
protein-1 (a transcription factor controlling response to
oxidative stress) and the antioxidative responsive element
that is present in the maize catalase (CAT) promoter.
However, cytosolic thiol-disulphide status reactions
are crucial in plant innate immune responses. GSSG
accumulation is often observed in close conjunction
with cell death and systemic acquired resistance (SAR)
responses resulting from exposure to pathogens
(Vanacker et al. 2000) or abiotic stresses (Gomez et al.
2004a). RNS are also important in the plant pathogen
responses, and the strong interaction between GSH and
NO in signal transduction process, as GSNO for example,
suggests that mutual upregulation of these signalling
components may occur in challenged cells.
An increase in tissue GSH contents activates the
expression of PR genes through the non-expressor of
PR protein 1 (NPR1) pathway (Mou et al. 2003,
Gomez et al. 2004a). The NPR1 protein is part of the
mechanism through which SA triggers SAR to pathogens.
While many aspects of NPR1 function with regard to
binding of the TGA transcription factors and movement
to the nucleus to facilitate SAR are not fully understood,
this glutathione-modulated ankyrin repeat protein is one
of the key regulators of SA-dependent gene expression
(Cao et al. 1994, Delaney et al. 1995). Such studies indicate a strong relationship between SA and glutathione
homeostasis in stress signalling. Increased oxidation
resulting from exposure to stress stimulates glutathione
accumulation. SA enhances this response presumably by
increasing oxidation possibly through inhibitory effects on
CAT and ascorbate peroxidase.
Cell death and the GSH/GSSG redox couple
The many varied functions of GSH and importance of
the GSH : GSSG redox couple as a sensitive signal for
cellular events necessitates strict GSH homeostasis. In
animal cells, substantial evidence implicates redox
potential as an important factor determining cell fate.
All aerobic organisms maintain thiols in the reduced
(-SH) state. GSH : GSSG ratios of 100:1 are typical
values for animal and plant cells. Departures from high
cellular GSH : GSSG ratios caused for example by
exposure to stress are potentially dangerous, as they
are a cell death trigger; low GSH : GSSG ratios initiating programmed cell death (PCD) (Schäfer and Buettner
2001). Many stresses cause an initial net oxidation of
the glutathione pool that is often followed by increases
in total glutathione (Smith et al. 1984, Sen Gupta et al.
1991, Willekens et al. 1997, Noctor et al. 2002b,
Gomez et al. 2004b). This effect is observed, because
oxidation of the glutathione pool stimulates of GSH
synthesis to restore cellular GSH : GSSG ratios. This is
386
a homeostatic mechanism by which [GSH] is increased
in an attempt to offset stress-induced decreases in the
[GSH] : [GSSG] ratio. The nature of the link between
redox-state perturbation and enhanced glutathione
accumulation is not fully resolved, but it may involve
multiple levels of control. For example, the activity of
adenosine phosphosulphate reductase, a key enzyme in
sulphate assimilation, is activated by decreases in
GSH/GSSG (Bick et al. 2001). In addition, H2O2 and/or
low GSH/GSSG ratios enhance translation of g-ECS
(Xiang and Oliver 1998). Significant deviations from
high GSH : GSSG ratios result in PCD (reviewed in
Noctor et al. 2002a, b). The significance of alterations to
the GSH : GSSG ratio can be quantified using the Nernst
equation through which the redox potential of a given
redox couple can be calculated: E 5 Em [–(RT/nF)]
ln([Ared]/[Aox]). This allows a quantitative appreciation of
the impact of changes in total glutathione content on the
redox state of the cell. The activity of GR is essential in
maintaining the high cellular GSH : GSSG ratios. Ectopic
expression of bacterial GR in the chloroplast or cytosol of
transgenic plants increased the GSH : GSSG ratio and
gave added protection against some stresses (Foyer et al.
1991, 1995, Aono et al. 1993, 1995, Creissen et al. 1996).
Plants that are deficient in CAT activity have also
been used to investigate oxidative signalling resulting
from decreased H2O2 detoxification (Vandenabeele
et al. 2002, 2004). CAT-deficient plants are particularly
interesting as overall levels of H2O2 accumulation are
relatively small, but there is a drastic increase in tissue
glutathione levels (Smith et al. 1984, Smith et al. 1989)
together with a low GSH : GSSG ratio (Noctor et al.
2002b). CAT-deficient plants have been used to identify
a complete inventory of genes induced or repressed in
H2O2-induced PCD, which appears as clusters of dead
palisade parenchyma cells along the leaf veins when
these plants are exposed to high light (Vandenabeele
et al. 2004). The stimulation of GSH synthesis and modified GSH : GSSG ratios are intrinsic features of ozone
and H2O2-dependent signalling. Thus, GSSG and thiolation may be important in adding specificity to the
signal and/or promoting second messenger signal transduction in these pathways (Rentel and Knight 2004,
Foyer and Noctor 2005).
Transport systems also fulfil important roles in determining the GSH : GSSG ratios of individual cell compartments (Foyer et al. 2001). Transport of GSSG into
the vacuole by the MRP transporters or across the plasmalemma (Jamaı̈ et al. 1996) contributes to
GSH : GSSG homeostasis in the cytosol. GSH degradation may also be an important factor (Storozhenko et al.
2002). However, the g-glutamyl cycle, which contributes to GSH metabolism in animals, remains to be
Physiol. Plant. 126, 2006
confirmed in plants (Storozhenko et al. 2002).
Overexpression of one putative component, g-glutamyl
transferase (GGT) in Arabidopsis, did not alter the
GSH : GSSG ratio or total tissue glutathione contents
(Storozhenko et al. 2002).
Thiol-disulphide exchange reactions are a universal
mechanism for perception of redox perturbation. In
addition, S-glutathionylation or thiolation is a further
crucial mechanism in perception of altered glutathione
redox state. Protein glutathionylation is a widespread
response to oxidizing conditions. It is becoming established as an important mechanism for reversible modification of protein cysteinyl residues in plants as it is in
animals. While relatively few thiolated proteins have
been identified to date, those that have been characterized include important metabolic enzymes such as
sucrose synthase and acetyl CoA carboxylase and
defence enzymes such as dehydroascorbate reductase
(Dixon et al. 2005). Thiolation can have a number of
effects on proteins, but it primarily protects cysteinyl
residues from irreversible oxidation to their sulfinic
and sulphonic acid derivatives. The process is reversed
by specific GRXs and/or TRXs, but much remains to be
characterized concerning these regulatory systems
(Noctor in press). Both types of redox-active proteins
are encoded by quite large gene families in plants
(Lemaire 2004). While relatively few plant thiolation
targets have been identified to date, many as yet unidentified targets are likely also to be thioredoxin targets,
which are becoming increasingly well characterized
(Buchanan and Balmer 2005).
Glutathione as a sink for reduced sulphur and
regulator of sulphur assimilation
Current evidence suggests that GSH has a role in sulphur homeostasis. GSH is an abundant low-molecular
weight thiol and hence a potentially important sulphur
sink as well as a predominant transported form of
reduced sulphur (reviewed in Hell 1997). This model
predicts that excess sulphur will be stored in GSH and
will be released when the cell experiences sulphur deficiency or requires increased sulphur availability.
Important mechanistic support came from studies
which show GSH is transported long distances in the
phloem and xylem (Rennenberg et al. 1979).
Furthermore, GSH is transported to developing tissues
from Maize scutella (Rauser et al. 1991). Herschbach
et al. (2000) showed that not only is GSH found in the
phloem and xylem but also that GSH levels control
sulphate loading into the xylem. Together with cysteine,
glutathione forms part of the repertoire of signals that
modulate sulphate uptake and assimilation (Kopriva and
Physiol. Plant. 126, 2006
Rennenberg 2004). GSH accumulation in cells has feedback effects on the pathway of primary sulphur assimilation (Kreuzwieser and Rennenberg 1998). The
addition of GSH to Arabidopsis roots caused a decrease
in adenosine 50 phosphosulphate reductase (APR) activity and transcript abundance (Vauclare et al. 2002) as
well as in ATP sulphurylase activity (Lappartient et al.
1999). Long distance GSH transport has also been suggested to decrease sulphur assimilation (Lappartient and
Touraine 1996) and poplar (Herschbach et al. 2000). A
simple regulatory mechanism has been proposed with
regard to the regulation of demand-driven sulphur
assimilation by positive signals such as o-acetylserine
and negative signals such as GSH (Kopriva and
Rennenberg 2004).
Effects of manipulation of GSH synthesis on
metabolism and development in mutant and
transgenic plants
Glutathione is synthesized in both photosynthetic and
non-photosynthetic cell types. However, green leaves
are often the major organs of GSH production and
export, and chloroplasts contain high (3–5 mM) GSH
concentrations (Foyer and Halliwell 1976, Smith et al.
1985). However, certain non-green cell types such as
trichomes also have a very high capacity to produce and
accumulate glutathione (Gutiérrez Alcalá et al. 2002).
Variable proportions of leaf glutathione are localized in
the chloroplasts, but values are generally considered to
be between 30 and 40% of the total (Klapheck et al.
1987) with 1–4 mM in the cytosol (Noctor et al. 2002a).
In nearly all organisms studied to date, GSH is synthesized from constituent amino acids in a two-step ATPdependent reaction sequence (Fig. 3) catalysed by
g-ECS and glutathione synthetase (GSH-S) (Hell and
Bergmann 1988, 1990, Meister 1988). With the exception of a novel enzyme from Streptococcus agalactiae
that catalyses both activities (Janowiak and Griffith
2005), g-ECS and GSH-S enzymes have been identified
as separate proteins and gene products from numerous
eukaryotes and gram-negative prokaryotes. The S. agalactiae enzyme has several features that are worthy of
note. This is not only the first bifunctional g-ECS-GSH-S
enzyme to be isolated but is also the first GSH synthesis
enzyme system to be identified from a gram-positive
organism (Janowiak and Griffith 2005). The g-ECSGSH-S sequence encodes an 85 kDA protein, which
when purified exhibits GSH-S activity with a similar
specific activity to that of g-ECS. It has similar Km values
for cysteine and ATP to the Escherichia. coli g-ECS, but
the streptococcal g-ECS-GSH-S has a lower affinity for
glutamate than the E. coli GSH-S. This may be
387
(γ-ECn)Gly - Heavy metals
Sequestration GS-X conjugates
GSSG
of harmful
(GSH)
compounds
on
ri
nd
o
och Cytochromes
t
MiReducing
Nucleus
NPR1
Vacuole
power for
energy chains
L-Glycine + ATP
GSH
γ-EC
PR genes
Redox
signaling/gene
expression
ER
GSH
2
Cytoplasm
Chloroplast
PMF
L-Cysteine
γ-Glutamylcysteine
(γ-EC)
2
L-Glycine
+
ATP
Glutathione
(GSH)
O
OH H HS
N
O
O
N
H
OH H HS
N
O
N
O H
O
OH
1
ATP
L-Glutamate
GSH
GSSG
GS-X
Pepides
(CysGly)
AAs
GSH
GSSG
GS-X
O
ROS Scavenging
(H2O2, 1O2)
OH
NH2
GSH-Ascorbate
cycle
Fig. 3. Glutathione synthesis and compartmentation. Reduced glutathione (GSH) synthesis proceeds by the sequential action of g-glutamyl cysteine
synthetase (g-ECS) (1) localized exclusively in the chloroplast and GSH-S, glutathione synthetase (GSH-S) (2) which is localized in both the chloroplastic
and cytosolic compartments. The many transporters capable of transporting GSH and related metabolites (shown in black) including the tonoplast
ATP-dependent MRP pumps, which can transport GSH, GSSG and GS-X compounds (see also Figs 2B,C), are shown. Other multidrug-resistant
transporters (MRPs) are shown on the plasma membrane. Alongside these are the proton-coupled pumps that can transport GSH, GSSG, GS-X, di/tripeptides and amino acids. Significant gaps remain in our current knowledge of GSH transporters, and it is likely that other as yet undescribed
transporters (red) must exist to allow effective GSH homeostasis.
explained by the fact that gram-positive bacteria maintain exceptionally high glutamate levels (of up to
100 mM). The GSH-S domain has a relatively small
molecular mass (31 kDaA) with an amino acid
sequence more related to D-Ala-ligase than GSH-S
(Janowiak and Griffith 2005). It is important to note
GSH inhibits neither the g-ECS nor the GSH-S activity
of the streptococcal g-ECS-GSH-S, allowing S. agalactiae to maintain much higher GSH concentrations than
for example E. coli, despite the fact that the overall
g-ECS activity is relatively lower. It is interesting to note
that S. agalactiae lack catalase and therefore rely heavily
on GSH-based enzymes for peroxide detoxification
(Janowiak and Griffith 2005).
The E. coli genes encoding g-ECS and GSH-S have
been used extensively in genetic engineering
approaches to enhance glutathione contents in plants
(Noctor et al. 1998a, Zhu et al. 1999). Targeting of the
bacterial g-ECS and GSH-S to either the chloroplast
or cytosol has led to marked increases in enzyme
activity. Increases in g-ECS but not GSH-S led to large
constitutive increases in leaf glutathione (Noctor et al.
1996, 1998a, Creissen et al. 1999). Moreover, glutathione was also increased in xylem sap, phloem
388
exudates and roots (Herschbach et al. 2000). The
identification of a g-ECS-GSH-S enzyme that facilitates
glutathione synthesis in S. agalactiais suggests that similar
bifunctional enzymes might occur in other gram-positive
bacteria where glutathione synthesis was thought to be
relatively uncommon. These bifunctional g-ECS-GSH-S
enzymes might be extremely useful in future plant
genetic engineering approaches to manipulate
glutathione for nutritional purposes. Similarly, specific
homologues of glutathione could be introduced for
example by ectopic expression of enzymes such as
homoglutathione synthetase.
The endogenous plant g-ECS and GSH-S enzymes display a high degree of sequence homology. In A. thaliana,
g-ECS is encoded by a single gene, GSH1, with a plastid
target signal (May and Leaver 1994). A recent analysis
using a g-ECS-GFP reporter system has indicated that
most of, if not all, the A. thaliana g-ECS protein is located
in the chloroplasts (Wachter et al. 2005). Targeting
information relative to GSH-S suggests that it is present
both in chloroplasts and the cytosol. The chloroplast
and cytosol thus cooperate in de novo GSH synthesis,
the chloroplast acting as the source of g-EC for
both chloroplastic and cytosolic GSH production.
Physiol. Plant. 126, 2006
These observations are surprising given that earlier studies localized g-ECS and GSH-S activities in the cystosol
as well as the chloroplasts (Klapheck et al. 1987, Hell
and Bergmann 1988, 1990). Moreover, the studies with
transgenic plants have shown that GSH synthesis can be
increased by targeting the synthetic enzymes to either
the chloroplastic or the cytosolic compartments (Noctor
et al. 1996, 1998a, Creissen et al. 1999). However,
kinetic analysis has revealed that the chloroplast and
cytosolic isoforms have similar properties (Noctor
et al. 2002a). More work is required to fully rationalize
the molecular genetic evidence with the biochemical
observations.
The targeting information indicating that g-EC is largely synthesized in the chloroplasts (Wachter et al.
2005) has implications for glutathione homeostasis.
Firstly, at least some of g-EC produced in the chloroplast
must be exported to the cytosol, as most of the GSH-S is
located in this compartment. Secondly, overexpression
of g-EC in the chloroplast enhanced not only leaf g-EC
and glutathione contents, but it also increased the abundance of amino acids synthesized in the chloroplasts
such as valine, leucine, isoleucine, lysine and tyrosine
(Noctor et al. 1998a). It is interesting to note that
increases in these amino acids was specific to chloroplast g-ECS overexpression and that the degree of
enhancement occurred in almost direct proportion to
increases in g-EC and glutathione (Noctor et al. 1998a)
GSH metabolism is more complex in C4 species such
as maize than in C3 species such as Arabidopsis,
tobacco and poplar. In maize leaves, cysteine synthesis
is exclusively localized in the bundle sheath (BS) tissues
(Burgener et al. 1998). g-ECS and GSH-S transcripts
and proteins are similar in abundance in maize leaf
mesophyll (M) and BS cells, and they were also found
in the epidermis and stomatal guard cells (Gomez et al.
2004b). It is important to note however that an earlier
study found that GSH-S activity primarily in the M cells
(Burgener et al. 1998).
A number of GSH-deficient A. thaliana mutants have
been described in the GSH1 locus. The analysis of
g-ECS-deficient mutants such as rml, cad and rax has
provided useful information concerning the effects of
glutathione depletion in plants (Cheng et al. 1995,
Howden et al. 1995, Vernoux et al. 2000, Ball et al.
2004). The mutants have rather different characteristics.
For example, the cad2-1, which has 15–30% of wildtype GSH, and the rax 1-1, which has 50% of wild-type
GSH, have different leaf transcriptome profiles (Ball
et al. 2004). It is possible that the GSH1 gene products
are themselves involved in signal transduction.
The extensive literature concerning the effects of
manipulation of g-ECS and GSH-S activities in plants
Physiol. Plant. 126, 2006
has already been reviewed (Noctor et al. 1998a, Noctor
et al. 2002a). The major factors controlling GSH accumulation in plants as in animals are abundance of g-ECS
and the availability of cysteine (Noctor et al. 1996,
1998a, 1998b, 2002a, Xiang and Oliver 1998, Xiang
and Bertrand 2000, Xiang et al. 2001). Overexpression
of enzymes such as g-ECS (Noctor et al. 1996), GR
(Foyer et al. 1991, 1995, Aono et al. 1993, 1995) or
serine acetyltransferase (Harms et al. 2000) enhances
tissue GSH contents. However, there are few attempts
to date to examine the effects of simultaneous expression of these enzymes. Increased tissue glutathione in
transformed plants has resulted in largely beneficial
effects (Foyer et al. 1995, Strohm et al. 1995, Noctor
et al. 1996, 1998a, Pilon-Smits et al. 1999, Zhu et al.
1999). The marked chlorotic phenotype produced by
chloroplastic E. coli g-ECS overexpression in transformed tobacco is unusual and may result from perturbed signal transduction rather than direct effects of
GSH or g-EC accumulation per se (Creissen et al. 1999).
Some of the effects of ectopic g-ECS expression are
illustrated in Fig. 4 using transformed Arabidopsis seedlings produced by Cobbett et al. (1998). The high level
of GSH1 transcripts observed in the transformed
Arabidopsis plants relative to controls (Fig. 4A) is
accompanied by increased total leaf glutathione
contents (Fig. 4B). Moreover, the enhanced capacity
for GSH synthesis arising from the increased expression
of g-ECS results in a much lower effect of BSO in terms
of its ability to deplete tissue glutathione (Fig. 4B). The
pronounced positive effect of GSH1 overexpression and
higher tissue GSH availability on Arabidopsis root
growth is illustrated in Fig. 4C. It is interesting to note
that in the presence of BSO, the wild-type plants have a
similar phenotype to the rml1 mutant as illustrated by
the absence of a post-embryonic root. However,
enhanced g-ECS activity (o/e line) alleviates this
inhibition of root development, presumably due to the
higher capacity for GSH synthesis even in the presence
of BSO, allowing the root to grow.
Sulphur uptake by the roots of transformed bacterial
g-EC-expressing poplar trees was enhanced to meet the
requirements of increased demand for sulphur caused
by enhanced GSH synthesis (Herschbach et al. 2000).
However, the glutathione contents of untransformed
and transformed poplar leaf discs were increased substantially by incubation with cysteine, particularly in the
light. This suggests that cysteine supply remains a key
limiting factor for glutathione synthesis even when
g-ECS activity is increased (Strohm et al. 1995, Noctor
et al. 1996, 1997). The leaf cysteine levels were maintained at values similar to the wild-type in transgenic
poplars with high g-ECS activities, but the activities of
389
A
20
Relative fold expression of GSH1
18
16
14
12
10
8
6
4
2
0
WT
GSH1 o/e
B 500
Regulation of glutathione synthesis and
accumulation
nmol GSH/g FW
400
300
200
100
0
WT
GSH1 o/e
C
WT
390
enzymes involved in sulphate reduction and assimilation were not increased (Noctor et al. 1998a,
b, Hartmann et al. 2004). Similarly, microarray analysis
of plants undergoing sulphate starvation did not reveal
effects on g-ECS or GSH-S transcripts suggesting little
coordination of the pathways of GSH synthesis in
response to sulphur availability (Hirai et al. 2003,
Maruyama-Nakashita et al. 2003). Hence, to remove
metabolic limitations in cysteine supply, it will be
essential to consider solutions that modulate sulphur
assimilation to further enhance the capacity of plant
tissues to synthesize and accumulate glutathione. For
example, modulation of oxylipin signalling may be
one approach, as methyl jasmonate stimulates transcription of both sulphur assimilation and GSH synthesis
genes (Harada et al. 2000).
GSH1 o/e
There is very little literature information available on the
coordinate regulation of expression of GSH1 and GSH2
in plants. It is worth noting the very high abundance of
GSH2 transcripts in the quiescent centre of Arabidopsis
roots, compared with surrounding cells (Nawy et al.
2005), and it may be that a high level of GSH is required
in these stem cells for antioxidant defence, because
quiescent centre cells are essentially devoid of reduced
ascorbate.
GSH1 and GSH2 mRNAs are increased by conditions
that require enhanced GSH abundance for metabolic
functions such as heavy metal sequestration. Neither
GSH nor GSSG exercise much control over GSH1 and
GSH2 transcription (Xiang and Oliver 1998). Similarly,
while H2O2 and CAT inhibitors increase tissue GSH,
they did not affect GSH1 or GSH2 transcript abundance
(Xiang and Oliver 1998). Exposure to oxidative stress
increased g-ECS activity and glutathione in Arabidopsis,
but GSH1 mRNA abundance was unchanged. In contrast, exposure to heavy metals such as cadmium and
copper increases GSH1 and GSH2 transcripts (Schäfer
et al. 1998, Xiang and Oliver 1998). While SA does not
regulate GSH1 or GSH2 transcripts, jasmonic acid (JA)
Fig. 4. Effect of g-glutamyl cysteine synthetase (g-ECS) overexpression
in Arabidopsis thaliana. Ten-day-old transgenic A. thaliana (Cobbett
et al. 1998) seedlings were compared with the wild-type. The effects
of constitutive g-ECS expression (g-ECS o/e) are demonstrated by semiquantitative RT-PCR (A) tissue reduced glutathione (GSH) contents (B)
and the stimulatory effect of the resultant high GSH levels on seedling
root growth (C). The higher activities of g-ECS activity in the transgenic
plants leads to greater GSH abundance (m) that persists even in the
presence of 0.7 mM 1-buthionine-SR-sulfoximine (M).
Physiol. Plant. 126, 2006
has a marked stimulatory effect (Xiang and Oliver
1998). Methyl jasmonate caused a rapid transient
increase in transcripts encoding sulphur assimilation
enzymes and GSH1 and GSH2 mRNA levels in
Arabidopsis (Harada et al. 2000). Although transcript
abundance was increased by heavy metals and JA, oxidative stress was required for the translation of the transcripts, implicating regulation at the post-transcriptional
level and a possible role for factors such as H2O2 or
modified GSH/GSSG ratios in de-repressing translation
of the existing mRNA (Xiang and Oliver 1998). Stressinduced increases in glutathione, such as those
observed in plants deficient in catalase, have shown
that glutathione accumulation is preceded or accompanied by a marked decrease in the reduction state of the
pool (Smith et al. 1984, Willekens et al. 1997). A similar
response was elicited by exposing poplar leaves to
ozone (Sen Gupta et al. 1991). The 50 untranslated
region (50 UTR) of the GSH1 gene was found to interact
with a repressor-binding protein that was released upon
addition of H2O2 or changes in the GSH/GSSG ratio
(Xiang and Bertrand 2000). A redox-sensitive 50 UTRbinding complex is thus suggested to control g-ECS
mRNA translation in A. thaliana (Xiang and Bertrand
2000).
It has long been accepted that post-translational regulation of g-ECS through end-product inhibition by GSH
may control tissue GSH concentrations (Hell and
Bergmann 1990, Schneider and Bergmann 1995, Jez
et al. 2004). Moreover, g-ECS activity is inhibited by
thiols in plant extracts (Noctor et al. 2002a) as well as
in the purified recombinant enzyme (Jez et al. 2004).
Such feedback control of g-ECS activity by GSH concentrations is easy to understand in terms of the exclusive localization of this enzyme in the chloroplast,
where localized GSH accumulation in the limited
space of the organelle would cause a specific feedback
limitation on g-ECS activity, until excess GSH could be
exported to the cytosol.
Post-translational control of g-ECS activity remains a
possibility (May et al. 1998), but protein phosphorylation similar to that reported in animals (Sun et al. 1996)
has not yet been found in plants. In animals, a smaller
regulatory subunit increases the catalytic potential of
the larger catalytic subunit by alleviating the effects of
feedback control (Huang et al. 1993, Mulcahy et al.
1995). A recent analysis in human cells has shown
that a number of enzymes affecting glutathione metabolism and breakdown are targets for of 14-3-3 proteins
(Pozuelo Rubio et al. 2004), and this may be also the
case in plants. Because the precise pathway of glutathione degradation in plants remains poorly resolved,
and many important plant enzymes are controlled by
Physiol. Plant. 126, 2006
interactions with 14-3-3 proteins (DeLille et al. 2001),
this type of regulation of GSH synthesis and/or degradation is a possibility that merits further investigation.
GSH homeostasis-transport
The physical separation of the rate-limiting step of GSH
synthesis catalysed by g-ECS from the second reaction
step suggests that g-EC and GSH transport have fundamental roles in glutathione homeostasis. Recent data
has implicated oligopeptide transport proteins (OPTs)
in GSH transport. However, none of the nine AtOPTs
has a high affinity for GSH transport, and the cellular
localizations of these proteins have not been resolved.
Chloroplastic transporters are particularly important, as
they are essential for the export of g-EC and GSH produced within the chloroplast. However, none of the
known transporters that have been implicated in GSH/
GSSG transport have been shown to be localized in the
chloroplast despite the fact that recent data suggests that
the chloroplast may be the major site of g-EC production. One or more high-affinity g-EC/GSH/GSSG transporters targeted to the chloroplast may be expected, as
illustrated in Fig. 3. Preliminary biochemical experimentation has shown that GSH is transported into isolated wheat chloroplasts. Noctor et al. (2002a) observed
that 35S-labelled GSH was taken up by chloroplasts in a
time-dependent linear fashion. There appeared to be
two independent systems for uptake with at least one
of those being an active transport system. This was
inferred by observing linear uptake of labelled GSH up
to 20–30 mM GSH showing saturation at a concentration of 100–200 mM, which was followed by an
increase in the rate of uptake until a concentration of
1 mM was reached. From these initial experiments,
there is evidently great potential for modulating GSH
concentration by transport in response to an altered
demand for GSH caused by either the enzymes of the
cytosol or ascorbate–glutathione cycle in the chloroplast.
GSH transport has not only been observed at the
chloroplast membrane. Early experiments with heterotrophic tobacco cells suggested that GSH could be
transported across plasma membranes of plants
(Schneider et al. 1992). In this study, both high-affinity
(Km 18 mM) and low-affinity (Km 780 mM) GSH transport systems were identified. Other biochemical studies
indicated that some transporters had preferences for
specific GSH metabolites. For example, the bean plasmalemma transporter exhibited a significantly greater affinity for GSSG compared with GSH (Jamaı̈ et al. 1996).
Taken together, the biochemical data from chloroplasts
and plasma membrane preparations suggest that there is
diversity in the GSH transport mechanisms.
391
To date, the best-characterized glutathione transporters are all members of the peptide transporter superfamily. Early research in yeast identified the Yeast
Cadmium Factor (YCF1) protein as a glutathione-S-conjugate (GS-conjugate) transporter (Szczypka et al. 1994;
Falcon-Perez et al. 1999). YCF1 is a vacuolar membrane
protein that transports Cd2þ.GSH2 and consequently is
essential for Cd2þ tolerance in yeast. YCF1 is a member
of the ABC-type transporters similar to the multiple
drug-resistance transporters (MRPs) in humans
(Decottignies and Goffeau 1997). A common feature of
many MRP proteins is the ability to transport GS-conjugates and, in some cases, cotransport of GSH and
drugs (Rappa et al. 1997). MRP homologues are also
found in plants (Kolukisaoglu et al. 2002). In
Arabidopsis, there are 14 genes (and one putative pseudogene) encoding MRP transporters (Kolukisaoglu et al.
2002, Martinoia et al. 2002). Among these, only
AtMRP1, AtMRP2, AtMRP3 and AtMRP5 have been
characterized. Transport of GS conjugates was observed
for AtMRP1, AtMRP2 and AtMRP3 and is consistent
with the phenotype observed in an AtMRP5 insertion
mutant (Lu et al. 1997, Lu et al. 1998, Tommasini et al.
1998, Gaedeke et al. 2001, Liu et al. 2001). More
recently, AtMRP3 has been implicated in Cd detoxification, as it is upregulated by Cd treatment which suggests
that it may be transporting GSH-Cd or PC-Cd complexes
(Bovet et al. 2005). However, the ability of the MRP
proteins to transport GSH and GSSG is variable. For
example, while only AtMRP2 and AtMRP4 exhibited
competitive inhibition of GS-conjugate transport by
GSH, all four showed competitive inhibition in the presence of oxidized glutathione (GSSG). Because GSSG
can be considered a GS-conjugate, this is consistent
with the MRP proteins being predominantly GS-conjugate transporters. However, the observed GSSG transport may also suggest that they play an important role in
removing oxidized glutathione as a mechanism to help
maintain the redox poise of the cell (Foyer et al. 2001).
A second peptide transporter belonging to a different
family of peptide transporters from yeast has provided
further insights into GSH transport. A high-affinity glutathione transporter (Hgt1p) was identified using the
hgt1 mutant of Saccharomyces cerevisiae which was
unable to transport GSH (Bourbouloux et al. 2000).
The Hgt1p protein is a member of the oligopeptide
transporter (OPT) family (Stacey et al. 2002). In
Arabidopsis, there are nine OPT orthologues of the
yeast OPT proteins (Hgt1p and Opt2p) (Koh et al.
2002). All of the OPT proteins are believed to be integral membrane proteins transporting small peptides (3–5
amino acids) (Koh et al. 2002). Heterologous expression
of the Arabidopsis OPT genes in S. cerevisiae has
392
confirmed that most are able to transport small peptides,
while only some exhibit glutathione transport capabilities and none exhibit high-affinity transport similar to
Hgt1p (Foyer et al. 2001, Koh et al. 2002). Thus,
although glutathione transport has been demonstrated
in plant cells, no GSH-specific transporters, like Hgt1p,
have been identified. Thus, while considerable movement of GSH/GSSG has been observed in biochemical
studies, little is know about the factors responsible for
this transport.
Conclusions and perspectives: prospects for
manipulation of glutathione metabolism for
improved nutritional quality
The ubiquity of GSH and its critical functions regulating
plant growth and as an important sulphur sink make
GSH an attractive target for manipulation to improve
crops for enhanced nutritional value as well as
improved crop growth, sustainability, predictability
and vigour. Fortunately, plant biology now has an
extensive toolbox available to help achieve this aim.
Many of the genes found in model plant systems such
as Arabidopsis have counterparts in important crop species, and hence it is expected that much of the knowledge on GSH gained in laboratory systems will be
readily transferable to important crop varieties. In addition, plant cell cultures could be used as a cheaper cell
synthetic factory for the production of g-EC, GSH, GSSG
and related homologues than the yeast system that is
currently used for GSH production. Glutathione has
potential for use in the food industry, for example as a
natural food additive with flavour-enhancing properties.
In whole plants, there is likely to be a ceiling on the
enhanced glutathione concentrations that can be
achieved, set by the level of feedback inhibition on
GSH synthesis within the chloroplast. However, this
ceiling GSH content may vary between plant species,
because it is known that some alpine plants hyperaccumulate other antioxidants particularly ascorbate, and
the same may be true of glutathione. There are essentially no metabolic arguments suggesting that GSH
hyperaccumulation in plants is impossible, because
GSH accumulation is simply a matter of compartmentation, such that the site of GSH accumulation is separate
from that of synthesis, preventing feedback inhibition.
This separation could easily be achieved in plant cell
culture systems where GSH synthesis and export were
enhanced such that GSH and related peptides were
transported into the culture medium from which they
could be continually harvested. Moreover, because the
bifunctional streptococcal g-ECS-GSH-S shows no feedback inhibition by GSH, plant cells transformed to
Physiol. Plant. 126, 2006
express this enzyme may be able to maintain much
higher GSH concentrations than those expressing the
plant or E. coli enzymes.
Manipulating GSH should affect three main phenotypic aspects (1) enhanced crop resilience and growth; (2)
improved innate immune responses and basal resistance
to pathogen attack and (3) increasing sulphur/cysteine
content in crop plants. A number of studies have
demonstrated the feasibility of enhancing plant glutathione synthesis and accumulation. Such plants have
been used successfully for phytoremediation purposes
and to reclaim arable land. All studies to date have
shown that increasing the GSH content of plants concomitantly increases the total organic sulphur content.
Comparatively, the manipulation of GSH partitioning,
both throughout the plant and between cellular compartments, has lagged behind, because no high-affinity
GSH transporters have been identified to date. The
identification of these will be a key step in combining
and interpreting current results in GSH research.
Moreover, while GSH transport mutants will allow for
a more detailed dissection of signalling and stress pathways, they will also provide for future targeted nutriomics
approaches where glutathione might be enriched in
specific cell types or in discrete compartments of the
plant cell.
The fully sequenced genomes of species such as
Arabidopsis and rice together with new breeding technologies and large scale ‘omics’, systems biology
approaches will allow increasingly rapid advances in
understanding and applications. For example, predictability of crop yield and hence income to the farmer and
reliable sources for the food industry are severely hampered by environmental factors. Collectively, the environmental abiotic stresses restrict plant vigour and create
a ‘yield gap’. This is the difference between the theoretical maximum or yield potential of the crop and the
actual yield achieved by the farmer. Over the last 50
years, plant breeders have improved the yield potential,
but the yield gap remains, and it is forecast to increase
as the world climate becomes less predictable. In
measurements made under field conditions, maize leaf
glutathione contents correlate well with chilling resistance
and yield. One factor determining the sensitivity of
maize to both short-term and long-term chilling is the
restriction over glutathione reduction and cycling
between the maize leaf BS and M cell types (Gomez
et al. 2004b). By understanding how glutathione is used
by the plant in acclimation responses to environmental
cues at the molecular level, we can use this information
to widen the range in which crop plants grow and
produce crops that perform closer to their theoretical
maximum. The quantitative trait loci (QTL) analysis in
Physiol. Plant. 126, 2006
plants such as maize is essential to determine chromosome regions carrying genes which control GSH accumulation and stress tolerance and other important traits
in crops. Mapping experiments at field sites have
allowed us to identify two major QTL for the total
amount of glutathione in maize, with a confidence
interval of about 30 cM. To date, little information on
QTL for glutathione has appeared in the literature.
Detailed fine maps of QTL regulating glutathione accumulation under field conditions in different plant species will be useful not only to the scientific community
but also to plant breeders and associated industries.
Acknowledgements – Rothamsted Research and the
Institute of Biotechnology receive grant-aided support from
the Biotechnology and Biological Sciences Research Council
of the UK [BB/C51508X/1, (C.F) and BB/C515047/1 (SM)].
Spencer Maughan is a Marie Curie International Incoming
Fellow of the EU (509962). We thank Dr Jeroen Nieuwland
for help with the mBBr-staining techniques and confocal
microscopy.
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