Physiologia Plantarum 126: 382–397. 2006 Copyright ß Physiologia Plantarum 2006, ISSN 0031-9317 REVIEW Engineering and genetic approaches to modulating the glutathione network in plants Spencer Maughana and Christine H. Foyerb,* aInstitute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QT, UK bCrop Performance and Improvement Division, Rothamsted Research, Harpenden, Herts, AL5 2JQ, UK Correspondence Reduced glutathione (GSH) is the most abundant low-molecular weight thiol *Corresponding author, in plant cells. It accumulates to high concentrations, particularly in stress e-mail: [email protected] situations. Because the pathway of GSH synthesis consists of only two Received 10 October 2005; revised 26 enzymes, manipulation of cellular glutathione contents by genetic interven- November 2005 tion has proved to be relatively straightforward. The discovery of a new bacterial bifunctional enzyme catalysing GSH synthesis but lacking feedback doi: 10.1111/j.1399-3054.2006.00684.x 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 50phosphosulphate reduc- tase; 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 and defence. Similarly, the analysis of mutants deficient in glutathione has greatly advanced current concepts of The thiol tripepide glutathione [g-Glu-Cys-Gly; reduced how GSH and glutathione disulphide (GSSG) regulate glutathione (GSH)] does not immediately come to mind cell signalling and development. Chemical inhibition of in considerations of human nutrition. However, this GSH synthesis using 1-buthionine-S, R-sulfoximine ubiquitous tripeptide thiol is a vital intracellular and (BSO), a specific inhibitor of g-ECS (Griffith and extracellular protective antioxidant against oxidative/ Meister 1979, Griffith 1982, Maughan and Cobbett nitrosative stresses, which play a key role in the control 2003), has been widely used to study the effects of of many human diseases. GSH is also important in GSH depletion in plants (Hell and Bergman 1990, immunity modulation in animals as well as in remodell- Vernoux et al. 2000). Analysis of Arabidopsis thaliana ing of the extracellular matrix, apoptosis and mitochon- T–DNA insertion and EMS mutants selected by their drial respiration in disease. Mammalian cancer cells ability to grow on BSO concentrations that are inhibi- often accumulate GSH, where there is also a metabolic tory to the wild-type has indicated that different switch to glycolysis rather than TCA cycle as a major mechanisms can confer BSO resistance (Maughan energy source (Pozuelo Rubio et al. 2004). Interactions 2003). of GSH with antiapoptotic factors such as Bcl-2 in can- Since its initial discovery in yeast over 125 years ago, cer cells have been linked to radiation and multidrug glutathione has been shown to have a prodigious number resistance (Ortega et al. 2003). However, because high of critical functions (Fig. 1). GSH is a powerful reducing GSH concentrations are essential for both antioxidant agent and is a key player in the plant redox-state (Noctor and immune defence systems in mammals, tissue GSH and Foyer 1998, Noctor et al. 2002a). Here we will levels can be regulated, particularly in malnourished focus on how manipulation of GSH synthesis and meta- patients through diet and nutrition before therapeutic bolism has advanced current concepts of glutathione treatments (Bray and Taylor 1993). homeostasis and function, concentrating on aspects Plants make abundant amounts of glutathione, and its related to cell signalling, development and defence synthesis and accumulation in either the chloroplast or that may be of importance in any consideration of nutri- cytosolic compartments of the plant cell can be tional genomics involving glutathione. The characteris- enhanced or decreased relatively easily by genetic engi- tics and functional importance of putative thiol-based neering approaches (Strohm et al. 1995, Creissen et al. reactive oxygen species (ROS) sensors and associated 1996, Noctor et al. 1998a, b, Zhu et al. 1999, Xiang redox-signalling cascades have recently been discussed 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 Biotic reductase (GR) (Foyer et al. 1991) in transgenic plants Abiotic ROS scavenging results in substantial increases in leaf glutathione. Heavy metal detoxification Systemic Acquired Resistance Glutathione contents have been increased by at least Xenobiotic detoxification Protein thiolation five-fold the values of wild-type plants without negative Salinity protection PCD Oxidative brust effects (Noctor et al. 1998b, 2002a). Only one report to Drought protection Calcium MAPK date has indicated that an enhanced capacity to gener- UV protection signaling cascades ate and accumulate the intermediate g-glutamyl Signaling cysteine (g-EC) can produce negative effects (Creissen et al. 1999). The increased capacity to generate glu- Cell cycle progression Root development tathione and enhance cellular glutathione pools leads to higher rates of sulphur assimilation, modified amino Sulfur storage acid metabolism and enhanced stress tolerance (Noctor Sulfur sensing et al. 1998a, b). Transformed poplar trees with an increased capacity to synthesize and accumulate glu- Carbon - Sulfur - Nitrogen tathione are currently being used in the field for bior- emediation purposes to purify and restore soils polluted Metabolism by human activities (Peuke and Rennenberg, 2005a, b). Genetic engineering approaches have led to an Fig. 1. The functions of glutathione in plants linked by essential signal- improved understanding of how compartment-specific ling functions that integrate plant growth, development and defence alterations in glutathione synthesis affect metabolism processes. Physiol. Plant. 126, 2006 383 (Foyer and Noctor 2005). In this context, we will con- checkpoint (Dewitte and Murray 2003, de Jager et al. centrate here on the implications for the plant of mod- 2005). The activity of the CDK/cyclin complexes is ulating glutathione homeostasis. regulated by a network of regulatory mechanisms Like ascorbate, glutathione limits the lifetime of ROS including transcription, proteolysis, phosphorylation/ and lipid peroxide signals. Moreover, GSH alters cal- dephosphorylation, interaction with regulatory proteins cium signalling in plants (Gomez et al. 2004a) and and intracellular trafficking. participates in the calcium-dependent pathways of In Arabidopsis, the embryo cells of the dry seed are ROS-signal transduction (Rentel and Knight 2004). arrested in the G1 phase of the cell cycle. Germination Through oxidation to GSSG, other signalling cascades is initiated by water uptake and the resumption of meta- can be initiated through thiol-disulphide exchange and bolism and cell division. Nitric oxide is a potent regu- thiolation. Moreover, the interaction between GSH and lator of germination in dormant seeds such as those of nitric oxide, catalysed by the enzyme formaldehyde Arabidopsis (Bethke et al. 2004, 2006). Germination is dehydrogenase (also known as class III alcohol dehy- stimulated by the presence of environmental nitrate drogenase), is also important in signal