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Cysteine is an indispensable molecule in the synthesis of various proteins. Biosynthesis of cysteine in the major sub-cellular compartments ensures sufficient availability of cysteine to fuel various functions of the cellular machinery. The reduced form of sulfur in cysteine, known as the thiol group (Cys-SH) is an important mediator of redox reaction. This is because sulfur is highly nucleophilic and an efficient donor of free electrons. The thiol group also enables di-sulphide bridge formation between Cys residues which aids in modification of the protein structure as well as protein-protein interactions. Further cysteine is also required in the synthesis of metabolites essential for plant growth as well as stress defence. For example, the thiol group of the Cys provides the reducing power of glutathione (GSH) which mitigates oxidative stress and metal toxicity in the cells.

(i) Glutathione

The tripeptide glutathione or GSH (L-γ-glutamyl-L-cysteinylglycine) is the most abundant sulfur-containing peptide in plants. GSH is involved in various stress migitation reactions in plants such as detoxification of ROS, reactive nitrogen species (RNS), xenobiotics and heavy metals.

The first step in GSH synthesis is carried out by the enzyme γ-glutamyl-cysteine ligase (GSH1 or γ-ECS). GSH1 is encoded by a single copy gene and catalyses the rate-limiting step in GSH biosynthesis. GSH1 is localised to plastids in many plants (Richman and Meister, 1975; May and Leaver, 1994) but in some species including pea and spinach, is present in both cytosol and plastids (Hell and Bergmann, 1990). GSH1 catalyses the synthesis of γ-glutamylcysteine (γ-EC) from two substrates, L-glutamate and L-cysteine. GSH1 acts as a monomeric protein and has not been found to interact with any other enzyme.

The final step in GSH biosynthesis involves the addition of glycine to γ-EC. This is catalysed by glutathione synthetase (GSH2 or GSH-S) (Rawlins et al., 1995). GSH2 is localised to both

cytosol and plastids in Arabidopsis. It is encoded by a single copy gene, which shows multiple transcriptional splicing patterns (Wachter et al., 2005). In absence of GSH2 localisation to plastids, the cytosolic GSH2 is sufficient for GSH biosynthesis in plants (Pasternak et al., 2007). This is because the precursor of GSH, γ-EC, can diffuse from plastids into the cytosol via a specific transporter and contribute to GSH biosynthesis. γ-EC transporters are localised to the plastidic membrane and are orthologs of the Plasmodium falciparum chloroquine-resistance transporter (CLT) (Maughan et al., 2010). Further the study also suggests that GSH can also be transported from plastids into cytosol via these transporters. GSH biosynthesis does not take place in mitochondria and depends upon import of GSH from the cytosol. Although plasma and plastidic membrane-specific transporters of GSH are suggested to be oligopeptide transporters (OPT) and CLTs in plants, respectively, mitochondrial specific transporters of GSH are yet to be revealed. The null GSH1 mutant lines show an embryo-lethal phenotype whereas GSH2 null lines are seedling lethal. These findings assert that γ-EC and GSH are required in plant development and viability (Vernoux et al., 2000; Cairns et al., 2006; Pasternak et al., 2008).

(ii) GSH functions in redox balance and resistance against ROS and RNS

The cysteine of GSH molecule provides the antioxidant capacity of GSH against ROS. ROS include various species such as superoxide and the hydroxyl radical. Excess concentration of ROS can damage the cellular system by oxidising proteins and enzymes as well as altering redox-sensitive reactions (Van Breusegem and Dat, 2006). ROS oxidise GSH and therefore GSH acts as a scavenger for these species to prevent them from oxidising important proteins and enzyme.

The oxidation of the thiol group in GSH results in the formation of a disulphide bridge between the two oxidised GS- anions which yields a stable molecule, GSSG (Foyer and Noctor, 2003; Noctor et al., 2012). GSSG is reduced back into GSH by Glutathione reductase (GR) which uses reducing equivalents from NADPH. The redox status in the cells is often measured by the ratio of GSH to GSSG, which is greater than 0.9 under non-stressed conditions. GR is localised to plastids, cytosol and mitochondria to ensure the reduction of GSSG. This maintains the

antioxidant capacity of plant cells and is therefore a crucial point in redox balance. Reduced biosynthesis of cysteine due to lack of OASTL-A1 has been shown to affect the cytosolic redox status and result in lower GSH:GSSG ratio which suggests that cytosolic cysteine production is an important player in modulating oxidative stress conditions (Lopez-Martin et al., 2008).

GSH also reacts with nitric oxide which results in the formation of S-nitrosoglutathione (GSNO). GSNO was unravelled to be a key player in signalling as it performs nitrosylation of cysteine residues which results in post-translational modification of the proteins (Lindermayr et al., 2005; Romero-Puertas et al., 2008; Lindermayr and Durner, 2009). Recently GSNO mediated post- translation modification was found to be a key step in ethylene biosynthesis (Lindermayr et al., 2006) and activation of defence signalling during disease resistance (Tada et al., 2008).

(iii) Role of GSH in immunity and disease resistance

Reduced GSH biosynthesis can lead to oxidised cytosolic environment which can negatively affect the metabolic processes. The role of GSH in plant immunity has been highlighted by the reduced resistance of pad2 and cad2 mutants against the avirulent pathogen strain of Pseudomonas syringae (Ball et al., 2004; Parisy et al., 2007). Moreover mutants lacking cytosolic GSH biosynthesis, either due to impaired γ-EC transport into the cytosol or lacking GS

enzymes showed reduced expression of PATHOGENESIS RELATED-1 defence genes and

enhanced disease susceptibility in Arabidopsis plants (Maughan et al., 2010). Similarly plants lacking cytosolic GR, which is involved in reducing GSSG to GSH, also showed reduced PR-1 transcript abundance and SA accumulation (Mhamdi et al., 2010). Redox changes in the cytosolic environment are shown to be involved in defence signalling in response to the disease resistance hormone salicylic acid (Tada et al., 2008). Therefore the cytosolic redox status is a key factor in defence signalling pressing the importance of cytosolic cysteine and thiol production in plant immunity (Foyer and Noctor, 2005). Moreover cysteine and GSH are also required in synthesis of plant defence compounds including indol-glucosinolates and camalexin which are actively involved in resistance against disease caused by bacterial and fungal pathogens as well as herbivory (Schlaeppi et al., 2008; Bednarek et al., 2009; Bottcher et al., 2009; Clay et al.,

2009; Su et al., 2011). Therefore the availability of cysteine and GSH seems a crucial factor in modifying plant-pathogen interactions (Rausch and Wachter, 2005).

(iv) Role of cysteine derived metabolites in resistance to metal toxicity

The role of cysteine and its derived metabolites γ-EC and GSH is already highlighted in protection against metal toxicity (Zhu et al., 1999a; Zhu et al., 1999b; Gullner et al., 2001; Dominguez-Solis et al., 2004; Lopez-Martin et al., 2008; Shirzadian-Khorramabad et al., 2010). Mutations in GSH1 (root meristemless 1/ rml 1 and cadmium sensitive -2/ cad-2) which causes reduced GSH levels lead to impaired root meristem development and shoot growth and enhanced susceptibility to cadmium toxicity (Cobbett et al., 1998; Vernoux et al., 2000). The GSH- mediated-antioxidant shield further prevents the damage in the cellular system caused by heavy- metal-induced ROS accumulation. GSH also acts as a scavenging molecule of heavy metals in form of phytochelatins. Phytochelatins are oligomers of GSH and synthesized by the cytosolic phytochelatins synthases (PCS) enzymes in Arabidopsis (Ha et al., 1999; Cazale and Clemens, 2001). Phytochelatins complex heavy metals which are transported to vacuoles for degradation (Cobbett and Goldsbrough, 2002).

(v) Cystathionine, Methionine and Fe-S cluster

Cysteine is converted in plastids into cystathionine by cystathionine γ-synthase (CgS) enzyme, which catalyses the substitution of the phosphate group of O-phospho-homoserine by the sulfhydryl group of cysteine (Ravanel et al., 1998). The synthesis of cystathionine from cysteine and O-phospho-homoserine is the starting point for the biosynthesis of the other sulfur containing amino acid, methionine. Cystathionine is converted into homocysteine by another enzyme Cystathionine β-lyase. Homocysteine is then methylated to form methionine. The three enzymatic reactions converting cysteine to methionine takes place in plastids (Ravanel et al., 2004). Methionine is transported from plastids into cytosol; however homocysteine can be converted into methionine in the cytosol as well. Methionine is further converted into S- adenosylmethionine (SAM) which is fed into the Yang-cycle that regulates ethylene

biosynthesis. Availability of reduced sulfur in the form of cysteine therefore plays a major role in methionine, SAM and ethylene biosynthesis. Cysteine is also required in the synthesis of Fe-S cluster which makes an important component of electron transport chain of chloroplast and mitochondria (Balk and Pilon, 2011).