## 4. ascorbate and Glutathione
### Biosynthesis of Glutathione  
The glutathione molecule is a tripeptide consisting of three amino acids: glutamate, cysteine, and glycine. Glutathione synthesis takes place in chloroplasts and cytosol [295,296]. Once synthesized, glutathione can be found in various cellular compartments, including mitochondria. Glutathione is primarily synthesized in chloroplasts, since both of the enzymes involved in this pathway, \(\gamma\)-glutamylcysteine synthetase (\(\gamma\)-ECS) and glutathione synthetase (GSHS), are located in this compartment. The biosynthesis of glutathione consists of two steps (Figure 6). In the first step, \(\gamma\)-ECS catalyzes the reaction between the \(\gamma\)-carboxyl group of glutamate and \(\alpha\)-amino group of cysteine, resulting in the formation of \(\gamma\)-glutamylcysteine, which is partially exported to the cytosol. In the second step, GSHS, which is located both in the cytosol and plastids, forms glutathione molecule by amide bond formation between the \(\alpha\)-carboxyl group of the cysteine moiety in \(\gamma\)-glutamylcysteine and the \(\alpha\)-amino group of glycine [297,298]. After that, glutathione is transported into the mitochondria or reimported into the plastids [299].  
The activities of various enzymes affect the reduced/oxidized glutathione ratio, which is also significantly affected by various stresses. The rate of glutathione synthesis is controlled by several factors with the most important one being the feedback inhibition of \(\gamma\)-ECS by glutathione due to its binding to the glutamate site of the enzyme [300]. The information about the genes encoding the main enzymes for glutathione biosynthesis in \(A\). _thaliana_ is given in Table 4.  
quenching rate is rather low [104]. Reduced glutathione and other thiols predominantly exhibit a chemical quenching mechanism of \({}^{1}\)O\({}_{2}\); the rate constant for glutathione is estimated to be 2.4 \(\times\) 10\({}^{6}\) M\({}^{-1}\) s\({}^{-1}\)[314].  
Asc and glutathione are involved not only in the water-water cycle operation and direct scavenging of ROS to minimize the consequences of the oxidative stress in plant cells, but also in regeneration and stabilization of several cell components. For example, Asc plays a role in the regeneration of \(\alpha\)-tocopherol from its radicals [11], which are produced during the detoxification of lipid peroxide radicals. Glutathione is also the substrate for phospholipid hydroperoxide glutathione peroxidase, which is one of the glutathione peroxidase (GPX) isoforms. Under stress conditions, this peroxidase is upregulated to protect against the accumulation of lipid hydroperoxides in various cell compartments [315].  
Glutathione regulates multiple metabolic functions. It is involved in detoxification, redox homeostasis, and acts as an antioxidant. It interacts with proteins in several ways, affecting their structure and activity through changes in the thiol-disulfide balance and preventing oxidative denaturation under stress conditions by protecting their thiol groups. Glutathione protects membranes by maintaining the reduced state of antioxidants such as \(\alpha\)-tocopherol and zeaxanthin. The mechanism of glutathione detoxification of various toxic compounds is based on the conjugation of the xenobiotics with glutathione, leading to the formation of less reactive products. This conjugation reaction can occur spontaneously, but in biological systems, it is presumably facilitated by glutathione S-transferase (GST) (for review, see [316]). Detoxified xenobiotics are then transported to vacuoles for their subsequent sequestration.