## 4. ascorbate and Glutathione
### The Approaches for Boosting Ascorbate and Glutathione Production
Overexpression of bacterial GR in chloroplasts of a poplar hybrid (_Populus tremula \(\times\) Populus alba_) led to a 100- to 500-fold increase in GR activity compared to wild-type plants [321]. This resulted in a two-fold increase in the total glutathione content in the leaves and an increase in the reduced fraction of glutathione. The engineered plants exhibited greater resistance to photoinhibitory conditions (1000 \(\upmu\)mol quanta/m\({}^{2}\) s, 5 \({}^{\circ}\)C) and oxidative stress induced by leaf incubation in the presence of MV [321]. Overexpression of tomato GR in tobacco plants also led to a 1.9- to 2.3-fold increase in GR activity compared to the wild-type plants [322]. The resulting transgenic plants exhibited higher germination rates and increased root length compared to the wild-type under normal conditions. Moreover, these plants demonstrated better growth under salinity conditions (100 mM NaCl), and lower hydrogen peroxide accumulation [322]. Arabidopsis explants overexpressing _AtGR1_ had a higher glutathione level and a higher reduced/oxidized glutathione ratio. Such transgenic plants exhibited greater resistance to the toxic effects of aluminum: H\({}_{2}\)O\({}_{2}\) production in transgenic plants was 26% lower compared to wild-type plants [323]. Noteworthy results were reported in the study by [PERSON] et al. In this research, tomato plants were engineered with a cassette of genes encoding enzymes of the ascorbate-glutathione cycle. Genes, which were used in this study, included _MDHAR_, _DHAR_, _GR_, _APX_, and _SOD_ from _Pennisetum glaucum_, under the control of stress-inducible promoters [324]. These transgenic tomato plants accumulated approximately 50% more ascorbate and 90% more DHA compared to the wild-type plants and demonstrated enhanced resistance to salinity (200 mM NaCl) and drought stress [324]. Furthermore, these engineered plants exhibited increased resistance to mercury (Hg) toxicity, accumulating significantly lower levels of H\({}_{2}\)O\({}_{2}\) and maintaining higher photosynthetic activity compared to wild-type plants [325]. Notably, the transgenic tomato plants accumulated 20% less Hg in their leaves but 40% more in their roots than the wild-type plants [325]. Similar to the case with flavonoids and ascorbate, a promising approach for glutathione level increase involves the regulation of transcription factor activity. For instance, it has been demonstrated that overexpressing the transcription factor NAC2 from _Solanum lycopersicum_ L. in Arabidopsis plants leads to increased expression of enzymes involved in glutathione biosynthesis (\(\gamma\)-ESC, GS, and GR) under abiotic stress conditions [326]. These engineered plants accumulated fewer ROS, exhibited improved growth, and demonstrated better water retention in tissues during drought and salinity stress compared to the wild-type plants [326]. As mentioned above, glutathione together with GST is involved in binding heavy metals. Thus, transgenic plants with increased glutathione content can be used to remediate soil contaminated with heavy metals. For instance, Indian mustard plants with overexpressionof glutathione synthetase from _E. coli_ and elevated glutathione levels accumulated three times more cadmium in their shoots than wild-type plants [327]. Rice plants overexpressing the _GST_ gene _OsGSTUS_ contained higher levels of glutathione compared to wild-type plants. In the presence of cadmium in the soil, these transgenic plants accumulated 1.5 times more cadmium than the wild-type plants [328]. Overexpression of _GST_ from _Suada salsa_ (halophytic plant adapted to saline-alkali soils) in Arabidopsis under the control of the 35S promoter led to a significant increase in the expression of GST and GPX [329]. The resulting plants also showed enhanced tolerance to salinity stress (100 mM NaCl).