Research Article

Evaluating the Performance of Genetically Engineered Serine acetyltransferase 4 (NtSAT4) Overexpression Brassica napus L. Lines under Xenobiotics Exposure

Fariha Qahar and Muhammad Sayyar Khan*

Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan.

Received | May 17, 2023; Accepted | September 22, 2023; Published | September 29, 2023

*Correspondence | Muhammad Sayyar Khan, Institute of Biotechnology and Genetic Engineering, The University of Agriculture, Peshawar, Khyber Pakhtunkhwa, Pakistan; Email: sayyar@aup.edu.pk

Citation | Qahar, F. and M.S. Khan. 2023. Evaluating the performance of genetically engineered Serine acetyltransferase 4 (NtSAT4) over expression Brassica napus L. lines under xenobiotics exposure. Sarhad Journal of Agriculture, 39(3): 765-772.

DOI | https://dx.doi.org/10.17582/journal.sja/2023/39.3.765.772

Keywords | Brassica napus, Glutathione, Xenobiotics, Oxidative stress, Detoxification, Transgenic

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Glutathione (GSH) is critical in protecting plants against several stresses, including heavy metals and xenobiotics. Their accumulation in plants triggers the abiotic defence response resulting in the production of GSH, which in turn sequesters/chelates these harmful substances to protect them (Liedschulte et al., 2010; Rajab et al., 2020; Foyer and Noctor, 2005; Mullineaux and Rausch, 2005). Conjugation of xenobiotics of different structures with GSH is a common detoxification pathway in higher plants. These GSH conjugates are catalyzed by GSH S transferases (GSTs) (Bártíková et al., 2015; Dasari et al., 2017). This conjugation generally occurs in the cytosol and is subsequently deposited in the vacuoles of plants and fungi to avoid toxicological effects (Verbruggen et al., 2009; Tong et al., 2004). Previous research has demonstrated that GSH availability is correlated with xenobiotic (herbicide) resistance (Nakka et al., 2017; Syguda et al., 2018; Ye et al., 2019; Dixon et al., 2002). Its availability is a critical factor in determining cell sensitivity and under standing herbicide selectivity (Marrs, 1996). Since conjugation of xenobiotics with GSH or its oligomers phytochelatins results in the fast depletion of cytosolic GSH pools, thereby, any such perturbation that causes the depletion of GSH can severely weaken a plant’s defense against xenobiotics. Therefore, the genetic manipulation of GSH biosynthesis in plants is a very promising approach to enhancing the plant’s tolerance potential against abiotic stresses (Pilon-Smits and Pilon, 2002; Rajab et al., 2020; Xiang et al., 2018; Khan et al., 2021). For this reason, the assimilatory sulfate reduction pathway has been genetically manipulated by overexpressing different rate-limiting genes to elevate in planta GSH in different plant species (Pilon-Smits et al., 1999; Rajab et al., 2020; Xia et al., 2018).

Cys availability is rate-limiting in GSH production; overexpression of a rate-limiting gene in the Cys synthesis pathway ATPS1 (ATP sulfurylase) has resulted in elevated GSH contents (Pilon-Smits et al., 1999). The GSH overproducer transgenic lines also showed improved tolerance to atrazine (Flocco et al., 2004). Serine acetyltransferase (SAT), which catalyzes the acetylation of L-serine, is one of the rate-limiting enzymes in the production of Cys. However, Cys synthesis is tightly regulated, and SAT activity is feedback inhibited by Cys itself. A Cysteine insensitive isoform of SAT from Nicotiana tabacum (NtSAT4) has been explored to be completely insensitive to Cys up-to 0.6 mM (Wirtz and Hell, 2003). The expression of this isoform in bacteria has resulted in elevated Cys and GSH contents.

The usage of Brassica napus L. for phytoextraction has gained importance in cleaning polluted lands (Van Ginneken et al., 2007; Zhang et al., 2018). It is a powerful phyto accumulator of heavy metals for environmental cleanup (Angelova et al., 2017). We have recently demonstrated that overexpression of Brassica napus L. with the feedback insensitive SAT (NtSAT4) in different subcellular compartments has resulted in up to 3.5-fold higher free Cys and 5.3-fold higher GSH contents. Overproduction of GSH in these lines led to improved tolerance against oxidative stress with hydrogen peroxide (H2O2) and the heavy metal cadmium (Cd) in overexpression lines (Rajab et al., 2020). These lines also translocated significantly greater amounts of iron (Fe) from roots to shoots. Since GSH-dependent GST-mediated detoxification is important in the bio transformations of several xenobiotics in plants to minimize their potential phytotoxicity (Bartha et al., 2014; Vontas et al., 2001), we tested the performance of these NtSAT4 GSH overproducer lines under selected herbicides stress in this study.

Materials and Methods

Plant material

The seeds of the single and double overexpression NtSAT4 transgenic B. napuslines recently produced at the Institute of Biotechnology and Genetic Engineering, The University of Agriculture Peshawar, were the starting material for this study. The seeds of the cultivar Oscar were used as a starting material for this project.

Plant growth conditions

The seeds of wild-type (Oscar) and single and double overexpression NtSAT4 transgenic lines plants were surface sterilized as mentioned before by (Syed et al., 2023), then treated at cold temperatures (2 days at 4 ºC) to break dormancy. The seeds were then germinated on Murashige and Skoog (MS) media (half-strength) on Petri plates for five days. The plantlets were then shifted to the soil in pots and allowed to acclimatize in the greenhouse. DNA was extracted (Rogers and Bendich, 1989) from the seedlings, and they were analyzed through PCR. The PCR-positive plants were allowed to grow and later used in the experiment.

Exposure of leaf discs to H2O2 and other xenobiotics

About 8-10 leaf discs from young leaves of single overexpressor NtSAT4 transgenic lines (P, M, and C), double overexpressors, and wild-type plants were subjected to 14% H2O2 for 24 hours (Rajab et al., 2020) and one set of leaf discs was kept as control and only subjected to distilled water. The plants were also subjected to xenobiotics such as herbicides, metolachlor, and atrazine. Leaf discs were exposed to 24 µM metolachlor stress for 24 hrs. While in the case of atrazine stress, whole NtSAT4 transformed and wild-type plantlets were subjected to 12 µM and 24 µM for 48 and 24 hours, respectively, in quarter-strength MS media. The control set of plants was kept in quarter-strength MS media. The performance of the NtSAT4 single and double overexpressor lines and wild-type plants was arbitrated by comparing the degree of chlorosis or wilting. Leaf damage in response to the above-mentioned stresses was also documented by taking into account the percentage of the total leaf area damaged in response to a particular stress in transgenic lines.

Results and Discussion

Performance of the NtSAT4 overexpression lines under H2O2 stress

In biological sciences, the leaf disc methodology is an established approach to documenting the response of plants to reactive oxygen species exposure (Rajab et al., 2020; Liedschulte et al., 2010). To essay the response of single and double overexpressor NtSAT4 lines and wild-type plants, the leaf discs taken from these lines grown under glasshouse conditions were exposed to14% H2O2. The leaf discs were kept at room temperature under light conditions for 24 hours. It was noted that the NtSAT4 (single and double) overexpressor lines exhibited lesser loss of pigments (chlorosis) as compared to the wild-type leaf discs after oxidative stress (Figure 1). The C264 and M193 showed lesser chlorosis as compared to the P230 amongst the single overexpressor lines and hence showed better performance. The double overexpressor NtSAT4 lines (M193×P230 and M193×C264) also performed better as compared to the wild-type leaf discs. Furthermore, amongst the transgenic lines, the double overexpressor lines exhibited distinctly reduced chlorosis as compared to NtSAT4 single overexpressor lines. Numerical data in the form of approximate percentage damage showed 95% of the leaf area turned necrotic in the wild-type when exposed to 14% H2O2; the single overexpressor lines showed 30-60% damage, whereas the least amount of damage was exhibited by double overexpressor lines (10-20%). The overall result of this experiment concluded that all the NtSAT4 transgenic lines showed lesser chlorosisas compared to the wild-type plants and hence showed moretolerance toH2O2 oxidative stress. Additionally, the double overexpressor NtSAT4 lines performed slightly superior to the single overexpression lines.

Performance of the overexpression lines under herbicide metolachlor stress

In another leaf disc assay, the leaf discs from NtSAT4 transgenic and wild-type greenhouse-grown plants were exposed to herbicide metolachlor. The leaf discs were subjected to 24 µM metolachlor and kept at room temperature under light conditions for 24 hours. The transgenic (single and double overexpression) lines indicated lesser chlorosis as compared to the non-transgenic leaf discs (Figure 2). In terms of percentage damage, the wild-type leaf discs showed 100% necrosis, whereas the single overexpressor lines exhibited 20-60%, and NtSAT4 double overexpressor lines revealed 15-20% necrosis.

 

 

Performance of the NtSAT4 overexpression lines under herbicide atrazine stress

To determine the effect of another xenobiotic on transgenic plants as compared to non-transgenic plants, four-week-old plantlets kept in growth chambers in hydroponic media were subjected to aherbicide known as atrazine. The transgenic (single and double overexpressor) lines and wild-type plants were distributed in three sets, one set was kept as control and subjected to 1⁄4 strength MS media without the herbicide, next set was exposed to 24 µl atrazine for 24 hours and the last set was exposed to 12 µl of atrazine for 48 hours. The single overexpression lines M193 and P230, and the double NtSAT4overexpression lines containing M193×C264 and M193×P230 exhibited higher tolerance compared to the wild-type plants in terms of lesser wilting (Figure 3). Among the NtSAT4 transgenic lines, M193 (single overexpressor line) and M×C (double overexpressor line) showed lesser wilting to both concentrations of atrazine stress.

 

Response of single and double overexpressionNtSAT4 lines to xenobiotics-induced oxidative stress

Heavy metals and herbicides such as metolachlor and atrazine have very harmful effects on plants and, ultimately, human health. Recently, it has been shown that genetically engineered plants can exhibit more tolerance to these xenobiotics as compared to their respective wild-type plants. These transgenic lines also showed an ability to remove these contaminants from the environment. After uptake from their surroundings via roots and shoots, these herbicides and heavy metals are converted to non-toxic forms through different biochemical pathways. These toxic metabolites chelate with GSH, amino acids, and sugars and are subsequently sequestered in the cell vacuoles (Linacre et al., 2003; Cao et al., 2019; Chandrakar et al., 2020; Asare et al., 2023).

These toxic environmental stimulants result in the production of reactive oxygen species (ROS), such as hydroxyl radicals, H2O2, and superoxide radicals, which are extremely damaging to the cell components and biochemical pathways (Ozougwu, 2016; Juan et al., 2021; O’Kane et al., 1996). The hydrogen peroxide produced triggers the plant’s defense mechanisms, and hence these toxic oxides are sequestered in the vacuole (Smirnoff and Arnaud, 2019; Sachdev et al., 2021; Foyer et al., 1997). The leaf disc assessment in biological sciences is a widely accepted approach in documenting plants responses to reactive oxygen species exposure (Rajab et al., 2020; Liedschulte et al., 2010). To document the antioxidant response of NtSAT4 single and double overexpression lines, their leaf discs were subjected to different xenobiotics. The leaf discs from single and double overexpression lines and wild-type plants were subjected to 14% H2O2 stress for 24 hours at room temperature under 16 hours of light conditions. The leaf discs of all the single overexpression lines exhibited lesser chlorosis as compared to leaf discs from wild-type plants. The double overexpressor lines M193×P230 and M193×C264 also performed significantly better as compared to the wild-type plants. Moreover, both the double overexpressor lines showed distinctly lesser pigment discoloration as compared to their single overexpressor counterparts as well. This better tolerance to ROS may be attributable to the higher potential for GSH production in the double overexpression transgenic lines. GSH is an important antioxidant and protects the cells from the damaging effects of ROS produced during different stresses by sequestrating them from the system (Gill and Tuteja, 2010; Hasanuzzaman and Fujita, 2011; Navari-Izzo et al., 1997).

To determine the response of NtSAT4 single and double overexpressor lines to organic contaminants such as herbicides, they were subjected to metolachlor and atrazine. The leaf discs were exposed to 24 µM metolachlor for 24 hours under light conditions at room temperature. The transgenic lines, both single and double overexpressors, exhibited lesser bleaching of pigments as compared to wild-type leaf samples. One-month-old NtSAT4 transgenic lines and wild-type plants were also subjected to another herbicide known as atrazine. Two concentrations of atrazine 12 µM for 48 hours and 24 µM for 24 hours were applied to the plants in hydroponic media. Both the single overexpressor lines (M193 and P230) and double overexpressor lines demonstrated significantly lesser wilting compared to the wild-type plants. GSH acts as a substrate of PCs biosynthesis, and PCs are involved in defense mechanisms of detoxification of toxic compounds (Liedschulte et al., 2010; Zhu et al., 1999). N. tabacum StGCL-GS overexpressors lines showed improved tolerance to herbicide paraquat-induced oxidative stress compared to the wild-type leaf discs (Liedschulte et al., 2010). Transgenic plants exhibited better tolerance to herbicides when expressed with P450 genes (Gorinova et al., 2005; Zheng et al., 2022; Ohkawa et al., 1998). Transformed Oryza sativa overexpressing CYP1A1, CYP2B, or CYP2C19 genes detoxify different herbicides (Kawahigashi et al., 2002). Whereas the pIKBACH transgenic plants detoxify many herbicides as these transgenics overexpress all three P450s (Kawahigashi et al., 2003, 2006). The leaf disc experiments indicated that NtSAT4 single and double overexpressing B. napus lines can potentially be used for phytoremediation of xenobiotics, including hydrogen peroxide, heavy metals, and herbicides from the environment.

Conclusions and Recommendations

The overexpression of NtSAT4 in single and double-overexpressing B. napus lineshas resulted in enhanced stress tolerance against H2O2, metolachlor, and atrazine-induced oxidative stresses. In terms of H2O2-inducedoxidative stresses, the double overexpression lines performed better compared to the single overexpression lines. The transgenic lines may be tested against a combination of heavy metals and oxidative stresses under different growth conditions.

Acknowledgments

The authors are thankful to the Higher Education Commission (HEC)-Pakistan for funding via Grant No. 20-2015/NRPU/R&D.

Novelty Statement

Unlike the previous approach for enhancing in planta GSH contents, we have addressed the issue of feedback inhibition by overexpressing the feedback-insensitive isoforms of SAT from tobacco, i.e., NtSAT4, to overproduce in planta GSH. Moreover, since Cys biosynthesis takes place in three subcellular compartments, therefore in this research, the expression of NtSAT4 was targeted to the mitochondria, plastids, and cytosol of the cell.

Authors contribution

Fariha Qahar: Conducted the research and wrote the first draft of the manuscript.

Muhammad Sayyar Khan: Designed the project and experiments.

Conflict of interest

The authors have declared no conflict of interest.

References

Angelova, V., R. Ivanova, J. Todorov and K. Ivanov. 2017. Potential of rapeseed (Brassica napus L.) for phytoremediation of soils contaminated with heavy metals. J. Environ. Prot. Ecol., 18(2): 468-478.

Asare, M.O., J. Száková and P. Tlustoš. 2023. The fate of secondary metabolites in plants growing on Cd-, As-, and Pb-contaminated soils. A comprehensive review. Environ. Sci. Pollut. Res., 30(5): 11378-11398. https://doi.org/10.1007/s11356-022-24776-x

Bartha, B., C. Huber and P. Schröder. 2014. Uptake and metabolism of diclofenac in Typha latifolia–how plants cope with human pharmaceutical pollution. Plant Sci., 227: 12-20. https://doi.org/10.1016/j.plantsci.2014.06.001

Bártíková, H., L. Skálová, L. Stuchlíková, I. Vokřál, T. Vaněk and R. Podlipná. 2015. Xenobiotic-metabolizing enzymes in plants and their role in uptake and biotransformation of veterinary drugs in the environment. Drug Metab. Rev., 47(3): 374-387.

Cao, Y.Y., C.D. Qi, S. Li, Z. Wang, X. Wang, J. Wang, S. Ren, X. Li, N. Zhang and Y.D. Guo. 2019. Melatonin alleviates copper toxicity via improving copper sequestration and ROS scavenging in cucumber. Plant Cell Physiol., 60(3): 562-574. https://doi.org/10.1093/pcp/pcy226

Chandrakar, V., B. Yadu, R. Xalxo, M. Kumar and S. Keshavkant. 2020. Mechanisms of plant adaptation and tolerance to metal/metalloid toxicity. Plant ecophysiology and adaptation under climate change: Mechanisms and perspectives II: Mechanisms of adaptation and stress amelioration. pp. 107-135. https://doi.org/10.1007/978-981-15-2172-0_6

Dasari, S., M. Ganjayi, L. Oruganti, H. Balaji and B. Meriga. 2017. Glutathione S-transferases detoxify endogenous and exogenous toxic agents. Mini review. J. Dairy, Vet. Anim. Res., 5(5): 00154. https://doi.org/10.15406/jdvar.2017.05.00154

Dixon, D.P., A. Lapthorn and R. Edwards. 2002. Plant glutathione transferases. Genome Biol., 3(3): 1-10. https://doi.org/10.1186/gb-2002-3-3-reviews3004

Flocco, C.G., S.D. Lindblom, A. Elizabeth and P. Smits. 2004. Overexpression of enzymes involved in glutathione synthesis enhances tolerance to organic pollutants in Brassica juncea. Int. J. Phytoremed., 6(4): 289-304. https://doi.org/10.1080/16226510490888811

Foyer, C.H. and G. Noctor. 2005. Redox homeostasis and antioxidant signaling: A metabolic interface between stress perception and physiological responses. Plant Cell, 17(7): 1866-1875. https://doi.org/10.1105/tpc.105.033589

Foyer, C.H., H. Lopez-Delgado, J.F. Dat and I.M. Scott. 1997. Hydrogen peroxide-and glutathione-associated mechanisms of acclamatory stress tolerance and signaling. Physiol. Plant. 100(2): 241-254. https://doi.org/10.1111/j.1399-3054.1997.tb04780.x

Gill, S.S. and N. Tuteja. 2010. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol. Biochem., 48(12): 909-930. https://doi.org/10.1016/j.plaphy.2010.08.016

Gorinova, N., M. Nedkovska and A. Atanassov. 2005. Cytochrome p450 monooxygenases as a tool for metabolizing of herbicides in plants. Biotechnol. Biotechnol. Equip., 19(3): 105-115. https://doi.org/10.1080/13102818.2005.10817290

Hasanuzzaman, M. and M. Fujita. 2011. Selenium pretreatment upregulates the antioxidant defense and methylglyoxal detoxification system and confers enhanced tolerance to drought stress in rapeseed seedlings. Biol. Trace Element. Res., 143: 1758-1776. https://doi.org/10.1007/s12011-011-8998-9

Juan, C.A., J.M. Pérez de la Lastra, F.J. Plou and E. Pérez-Lebeña. 2021. The chemistry of reactive oxygen species (ROS) revisited: Outlining their role in biological macromolecules (DNA, lipids and proteins) and induced pathologies. Int. J. Mol. Sci., 22(9): 4642. https://doi.org/10.3390/ijms22094642

Kawahigashi, H., S. Hirose, E. Hayashi, H. Ohkawa and Y. Ohkawa. 2002. Phytotoxicity and metabolism of ethofumesate in transgenic rice plants expressing the human CYP2B6 gene. Pestic. Biochem. Physiol. 74(3): 139-147. https://doi.org/10.1016/S0048-3575(02)00153-0

Kawahigashi, H., S. Hirose, H. Ohkawa and Y. Ohkawa. 2003. Transgenic rice plants expressing human CYP1A1 exude herbicide metabolites from their roots. Plant Sci., 165(2): 373-381. https://doi.org/10.1016/S0168-9452(03)00197-3

Kawahigashi, H., S. Hirose, H. Ohkawa and Y. Ohkawa. 2006. Phytoremediation of the herbicides atrazine and metolachlor by transgenic rice plants expressing human CYP1A1, CYP2B6, and CYP2C19. J. Agric. Food Chem., 54(8): 2985-2991. https://doi.org/10.1021/jf052610u

Khan, A.D., M.S. Khan, S.A.K. Bangash, K. Naeem, A. Jalal and M. Tayyab. 2021. Cadmium and arsenic provoke mostly distinct but partly overlapping responses in Brassica juncea. Crop Pasture Sci., https://doi.org/10.1071/CP21157

Liedschulte, V., A. Wachter, A. Zhigang and T. Rausch. 2010. Exploiting plants for glutathione (GSH) production: uncoupling GSH synthesis from cellular controls results in unprecedented GSH accumulation. Plant Biotechnol. J., 8(7): 807-820. https://doi.org/10.1111/j.1467-7652.2010.00510.x

Linacre, N.A., S.N. Whiting, A.J. Baker, J. Scott Angle and P.K. Ades. 2003. Transgenics and phytoremediation: the need for an integrated risk assessment, management, and communication strategy. Int. J. Phytoremed., 5(2): 181-185. https://doi.org/10.1080/713610179

Marrs, K.A., 1996. The functions and regulation of glutathione s-transferases in plants. Ann. Rev. Plant Biol., 47(1): 127-158. https://doi.org/10.1146/annurev.arplant.47.1.127

Mullineaux, P.M. and T. Rausch. 2005. Glutathione, photosynthesis and the redox regulation of stress-responsive gene expression. Photosynth. Res., 86(3): 459-474. https://doi.org/10.1007/s11120-005-8811-8

Nakka, S., A.S. Godar, C.R. Thompson, D.E. Peterson and M. Jugulam. 2017. Rapid detoxification via glutathione S‐transferase (GST) conjugation confers a high level of atrazine resistance in Palmer amaranth (Amaranthus palmeri). Pest Manage. Sci., 73(11): 2236-2243. https://doi.org/10.1002/ps.4615

Navari‐Izzo, F., S. Meneguzzo, B. Loggini, C. Vazzana and C. Sgherri. 1997. The role of the glutathione system during dehydration of Boea hygroscopica. Physiol. Plant., 99(1): 23-30. https://doi.org/10.1034/j.1399-3054.1997.990104.x

Ohkawa, H., H. Imaishi, N. Shiota, T. Yamada, H. Inui and Y. Ohkawa. 1998. Molecular mechanisms of herbicide resistance with special emphasis on cytochrome P450 monooxygenases. Plant Biotechnol., 15: 173-176. https://doi.org/10.5511/plantbiotechnology.15.173

O’Kane, D., V. Gill, P. Boyd and R. Burdon. 1996. Chilling, oxidative stress and antioxidant responses in Arabidopsis thaliana callus. Planta, 198(3): 371-377. https://doi.org/10.1007/BF00620053

Ozougwu, J.C., 2016. The role of reactive oxygen species and antioxidants in oxidative stress. Int. J. Res., 1(8).

Pilon-Smits, E. and M. Pilon. 2002. Phytoremediation of metals using transgenic plants. Crit. Rev. Plant Sci., 21(5): 439-456. https://doi.org/10.1080/0735-260291044313

Pilon-Smits, E.A., S. Hwang, C.M. Lytle, Y. Zhu, J.C. Tai, R.C. Bravo, Y. Chen, T. Leustek and N. Terry. 1999. Overexpression of ATP sulfurylase in Indian mustard leads to increased selenate uptake, reduction, and tolerance. Plant Physiol., 119(1): 123-132. https://doi.org/10.1104/pp.119.1.123

Rajab, H., M.S. Khan, M. Wirtz, M. Malagoli, F. Qahar and R. Hell. 2020. Sulfur metabolic engineering enhances cadmium stress tolerance and root to shoot iron translocation in Brassica napus L. Plant Physiol. Biochem., 152: 32-43. https://doi.org/10.1016/j.plaphy.2020.04.017

Rajab, H., M.S. Khan, S.H. Shah and S.M.A. Shah. 2019. Genetic transformation of tobacco serine acetyltransferase 4 (NtSAT4) gene in Brassica napus L. Sarhad J. Agric., 35(4): 1224-1233. https://doi.org/10.17582/journal.sja/2019/35.4.1224.1233

Rogers, S.O. and A.J. Bendich. 1989. Extraction of DNA from plant tissues. Plant Mol. Biol. Manual, pp. 73-83. https://doi.org/10.1007/978-94-009-0951-9_6

Sachdev, S., S.A. Ansari, M.I. Ansari, M. Fujita and M. Hasanuzzaman. 2021. Abiotic stress and reactive oxygen species: Generation, signaling, and defense mechanisms. Antioxidants, 10(2): 277. https://doi.org/10.3390/antiox10020277

Smirnoff, N. and D. Arnaud. 2019. Hydrogen peroxide metabolism and functions in plants. New Phytol., 221(3): 1197-1214. https://doi.org/10.1111/nph.15488

Syed, S., M.S. Khan, A. Jalal and Z. Iqbal. 2023. Inoculation of Serratia sp. under cadmium stress significantly affected Brassica juncea growth attributes and glutathione levels. Pak. J. Agric. Sci., 60(1).

Syguda, A., A. Gielnik, A. Borkowski, M. Woźniak-Karczewska, A. Parus, A. Piechalak, A. Olejnik, R. Marecik, L. Lawniczak and L. Chrzanowski. 2018. Esterquat herbicidal ionic liquids (HILs) with two different herbicides: Evaluation of activity and phytotoxicity. New J. Chem., 42(12): 9819-9827. https://doi.org/10.1039/C8NJ01239C

Tong, Y.P., R. Kneer and Y.G. Zhu. 2004. Vacuolar compartmentalization: A second-generation approach to engineering plants for phytoremediation. Trends Plant Sci., 9(1): 7-9. https://doi.org/10.1016/j.tplants.2003.11.009

Van Ginneken, L., E. Meers, R. Guisson, A. Ruttens, K. Elst, F.M. Tack, J. Vangronsveld, L. Diels and W. Dejonghe. 2007. Phytoremediation for heavy metal‐contaminated soils combined with bioenergy production. J. Environ. Eng. Lands. Manage., 15(4): 227-236. https://doi.org/10.3846/16486897.2007.9636935

Verbruggen, N., C. Hermans and H. Schat. 2009. Molecular mechanisms of metal hyperaccumulation in plants. New Phytol., 181(4): 759-776. https://doi.org/10.1111/j.1469-8137.2008.02748.x

Vontas, J.G., G.J. Small and J. Hemingway. 2001. Glutathione S-transferases as antioxidant defense agents confer pyrethroid resistance in Nilaparvata lugens. Biochem. J., 357(1): 65-72. https://doi.org/10.1042/bj3570065

Wirtz, M. and R. Hell. 2003. Production of cysteine for bacterial and plant biotechnology: application of cysteine feedback-insensitive isoforms of serine acetyltransferase. Amino Acids, 24(1): 195-203. https://doi.org/10.1007/s00726-002-0313-9

Xia, Z., Z. Xu, Y. Wei and M. Wang. 2018. Overexpression of the maize sulfite oxidase increases sulfate and GSH levels and enhances drought tolerance in transgenic tobacco. Front. Plant Sci., 9: 298. https://doi.org/10.3389/fpls.2018.00298

Xiang, X., Y. Wu, J. Planta, J. Messing and T. Leustek. 2018. Overexpression of serine acetyltransferase in maize leaves increases seed-specific methionine-rich zeins. Plant Biotechnol. J., 16(5): 1057-1067. https://doi.org/10.1111/pbi.12851

Ye, F., Y. Zhai, K.-L. Guo, Y.-X. Liu, N. Li, S. Gao, L.-X. Zhao and Y. Fu. 2019. Safeners improve maize tolerance under herbicide toxicity stress by increasing the activity of enzymes in vivo. J. Agric. Food Chem., 67(42): 11568-11576. https://doi.org/10.1021/acs.jafc.9b03587

Zhang, F., X. Xiao, G. Yan, J. Hu, X. Cheng, L. Li, H. Li and X. Wu. 2018. Association mapping of cadmium-tolerant QTLs in Brassica napus L. and insight into their contributions to phytoremediation. Environ. Exp. Bot., 155: 420-428. https://doi.org/10.1016/j.envexpbot.2018.07.014

Zheng, T., X. Yu, Y. Sun, Q. Zhang, X. Zhang, M. Tang, C. Lin and Z. Shen. 2022. Expression of a cytochrome P450 gene from bermuda grass Cynodon dactylon in soybean confers tolerance to multiple herbicides. Plants, 11(7): 949. https://doi.org/10.3390/plants11070949

Zhu, Y.L., E.A. Pilon-Smits, L. Jouanin and N. Terry. 1999. Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiolo., 119(1): 73-80. https://doi.org/10.1104/pp.119.1.73