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

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

Muhammad Sayyar Khan: Designed the project and experiments.

The authors have declared no conflict of interest.