Overexpression of bacterial γ-glutamylcysteine synthetase increases toxic metal(loid)s tolerance and accumulation in Crambe abyssinica

Abstract

Transgenic Crambe abyssinica lines overexpressing γ-ECS significantly enhance tolerance to and accumulation of toxic metal(loid)s, improving phytoremediation potential and offering an effective solution for contaminated soil management. Phytoremediation is an attractive environmental-friendly technology to remove metal(loid)s from contaminated soils and water. However, tolerance to toxic metals in plants is a critical limiting factor. Transgenic Crambe abyssinica lines were developed that overexpress the bacterial γ-glutamylcysteine synthetase (γ-ECS) gene to increase the levels of non-protein thiol peptides such as γ-glutamylcysteine (γ-EC), glutathione (GSH), and phytochelatins (PCs) that mediate metal(loid)s detoxification. The present study investigated the effect of γ-ECS overexpression on the tolerance to and accumulation of toxic As, Cd, Pb, Hg, and Cr supplied individually or as a mixture of metals. Compared to wild-type plants, γ-ECS transgenics (γ-ECS1-8 and γ-ECS16-5) exhibited a significantly higher capacity to tolerate and accumulate these elements in aboveground tissues, i.e., 76-154% As, 200-254% Cd, 37-48% Hg, 26-69% Pb, and 39-46% Cr, when supplied individually. This is attributable to enhanced production of GSH (82-159% and 75-87%) and PC2 (27-33% and 37-65%) as compared to WT plants under AsV and Cd exposure, respectively. The levels of Cys and γ-EC were also increased by 56-67% and 450-794% in the overexpression lines compared to WT plants under non-stress conditions, respectively. This likely enhanced the metabolic pathway associated with GSH biosynthesis, leading to the ultimate synthesis of PCs, which detoxify toxic metal(loid)s through chelation. These findings demonstrate that γ-ECS overexpressing Crambe lines can be used for the enhanced phytoremediation of toxic metals and metalloids from contaminated soils.

Keywords: Crambe abyssinica; Glutamylcysteine synthetase; Heavy metals; Metal tolerance; Overexpression; Phytoremediation.

Figure 1.

Transformation and expression analysis of γECS in Crambe abyssinica. (a) Structure of ACT2pt::γ-ECS construct in plasmid pCAMBIA1300 (b & c) Confirmation of expression level in T₂ homozygous seedlings of two γ-ECS overexpression lines (1–8 and 16–5) in shoots and roots. Data are mean ± SE, n = 3

Figure 2.

Levels of thiol-containing peptides, (a) cysteine (Cys), (b) gamma-glutamylcysteine (γ-EC), (c) glutathione (GSH), and (d) phytochelatins (PCs) in shoots of wild-type (WT) and γ-ECS OE lines. Overexpression lines showed increased levels of Cys, γ-EC, GSH, and PC peptides compared to WT plants when exposed to no metal/metalloids or 250 µM arsenate (As) or 150 µM CdCl₂ (Cd)) for three weeks. The data shown are mean±SD, n=3. Significant differences between the γ-ECS and WT plants treated under the same conditions are indicated by asterisks * (p<0.05) and ** (p<0.01).

Figure 3.

Phenotypic images of WT and γ-ECS OE lines on media containing toxic metals/metalloids. (A) Image showing phenotypes of 21-day-old WT and Ca γ-ECS OE lines 1–8, and 16–5 on ½ × MS (control) and ½ × MS + AsV (300 µM), CdCl₂ (200 µM), HgCl₂ (150 µM), PbNO₃ (800 µM), and K₂CrO₄ (600 µM) treatments. (B) Graph represents fresh shoot biomass of WT and Ca γ-ECS OE lines 1–8, and 16–5. Data are mean ± SE, n = 4. The asterisks represent the significant differences between WT and Ca γ-ECS OE lines within treatments (*P < 0.05, and **P < 0.01).

Figure 4.

Phenotypic images of WT and γ-ECS OE lines on media containing a mixture of metals. (A). Image showing phenotypes of 21-day-old WT and γ-ECS OE lines 1–8 and 16–5 on ½ × MS (control) and ½ × MS + mix metals (200µM Arsenate, 100 µM Cd, 100µM Hg, 600 µM Pb and 400 µM Cr treatments. (B). Graph represents fresh shoot biomass of WT and γ-ECS OE lines 1–8 and 16–5. Data are mean ± SE, n = 4. The asterisks represent the significant differences between WT and γ-ECS OE lines within treatments (*P < 0.05, and **P < 0.01).

Figure 5.

Toxic metal and metalloid accumulation in WT and γ-ECS OE lines exposed to different elements (a) AsV (300 µM), (b) CdCl₂ (200 µM), (c) HgCl₂ (150 µM), (d) PbNO₃ (800 µM)and (e) K₂CrO₄, (600 µM),. Values and bars represent means ± SD (n=3). The asterisks indicate the significant differences between WT and γ-ECS OE lines within treatment (*P < 0.05, and **P < 0.01).

Figure 6.

Translocation factors (TF) in WT and γ-ECS OE lines exposed to different toxic elements (a) AsV (300 µM), CdCl₂ (200 µM), HgCl₂ (150 µM), PbNO₃ (800 µM), or K₂CrO₄ (600 µM), separately, (b) mixed metal treatment of AsV (200 µM) + CdCl₂ (100 µM) + HgCl₂ (100 µM) + PbNO₃ (600 µM) + K₂CrO₄ (400 µM).

Figure 7.

Toxic element accumulation in WT and γ-ECS OE lines exposed to a mixture of metal treatments of AsV (200 µM) + CdCl₂ (100 µM) + HgCl₂ (100 µM) + PbNO₃ (600 µM) + K₂CrO₄ (400 µM). (a) As, (b) Cd, (c) Hg, (d) Pb, and (e) Cr. Values and bars represent means ± SD (n=3). The asterisks indicate the significant differences between WT and γ-ECS OE lines within treatment (*P < 0.05, and **P < 0.01).

Figure 8.

A schematic illustration of role of γ-ECS and the phytochelatin biosynthesis pathway for enhancing tolerance to and accumulation of heavy metals, and its contribution to phytoremediation. GS- glutathione synthetase, PCS- phytocheltatin synthase, γ-ECS- gamma glutamylcysteine synthase, γ-EC- gamma glutamylcysteine, GSH- glutathione, PC- phytochelatins, HM- heavy metals, ROS- reactive oxygen species.