Page 262 - Plant-Based Remediation Processes

12 Transgenic Approaches to Enhance Phytoremediation of Heavy Metal-Polluted Soils 255

control WT. The same hold true for hybrid poplar (Populus tremula P. alba),
in which production of E. coli γ-ECS enhanced foliar GSH content 2- to 4-folds
(Arisi et al. 1997). It promoted accumulation of Cd, but not of Zn, particularly in
young leaves of plants grown in soils complemented with 225 mg kg 1 Cd; they
contained 2.5–3.0 higher levels than WT (Koprivova et al. 2002; Bittsa ´nszky et al.
2005). The significance of glutathione reductase (GR) in Cd accumulation and
tolerance was recorded in transgenic B. juncea producing the GR of E. coli in the
cytosol and plastids (Pilon-Smits et al. 2000). Only plastidic heterologous GR,
improving natural enzyme levels 20- to 50-fold, doubled GSH levels in roots.
In contrast to the WT control, the plastidic transformants showed no chlorosis
when treated with 100 μM Cd; however, the shoot Cd accumulation was only a
half of that of control WT plants.
As indicated above, the overproduction of PCs followed by an exhaustion of the
GSH pool in A. thaliana had a negative impact on the ability of transgenes to
tolerate and accumulate Cd (Lee et al. 2003; Li et al. 2004). This phenotype was
converted to the tolerant on expression of yeast GSH1-encoded GS in A. thaliana
lineages producing AsPCS1 of garlic Allium sativum (Guo et al. 2008a). Combina-
tion of both transgenes further enhanced by four times the natural capacity of
A. thaliana to accumulate Cd from media with 30 mg kg 1 Cd. The sulfur
assimilatory mechanism and subsequent production of the antioxidant and PC
precursor GSH in plants is known to be highly induced by heavy metal exposure
(Foyer and Noctor 2005). In the respective pathways, the overall rate of GSH
biosynthesis and the capacity to maintain an elevated GSH pool limited by the
activity of cysteine synthase (O-acetylserine [thiol] lyase, OAS-TL), which
substitutes the acetate of O-acetyl-L-serine (OAS) with sulfide (Barroso et al.
1995; Meyer and Fricker 2002). Indeed, constitutive overexpression of Atcys-3A
encoding intrinsic OAS-TL in A. thaliana increased intracellular cysteine and GSH
levels, allowing transgenes to survive at 400 μM Cd stress (Domı ´nguez-Solı ´s et al.
2004). Over a 14-day period, OAS-TL Arabidopsis accumulated 72 % more metal
than WT control plants from a medium containing 250 μM Cd, the highest Cd
content being detected in the trichomes. Kawashima et al. (2004) reported a
substantial improvement in Cd and Ni tolerance in N. tabacum overproducing
OAS-TL from spinach (Spinacia oleracea). The authors also determined the Cd
accumulation potential of the best performing transgenic line and found that the Cd
concentration was reduced in roots (4 times) and slightly higher (1.4-fold) in shoots
compared to the WT control, indicating the onset of promoted metal translocation.
Moreover, due to highly improved biomass yields on media with 100 μM Cd, shoots
of 3-weeks-old transgenic plants accumulated 2.8 times higher net amount of the
metal than shoots of WT plants.
Improved supply of O-acetyl-L-serine (OAS) to the OAS-TL enzyme has also
been shown as an effective method to increase the rate and yield of GSH synthesis.
OAS synthesis from L-serine and acetyl-CoA is catalyzed by serin-O-
acetyltransferase (SAT). Overproduction of mitochondrial SAT encoded by
TgSATm of tiny wild mustard T. goesingense promoted accumulation of GSH in
leaves of A. thaliana, providing increased tolerance to Ni, Co, Zn, and Cd,