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254 P. Kotrba

12.4.3 Plants Engineered for Enhanced Metal Ligand Production

As explained in Sect. 12.3.2, PCs are synthesized by phytochelatin synthase (PCS)
enzyme from glutathione or its homologues. Although the overexpression of
intrinsic AtPCS1 in A. thaliana resulted in 25 times higher levels of the transcript
and up to a twofold increased production of PCs, AtPCS1-transformed lines para-
doxically showed hypersensitivity to Cd and Zn (Lee et al. 2003). Such a phenotype
could be attributed to a rapid nonphysiological decrease in the intracellular GSH
pool due to the synthesis of supraoptimal levels of PCs. In contrast, expression of
TaPCS1 encoding PCS from wheat in shrub tobacco N. glauca substantially
increased its tolerance of transgenic plants to Pb and Cd (Gisbert et al. 2003).
Moreover, TaPCS1 N. glauca accumulated, respectively, 6, 3.3, 4.8, 18.2, and 2.6
times more Pb, Cd, Zn, Cu, and Ni from industrial soil than did the WT plant
(Martı ´nez et al. 2006). Also certain lines of transgenic aspen (Populus tremula
tremuloides cv. Etrepole) expressing the same TaPCS1 gene showed better growth
than the parental plant and accumulated more Pb from mining soil (Couselo et al.
2010). Transgenic plants also showed higher biomass and by 70 % higher Pb levels
than WT in exposures to up to 1.5 mM Pb concentrations in the hydroponic growth
media. Since GSH molecule is involved in many aspects of the plant response to
heavy metal ions, many efforts have been directed towards engineering its biosyn-
thesis pathway. Attempts to increase GSH production in plants, by the implemen-
tation of enzyme activities involved in its synthesis and recycling, have aimed
mainly at the promotion of increased PC levels under metal stress. GSH is
synthesized from its constituent amino acids in two sequential, ATP-dependent
enzymatic reactions catalyzed by γ-glutamylcysteine synthetase (γ-ECS) and glu-
tathione synthetase (GS), respectively. Constitutive production of the E. coli gshI
gene and targeting of encoded γ-ECS in plastids in B. juncea increased GSH levels
in hydroponically grown transformants threefolds (Zhu et al. 1999b). Consequently,
the PC2 levels of shoots and PC2, PC3, and PC4 levels in roots of γ-ECS B. juncea
stressed at 200 μM Cd increased, compared to WT plants, by 30 %, which resulted
in higher Cd tolerance. In 50 μM Cd exposures, overexpression of gshI enhanced
the natural capacity of B. juncea to accumulate Cd in shoots nearly twofold. The
effect of cytosolic overexpression of gshII encoding GS on Cd tolerance and
accumulation from a hydroponic solution was less pronounced, although
transformed plants stressed at 100 μM Cd had 2.3 and 1.7 times higher PC2
compared to the WT control (Zhu et al. 1999a). Both gshI and gshII were later
demonstrated to enhance the capacity of B. juncea to accumulate from hydroponic
solutions and tolerate a variety of metals and metalloids (particularly As, Cd, and
Cr) as well as mixed-metal(loid) combinations (Reisinger et al. 2008). Bennett et al.
(2003) further demonstrated that overexpression of gshI and gshII can indeed
promote phytoextraction with B. juncea in soils from a mine tailings: plants
expressing gshI accumulated in shoots, respectively, 3.5, 2.0, 1.54, and 2.0 times
higher levels of Pb, Zn, Cd, and Cu than the WT plants and those expressing ghsII
contained in shoots 1.5 times higher concentrations of Cd and Zn than the