The role of gamma-ECS and GS in PC synthesis.

Zhu et. al. has examined two enzymes in the phytochelatin synthesis pathway: °-glutamyl-Cys synthetase (°-ECS) and glutathione synthetase (GS).  g-ECS plays an important role in glutathione synthesis and is thought to be rate limiting in the absence of heavy metals.  °-ECS is thought to be rate limiting because it is feedback regulated by glutathione and dependent on the availability of Cys .  In support of this hypothesis, Arisi et. al. demonstrated that overexpression of an E. coli gshI gene (°-ECS) did increase glutathione levels .

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g-ECS

Rationale:  Overexpression of g-ECS would increase the amount of g-glutamylcysteine, increasing the amount of glutathione, which will increase the amount of phytochelatins.  Phytochelatins are known to bind heavy metals and confer tolerance for plants.

Method:  The E. coli gene, gshI, encoding the g-ECS enzyme was overexpressed in Brassica juncea (Indian Mustard).

Results:  g-ECS plant growth was less inhibited by Cd, and when grown at 0.05mM external Cd, the shoot Cd concentrations were up to 90% higher than in WT plants.  Zhu et. al. concluded that g-ECS expressing plants exhibited increase tolerance and accumulation of Cd as a result of enhanced phytochelatin production.  Additionally, their results supported the view that g-ECS is rate limiting in glutathione synthesis of unstressed plants because glutathione levels increased in g-ECS plants in the absence of Cd stress.

Rationale:  Under Cd stress, PCS quickly uses the supply of glutathione and GS becomes rate limiting.  Therefore, by overexpressing GS, more glutathione will be produced.

Method:  Several plant lines were constructed overexpressing gshII (E. coli GS) in Brassica juncea and analyzed by PCR, western blot analysis, and tolerance to cadmium.

Results:  The transgenic Indian Mustard demonstrated improved Cd2+ accumulation and tolerance in seedlings and mature plants.  Cadmium treated plants had higher levels of glutathione, phytochelatins, thiols, sulfur, and calcium.  Zhu et. al. concluded "that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of glutathione and phytochelatins, and that the level of glutathione and/or phytochelatins determine the plant’s capacity accumulate and tolerate Cd."

• Clemens et. al. pursued identifying the genes involved in Cd2+ resistance, particularly, phytochelatin synthases.  Saccharomyces cerevisiae was transformed with wheat root library cDNA restricting fragments < 1.5 Kb that may include small metal binding peptides such as metallothioneins.  The yeast expression system was then grown on Cd2+ contaminated medium and resistant colonies were selected and grown to saturation.  Restriction analysis revealed a single DNA clone, TaPCS1: Triticum aestivum phytochelatin synthase.  Further sequence analysis revealed homologues in A. thaliana, S. pombe,  and C. elegans.  These clones also conferred Cd2+ tolerance in yeast.  Clemens further demonstrated that the Cd2+ was correlated with phytochelatin synthesis for each of the clones.  Specifically, S. pombe knockout strains lacked PCS activity but PC2 was clearly detected in control cells.  RT-PCR revealed that TaPCS1 is found in the roots and the shoots of seedlings.

• Conclusions:   Clemens concluded that they i) isolated a new family of metal tolerance genes from several different organisms.  ii) TaPCS1 was sufficient for PC synthesis from glutathione in an organism that does not contain a PCS homologue suggesting a role in PC synthesis.  Finally, Clemens concluded that iii) PCs represent a significant cyotsolic buffer for metal ions in organisms and iv) provided molecular evidence that phytochelatins play a crucial role in metal tolerance.