Another important aspect of PC-mediated tolerance of plants to heavy metals is probably the effective transportation of the metal to vacuoles for storage in which they could be playing an important role. Arguments in favor of this come from several observations. Vogeli-Lange and Wagner  isolated mesophyll protoplast from tobacco exposed to Cd and showed that the vacuoles contained 110 ± 8% of the protoplast Cd and 104 ± 8% of the protoplast PCs. These workers envisioned the synthesis of PCs in cytosol and transfer of Cd and the peptides, perhaps as complex, across the tonoplast into the vacuole, where the metal is chelated by the peptides and organic acids.
Working on tomato cells, Gupta and Goldsbrough  observed the highest level of PCs after 4 days of their exposure to Cd, which coincided with the peak of cellular Cd concentration (0.6 mM). At this time, there was an eightfold molar excess of PC over Cd. However, the PCs could not be detected after 12 days and the cellular concentration of Cd was still 0.2 mM (the intracellular concentration of Cd decreased as a result of increase in the cell mass). This led them to suggest that PCs possibly function as transport carriers for Cd into the vacuole, where the acidic pH favors dissociation of the Cd-PC complexes, followed by breakdown of the PCs and possible sequestration of the metal in some other form, in agreement with the model proposed by Vogeli-Lange and Wagner .
Later, while working on Cd-tolerant and Cd-sensitive plants of Silene vulgaris, De Knecht et al.  observed that, in response to a range of Cd concentrations, the root tips of Cd-tolerant plants exhibited a lower rate of PCs production accompanied by a lower rate of larger chain PC synthesis than those of Cd-sensitive plants, although both the plants (root tips) accumulated nearly similar levels of Cd at a particular metal-exposure concentration. Second, the tolerant plants reached the same PC concentration as the sensitive plants only after exposure to high Cd concentrations, and at an equal PC concentration the composition of PC and the amount of sulfide incorporated per unit PC-thiol were the same in both the populations.
The authors concluded that the lower concentration of PCs in the Cd-tolerant plants than in the Cd-sensitive plants could be because of greater transport of Cd-PC complexes in vacuoles in the former and, as suggested by Vogeli-Lange and Wagner  and Gupta and Goldsbrough , the PC-Cd complexes might be getting dissociated in the vacuole because of its acidic pH, followed by breakdown of the PCs or their reshuttling into the cytoplasm. Thus, the observed lower PC concentration in Cd-tolerant plants might be a result of a lower Cd concentration in the cytoplasm caused by (1) a faster transport of the metal into the vacuole when compared to that in the Cd-sensitive plants; and (2) return of the dissociated PCs (in the vacuole) into the cytoplasm, obviating the need of their fresh synthesis for the additional Cd uptake.
Because the enzymes involved in PC synthesis are present in cytoplasm but PCs are also found in the vacuole, a transport mechanism must be involved, and an insight into this comes from the work on S. pombe. A Cd-hypersensitive mutant, deficient in producing HMW complex, was observed . This was found to be as a result of mutation within the hmtl (heavy metal tolerance 1) gene encoding an ATP-binding cassette (ABC)-type protein associated with vacuolar membrane . ABC-type proteins represent one of the largest known families of membrane transporters. They can mediate tolerance to a wide diversity of cytosolic agents. The presence of HMT1 protein in the vacuolar membrane suggests the possibility of an ABC-type transporter-mediated resistance to Cd, by its sequestration in the vacuole .
The yeast hmtl- mutant harboring hmtl-expressing multicopy plasmid (pDH35) exhibited enhanced resistance to Cd compared to the wild-strain (hmtl- mutant) and accumulated more Cd with HMW complex formation . The vacuolar vesicle derived from the hmtl- mutant complemented with hmtl cDNA (hmtl-/pDH35), i.e., HMT1 hyperproducer exhibited ATP-dependent uptake of LMW apophytochelatin and LMW-Cd complexes, but that from the hmtl- mutant did not show any such activity. HMW-Cd complex was not an effective substrate for the transporter proteins. The vacuolar uptake of Cd2+, which was ATP dependent, was also observed, but was not attributable to HMT1. The electrochemical potential generated by vacuolar ATPase did not drive transport of peptides or complexes.
The observation of Ortiz et al.  is also supported by work on oat tonoplast vesicles [128,129]. Tonoplast vesicles from oat roots have a Cd2+/H+ antiporter . The vesicles also show MgATP-dependent transport of PCs and Cd-PC complex , and the peptide transport is not driven by electrochemical potential generated by the vacuolar ATPase. Based on the information available, Rauser  proposed a model, somewhat similar to that proposed by Ortiz et al. , for the transport of Cd and Cd-binding complexes across the tonoplast. PCs synthesized in the cytosol combine with Cd to form LMW complex that is moved across the tonoplast by ABC-type transporters. Apo-PCs are also transported by them. The energy required for the transport is derived from ATP.
Once inside the vacuole, more Cd, transported by Cd2+/H+ antiporter, is added to the LMW, along with Apo-PCs and sulfide complexes, to produce HMW complexes. Genetic and biochemical analyses suggest that the formation of sulfide moiety in the HMW PC-Cd-S2- complex involves purine metabolism, which serves as the source of sulfide [130,131]. The sulfide-rich HMW complex is more stable in the acidic environment of the vacuole and has a higher Cd-binding capacity than the LMW complex. The LMW complex functions as a cytosolic carrier and the vacuolar HMW complex is the major storage form of cellular Cd. Whether LMW and HMW complexes in plants are compartmentalized as depicted in the model and are of the same peptide composition, however, awaits direct evaluation. Nevertheless, the studies [127,128] do indicate a central role of vacuole in sequestration and detoxification of Cd, and maybe heavy metals in general, and that tolerance to metals could also be due to increased ability of plants to transport them into the vacuole (see Figure 15.2 in Chapter 15).
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