Date of Award
Doctor of Philosophy
Accumulation of a given metal in plants depends on a delicate and precise balance of various biological processes. Some plants have developed strategies that allow them to tolerate heavy metals in extreme conditions without suffering toxicity. This research focuses on the characterization of two metal transporters, a member of the P1B-type (ATPase) transporter family (HMA3) and a member the Natural Resistance Associated Macrophage Protein (NRAMP) Nramp1 family. These transporters have proposed roles in ion homeostasis and mineral nutrition. The work here sought to determine if these transporters might have characteristics that suggest a role in heavy metal transport and tolerance in metal hyperaccumulating plants. These proteins are very well conserved among different taxa. Nonetheless, as little as a single amino acid change has the potential to modify their capacity to take up non essential metals such as Cd, or Pb, and/or increase affinity for other mineral nutrients. These transporters were cloned from a non-accumulator (Arabidopsis thaliana L) and two ecotypes (Prayon and Ganges) of the hyperaccumulator Noccaea caerulescens (formerly= Thlaspi caerulescens). The full cDNA of an ortholog of either Nramp1 or HMA3 was expressed in yeast in order to provide a heterologous model to elucidate how polymorphisms between the orthologs might translate into functional differences between the protein sequences. A comparison of the HMA3 sequences to each other, or the Nramp1 sequences to each other, demonstrated that major motifs and domains in each protein were highly conserved but that there were numerous single amino acid polymorphisms. Few of these polymorphisms corresponded to positions in a protein that are known to be critical for transporter function. However, metal accumulation, tolerance and cell growth assays showed that the Nramp1 and HMA3 genes from Arabidopsis encoded proteins with the expected broad selectivity for divalent ion transport. In contrast, the genes from the Thlaspi ecotypes encoded proteins that showed more selectivity for ion transport. The Thlaspi ecotypes showed high selectivity for cadmium but the accumulation of other elements differed between the Thlaspi orthologs. These results suggest that the polymorphisms present in the Thlaspi sequences have produced differences in the transport characteristics of both the HMA3 and the Nramp1 transporters.
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