22001:

Towards a molecular understanding of the mechanism of Ni hyperaccumulation in Thlaspi.

Authors:	Salt, David, E.(A)
Affiliations:	(A): Northern Arizona University
Presenter:	Salt, David E., david.salt@nau.edu

Thlaspi goesingense Hálácsy, a plant found on serpentine soils in Austria has been observed to contain up to 1% of its shoot dry biomass as Ni. A comparison of root exudates, Ni transport rates and Ni tolerance in hydroponically cultured T. goesingense and the non-accumulator Thlaspi arvense has revealed that the primary mechanism underlying the Ni hyperaccumulation phenotype in T. goesingense is an enhanced cellular Ni tolerance mechanism. In order to understand the mechanisms involved in this enhanced Ni tolerance we have investigated both the in planta speciation and the cellular localization of Ni. Using X-ray absorption spectroscopy we have determined what ligands are involved in binding Ni within these two Thlaspi species. In the hyperaccumulator the majority of intracellular shoot Ni appears to remain complexed with citrate where as in the non-accumulator histidine-like ligands appear to play an increasing role as Ni accumulates to toxic concentrations. We have also demonstrated that T. goesingense has an enhanced ability to accumulate Ni in the vacuoles of shoot cells, when compared to T. arvense. Both the chemical speciation and localization data therefore support the conclusion that Ni tolerance in shoots of T. goesingense is mediated by the efficient accumulation of Ni-citrate within the vacuole. However, the question remains as to what is the molecular mechanism of this enhanced accumulation. To address this we are investigating the role of both putative "shuttle-molecules" involved in carrying Ni across the cytoplasm to the vacuolar, and tonoplast transport proteins involved in pumping Ni into the vacuole. Recent reports have suggested that free histidine plays a role in Ni hyperaccumulation in certain Alyssum species by chelating Ni in planta. By cloning three key histidine biosynthetic genes from T. goesingense, and measuring the concentrations of free histidine in roots, shoots and xylem sap of both accumulator and non-accumulator Thlaspi species we have been able to demonstrate that free histidine does not play a key role in Ni hyperaccumulation in T. goesingense. At present we are screening a T. goesingense cDNA library for cDNA's with the capacity to confer Ni tolerance in E. coli and yeast. We are also using Differential Display RT-PCR (DD RT-PCR) to identify genes in T. goesingense which are regulated by Ni. With these different approaches we hope to shed light on the molecular basis of Ni hyperaccumulation in T. goesingense.