Poster: Heavy metals & phytoremediation

Abs # 50: Functional characterization of Arabidopsis metallothioneins: expression in yeast and analysis of T-DNA insertion knockouts

Presenter:	Guo, Woei-Jiun , wguo@purdue.edu
Authors	Guo, Woei-Jiun  (A)   Meetam, Metha  (A)   Goldsbrough, Peter B (A)
Affiliations:	(A): Purdue University

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins that participate in heavy metal homeostasis and provide oxidative stress protection in animals and yeast. To examine the functions of plant MTs, Arabidopsis MTs have been expressed in yeast where they complemented mutants that were hypersensitive to Cu or Zn. These results indicate that all Arabidopsis MTs are functional metal binding proteins and can increase the capacity for heavy metal accumulation. A similar strategy is used to determine if plant MTs can scavenge reactive oxygen species in vivo. To investigate the functions of MTs in plants, Arabidopsis mutants with insertions in all MT genes have been identified. For mt1a, mt2a and mt2b, MT RNA expression has been eliminated in the mutants. Interestingly, RNA expression of MT1c, is increased 7-fold in the mt1a-2, suggesting an interaction between MT1a and MT1c. Characterization of mutants deficient in just one MT gene show no obvious phenotypes suggesting there is functional redundancy between MT genes. These results are consistent with results from other Arabidopsis plants (antisense and RNAi) with reduced expression of MT1a, MT2b or MT3. To dissect the functional redundancy, we are developing and characterizing mutants that are defect in 2, 3 or 4 MT genes. Transgenic Arabidopsis expresing a MT2b-GFP fusion protein have been produced to examine the localization and potential transport of MTs in plants. Localization of GFP expression indicates that MT2b proteins function mainly in the phloem. Grafting experiments and analysis of MT2b-GFP will be used to demonstrate if MT2b is involved in Cu transport in the phloem. Taken together, these approaches will provide fundamental insights into the function of Arabidopsis MT genes.