Subcellular Compartmentation And Heavy Metal Resistance Of Plant Metallothionein Type 2

In eukaryotic organisms, several specialized peptides are involved in heavy metal ion homeostasis and detoxification. To achieve these functions, higher plants make use of two types of peptides: the protein family of gene-encoded metallothioneins and the phytochelatins, which are enzymatically formed from glutathione.Metallothionein is a low molecular weight and cystein rich protein, which has constantly been found in mammalian plants and microorganisms. It has been understood so far that the metallothioneins play important roles in heavy metals chelating and ions transformation essential for cellular metabolism. In this study, we applied the plant metallothionein type 2 gene isolated from Brassica Juncea as a heterogeneous gene transformed into Arabidopsis thaliana for functional analysis of heavy metal resistance and investigating the cellular compartmentation of expressed protein. Although it is widely accepted that metallothionein play an important role for copper (and possibly zinc) homeostasis, only few studies have further explored their in vivo function in plants.When BjMT2 cDNA was expressed in Arabidopsis thaliana under the regulation of the 35S-promoter, seedlings exhibited an increased tolerance against Cu and Cd as based on shoot growth and chlorophyll content. CLSM analysis of transiently transformed cells of A. thaliana and tobacco leaves revealed exclusive cytosolic localization of a BjMT2::EGFP fusion protein in control and heavy metal-exposed plant cells. The heavy metal exposure did not affect the subcellular distribution of BjMT2 protein, the protein remaining evenly distributed throughout the cytoplasm.Ectopic expression of BjMT2 reduced root growth in the absence of heavy-metal exposure, whereas in the presence of 50 or 100 μM Curoot growth in control and transgenic lines was identical. The results indicate that in A. thaliana, root and shoot development are differentially affected by ectopic expression of BjMT2.In the present we have readdressed the role of MT2 in plants for copper tolerance. In particular, we have analyzed the effect of MT2 overexpression on seedling growth and development in the absence or presence of toxic copper levels, an important aspect not yet addressed in previous studies. Following proof of function of BjMT2 in E. coli, we have analyzed shoot and root development of transgenic A. thaliana seedlings.