The Subcellular Targeting and In Vivo Metal Binding Characteristics of AgNt84 using Transgenic Tobacco and BY-2 Suspension Cell Cultures

AgNt84 is a nodulin protein expressed in Alnus glutinosa, an actinorhizal tree that participates with the actinomycete Frankia in symbiotic nitrogen fixation. Expression of AgNt84 mRNA is exclusively found within Zone 2 of the nodules, which is the zone that contains cells in the process of being infected by Frankia. A truncated form of the protein was found to bind Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Cd²⁺, and Hg²⁺ in vitro. AgNt84 was predicted to be targeted extracellularly based on the presence of an N-terminal signal sequence. The purpose of this research was to determine the subcellular targeting and in vivo metal binding properties of AgNt84. Transient transformation of onion and tobacco tissue and stable transformation of tobacco and tobacco BY-2 cells were used to accomplish these goals.

Transient expression of AgNt84-GFP fusion protein in onion epidermal cells confirmed that AgNt84 is targeted to the endoplasmic reticulum. The fusion protein was not found in the cytosol. Expression of AgNt84-GFP with an HDEL retention tag resulted in the accumulation of the fusion protein at what appear to be the plasmadesmata as well as its presence in the endoplasmic reticulum. The presence of AgNt84-GFP in the endoplasmic reticulum is consistent with the pathway that a protein targeted to the plasma membrane would follow. However, the possibility that the protein may be targeted to other organelles or remain in the endoplasmic reticulum remains. To determine the ability of AgNt84 to bind metal in vivo, three hydroponically grown tobacco lines transgenic for AgNt84 and wild-type tobacco plants were incubated in 5 mM MES buffer containing 200 μM Cd(NO₃)₂4H₂O. The amount of cadmium in the plants at day 1 and day 3 was analyzed by inductively coupled argon plasma spectrometry (ICP). Two of the transgenic tobacco lines tested had significantly more cadmium in the roots than wild-type tobacco at day 1 and day 3. One of these, T10, was a transgenic line expressing AgNt84, and the expression of AgNt84 in the other tobacco lines remains to be confirmed. Histochemical staining of tobacco tissue using dithizone supported ICP measurements of cadmium content.