Intracellular localization of “soluble” adenylate cyclase in potato plants

Abstract

In animal cells, a key enzyme of the adenylate cuclase system, adenylate cyclase (AC), is represented by its transmembrane (tmAC) and soluble (sAC) forms [1]. Earlier, we have established that both forms of this enzyme are present in plants as well (potato), in nuclei and chloroplasts [2]. Using immunoprecepitation and Western blotting with antibody against animal sAC, we have found five sAC isoforms with mol wts of 225, 193, 92, 68, and 60 kD [3]. However, it is not clear until now, whether sAC is present only in nuclei and chloroplasts or it is localized also in other cell compartments, which could provide for more rapid transduction of external signals. Therefore, the objective of our work was to analyze intracellular sAC localization at the level of the cell ultrastructure. In this study, we applied for the first time the method of immuno-electronic cytochemistry for the studying of sAC intracellular localization in various potato tissues and its re-dislocation in the response to biotic stress. Tube-grown potato plantlets of two cultivars contrasting in their resistance to the causal agent of the ring rot were used in experiments: cv. Lugovskoi, resistant, and cv. Luk’yanovskii, susceptible. The root segments and leaf pieces from the upper part of the shoot of control plants (with roots not treated with extracellular polysaccharides (EPS) of the pathogen) and experimental plants kept for 2 h in the 0.1% EPS solution were fixed for 2 h in the mixture of 4% formaldehyde and 1% glutaraldehyde (Sigma, United States) prepared in 0.1 M Na-phosphate buffer, pH 7.2. Thereafter, samples were postfixed in 0.8% OsO 4 in the same buffer (2 h), dehydrated by the routine procedure, and embedded in the mixture of epoxide resins, Epon and Araldite. Immunocytochemistry was performed on ultrathin sections mounted on nickel grids, using primary rabbit polyclonal antibodies (PAB) against animal sAC at 1 : 200 dilution conjugated with 10-nm colloidal gold particles and secondary goat antibodies against rabbit immunoglobulins (SAB, Sigma, United States) at 1 : 30 dilution. All procedures were performed as described in [4], with the exception of the usage of the hoarse serum at 1 : 5 dilution as a blocking solution. Two control treatments were applied: (a) ultrathin sections not treated with PAB and (b) treatment with nonimmune rabbit serum instead PAB and then with SAB saturated with such serum. Specimens stained with uranyl acetate were examined with an LEO 906E electron microscope (Carl Zeiss, Germany), and the images were shown on the computer display. To evaluate sAC partition between cell compartments, we counted the number of gold particles in them. In each treatment, we analyzed 25 images; the total number of particles on the area of each compartment was calculated per 1 µ m 2 . The results were processed statistically. We observed the presence of SAB, reflecting sAC localization, in many intracellular compartments. Thus, in the cells of the root meristem and elongation zone, gold particles were seen in the cytosol, mitochondria,