Cellular and Subcellular Immunohistochemical Localization and Quantification of Cadmium Ions in Wheat (Triticum aestivum)

The distribution of metallic ions in plant tissues is associated with their toxicity and is important for understanding mechanisms of toxicity tolerance. A quantitative histochemical method can help advance knowledge of cellular and subcellular localization and distribution of heavy metals in plant tissues. An immunohistochemical (IHC) imaging method for cadmium ions (Cd(2+)) was developed for the first time for the wheat Triticum aestivum grown in Cd(2+)-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd(2+). The ITCB-EDTA/Cd(2+) complex was fixed with proteins in situ via the isothiocyano group. A new Cd(2+)-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd(2+)-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd(2+)-positive roots were compared with those of Cd(2+)-negative roots under a laser confocal scanning microscope, and the location of colloidal gold particles was determined with a transmission electron microscope. The results enable quantification of the Cd(2+) content in plant tissues and illustrate Cd(2+) translocation and cellular and subcellular responses of T. aestivum to Cd(2+) stress. Compared to the conventional metal-S coprecipitation histochemical method, this new IHC method is quantitative, more specific and has less background interference. The subcellular location of Cd(2+) was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd(2+) in plant tissues and can be extended to other heavy metallic ions.