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

Abstract

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 (Cd2+) was developed for the first time for the wheat Triticum aestivum grown in Cd2+-fortified soils. Also, 1-(4-Isothiocyanobenzyl)-ethylenediamine-N,N,N,N-tetraacetic acid (ITCB-EDTA) was used to chelate the mobile Cd2+. The ITCB-EDTA/Cd2+ complex was fixed with proteins in situ via the isothiocyano group. A new Cd2+-EDTA specific monoclonal antibody, 4F3B6D9A1, was used to locate the Cd2+-EDTA protein complex. After staining, the fluorescence intensities of sections of Cd2+-positive roots were compared with those of Cd2+-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 Cd2+ content in plant tissues and illustrate Cd2+ translocation and cellular and subcellular responses of T. aestivum to Cd2+ 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 Cd2+ was also confirmed with energy-dispersive X-ray microanalysis. The IHC method is suitable for locating and quantifying Cd2+ in plant tissues and can be extended to other heavy metallic ions.

Unraveling the role of dark septate endophyte (DSE) colonizing maize (Zea mays) under cadmium stress: physiological, cytological and genic aspects

“…The distribution of metallic ions in plant tissues is associated with their toxicity, and selective distribution of toxins is of crucial significance in determining the survival of plants in toxic metal stress 27 . At subcellular levels, Cd can be accumulated in specific cell sites which correspond to less metabolically active areas, e.g.…”

Endophytic Paecilomyces formosus LHL10 Augments Glycine max L. Adaptation to Ni-Contamination through Affecting Endogenous Phytohormones and Oxidative Stress

“…To further assess the beneficial effect of P. formosus inoculation, it is important to determine the distribution and accumulation of metal uptake in the roots and shoots when evaluating the survival of plants subjected to metal stress ( Gao et al, 2015 ). Here, we found mitigating effects in inoculated soybean plants in terms of a lower accumulation in shoots followed by roots compared with non-inoculated plants.…”

Tools for the Discovery of Hyperaccumulator Plant Species and Understanding Their Ecophysiology

“…The main disadvantage is that the sulphide reaction is non-specific and precipitates a range of transition elements, although at neutral pH the selective formation of ZnS nanocrystals is favoured (McRae et al 2009). Selectivity may also be achieved using immunohistochemical methods via monoclonal anti-bodies to specifically bind chelated target ions, as demonstrated for Cd 2+ in plant tissue (Gao et al 2015).…”

“…Delivery of cell permeable fluorophores into a nutrient solution, followed by uptake in the plant, could circumvent this issue, but avoiding re-distribution during sectioning remains difficult. Attempts have also been made at fixing mobile target ions (Cd 2+ or Zn 2+ ) by in situ precipitation as metal sulphides or with strong chelators before microtoming and microscopy (Hu et al 2009(Hu et al , 2012Gao et al 2015). There is a need for independently validating the use of fluorophores for mapping metal(loid) ions in hyperaccumulator plants, for instance by mapping of fluorophore labelled tissues with other micro-analytical techniques, such as XFM or PIXE.…”