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.

Fig 1. Proposed mechanism of immunohistochemical prefixation of Cd²⁺ in plant tissues.

Fig 2. Quantitative immunohistochemical images (I) and quantitative relation (II).

(I) Fluorescence microscopic images of Cd²⁺ distribution in the roots of wheat plants exposed to Cd²⁺ at 0, 1, 5, 25, 50 and 200 μg g^-1 (A-F, respectively), image of no primary antibody control tests (G), and corresponding bright field images (a-g). Cd²⁺ was immunohistochemically localized with mAb4F₃B₆D₉A₁ and ITCB-EDTA. (II) The quantitative relation between the Cd²⁺ content and relative fluorescent value measured in A-F. The error bars represent standard derivations of triplicate measurements.

Fig 3. Energy-dispersive X-ray (EDX) analysis of the immunohistochemical method.

EDX spectra of metals in xylem cells (A), endodermis cells (B), curdled macromolecular substances in the vacuole (C) and a region where there were no gold particles, indicating no Cd²⁺ deposition (D). The EDX spectra show the specific detection of Cd²⁺ in the different compartments of wheat plants grown in soil supplemented with 100 μg g^-1 Cd²⁺. The Al and Ni peaks were from the Al holder and Ni grid, respectively.

Fig 4. Cd²⁺ distribution in the stele cells of wheat roots with metal-S coprecipitation method.

Electron microscope images of Cd²⁺ distribution in the cell walls (A and B) and plasma membrane (C and D) of root stele cells of wheat plants grown in soil fortified with 0 (A and C) and 100 μg g^-1 Cd²⁺ (B and D). Cd²⁺ was localized with the conventional metal-S coprecipitation histochemical method. Arrowheads indicate Cd²⁺ deposits.

Fig 5. EDX analysis of the metal-S coprecipitation method.

EDX spectra of Cd²⁺ deposits near the cell wall (A and B) and curdled macromolecular substances (C and D) of wheat plants grown in soil fortified with 0 (A and C) and 100 μg g^-1 Cd²⁺ (B and D). Cd²⁺ was localized with the conventional metal-S coprecipitation histochemical method. Note: The EDX spectra suggest that the illustration of Cd²⁺ deposition detected with the traditional histochemical method as shown in Fig 4 may be interfered by other metal ions such as Mg²⁺.

Fig 6. Dynamic distribution of Cd²⁺ in wheat roots.

Fluorescence microscope images of the cellular distribution of Cd²⁺ in wheat roots 2, 4, and 15 days after germination (A, B and C, respectively) and the corresponding bright field images (a, b and c, respectively). Cd²⁺ was localized with the IHC method using mAb4F₃B₆D₉A₁ and ITCB-EDTA.

Fig 7. Immunoelectronic microscope images of wheat root transverse sections.

(A) and (B) are a Cd²⁺-negative plant and one exposed to 100 μg g^-1 Cd²⁺, respectively. Note: curdled macromolecular substances in the vacuole (a), cell wall fracture (b), and lignification (c) are shown in (B).

Fig 8. Cd²⁺ subcellular distribution in different cellular compartments of wheat roots.

Comparison of immunoelectronic microscope images of Cd²⁺ distribution in the cell walls of the cortex (A, B), outer tangential wall (C, D) and inner tangential wall (E, F) of endodermal cells, the cell membrane of xylem cells (G and H), plasmodesmata (PD) of phloem cells (I and J), and curdled macromolecular substances in the vacuole (K and L) of root vascular cells of wheat roots from a Cd²⁺-negative plant (A, C, E, G, I and K) and a plant treated with 100 μg g^-1 Cd²⁺ (B, D, F, H, J and L). Cd²⁺ was detected with the IHC method. Colloidal gold particles were only observed in the cortical tissue of the middle lamella (ML) and the outer surface of the cell wall in the intercellular space (ICS) (B), the outer tangential endodermis wall (D), the membrane along the inner tangential cell wall (F), the plasma membrane near the xylem cell wall (H), PD (J) and curdled proteinaceous material (L) in the vacuole and membrane-bound organelles (M and N) of phloem cells in Cd²⁺-positive plants. ICS is the intercellular space, and ML is the middle lamella. The insets display the magnified regions, and the arrowheads indicate Cd²⁺ depositions.