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 (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

An evaluation of Astragali Radix with different growth patterns and years, based on a new multidimensional comparison method

IntroductionWith the depletion of wild Astragali Radix (WA) resources, imitated-wild Astragali Radix (IWA) and cultivated Astragali Radix (CA) have become the main products of Astragali Radix. However, the quality differences of three growth patterns (WA, IWA, CA) and different growth years of Astragali Radix have not been fully characterized, leading to a lack of necessary scientific evidence for their use as substitutes for WA.MethodsWe innovatively proposed a multidimensional evaluation method that encompassed traits, microstructure, cell wall components, saccharides, and pharmacodynamic compounds, to comprehensively explain the quality variances among different growth patterns and years of Astragali Radix.Results and discussionOur study showed that the quality of IWA and WA was comparatively similar, including evaluation indicators such as apparent color, sectional structure and odor, thickness of phellem, diameter and number of vessels, morphology of phloem and xylem, and the levels and ratios of cellulose, hemicellulose, lignin, sucrose, starch, water-soluble polysaccharides, total-saponins. However, the content of sucrose, starch and sorbose in CA was significantly higher than WA, and the diameter and number of vessels, total-flavonoids content were lower than WA, indicating significant quality differences between CA and WA. Hence, we suggest that IWA should be used as a substitute for WA instead of CA. As for the planting years of IWA, our results indicated that IWA aged 1-32 years could be divided into three stages according to their quality change: rapid growth period (1-5 years), stable growth period (6-20 years), and elderly growth period (25-32 years). Among these, 6-20 years old IWA exhibited consistent multidimensional comparative results, showcasing elevated levels of key active components such as water-soluble polysaccharides, flavonoids, and saponins. Considering both the quality and cultivation expenses of IWA, we recommend a cultivation duration of 6-8 years for growers. In conclusion, we established a novel multidimensional evaluation method to systematically characterize the quality of Astragali Radix, and provided a new scientific perspective for the artificial cultivation and quality assurance of Astragali Radix

A Highly Sensitive and Rapid Colloidal Gold Immunoassay for Puerarin Detection

Radix Puerariae is a traditional Chinese medicinal material with a rich history of use in East and Southeast Asia. Puerarin, a unique component of the Pueraria genus, serves as a quality control marker for herbal medicines like Pueraria lobata and Pueraria thomsonii in China, displaying diverse pharmacological properties. This study developed puerarin colloidal gold immunoassay dipsticks utilizing an anti-puerarin monoclonal antibody, resulting in a fast and sensitive detection method with a limit of 500–1000 ng·mL–1. Evaluation using tap water-extracted P. lobata and P. thomsonii samples showed consistent results compared to LC-MS analysis. Cross-reactivity assessments of puerarin analogs revealed minimal interference, affirming the dipstick’s reliability for distinguishing between the two species

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

<p>Proposed mechanism of immunohistochemical prefixation of Cd²⁺ in plant tissues.</p

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

<p>Comparison of immunoelectronic microscope images of Cd^<b>2+</b> 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 Cd^<b>2+</b>-negative plant (A, C, E, G, I and K) and a plant treated with 100 μg g^<b>-1</b> Cd^<b>2+</b> (B, D, F, H, J and L). Cd^<b>2+</b> 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 Cd^<b>2+</b>-positive plants. ICS is the intercellular space, and ML is the middle lamella. The insets display the magnified regions, and the arrowheads indicate Cd^<b>2+</b> depositions.</p

Immunoelectronic microscope images of wheat root transverse sections.

<p>(A) and (B) are a Cd^<b>2+</b>-negative plant and one exposed to 100 μg g^<b>-1</b> Cd^<b>2+</b>, respectively. Note: curdled macromolecular substances in the vacuole (a), cell wall fracture (b), and lignification (c) are shown in (B).</p

EDX analysis of the metal-S coprecipitation method.

<p>EDX spectra of Cd^<b>2+</b> 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^<b>-1</b> Cd^<b>2+</b> (B and D). Cd^<b>2+</b> was localized with the conventional metal-S coprecipitation histochemical method. Note: The EDX spectra suggest that the illustration of Cd^<b>2+</b> deposition detected with the traditional histochemical method as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123779#pone.0123779.g004" target="_blank">Fig 4</a> may be interfered by other metal ions such as Mg^<b>2+</b>.</p

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

<p>(I) Fluorescence microscopic images of Cd^<b>2+</b> distribution in the roots of wheat plants exposed to Cd^<b>2+</b> at 0, 1, 5, 25, 50 and 200 μg g^<b>-1</b> (A-F, respectively), image of no primary antibody control tests (G), and corresponding bright field images (a-g). Cd^<b>2+</b> was immunohistochemically localized with mAb4F₃B₆D₉A₁ and ITCB-EDTA. (II) The quantitative relation between the Cd^<b>2+</b> content and relative fluorescent value measured in A-F. The error bars represent standard derivations of triplicate measurements.</p

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

<p>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 Cd^<b>2+</b> deposition (D). The EDX spectra show the specific detection of Cd^<b>2+</b> in the different compartments of wheat plants grown in soil supplemented with 100 μg g^<b>-1</b> Cd^<b>2+</b>. The Al and Ni peaks were from the Al holder and Ni grid, respectively.</p