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

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

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

Dynamic distribution of Cd²⁺ in wheat roots.

<p>Fluorescence microscope images of the cellular distribution of Cd^<b>2+</b> 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). Cd^<b>2+</b> was localized with the IHC method using mAb4F₃B₆D₉A₁ and ITCB-EDTA.</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

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

<p>Electron microscope images of Cd^<b>2+</b> 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^<b>-1</b> Cd^<b>2+</b> (B and D). Cd^<b>2+</b> was localized with the conventional metal-S coprecipitation histochemical method. Arrowheads indicate Cd^<b>2+</b> deposits.</p

DataSheet_1_An evaluation of Astragali Radix with different growth patterns and years, based on a new multidimensional comparison method.docx

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.</p