Icap 7400

UV-vis absorption spectra were performed using a SHIMADZU UVmini-1240 UV-VIS spectrophotometer. PL spectra and lifetime were measured by state/transient fluorescence spectrometer (FLS1000, Edinburgh). FT-IR spectra were taken on a Nicolet IS50 (Thermo Fisher, Waltham, MA, USA) FT-IR spectrophotometer. The TEM and HR-TEM images were recorded using FEI, TECNAI TF20 field emission electron microscope. X-ray photoelectron spectroscopy (XPS) experiments were performed using electronic spectrometer (ESCALAB XI+, Thermo Fisher). The results (binding energies) were calibrated using the C 1s peak for C-C at 284.8 eV, and fitted using the software Thermo Advantage v5.967 (Thermo Fisher Scientific Inc., USA). The determination of calcium content was performed by Inductively coupled plasma spectrometer (iCAP 7400, Thermo Fisher).

The sequential P extractions from cB, gB, gBM, cBM and cBS ashes followed the protocol of Hieltjes and Lijklema (1980) (link), modified by Qian et al. (2009) (link). In brief, 0.5 g of each ash were sequentially extracted with 20 mL of distilled deionized water (ddH₂O), 1 M ammonium chloride (NH₄Cl) at pH 7, 0.1 M sodium hydroxide (NaOH), and twice with 0.5 M hydrochloric acid (HCl) solutions. Samples were shaken (Turbula T2c, Willy A. Bachofen AG, Muttenz, Switzerland) for 2 hours (h), 2 h, 17 h, and 2x 24 h, respectively, and centrifuged at 15000 g (Hermle Z326K, HERMLE Labortechnik GmbH, Wehingen, Germany) for 5 min. before collecting supernatants. Phosphorus in supernatants was measured using inductively coupled plasma atomic emission spectroscopy (Thermo iCAP 7400, Dreieich, Germany) according to Brod et al. (2015) (link).

Dried shoot material, including stem and leaves inclusive of lost leaves (subsample of 200–300 mg), were digested with 5 mL concentrated HNO₃ (65%) and 2 mL H₂O₂ (30%) at 200 °C for 15 min in a microwave (MARSXpress 250/50; CEM Corporation, Charlotte, NC, USA), filtrated and taken up to 50 mL with distilled water. The concentrations of P, Fe and Zn were measured in the filtrate using an ICP-OES analyzer (Thermo Scientific iCAP™ 7400; Thermo Fisher, Taufkirchen, Germany) separate for element-specific wavelength according to the manufacturer’s instructions. To quantify N concentrations in the shoot, approximately 15 mg of the pulverized plant material, including stem and leaves inclusive of lost leaves, was analyzed after dry oxidation in an elemental analyzer (Elementar Vario EL, Elementar, Germany) following the Dumas method.
Data with a normal distribution (Kolmogorov-Smirnov test; p > 0.05) and homogeneity of variance (Levene’s test; p > 0.05) were subjected to factorial analysis of variance (ANOVA). The multiple comparison Tukey’s test was used to assign significant differences between means, applying a cut-off of p < 0.05. Data that were not normally distributed were subjected to the Kruskal-Wallis test (p < 0.05). Statistics were performed using the STATISTICA program version 12 (StatSoft Inc., Tulsa, OK, USA).

The dried shoots and roots were ground to a powder. Total-N concentration was measured by Kjeldahl’s method. The concentrations of Cd, P, K, Ca, Cu, Mg, Fe and Zn were analyzed with ICP-OES (iCAP™ 7400; Thermo Fisher Sci., Cambridge, UK).

Fine powder (20 mg) of the freeze-dried samples was digested in 2 mL of 60% HNO₃ at 110°C in a DigiTUBE tube (SCP Science, Montreal, Canada) for approximately 2 h. After the samples had cooled to room temperature, 2 mL of 60% HClO was added, and the samples were heated at 110°C for approximately 2 h. Once digestion was complete, the samples were cooled and the solution volume was adjusted to 10 mL by the addition of ultra-pure water. The total concentrations of magnesium (Mg) and iron (Fe) were measured at 285.213 and 259.940 nm, respectively, using an inductively coupled plasma–optical emission spectrometer (iCAP 7400, Thermo Fisher Scientific, Waltham, MA, United States).
Total N and C contents were measured by dry combustion using an NC analyzer (SUMIGRAPH NC-95A, Sumika Chemical Analysis Service, Tokyo, Japan). Acetanilide was used as a standard for total C and N analyses.

Anhydrous copper sulfate is dissolved in deionized water to a concentration of 60 ppm Cu²⁺ as reference. Three magnetic hydrogel rotors (r = 1000 µm, h = 790 µm and n = 8) were placed in 5 ml 60 ppm solution and rotated for 30 min without applying a magnetic field. As a comparison, another three magnetic hydrogel rotors with the same parameters were placed in 5 ml 60 ppm solution and revolved for 30 min under the magnetic field of a cylindrical magnet. The Cu²⁺ concentrations of the solutions were measured by inductively coupled plasma-optical emission spectrometry (iCAP 7400, Thermo, USA).

To reconstitute the protein with Cd^II, aliquots of thionein in 5 mm HCl were mixed with cadmium sulfate at a molar ratio of 1:9 under a nitrogen blanket, and the pH adjusted to 8.6 with 1 m solution of Tris base and purified on an SEC‐70 gel filtration column equilibrated with 20 mm Tris‐HCl buffer, pH 8.6. The concentration of the purified protein was obtained spectrophotometrically, with DTNB and PAR assays for the thiol and Cd^II concentration, respectively.86, 87 Additionally, samples were analyzed by inductively coupled plasma (ICP) analysis (ICP‐AES iCAP 7400, Thermo Scientific) to confirm the spectrophotometric results.