710 icp oes

For all treatments, the kernels of plants in 9 pots (each replicate constituted 3 pots) were harvested at maturity, and after being air dried, the kernels were ground into whole wheat flour for the test of mineral components.
Samples were subjected to high-temperature digestion with an HNO₃/HClO₄ acid mixture (Johnson and Ulrich, 1959 ), and the mineral component (P, K, Ca, Fe, and Zn) contents were determined by inductively coupled plasma emission spectrometry (ICP−OES 710; Agilent Technologies, USA) (Tracy et al., 1991 (link)).
The accumulation of mineral components in wheat grains was calculated by multiplying the mineral component concentration by the grain weight. The wheat grain weight under different low-temperature treatments is shown in Supplementary Figure S1.

For the plant total P concentration assay, fresh plant samples were heated at 75°C until completely dry, and then ground into powder separately. Approximately 0.05 g of the plant dry samples were weighed and digested with H₂SO₄ and H₂O₂. After cooling, the digested samples were diluted to 100 ml with distilled water. Then, the concentration of P in the solution was determined with 710 ICP-OES (Agilent Technologies, Santa Clara, CA, United States). Each experiment was repeated three times.
For the soluble Pi concentration assay, Tissue Inorganic Phosphorus Content Detection Kit (Solarbio, Beijing, China) was used. Each sample consisted of five seedlings, which were ground into fine powder in liquid nitrogen, repeated three times.

For solubilisation experiments, a modified version of the methods described by Altomare et al. [7 (link)] was used. The minerals (20 mg) used in the present study were Ca₃(PO₄)₂, Fe₂O₃, MnO₂, CuO and metallic Zn which were added in powder form to 50 ml of sucrose-yeast extract (SY) medium separately. Three replicate flasks were sampled each day for 9 days to determine the pH values and soluble mineral contents in the culture medium. The mycelium was removed by filtration (0.45 μm), and the P, Fe, Mn, Cu and Zn contents in the filtrates were determined using an inductively coupled plasma optical emission spectrometer (710 ICP-OES, Agilent Technologies, California, USA). Uninoculated media in flasks that were processed in the same way were used as the control.

Roots and leaves were harvested from 6-week-old plants, washed three times with tap water and rinsed twice (5 min each) with deionised water to remove any adhering nutrients. The leaves and roots were dried in a forced-air oven at 70 °C for ~48 h to a constant weight for dry weight measurements (Figs. 2b and 3b and Supplementary Fig. 4a). The dried samples were ground and passed through a 1.0-mm screen. Total N/C contents (Figs. 2g, h and 3g, h) were determined via the dry combustion method using an Element Analyser (vario EL, Elementar, Langenselbold, Germany). For the analysis of mineral elements, the dry biomass was digested in H₂SO₄ or HClO₄. P concentrations were determined using the molybdate yellow method and K concentrations were determined by flame emission photometry (Supplementary Fig. 6)^{54 (link),55 (link)}. The other nutrient elements were measured by ICP (Agilent 710 ICP-OES). At least three plants per treatment were harvested, and three independent biological replicates were analysed for each treatment.