Xepos

Dried leaf samples were ground using 4 mm diameter inox beads in an oscillating grinder (Mixer Mill MM400, Retsch, Haan, Germany). The concentrations of most elements (Mg, P, S, K, Ca, B, Mn, Fe, Ni, Cu, Zn, Mo Na, Co, V, and Se) were quantified using the procedure previously described [102 (link)] using 40 mg of dry powder per sample that was submitted to acid digestion and mineralization. The resulting solutions were then analyzed by high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS, Element 2^TM, Thermo Fisher Scientific, Bremen, Germany).
The total N concentration was determined with 1.5 mg of fine powder placed in tin capsules before analysis with an isotope-ratio mass spectrometer (IRMS, Isoprime, GV Instruments, Manchester, UK) linked to a C/N/S analyzer (EA3000, Euro Vector, Milan, Italy).
The remaining elements (Cl, Si, and Al) were quantified with approximately 1 g of dry weight powder analyzed with an X-ray-fluorescence spectrometer (XEPOS, Ametek, Berwyn, PA, USA) using calibration curves obtained from international standards with known concentrations.
In this study, elemental content is expressed as the ratio WD/control plants following the calculation method described [61 (link)].

In roots and shoots from unlabeled plants, Si and S contents were quantified with approximately 1 g of dry weight powder analyzed with an X-ray-fluorescence spectrometer (XEPOS, Ametek, Berwyn, PA, United States) using calibration curves obtained from international standards with known contents.

For S and Si concentration determinations, approximately 1 g of the dried powder was analyzed with an X-ray-fluorescence spectrometer (XEPOS, Ametek, Berwyn, PA, USA) using calibration curves obtained from international standards.
To determine the total N and ¹⁵N concentrations, 1.5 mg of each dried powder was precisely weighed and placed into tin capsules before analysis with a continuous flow isotope ratio mass spectrometer (IRMS, Horizon, NU Instruments, Wrexham, UK) linked to a C/N/S analyzer (EA3000, Euro Vector, Milan, Italy). The total N amount (Ntot) in each organ or in the whole plant was calculated as: $$Ntot=\frac{\%N x W}{100}$$
The amount of ¹⁵N (¹⁵N) was determined by the following: $${}^{15}N=\frac{{\%}^{15}\mathrm{N} x Ntot}{100}$$
As the nutrient solution was labeled with a 0.5% isotopic excess, it was possible to calculate the total amount of nitrogen taken up from the nutrient solution (N_ns): $$N_{ns}=\frac{{}^{15}\mathrm{Ntotal} x 100}{0.5}$$
The amount of nitrogen from dinitrogen (N₂) atmospheric fixation (N_atm) was then obtained by subtracting the amount of nitrogen taken up from the nutrient solution from the total nitrogen in the plant. Then, the proportion of each N source (% of N_ns or % N_atm) was calculated as follows: $${\%N}_{ns}(\mathrm{or} {\%N}_{atm})=\frac{N_{ns}\left(\mathrm{or} N_{atm}\right) x 100}{N_{tot (\mathrm{whole} \mathrm{plant})}}$$