*2.3. Hydroponic Culture, Physiological Characterization, and Nitrate Uptake Assays*

The hydroponic culture was performed in a growth chamber at a constant temperature of 20 ◦C, a photoperiod of 8 h light (150 μmol photons m−<sup>2</sup> s−1)/16 h darkness and a relative humidity of 60%. First, seeds of 12 selected cultivars were sown in peat-based soil (DCM Maison et Jardin, EAN:5413448070033) and stratified during two days at 4 ◦C. After five days, the roots were carefully rinsed with water and seedlings transferred for seven days into hydroponic containers, with dimensions 35 cm × 25 cm × 13.5 cm and filled with 8 L of nutrient solution (Figure 1b). Plants were first fed during nine days with a nutrient solution containing 1 mM Ca(NO3)2 [43,44], then during seven days with solutions containing 0.1 mM Ca(NO3)2 + 1.9 mM CaCl2 (N−) or 1 mM Ca(NO3)2 (N+). Five plants per cultivar and per N condition were cultivated. Plants were harvested after nine days of treatment, corresponding to 21 days after germination. Roots and leaves were separated and scanned at a resolution of 400 dpi. Total root lengths were measured with the image analysis software Optimas 6.0 (Meyer Instruments, Inc., Houston, TX, USA). Organs were dried at 60 ◦C and dry weight was measured. Between 10 and 50 mg of crushed organ samples were analyzed with a Vario MAX Cube (Elementar, Langenselbold, Germany) for simultaneous carbon and nitrogen determination at the Centre pour l'Agronomie et l'Agro-Industrie de la province de Hainaut (CARAH), 11 rue Paul Pastur, 7800 Ath, Belgium.

The 15N labelling procedure for the nitrate uptake assay was conducted as described in [45]. Plants were fed during 21 days with a nutrient solution containing 1 mM Ca(NO3)2 [43,44]. Then, they were incubated for 90 min in a solution containing 0.1 mM or 5 mM KNO3. Roots were sequentially bathing for 1 min in 10 mL of CaSO4 solution (0.1 mM), for 5 min in 10 mL of a solution containing 0.1 mM or 5 mM K15NO3 (Sigma-Aldrich, Saint-Quentin-Fallavier, France) and for 1 min in 10 mL CaSO4 solution (0.1 mM). Root and shoot organs were immediately separated and dried at 60 ◦C, prior to dry weight measurement. Samples were analyzed with an integrated system for continuous flow isotope ratio mass spectrometry (Euro-EA elementar Analyzer, EuroVector IRMS Isoprime Elementar) at the

Stable Isotope Analytical platform of the Biochemistry and Plant Molecular Physiology, INRAE Montpellier, Place Viala, CEDEX 1, 34060 Montpellier, France.

#### *2.4. Soil Culture and Micro-Scale Computed Tomography Imaging*

The soil culture was conducted between 20 April and 18 May 2015, in a greenhouse located on the Sutton Bonington campus, Nottingham University (UK), with the control on light and temperature levels. Seeds of four selected cultivars were germinated in soil-filled pipes (d = 5 cm, h = 30 cm) for the purpose of X-ray computed tomography (Figure 1c). The soil was collected on a site (50◦36 47 N, 3◦46 12 E) without recorded agricultural activity during the last decades. The loamy soil characteristics were total C = 1.05%, organic N = 1.13‰, N-NH4 <sup>+</sup> = 0.73 mg kg−1, N-NO3 <sup>−</sup> = 10.48 mg kg−1, Ca = 224 mg 100 g−1, K = 31.76 mg 100 g−1, Mg = 10.89 mg 100 g−<sup>1</sup> and *p* = 16.08 mg 100 g−1. The columns were watered from the top with 500 mL of a nutrient solution without nitrate (N−) or with 50 mM KNO3 (N+) [43,44]. Watering was stopped two days prior to scanning. All images were acquired using a Phoenix v|tome|x m scanner (GE Measurement and Control Solutions, Billerica, MA, USA). The accelerating voltage was 150 kV and the current 160 μA. The X-rays were filtered through a 0.1 mm copper plate. For each sample, there were 4 × 2160 projections taken with an exposure time of 200 ms, and signal averaging of two and one skip per projection. These were then assembled into a single 3D image with a 58 μm spatial resolution. The root images were segmented and measured manually using VGStudio MAX.
