Metabolite profiling
For the primary metabolites quantification, four plants of the selected set of accessions (Tables S6 and S7) were grown on the same conditions used for the N screening. When the plants reached the ten-leaf stage, they were collected and frozen in liquid nitrogen. Another four plants in two pots were harvested and frozen to liquid nitrogen at mid-day for metabolites analysis at ten-leaf stage. Extraction and analysis by gas chromatography–mass spectrometry (GC-MS) was performed as described in (Lisec, Schauer, Kopka, Willmitzer, & Fernie, 2006). Briefly, frozen ground material was homogenized in 300 μl of methanol at 70 °C for 15 min and 200 μl of chloroform followed by 300 μl of water was added. The polar fraction was dried under vacuum, and the residue was derivatized for 120 min at 37 °C (in 40 μl of 20 mg ml−1methoxyamine hydrochloride (Sigma-Aldrich, cat. no. 593-56-6) in pyridine followed by a 30 min treatment at 37 °C with 70 μl of N-methyl-N (trimethylsilyl)trifluoroacetamide (MSTFA reagent; Macherey-Nagel, cat. no. 24589-78-4). The GC‐MS system used was a gas chromatograph coupled to a time‐of‐flight mass spectrometer (Leco Pegasus HT TOF‐MS). An auto sampler Gerstel MultiPurpose system injected the samples. Chromatograms and mass spectra were evaluated by using Chroma TOF 4.5 (Leco) and Xcalibur 2.1 software, peak area was normalized by comparison to an internal standard (ribitol; CAS488‐81‐3) and the fresh weight of the sample used for extraction.