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.