Gene expression analysis
For each line, five pools each containing two rosette of plants grown under optimal or limiting N were harvested to liquid nitrogen 10 days after pricking. Total RNA was extracted using TRIzol reagent (Invitrogen, catalog 15596026) and DNA was removed using TURBO DNA-free kit (Invitrogen, catalog AM1907). cDNA was synthesized with the ImProm-II Reverse Transcription System (Promega, catalog A3800). Quantitative real-time PCR reactions were performed using Power SYBR Green PCR-Master-Mix (Applied Biosystems, catalog 4367659) and the ABI PRISM 7900HT (Applied Biosystems) system. EF1ALPHA (At5g60390),PP2AA3 (At1g13320), and SAND (At2g28390) were used as housekeeping genes (Table S9). For each sample, the target gene levels were normalized by the delta-Ct to the average of the three housekeeping genes. The complete list of primers used for gene expression quantification is provided in Table S9.