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.