Gene expression analysis by qRT-PCR
For gene expression analysis, seedlings of same growth status were treated with 0.5 mM KNO3 + 50 µM Fe, 15 mM KNO3 + 50 µM Fe, 0.5 mM KNO3 -Fe (-Fe solution supplemented with 200 µM ferrozine) or 15 mM KNO3 -Fe (-Fe solution supplemented with 200 µM ferrozine) solutions for 1, 3, 5 and 7 days, respectively. Roots were collected, washed and frozen in liquid nitrogen. The roots were grinded into powder with a mortar and pestle. Total RNA was extracted with an Omini Plant RNA Kit (CWBIO, Beijing, China) following the manufacturer’s protocol. cDNA was prepared from 1 µg total RNA using the PrimeScriptTM RT reagent Kit (Takara, Dalian, China). An Ultra SYBR Mixture (CWBIO, Beijing, China) was used to detect the expression levels of genes using qRT-PCR (ABI, StepOnePlus, USA).MdActin (GenBank accession No.:CN938023) was selected as an internal control gene. Results were based on the average of three replicate experiments. All primers used in this study was shown in Table 1.