Plant culture conditions
The cultivar of peanut used in the pot and field trials was Luhua 14. The pot assay was carried out in a greenhouse at China Agricultural University (Beijing, China). After germination for 2 days, six peanut seeds were transferred to each pot, each filled with 8 kg of calcareous soil. The soil pH was 7.5, and the available Fe was 3.61 mg·kg−1. The basic fertilizer contents (mg·kg−1) were Ca(NO3)2·4H2O, 10; KH2PO4, 150; KCl, 100; MgSO4·7H2O, 50; CuSO4·5H2O, 5; and ZnSO4·7H2O, 5. The treatments (PDMA, PDMA–Fe, and EDTA–Fe) were prepared as 40 µM aqueous solutions. The treatments (300 ml) were irrigated into the peanut rhizosphere of each pot at 30, 48, 65, and 83 days post-sowing (dps), which matched the flowering stage, early pod-bearing stage, late pod-bearing stage, and fruit-ripening stage of peanut, respectively. The same amount of water was irrigated as a control. The soil plant analysis development (SPAD) value in expanded young leaves was recorded using a chlorophyll meter (SPAD-502Plus; Konica Minolta, Japan) at 7 days after treatment application. At 90 dps, we took photographs of the peanuts under the different treatments and then harvested the peanuts. A portion of the fresh young leaves was collected to determine the active Fe concentration. The fresh root was divided into two parts. One part was used to measure the activity of ferric reductase, and the other part was stored in a −80°C freezer to examine gene expression. Rhizosphere soil was collected to determine the available Fe content. The other parts of the peanut plants were dried in a drying oven (ULM700; Memmert, Germany) until constancy and then weighed with a balance.
The field experiment was conducted in the village of Xichang in Beijing, China (116°10ʹ E, 39°10ʹ N). The average temperature from May to September is 22.9°C, and the annual precipitation is 603 mm. The soil in the field is a calcic Cambisol. The soil pH, available Fe, total nitrogen (N), available phosphorus (P), and available potassium (K) were 7.7, 3.0 mg·kg−1, 1.4 g·kg−1, 112.3 mg·kg−1, and 157.2 mg·kg−1, respectively. Organic fertilizer (N:P:K contents of 25.8 g·kg−1, 5.7 g·kg−1, and 17.1 g·kg−1, respectively) was applied at 20 t·hm−1 before peanut planting. The plant spacing was 30 cm, and the row spacing was 60 cm. The treatments in the field trial were the same as those in the pot trial, with three districts used in each treatment. Each district contained 16 holes, and two peanut kernels were planted in each hole. The treatments were prepared as 40 μM aqueous solutions, and 100 ml of solution was irrigated into the peanut rhizosphere in each hole. The same amount of water was irrigated as a control. In the field condition, the treatments were added four times during the growth stage at 30, 52, 72, and 93 dps, matching the flowering stage, early pod-bearing stage, late pod-bearing stage, and fruit-ripening stage of peanut. The SPAD value in expanded young leaves was recorded at 67 and 107 dps using a chlorophyll meter (SPAD-502Plus; Konica Minolta). Eight SPAD values were measured in each district. At 107 dps, the peanut plants were photographed and then harvested. A portion of the fresh young leaves was collected to determine the active Fe concentration. The fruits of the peanuts were harvested, dried until constant weight, and then the yield, kernel yield, fruit weight per plant, and fruit number per plant were determined.