Plant material and growth conditions
Pinyiensis ’ (Malus hupehensis Rehd. ) andArabidopsis thaliana (Columbia ecotype) were used in this study. ‘Pinyiensis ’ fruits were collected from National Engineering Research Center for Apple of Shandong Agricultural University and the seeds were harvest. ‘Pinyiensis ’ seeds were first laminated for 30 days using wet sand in 4℃, dark. Then the seeds were planting onto the nursery (substrate: meteorite 3:1) for germination at 25℃. After germination the seedlings were cultivated on nursery for 2 weeks and then transferred into vermiculite moistened with the complete nutrient. The complete nutrient was composed of following components:10 µM MnSO4; 100 µM H3BO3; 0.1 µM CuSO4; 0.1 µM Na2MoO4; 30 µM ZnSO4; 5 µM KI; 0.1 µM CoCl2; 4 mM CaCl2; 1 mM MgSO4; 1 mM KH2PO4; 50 µM Fe(Ⅲ)-EDTA; 5 mM KNO3. For phenotypic analysis, 6-week-old seedlings with the same growth status were transplanted to a new vermiculite pot and subjected with following treatments for 3 weeks: 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), with KCl was added to maintain the same K+concentration (15 mM). Seedlings were cultivated in a controlled environment: 25℃, 60% relative humidity and a 16h day/8h night rhythm. The treatment solutions were renewed every 7 days.
Columbia ecotype Arabidopsis thaliana seeds were surface-sterilized and germinated on substrate containing agar and 1/2 MS (Murashige & Skoog, 1962) nutrient. After 1 week of growth, seedlings were transferred to vermiculite moistened with complete nutrient for 1 week. The vermiculite was washed with deionized water from top to bottom for 4 times before subjected with following treatments: 0.2 mM KNO3 + 50 µM Fe, 20 mM KNO3 + 50 µM Fe, 0.2 mM KNO3 -Fe ( -Fe solution supplemented with 200 µM ferrozine), 20 mM KNO3 -Fe (-Fe solution supplemented with 200 µM ferrozine), with KCl was added to maintain the same K+ concentration (20 mM). Seedlings of similar rosette diameters were picked for treatment. The seedlings were cultivated in a growth room at 22℃, 65% relative humidity and a 16h day/8h night rhythm. The treatment solutions were renewed every 3 days.