RESULTS
Nitrate inhibits iron accumulation in the newly growing leaves of apple
Two-year-old trees of the apple cultivar ‘Fuji’ were treated with 10, 30, 60, or 120 g nitrate fertilizer per plant. We found that increased nitrate application resulted in more severe iron deficiency symptoms in new leaves (Fig. S1), while chlorophyll content gradually decreased (Fig. S2). To further explore the effects of nitrate on iron uptake in apple, apple seedlings were treated with +Fe and -Fe nutrient solutions containing high concentration of nitrate (HN, 15 mM) and low concentration of nitrate (LN, 0.5 mM). To create an ideal experimental environment, we chose vermiculite rather than soil to cultivate the seedlings, which has no soluble Fe but contains Fe2O3. The application of ferrozine (FRZ) in -Fe treatments enabled chelation of Fe2+which limits the uptake of Fe2+. After 15 days, the newly growing leaves showed severe chlorosis and reduced chlorophyll content under HN-Fe treatment (Fig. 1a, b, c). By contrast, slight chlorosis was observed under LN-Fe treatment. Additionally, when there was no iron shortage, LN treatment seedlings had a lower chlorophyll content than HN treatment seedlings (Fig. 1a, c). These results suggested that LN treatment alleviates iron deficiency in seedlings. Meanwhile, the soluble Fe content of both newly growing leaves (Fig. 1d) and roots (Fig. 1e) significantly increased under LN-Fe treatment, even though vermiculite has very little soluble Fe. Additionally, soluble Fe content in young leaves of seedlings which were treated with LN solution with sufficient iron supplementation was higher than seedlings which were treated with HN solution with sufficient iron supplement treatment (Fig. 1d). These results implied that LN-triggered alleviation of Fe deficiency may be due to an increase in iron reutilization.