Citrate participates in nitrate-alleviated iron deficiency
Since citrate is known to function in the transport of Fe from root to shoot (Grotz & Guerinot. 2006), it seems likely that it may participate in the nitrate-regulated iron deficiency response in apple. To assess this possibility, we measured the contents of organic acids, including citrate (CIT) and malate (Mal), in apple roots. When Fe was adequate, HN treatment promoted citrate accumulation (Fig. 4a) but had no effects on malate content (Fig. 4b) in roots. Under Fe deficiency conditions, LN-treated roots showed a higher citrate content compared to HN-treated roots (Fig.4a), while no significant difference was observed in malate content (Fig. 4b). Citrate and malate content showed no significant difference in leaves under HN-Fe and LN-Fe treatment (Fig. S4a, b). These results indicated that low nitrate affects citrate content but not malate accumulation in roots.
To further verify whether citrate participated in LN-alleviated Fe deficiency, exogenous citrate was applied to Fe deficient plants. As shown in Fig. 4, exogenous citrate noticeably alleviated HN-mediated Fe deficiency, resulting in an increase in chlorophyll content (Fig. 4c, d), increased total Fe content in newly growing leaves (Fig. 4e), and decreased total Fe content in roots (Fig. 4f). The above results suggested that exogenous citrate was able to alleviate Fe deficiency by facilitating Fe transportation from root to shoot.