Transcriptome profiling of genes related to iron utilization in response to nitrate treatment
To better understand and explain the mechanism surrounding nitrate’s effects on iron deficiency responses, an RNA-seq analysis was carried out using seedlings of crabapple ‘Pinyiensis ’ treated with different nitrate concentrations. All seedlings were pre-treated with KCl (15 mM) for 3 days. Then, half of the seedlings were exposed to HN (15 mM KNO3, 5 µM Fe) treatment for 12 h, while another half were still treated with KCl (15mM, 5 µM Fe). RNA-seq analysis demonstrated that the expression levels of over 1600 genes changed in root samples (Fig. 5a). Among them, many genes were related to Fe uptake and transportation as well as Fe-related transcription regulators (Fig. 5b). The qRT-PCR analysis found that the plasma membrane H+-ATPase gene MdAHA2 was significantly induced by LN treatment, especially during the first day of iron deficiency (Fig. 6g). Moreover, the master transcriptional regulator of iron deficiency, MdFIT , was also induced by LN treatment (Fig. 6a). In the early stages of iron deficiency (1 day), the expression levels ofMdYSL1 , MdYSL3 , MdFER1 and MdNAS1 were higher in LN treatment than in HN treatment (Fig. 6d, c, b; Fig. 3f). A vacuole Fe transporter gene, MdNRAMP1 which transports Fe from the vacuole to the cytosol, was also significantly induced by LN treatment (Fig. 6f). These results indicated that the effect of LN on iron deficiency involved transcriptional regulation of multiple genes, including MdAHA2 , which is involved in rhizosphere acidification, Fe transporter genes (MdMATE43 , MdNAS1 , MdYSL1 ) andMdFIT promote the expression of downstream genes related to iron deficiency. Further analysis also showed that the expression of some other metal ion related genes were affected by nitrate, including genes associated with Zn, Al, and Cu (Fig. S5a).