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).