3.6 MT promoted nitrate accumulation and transcription ofMdHY5
Previous studies have shown that exogenous MT treatment enhances N uptake during N deficiency (Du et al. , 2022; Qiao et al. , 2019). To investigate the function of MdASMT9 in N absorption, the 15N-NO3 influx rate in WT and MdASMT9 -OE plants were measured. We found that under N-deficiency conditions, the15N-NO3 influx rate of MdASMT9 -OE plants was substantially higher compared to WT plants (Figure 5b). Under normal conditions, the NO3 concentration in the roots of OE lines were similar to those of WT plants (Figure 5a). However,MdASMT9 -OE plants exhibited more NO3 in their roots under N-deficiency conditions. NRT1.1 and NRT2 proteins also play key roles in NO3 absorption in response to the low NO3 conditions (Okamoto et al. , 2003; You et al. , 2022), so we analyzed the expression of MdNRT1.1 , MdNRT2.1 , MdNRT2.2 ,MdNRT2.3 , MdNRT2.4 , and MdNRT2.5 in the roots of WT and MdASMT9 transgenic plants under low-N conditions as well (Figure 5c,d; Figure S1). The results showed that MdASMT9 OE plants exhibited higher expression of MdNRT2.1 andMdNRT2.4 in roots than that of WT plants during low-N treatment (Figure 5c,d).
Previous studies have reported that HY5 positively regulates root nitrate absorption by activating NRT2.1 (Chen et al. , 2016; Huang et al. , 2015). Yao et al. (2021) found that exogenous MT and altered MT biosynthesis in Arabidopsis enhance the expression of HY5 to protect the plant from oxidative stress. Therefore, we examined whether MT increased the expression of MdHY5 under N-deficiency conditions (Figure 6a). MdHY5 expression was detected in apple plants treated with 0.5 μM melatonin (WT+MT) and overexpressed with MdASMT9 under N-deficiency conditions. The expression of MdHY5 in the roots of WT, WT+MT, OE-3, and OE-4 plants first increased and then decreased, reaching the maximum value at 16 d of low-N stress. Starting from the fourth day of low-N treatment, WT+MT, OE-3, and OE-4 plants had higher expression of MdHY5 than WT. Exogenous MT treatment and overexpression of MdASMT9 in apple plants thus appeared to enhance the expression of MdHY5 during low-N treatment.