3.7 MdHY5 plays a positive role in N-deficiency tolerance
Previous studies have shown that shoot-derived HY5 moves to roots promotes NO3 uptake by activatingNRT2.1 (Chen et al. , 2016). To investigate whether MdHY5 binds to the promoters of MdNRT2.1 and MdNRT2.4 , their promoters were analyzed (Table S3). The results showed that theMdNRT2.1 promoter contains MdHY5 binding elements P1 and P2 (CACGTC), while the MdNRT2.4 promoter contains MdHY5 binding elements P3 (TACGTA) and P4 (CACGTA) (Figure 6b,c). EMSA results indicated that MdHY5 directly and specifically binds to the promoters of the MdNRT2.1 and MdNRT2.4 . Further, a transient expression assay showed that LUC were activated by the co-expression ofMdHY5 with MdNRT2.1 or MdNRT2.4 reporters significantly beyond the control (Figure 6d,e). These findings demonstrated that MdHY5 protein can directly and specifically bind toMdNRT2.1 and MdNRT2.4 promoters, thereby activating their expression.
To examine the function of MdHY5 in low-N stress responses, transgenic calli overexpressing MdHY5 (MdHY5 -OE) and interfering withMdHY5 (MdHY5 -RNAi) were generated (Figure 7a; Figure S2). After low-N treatment, the MdHY5 -OE apple callus had higher fresh weight (FW) and total N content than WT, while the FW and total N content of the MdHY5 -RNAi apple callus were significantly lower than WT (Figure 7a-c). In addition, under low-N conditions,MdHY5 -OE apple calli showed higher expression of MdNRT2.1and MdNRT2.4 than WT and significantly lower the expression ofMdNRT2.1 and MdNRT2.4 in MdHY5 -RNAi apple callus than WT (Figure 7d,e). These results indicated that MdHY5 enhanced the expression ofMdNRT2.1 and MdNRT2.4 , thereby improving the tolerance to low-N stress.