Figure 7.

MdNAC5^A and MdNAC5^T differentially interact with MdERF3 to regulate MdACS1. (A) The structure of MdNAC5^A and MdNAC5^T. (B) Docking prediction of MdNAC5^A and MdNAC5^T on MdERF3. (C) Luciferase complementation imaging assays showed that MdNAC5^A and MdNAC5^T interact with MdERF3. (D) BiFC assays showed that MdNAC5^A and MdNAC5^T interact with MdERF3 (indicated by the fluorescence observed in the nucleus of protoplasts). The YFP fluorescence coexpressed by MdNAC5^A/MdNAC5^T and MdERF3 Arabidopsis thaliana protoplasts was detected by confocal laser scanning microscope. From left to right, YFP, chloroplast, bright field, and merged field images. Bars = 10 μm. (E) Y2H assays showing that MdNAC5^A and MdNAC5^T interact with MdERF3. The fusion vectors pGBKT7-MdNAC5^A_△175–364/MdNAC5^T_△175–364 and pGADT7-MdERF3 were co-transformed in Y2H on SD/−Leu/−Trp (−LT) medium. Single colonies were dissolved in 10 μl of sterile 0.1% NaCl, coated, and transferred to SD/−Ade/–His/−Leu/−Trp/X-α-Gal (–AHLT) and SD/−Ade/–His/−Leu/−Trp/X-α-Gal (–AHLT/X) medium. Positive control: pGADT7-T/pGBKT7–53; negative control: pGADT7-T/pGBKT7-Lam. (F) MdERF3 was overexpressed in OE-MdNAC5^A and OE-MdNAC5^T apple calli. (G, H) The protein level and RNA level of OE-MdNAC5^A + OE-MdERF3 and OE-MdNAC5^T + OE-MdERF3 were identified. (I) The expression levels of MdACS1 in transgenic calli. Values are means of three replicates ± SD (Turkey’s test, ^***  P < 0.001, ^**  P < 0.01, ^*  P < 0.05).