3.7 MT increased stomatal aperture through promoting MdWRKY33-mediated transcriptional inhibition of MdNCEDs
Because MdWRKY33 negatively regulate ABA biosynthesis, andMdASMT9 enhances MdWRKY33 transcription, we speculated that melatonin inhibited ABA biosynthesis through anMdWRKY33 -mediated pathway. Therefore, MdWRKY33 was transiently suppressed in WT apple plants using a tobacco rattle virus (TRV) system (TRV-MdWRKY33 ), validated by detected expression level of MdWRKY33 (Figure 7B). After exposure to 48°C for 8 h, plants infiltrated by empty TRV (EV) pretreated with 100 μM MT had less dehydration symptoms and lower REL than those without MT treatment (Figure 7A and 7D). TRV-MdWRKY33 plants had higher degree of wilting and higher REL than EV plants with or without MT. The concentration of MT used in this experiment was determined through concentration gradient analysis (unpublished data).
After exposure to heat stress, EV plants pretreated with 100 μM MT had higher stomatal aperture than those without MT treatment, but there was no significant difference in stomatal aperture between TRV-MdWRKY33 plants pretreated with MT and TRV-MdWRKY33plants not treated with MT (Figure 7E and 7F). As shown in Figure 7G,H,I, exogenous application of MT reduced the content of ABA and the expression of MdNCED1 /3 in EV plants under heat stress. However, there was no significant difference in ABA contents andMdNCED1 /3 expression levels between TRV-MdWRKY33plants pretreated with MT and TRV-MdWRKY33 plants not treated with MT.
To further explore whether MdWRKY33 plays an important role in endogenous MT-mediated stomatal movement under heat stress,MdWRKY33 was transiently suppressed in WT and MdASMT9 -OE plants before the plants underwent heat treatment (WT/MdWRKY33RNAi, OE-3/MdWRKY33 RNAi, OE-4/MdWRKY33 RNAi, respectively), validated by detected expression level of MdWRKY33(Figure 8A and 8B). Under high-temperature conditions, the REL of OE-3/EV and OE-4/EV was significantly lower than that of WT/EV, while the REL of OE-3/MdWRKY33 and OE-4/MdWRKY33 is not significantly different from that of WT/MdWRKY33 (Figure 8C). After exposure to heat stress, OE-3/EV and OE-4/MdWRKY33 had higher stomatal aperture than WT/EV, while the stomatal aperture of OE-3/MdWRKY33 and OE-4/MdWRKY33 is not significantly different from that of WT/MdWRKY33 (Figure 8D and 8E). OE-3/EV and OE-4/MdWRKY33 had lower ABA contents andMdNCED1 /3 expression levels than WT/EV under heat treatment (Figure 8F-H). However, ABA contents andMdNCED1 /3 expression levels of OE-3/MdWRKY33 and OE-4/MdWRKY33 is not significantly different from that of WT/MdWRKY33 . The above results indicate that MT increased stomatal aperture through promoting MdWRKY33-mediated transcriptional inhibition of MdNCED1 and MdNCED3 .