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concentration gradient analysis (unpublished data).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 .