3.6 MdWRKY33 negatively regulates the transcription ofMdNCED1 and MdNCED3
Analysis of the transcriptome data demonstrate that the expression of
9-cis-epoxycarotenoid dioxygenase (MdNCED1 ) (MD10G1194200) andMdNCED3 (MD05G1207300), involved in ABA biosynthesis, were
down-regulated in OE-3 and OE-4 (Figure 5D). QRT-PCR results showed that
after 1 h of heat stress, the MdNCED1 and MdNCED3expressions in MdASMT9 -OE lines were remarkably lower than in the
WT (Figure 6A and 6B).
Among these differentially expressed TFs, MdWRKY33 (MD04G1167700)
was significantly upregulated in OE-3 and OE-4 lines, with
log2FC values of 1.75 and 1.62, respectively (Figure
5D). Earlier studies have shown that WRKY33 acts on theNCED3/NCED5 promoter, thereby inhibiting its expression and
negatively impacting ABA biosynthesis in Arabidopsis (Liu et al.,2015). To investigate whether endogenous MT negatively regulated ABA
biosynthesis through MdWRKY33 , the MdWRKY33 expression
pattern in WT and MdASMT9 -OE lines under heat stress was
determined. QRT-PCR showed that MdWRKY33 expression increased
then decreased, reaching its highest level after 2 h of heat treatment
(Figure 6C). In addition, OE-3 and OE-4 lines had remarkably higherMdWRKY33 expression than the WT during high-temperature stress.
This confirms the MdASMT9 -driven activation of MdWRKY33expression under heat stress.
Analysis of the MdNCED1 and MdNCED3 promoters showed that
both contained the MdWRKY33 binding elements ‘TTGAAT’ (Figure 6D and
6E). To verify whether MdWRKY33 binds to MdNCED1 and MdNCED3promoters and assesses its effect on their expression, electromobility
shift assays ( EMSA) and dual-luciferase (LUC) reporter assays
were conducted. MdWRKY33 could bind to MdNCED1 promoter-probe P1
and MdNCED3 promoter-probe P2. MdWRKY33 cannot bind to mutated
probes. These findings suggest that MdWRKY33 specifically recognizes
‘TTGAAT’ elements in the MdNCED1 and MdNCED3 promoters in
vitro. Transient expression assays were performed in tobacco
(Nicotiana benthamiana ) leaves transformed withAgrobacterium tumefaciens to verify transcriptional activity. The
luminescence intensity of cells co-expressing 35S::MdWRKY33 andNCED1 pro::LUC was higher than in cells co-expressing empty vector
and NCED1 pro::LUC (Figure 6F and 6H). Similarly, cells
co-expressing 35S::MdWRKY33 and NCED3 pro:: LUC had higher
luminescence intensity than cells co-expressing empty vector andNCED3 pro::LUC (Figure 6G and 6I). These results demonstrated that
MdWRKY33 binds to MdNCED1 and MdNCED3 promoters and
inhibits their expression. This result preliminarily indicated that
MdWRKY33 negatively regulates the transcription of MdNCED1 andMdNCED3 .