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 .