Abstract
Topping damage can induce the nicotine synthesis in tobacco roots, which involves the activation of JA and auxin signal transduction. It remains unclear how these hormone signals are integrated to regulate nicotine synthesis. Here we isolated a transcription factor NtWRKY-R1 from the group IIe of WRKY family and it had strong negative correlation with the expression of putrescine N-methyltransferase, the key enzyme of nicotine synthesis pathway. NtWRKY-R1 was specifically and highly expressed in tobacco roots, and it contains two transcriptional activity domains in the N- and C-terminal. The promoter region of NtWRKY-R1 contains two cis-elements which are responding to JA and auxin signals, respectively. Deletion of NtWRKY-R1 promoter showed that JA and auxin signals were subdued by NtWRKY-R1, and the expression of NtWRKY-R1 was more sensitive to auxin than JA. Furthermore, Yeast two-hybrid experiment demonstrated that NtWRKY-R1 can interact with the actin-binding protein. Our data showed that the intensity of JA and auxin signals can be translated into the expression of NtWRKY-R1, which regulates the balance of actin polymerization and depolymerization through binding actin-binding protein, and then regulates the expression of genes related to nicotine synthesis. The results will help us better understand the function of the WRKY-IIe family in the signaling crosstalk of JA and auxin under damage stress.
Highlights
Nicotine is a secondary metabolite exclusively synthesized in tobacco roots
The Open Reading Frame (ORF) of the cDNA was predicted by ORF Finder, and this gene was named as NtWRKY-R1 and the coding sequence was amplified by primers NtWRKY-R1-F/R (Supplementary Table 1)
To understand whether NtWRKY-R1 participates in JA- and auxin-mediated signal transduction and involves in topping damage-induced nicotine biosynthesis, the expression of NtWRKY-R1, putrescine N-methyltransferase (PMT), Auxin-induced protein 13 (IAA13), and PDF1.2 were determined in tobacco roots at 24 h after topping by qRT-PCR (Figure 1)
Summary
Nicotine is a secondary metabolite exclusively synthesized in tobacco roots. Topping, removal of the flowering head and adjacent young leaves, is an important cultivating measure for fluecured tobacco. Topping damage occurs in over-ground part of plant and changes the hormonal balance including the jasmonate (JA) and auxin (Baldwin et al, 1994), which leads to the increase of nicotine synthesis in tobacco roots (Hibi et al, 1994; Li et al, 2016). Both JA and auxin are important for plant response to wounding attack (Hibi et al, 1994; Machado et al, 2013; Zhang et al, 2016).
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