MdMPK3 and MdMPK6 fine-tune MdWRKY17-mediated transcriptional activation of the melatonin biosynthesis gene MdASMT7

Abstract

Melatonin (N-acetyl-5-methoxytryptamine) is a potent reactive oxygen species (ROS) scavenger that increases the biotic and abiotic stress tolerance in plants. The signaling and regulation pathways of melatonin in plants remain elusive. Here, we report that transgenic apple (Malus domestica) plants overexpressing the transcription factor gene, MdWRKY17, have higher melatonin contents and lower ROS levels than those of control, while the MdWRKY17 RNA interference (RNAi) lines show the reversed phenotype. The binding of MdWRKY17 to N-acetylserotonin O-methyltransferase7 (MdASMT7) directly promotes the MdASMT7 expression in the in vitro and in vivo. MdASMT7 is a melatonin synthase that localizes to the plasma membrane. MdASMT7 overexpression rescued the lower melatonin contents of MdWRKY17-RNAi lines, confirming the role of MdWRKY17-MdASMT7 module in melatonin biosynthesis in apple. Furthermore, melatonin treatment activated the mitogen-activated kinases (MPKs) MdMPK3 and MdMPK6, which phosphorylate MdWRKY17 to promote transcriptional activation of MdASMT7. RNAi-mediated silencing of MdMPK3/6 decreases MdASMT7 expression in transgenic apple plants overexpressing MdWRKY17, which further confirms MdMPK3/6 fine-tunes MdWRKY17-mediated MdASMT7 transcription. This also forms a positive loop that melatonin activates MdMPK3/6 and thus accelerates the biosynthesis of itself via triggering MdMPK3/6-MdWRKY17-MdASMT7 pathway. This novel melatonin regulatory pathway not only have dissected the molecular mechanisms of melatonin biosynthesis but also provided an alternative approach for generating transgenic melatonin-rich apples which may benefits to human health.

Keywords: MdMPK3/6; MdWRKY17; biosynthesis; melatonin; phosphorylation; transcriptional regulation.