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.

中文翻译：

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MdMPK3 和 MdMPK6 微调 MdWRKY17 介导的褪黑激素生物合成基因 MdASMT7 的转录激活

Melatonin ( N-乙酰基-5-甲氧基色胺) 是一种有效的活性氧 (ROS) 清除剂，可提高植物对生物和非生物胁迫的耐受性。植物中褪黑激素的信号传导和调节途径仍然难以捉摸。在这里，我们报道过表达转录因子基因MdWRKY17的转基因苹果 ( Malus Domestica ) 植物比对照植物具有更高的褪黑激素含量和更低的 ROS 水平，而MdWRKY17 RNA 干扰 (RNAi) 品系则显示出相反的表型。MdWRKY17 与N-乙酰血清素O-甲基转移酶 7 ( MdASMT7 )的结合直接促进MdASMT7体外和体内的表达。MdASMT7 是一种定位于质膜的褪黑激素合酶。MdASMT7 过表达挽救了 MdWRKY17-RNAi 品系较低的褪黑激素含量，证实了 MdWRKY17-MdASMT7 模块在苹果褪黑激素生物合成中的作用。此外，褪黑激素治疗激活了丝裂原激活激酶（MPK）MdMPK3和MdMPK6，它们磷酸化MdWRKY17以促进MdASMT7的转录激活。RNAi介导的MdMPK3/6沉默降低了过表达MdWRKY17的转基因苹果植物中MdASMT7的表达，这进一步证实了MdMPK3/6微调MdWRKY17介导的MdASMT7转录。这也形成了褪黑素激活MdMPK3/6从而通过触发MdMPK3/6-MdWRKY17-MdASMT7途径加速其自身生物合成的正循环。这种新颖的褪黑激素调节途径不仅剖析了褪黑激素生物合成的分子机制，而且为生产富含褪黑激素的转基因苹果提供了一种替代方法，可能有益于人类健康。