Melatonin is widely involved in growth, development, and stress responses in plants. Although the melatonin synthesis enzymes have been identified in various plants, their interacting proteins remain unknown. Herein, overexpression of tryptophan decarboxylase 2 (MeTDC2)‐interacting proteins, N‐acetylserotonin O‐methyltransferase 2 (MeASMT2) interacting proteins, and N‐acetylserotonin O‐methyltransferase 3 (MeASMT3) in cassava leaf protoplasts resulted in more melatonin than when other enzymes were overexpressed. Through yeast two‐hybrid, 14 MeTDC2‐interacting proteins, 24 MeASMT2 interacting proteins, and 9 MeASMT3‐interacting proteins were identified. Notably, we highlighted MeWRKY20 and MeWRKY75 as common interacting proteins of the 3 enzymes, as evidenced by yeast two‐hybrid, and in vivo bimolecular fluorescence complementation (BiFC). Moreover, co‐overexpression of MeTDC2/MeASMT2/3 with MeWRKY20/75 in cassava leaf protoplasts did not only activated the transcriptional activities of MeWRKY20 and MeWRKY75 on W‐box, but also induced the effects of MeTDC2, MeASMT2/3 on endogenous melatonin levels. Taken together, 3 melatonin synthesis enzymes (MeTDC2, MeASMT2/3) interact with MeWRKY20/75 to form a protein complex in cassava. This information significantly extends the knowledge of the complex modulation of plant melatonin signaling.

中文翻译：

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褪黑素生物合成酶募集WRKY转录因子来调节木薯W-box上的褪黑素积累和转录活性

褪黑激素广泛参与植物的生长，发育和胁迫反应。尽管已经在多种植物中鉴定了褪黑激素合成酶，但它们的相互作用蛋白仍是未知的。在此，色氨酸脱羧酶2（MeTDC2）相互作用蛋白，N乙酰基5-羟色胺O甲基转移酶2（MeASMT2）相互作用蛋白和N乙酰基5-羟色胺O的过表达与过量表达其他酶相比，木薯叶原生质体中的甲基转移酶3（MeASMT3）产生更多的褪黑激素。通过酵母双杂交，鉴定出14种与MeTDC2相互作用的蛋白质，24种与MeASMT2相互作用的蛋白质和9种与MeASMT3相互作用的蛋白质。值得注意的是，我们强调了MeWRKY20和MeWRKY75是这3种酶的常见相互作用蛋白，这通过酵母双杂交和体内双分子荧光互补（BiFC）得以证明。此外，在木薯叶原生质体中MeTDC2 / MeASMT2 / 3与MeWRKY20 / 75的共表达不仅激活了MeWRKY20和MeWRKY75在W-box上的转录活性，而且还诱导了MeTDC2，MeASMT2 / 3对内源性褪黑激素水平的影响。总而言之，3种褪黑激素合成酶（MeTDC2，MeASMT2 / 3）与MeWRKY20 / 75相互作用，在木薯中形成蛋白质复合物。