National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
Plant Commun. 2020 Apr 18;1(3):100046. doi: 10.1016/j.xplc.2020.100046. eCollection 2020 May 11.
ETHYLENE INSENSITIVE2 (EIN2) is a key component of ethylene signaling whose activity is inhibited upon phosphorylation of Ser and Ser by the Raf-like CONSTITUTIVE TRIPLE-RESPONSE 1 (CTR1) in the absence of ethylene. Ethylene prevents CTR1 activity and thus EIN2 phosphorylation, and subcellular trafficking of a proteolytically cleaved EIN2 C terminus (EIN2-C) from the endoplasmic reticulum to the nucleus and processing bodies triggers ethylene signaling. Here, we report an unexpected complexity of EIN2-activated ethylene signaling. EIN2 activation in part requires ethylene in the absence of CTR1-mediated negative regulation. The mutant was complemented by the transgenes encoding EIN2, EIN2 variants with mutations that either prevent or mimic Ser/Ser phosphorylation, or EIN2-C; and all the transgenic lines carrying these EIN2-derived transgenes responded to ethylene. Furthermore, we found that the fluorescence protein-tagged EIN2 and its variants were affected little by ethylene and exhibited similar subcellular distribution patterns: in the cytosolic particles and nuclear speckles. Of note, the subcellular localization patterns of EIN2 proteins fused with a fluorescence protein either at the N or C terminus were similar, whereas EIN2-C-YFP was primarily observed in the cytosol but not in the nucleus. Western blots and mass spectrum analyses suggested a high complexity of EIN2, which is likely proteolytically processed into multiple fragments. Our results suggested a nuclear localization of the full-length EIN2, weak association of the EIN2 phosphorylation status and ethylene signaling, and the complexity of ethylene signaling caused by EIN2 and its proteolytic products in different subcellular compartments. We propose an alternative model to explain EIN2-activated ethylene signaling.
乙烯不敏感 2 型（EIN2）是乙烯信号转导的关键组成部分，在没有乙烯的情况下，其活性会被 Raf 样组成型三重反应 1（CTR1）磷酸化 Ser 和 Ser 所抑制。乙烯阻止 CTR1 活性，从而阻止 EIN2 磷酸化，以及蛋白酶切割的 EIN2 C 末端（EIN2-C）从内质网到细胞核和处理体的亚细胞易位触发乙烯信号转导。在这里，我们报告了 EIN2 激活的乙烯信号转导的一个意外的复杂性。EIN2 的激活部分需要在没有 CTR1 介导的负调控的情况下需要乙烯。在缺乏 CTR1 介导的负调控的情况下， 突变体被编码 EIN2、具有阻止或模拟 Ser/Ser 磷酸化的 EIN2 变体或 EIN2-C 的转基因所互补；并且所有携带这些 EIN2 衍生转基因的 转基因系都对乙烯有反应。此外，我们发现荧光蛋白标记的 EIN2 及其变体受乙烯的影响很小，表现出相似的亚细胞分布模式：在细胞质颗粒和核斑点中。值得注意的是，与荧光蛋白融合的 EIN2 蛋白的亚细胞定位模式在 N 端或 C 端相似，而 EIN2-C-YFP 主要在细胞质中观察到，而不在细胞核中。Western blot 和质谱分析表明 EIN2 具有很高的复杂性，可能被蛋白酶切割成多个片段。我们的结果表明全长 EIN2 的核定位、EIN2 磷酸化状态与乙烯信号转导的弱相关性，以及 EIN2 和其在不同亚细胞区室中的蛋白水解产物引起的乙烯信号转导的复杂性。我们提出了一个替代模型来解释 EIN2 激活的乙烯信号转导。
