ETHYLENE INSENSITIVE2 (EIN2) is a key component of ethylene signaling whose activity is inhibited upon phosphorylation of Ser⁶⁴⁵ and Ser⁹²⁴ by the Raf-like CONSTITUTIVE TRIPLE-RESPONSE 1 (CTR1) in the absence of ethylene. Ethylene prevents CTR1 activity and thus EIN2^{Ser645/Ser924} 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 ein2 mutant was complemented by the transgenes encoding EIN2, EIN2 variants with mutations that either prevent or mimic Ser⁶⁴⁵/Ser⁹²⁴ 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^{Ser645/Ser924} 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.

ETHYLENE INSENSITIVE2 (EIN2) 是乙烯信号传导的关键成分，在没有乙烯的情况下，Raf 样组成性三重响应 1 (CTR1)磷酸化 Ser ⁶⁴⁵和 Ser ^{924 时}抑制其活性。乙烯会阻止 CTR1 活性，从而阻止 EIN2 ^{Ser645/Ser924}磷酸化，并且蛋白水解切割的 EIN2 C 末端 (EIN2-C) 从内质网到细胞核的亚细胞运输会触发乙烯信号传导。在这里，我们报告了 EIN2 激活的乙烯信号的意外复杂性。在缺乏 CTR1 介导的负调控的情况下，EIN2 的激活部分需要乙烯。该EIN2突变体由编码 EIN2 的转基因补充，EIN2 变体具有阻止或模拟 Ser ⁶⁴⁵ /Ser ^{924 的}突变磷酸化，或 EIN2-C；并且所有携带这些 EIN2 衍生转基因的转基因品系都对乙烯有反应。此外，我们发现荧光蛋白标记的 EIN2 及其变体几乎不受乙烯的影响，并表现出类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，在 N 或 C 末端与荧光蛋白融合的 EIN2 蛋白的亚细胞定位模式是相似的，而 EIN2-C-YFP 主要在细胞质中观察到，而不是在细胞核中。蛋白质印迹和质谱分析表明 EIN2 的高度复杂性，它很可能被蛋白水解加工成多个片段。我们的结果表明全长 EIN2 的核定位，EIN2 ^{Ser645/Ser924 的}弱结合磷酸化状态和乙烯信号，以及由 EIN2 及其不同亚细胞区室中的蛋白水解产物引起的乙烯信号的复杂性。我们提出了一个替代模型来解释 EIN2 激活的乙烯信号。