Uncertainty of EIN2^{Ser645/Ser924} Inactivation by CTR1-Mediated Phosphorylation Reveals the Complexity of Ethylene...

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.