Uncovering the versatility of CTR1: Newly Identified Protein Interactors, Subcellular Localizations, and Phosphorylation Substrates

Ethylene is a crucial phytohormone regulating plant development and stress responses. In the absence of ethylene, the kinase Constitutive Triple Response 1 (CTR1) phosphorylates Ethylene Insensitive 2 (EIN2), leading to its degradation and repression of ethylene signaling. Ethylene binding inactivates CTR1, allowing EIN2 to translocate into the nucleus and activate ethylene-responsive genes. While CTR1 was traditionally thought to be restricted to the endoplasmic reticulum (ER), recent evidence indicates its translocation to the nucleus, where it stabilizes EIN3 and enhances ethylene signaling.

To further elucidate the diverse roles of CTR1, we employed TurboID-based proximity labeling and mass spectrometry in Nicotiana benthamiana, identifying its proximal proteome. This analysis revealed interactions not only with core ethylene receptor Ethylene Response 1 (ETR1) but also with proteins linked to mitochondrial respiration, mRNA metabolism, and organelle biogenesis, suggesting broader cellular functions for CTR1.

Beyond ethylene signaling, we uncovered a novel role for CTR1 in chloroplast biogenesis. Independently of ethylene, CTR1 phosphorylates Toc33, a key component of the TOC (Translocon at the Outer Envelope of Chloroplasts) complex, which facilitates the import of nuclear-encoded photosynthetic proteins. Phosphorylation by CTR1 stabilizes Toc33 by preventing its ubiquitination-mediated degradation. Loss of this regulation disrupts photosynthetic protein import, compromising chloroplast structure and organelle development.

Our findings establish CTR1 as a multifunctional kinase that operates beyond ethylene signaling to regulate essential cellular processes. Its role in chloroplast biogenesis, independent of ethylene, highlights a previously unrecognized function in organelle development. This expanded regulatory scope underscores CTR1’s broader significance in plant physiology, revealing new layers of control in cellular and developmental processes.