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Chloroplast biogenesis, essential for photosynthesis, depends on the import of nuclear-encoded proteins through the translocon at the outer envelope of chloroplasts (TOC) complexes. Despite its importance, the mechanisms regulating this process remain largely elusive. We identify serine-260 (S260) as a critical phosphorylation site in Toc33, a core TOC component. This phosphorylation stabilizes Toc33 by preventing its ubiquitination and degradation. Constitutive triple response 1 (CTR1), a negative regulator of ethylene signaling, and its paralog RAF-like kinase are involved in phosphorylating Toc33. Disruption of Toc33 phosphorylation impairs its stability and photosynthetic protein import, consequently affecting chloroplast structural integrity and biogenesis. Our findings underscore the essential role of TOC phosphorylation in chloroplast biogenesis and reveal an unexpected regulatory network involving RAF-like kinases in organelle development. 

Abstract The phytohormone ethylene controls plant growth and stress responses. Ethylene-exposed dark-grown Arabidopsis seedlings exhibit dramatic growth reduction, yet the seedlings rapidly return to the basal growth rate when ethylene gas is removed. However, the underlying mechanism governing this acclimation of dark-grown seedlings to ethylene remains enigmatic. Here, we report that ethylene triggers the translocation of the Raf-like protein kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1), a negative regulator of ethylene signaling, from the endoplasmic reticulum to the nucleus. Nuclear-localized CTR1 stabilizes the ETHYLENE-INSENSITIVE3 (EIN3) transcription factor by interacting with and inhibiting EIN3-BINDING F-box (EBF) proteins, thus enhancing the ethylene response and delaying growth recovery. Furthermore, Arabidopsis plants with enhanced nuclear-localized CTR1 exhibited improved tolerance to drought and salinity stress. These findings uncover a mechanism of the ethylene signaling pathway that links the spatiotemporal dynamics of cellular signaling components to physiological responses. 

Ethylene influences plant growth, development, and stress responses via crosstalk with other phytohormones; however, the underlying molecular mechanisms are still unclear. Here, we describe a mechanistic link between the brassinosteroid (BR) and ethylene biosynthesis, which regulates cellular protein homeostasis and stress responses. We demonstrate that as a scaffold, 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS), a rate-limiting enzyme in ethylene biosynthesis, promote the interaction between Seven-in-Absentia of Arabidopsis (SINAT), a RING-domain containing E3 ligase involved in stress response, and ETHYLENE OVERPRODUCER 1 (ETO1) and ETO1-like (EOL) proteins, the E3 ligase adaptors that target a subset of ACS isoforms. Each E3 ligase promotes the degradation of the other, and this reciprocally antagonistic interaction affects the protein stability of ACS. Furthermore, 14–3-3, a phosphoprotein-binding protein, interacts with SINAT in a BR-dependent manner, thus activating reciprocal degradation. Disrupted reciprocal degradation between the E3 ligases compromises the survival of plants in carbon-deficient conditions. Our study reveals a mechanism by which plants respond to stress by modulating the homeostasis of ACS and its cognate E3 ligases. 

Abstract Protein‐protein interactions play a crucial role in driving cellular processes and enabling appropriate physiological responses in organisms. The plant hormone ethylene signaling pathway is complex and regulated by the spatiotemporal regulation of its signaling molecules. Constitutive Triple Response 1 (CTR1), a key negative regulator of the pathway, regulates the function of Ethylene‐Insensitive 2 (EIN2), a positive regulator of ethylene signaling, at the endoplasmic reticulum (ER) through phosphorylation. Our recent study revealed that CTR1 can also translocate from the ER to the nucleus in response to ethylene and positively regulate ethylene responses by stabilizing EIN3. To gain further insights into the role of CTR1 in plants, we used TurboID‐based proximity labeling and mass spectrometry to identify the proximal proteomes of CTR1 inNicotiana benthamiana. The identified proximal proteins include known ethylene signaling components, as well as proteins involved in diverse cellular processes such as mitochondrial respiration, mRNA metabolism, and organelle biogenesis. Our study demonstrates the feasibility of proximity labeling using theN. benthamianatransient expression system and identifies the potential interactors of CTR1 in vivo, uncovering the potential roles of CTR1 in a wide range of cellular processes.