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Abstract RAF-like kinases, members of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, are central integrators of external and internal signals in plant stress responses and growth regulation. These kinases mediate signaling through multiple hormone pathways, including abscisic acid-dependent and -independent pathways, ethylene signaling, and rapid auxin responses. Unlike typical MAPKKKs that function through kinase cascades, RAF-like kinases primarily employ direct phosphorylation of downstream targets and dynamic subcellular localization to mediate specific physiological responses. Here, we review the emerging roles of RAF-like kinases in Arabidopsis thaliana, highlighting their integrative functions in hormone signaling, stress responses, and growth control. The complex interplay between different RAF-like kinase subgroups and their diverse cellular targets underscores the intricate regulatory mechanisms plants have evolved to coordinate environmental responses with development. 

Abstract Elucidating kinase–substrate relationships is pivotal for deciphering cellular signaling mechanisms, yet it remains challenging due to the complexity of kinase networks. Herein, we report the development of a versatile DNA-based kinase assay platform for high-throughput profiling of plant protein kinase activities and substrate preferences. Our approach employs DNA-linked peptide substrates, facilitating quantitative and specific kinase activity detection through next-generation DNA sequencing. Leveraging DNA barcodes as quantitative readouts, our approach establishes a high-throughput, sensitive, and specific platform for dissecting kinase–substrate networks in plants, representing a powerful tool for elucidating signaling mechanisms in plants. 

Abstract Volatile compounds, such as nitric oxide and ethylene gas, play a vital role as signaling molecules in organisms. Ethylene is a plant hormone that regulates a wide range of plant growth, development, and responses to stress and is perceived by a family of ethylene receptors that localize in the endoplasmic reticulum. Constitutive Triple Response 1 (CTR1), a Raf‐like protein kinase and a key negative regulator for ethylene responses, tethers to the ethylene receptors, but undergoes nuclear translocation upon activation of ethylene signaling. This ER‐to‐nucleus trafficking transforms CTR1 into a positive regulator for ethylene responses, significantly enhancing stress resilience to drought and salinity. The nuclear trafficking of CTR1 demonstrates that the spatiotemporal control of ethylene signaling is essential for stress adaptation. Understanding the mechanisms governing the spatiotemporal control of ethylene signaling elements is crucial for unraveling the system‐level regulatory mechanisms that collectively fine‐tune ethylene responses to optimize plant growth, development, and stress adaptation. 

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.