Mechanistic insights into proteolytic activation ofmembrane-bound NAC transcription factor SlNACMTF3 intomato plants under heat stress

Stress is perceived at the cellular membrane, where membrane-bound transcription factors (MTFs) are activated, initiating a rapid gene-regulation cascade. We previously demonstrated the multifaceted role of tomato MTF, SlNACMTF3, in regulating expression of biotic and abiotic stress responsive genes. However, the molecular mechanism underlying its activation following stress perception remained unknown. Here, we show that the membrane rhomboid protease SlRP3 recognizes an ISK motif near the transmembrane domain of SlNACMTF3 for cleavage and releasing the transcription factor from the membrane. SlRP3 and SlNACMTF3 co-localize within the same plasma membrane microdomain and interact directly. Molecular dynamics simulations supported the proposed mechanism, in which heat-induced conformational switching of SlRP3, driven by reorientation of the L5 loop, enhances substrate accessibility and proteolytic activity. Furthermore, mutation of key SlRP3 residues involved in substrate interaction impaired SlNACMTF3 activation and reduced the expression of stress33 responsive genes. Analysis with SlNACMTF3-overexpressing tomato lines and SlRP3 transient knockdown leaves further revealed that transcription factor activation, stress35 responsive gene regulation, and cellular ROS detoxification occur coordinately. Collectively, these findings provide mechanistic insight into stress-triggered SlNACMTF3 activation and identify SlRP3 as a novel membrane-associated stress sensor that activates a major transcriptional regulator to enhance stress preparedness in economically important tomato plants.