A natural small molecule 2-amino-3-methylhexanoic acid enhances tomato thermotolerance via stabilization of the key mediator SlGAD4.

Plant resistance inducers (PRIs) are bioactive compounds that trigger protective responses, offering promise for reducing stress-induced yield losses in agriculture. The natural PRI (2S, 3S)-2-amino-3-methylhexanoic acid (AMHA) markedly improves tomato (Solanum lycopersicum) tolerance to high temperature (HT), but its molecular target and mechanism of action remain elusive. Here, we show that AMHA improved tomato thermotolerance by sustaining photosynthetic efficiency, increasing osmoprotectant accumulation (proline, soluble proteins, and sugars), and alleviating oxidative damage through reduced reactive oxygen species accumulation and enhanced antioxidant capacity. Integrated transcriptomic analysis, together with AlphaFold3-based structural prediction and molecular docking, identified S. lycopersicum glutamate decarboxylase 4 (SlGAD4) as a putative target of AMHA. Lys369 and Leu375 were pinpointed as key residues mediating AMHA-SlGAD4 binding, as validated by isothermal titration calorimetry, microscale thermophoresis, and electron circular dichroism. Functional assays, including liquid chromatography-mass spectrometry, Western blotting, and gene silencing, demonstrated that AMHA thermostabilizes SlGAD4 without altering its catalytic activity, thereby maintaining γ-aminobutyric acid (GABA) levels under HT stress. This stabilization reinforces photosynthesis, osmoprotectant accumulation, and redox homeostasis. Phylogenetic analysis revealed that the AMHA-binding residues are evolutionarily conserved among GAD family proteins across plant species, implying that these conserved GAD proteins may be potential targets for AMHA in diverse plants. These findings provide mechanistic insight into AMHA action and support further investigation of AMHA-based strategies for improving plant thermotolerance.