Methyl salicylate/methyl-β-cyclodextrin inclusion complex modulates plant defense against cold and heat stress

Temperature fluctuations beyond optimal limits such as heat or cold severely impair plant growth and productivity. Biostimulants are emerging as sustainable tools to enhance plant resilience under stress. Methyl salicylate (MeSA), a known defense modulator, holds promise as a biostimulant; however, its volatility and poor aqueous solubility limit its applications. To overcome these drawbacks, we have developed methyl-β-cyclodextrin (M-β-CD) based inclusion complex (IC) of MeSA. This study evaluated MeSA/M-β-CD-IC for improving temperature tolerance in Arabidopsis thaliana, offering a novel and environmentally compatible strategy for stress mitigation. Phase solubility analysis revealed that modified β-cyclodextrin (M-β-CD) enhanced MeSA solubility 4.41-fold, with a 1:1 inclusion stoichiometry. Spectroscopic, morphological and thermal analysis (FTIR, NMR, SEM and TGA) confirmed successful complexation and improved thermal stability. The in vitro release profile of MeSA/M-β-CD-IC indicated ~ 91% cumulative MeSA release at 120 min, validating enhanced aqueous release. Biologically, MeSA inhibited seed germination at ≥ 2.5 mM, whereas M-β-CD promoted germination at low concentrations. Notably, the MeSA/M-β-CD-IC alleviated MeSA-induced inhibition, enabling successful germination across all concentrations. Under cold and heat stress, plants treated with M-β-CD showed robust growth and biomass, while the MeSA/M-β-CD-IC treatment achieved intermediate yet significant protection compared with MeSA alone. Photosynthetic efficiency (Φmax, Fv/Fm, NPQ) and pigment contents were improved in IC-treated plants, reflecting enhanced photoprotection. Cold stress induced higher oxidative damage than heat, but MeSA/M-β-CD-IC markedly reduced reactive oxygen species and malondialdehyde accumulation. Molecularly, MeSA/M-β-CD-IC pre-priming enhanced the expression of cold-responsive (CBF, COR) and heat-responsive (HSFA, HSP) genes, along with major antioxidant genes (APX, CAT, GR, POD, SOD), indicating coordinated activation of stress signaling and tolerance pathways. Encapsulation of MeSA within M-β-CD substantially improves its aqueous solubility and biological efficacy. The inclusion complex strengthens Arabidopsis tolerance to cold and heat through activation of antioxidant and thermoprotective mechanisms. This work highlights cyclodextrin-based encapsulation as a sustainable, scalable approach for delivering volatile biostimulants to enhance crop resilience under climate stress.