Exploring plant-derived matrices for proline encapsulation to enhance proline-mediated heat stress tolerance

Proline plays a central role in plant adaptation to abiotic stress, but its exogenous application at high concentrations may disrupt proline metabolism and promote oxidative damage. This study evaluated whether controlled foliar delivery of proline using biodegradable, plant-derived matrices could prevent metabolic overload and improve plant tolerance to heat stress. Proline was encapsulated by spray drying using gum arabic, carboxymethylcellulose, maltodextrin and pectin, alone or in combination. The resulting formulations were characterized for their physicochemical properties, encapsulation efficiency, thermal stability and release kinetics. Based on their release behaviour and powder properties, CMC-Ara (1:1) and Malt-Pec (1:0.25) were selected for biological evaluation. Their effects were assessed in Arabidopsis thaliana wild-type plants and in sid2–1, apx1 and p5cdh mutants, which are impaired in salicylic acid biosynthesis, antioxidant metabolism and proline catabolism, respectively. Plant growth, photosynthetic performance, oxidative markers and proline metabolism were analysed under non-stress and heat stress conditions. Malt-Pec showed the slowest proline release and provided greater thermal protection than free proline. Under non-stress conditions, free proline and CMC-Ara reduced photosynthetic performance and increased H₂O₂, malondialdehyde, endogenous proline and pyrroline-5-carboxylate accumulation. In contrast, Malt-Pec maintained growth and oxidative parameters close to control levels and promoted a more coordinated regulation of proline biosynthesis and catabolism. Under heat stress, Malt-Pec improved rosette growth and reduced leaf damage in all genotypes compared with untreated and free-proline-treated plants. These responses were associated with moderate endogenous proline accumulation and lower pyrroline-5-carboxylate and H₂O₂ levels, including in the stress-sensitive apx1 and p5cdh mutants. Overall, the Malt-Pec matrix enabled a gradual proline supply that limited metabolic and oxidative overload while enhancing proline-mediated heat stress tolerance. This biodegradable formulation therefore represents a promising foliar strategy for improving plant resilience to high-temperature stress.