Abstract: Soil salinization is a major abiotic stress factor that reduces soybean production. Studies have shown that application of Chlorella promotes plant growth; however, its potential to mitigate salt stress, and the active components involved and the underlying mechanisms, remains unclear. In this study, the application of Chlorella vulgaris significantly mitigated salt stress in soybean seedlings. To identify the active components, we fractionated C. vulgaris and found that the extracellular secretions were primarily responsible for the mitigation. Furthermore, we isolated and characterized exosomes from these secretions. Phenotypic and physiological assessments confirmed that C. vulgaris exosomes alleviated salt stress in soybean seedlings to an extent similar to the intact organism. We identified the key metabolites (linolenic acid and inosine) as the active components within these exosomes. Notably, when applied in combination, they showed a strong synergistic effect, collectively promoting seedling growth, restoring ion homeostasis, and improving redox homeostasis under salt stress. To investigate the molecular mechanisms of salt stress alleviation, the transcriptomes of soybean seedlings subjected to different treatments were analyzed. Salt stress induced widespread changes in gene expression; however, application of linoleic acid (LA), inosine, and especially their combination, led to substantial transcriptomic reprogramming. Enrichment analysis indicated that these modifications were coordinated across energy metabolism, redox homeostasis, carbohydrate metabolism, and ion transport. Importantly, the LA + inosine combination induced a synergistic rather than simply additive transcriptional response, which explains its superior effect on salt tolerance at the molecular level. In this study, we successfully isolated and identified, for the first time, exosomes as the functional components through which C. vulgaris alleviates salt stress in soybean seedlings. Further analysis identified LA and inosine within the exosomes as the key active metabolites. These findings provide a novel theoretical basis for improving crop salt tolerance and are important for developing novel algal nanovesicle-based biostimulants.

Key words: Chlorella, exosomes, inosine, linolenic acid, salt stress, soybean