CaO nanoparticles improve salt stress tolerance in diploid and tetraploid alfalfa (Medicago sativa L.) by modulating miRNA expression and physiological responses

Abstract This study investigated the effects of salt stress (NaCl) and CaO nanoparticles (CaO NP), as well as their combinations, on the expression levels of miRNAs in diploid (Bilensoy) and tetraploid (Alsancak) alfalfa varieties, while also assessing physiological and biochemical parameters. MiR159a, miR166a and miR393a have been downregulated by all applications in the leaves of both varieties. In the roots, following all treatments, only miR393a was downregulated in the diploid Bilensoy whereas in Alsancak, miR166a and miR393a were downregulated. MiR156a was the only miRNA consistently upregulated in both leaves and roots in all treatments in both varieties. Similarly, miR393a was found to be downregulated in all treatments, with the exception of one. The morphology of leaves, roots and stems that changed in both varieties with salt stress showed a tendency to improve with NP and combined applications. In both alfalfa varieties, the content of H 2 O 2 , soluble sugar, MDA and proline and the activities of POD and SOD increased in both leaves and roots with salt stress. However, combined applications showed varying trends, particularly notable decreases in some cases, in leaves and roots. These effects were more pronounced in Alsancak variety, particularly in the enzymatic activities observed in the roots. Photosynthetic pigments (chlorophyll and carotenoid), which decreased in the leaves of both varieties with salt stress compared to the control, also increased with combined applications. The supportive miRNA gene expressions, along with the observed morphological and physiological responses, highlighted the healing effect of CaO NPs in mitigating the impacts of salt stress in both alfalfa varieties.