The effect of salicylic acid and magnesium oxide nanoparticle applications on cold stress tolerance and mirna156, miRNA173 gene expression levels in alfalfa (Medicago sativa L.) genotypes under cold stress

This study aims to evaluate the responses of alfalfa (Medicago sativa L.) genotypes (Van/Denizli) to cold stress and investigate the effects of exogenous salicylic acid (SA), magnesium oxide nanoparticles (MgO NPs), and their combination (SA+MgO NPs) on these responses. For this aim, alfalfa genotypes grown for 30 days were subjected to exogenous application of SA (1 and 2 mM), MgO NPs (5 and 20 ppm), and SA+MgO NPs, and the plants were exposed to cold stress at 10 °C and 4 °C for 14 days each, respectively. To determine the alleviating effects of the treatments on cold damage, changes in some biochemical parameters, scanning electron microscope (SEM) image analysis, and alterations in miRNA (miRNA156 and miRNA173) gene expression levels were examined. Both low-temperature applications increased total sugar, lipid peroxidation (LPO), H2O2, and proline contents compared to the control. Under the cold conditions, SA and MgO NPs alone led to a significant decrease in LPO and H2O2 contents and an increase in proline content, while not affecting total sugar content. The most effective application in alleviating the adverse effects of cold stress at both temperatures was the high concentration combination containing 2 mM SA+20 ppm MgO NPs. When evaluating miRNA gene expression levels, miR156 was down-regulated in the Van genotype at 10 °C and in the Denizli ecotype at 10 °C / 4 °C, while it was up-regulated in the Van genotype at 4 °C. miR173 was up-regulated in the Van genotype at 10 °C and in the Denizli ecotype at 10 °C / 4 °C, while it was down-regulated in the Van genotype at 4 °C. The application of 2 mM SA+20 ppm MgO NPs resulted in up-regulation of miR156 and down-regulation of miR173 at both low temperatures (10 / 4 °C) in both genotypes. The results of this study indicate that the combination of 2 mM SA+20 ppm MgO NPs enhances some biochemical and genetic adaptation mechanisms of the studied alfalfa genotypes to low temperatures, providing effective protection against cold stress.