Zinc Oxide Nanoparticles Enhance Salt Stress Tolerance in Onobrychis Viciifolia by Modulating miRNA164a and miRNA399b Expression and Associated Physiological Mechanisms

Abstract

Nanoparticles have emerged as critical mediators of plant adaptive responses to abiotic stressors, particularly salinity. Here, we demonstrate that zinc oxide nanoparticles (ZnO NPs) mitigate NaCl-induced stress in two cultivars of Onobrychis viciifolia (A & ccedil;& imath;kyol and G & uuml;rp & imath;nar) through coordinated regulation of oxidative stress markers, osmolytes, and microRNA expression. O. viciifolia seeds were placed on MS medium with varying salt concentrations (50-150 mM), different ZnO NPs concentrations (0.5 and 1.5 ppm), and a combination of NaCl and ZnO NPs for 1 month. Salt stress caused a decrease in root and leaf length, but ZnO NPs application increased these lengths. Malondialdehyde (MDA) and Hydrogen peroxide (H2O2) levels rose with increasing salt stress. Specifically, in MDA content, 50 mM NaCl + 0.5 ppm ZnO NPs was observed in A & ccedil;& imath;kyol, and 150 mM NaCl + 1.5 ppm ZnO NPs in G & uuml;rp & imath;nar. For H2O2, cell damage was lowest at 50 mM NaCl + 1.5 ppm ZnO NPs in both cultivars. Total sugar content was highest at 1.5 ppm ZnO NPs in A & ccedil;& imath;kyol, and at 50 mM NaCl + 1.5 ppm ZnO NPs in G & uuml;rp & imath;nar. Proline levels increased with higher salinity in both cultivars, while chlorophyll levels decreased. Changes in miRNA164a and miRNA399b expression levels showed gene repression as salinity stress increased. In A & ccedil;& imath;kyol, miRNA164a and miRNA399b were overexpressed at 150 mM NaCl + 0.5 ppm ZnO NPs. In G & uuml;rp & imath;nar, miR164a was overexpressed at 50 mM NaCl + 0.5 ppm ZnO NPs, and miRNA399b under the same conditions. Laser scanning confocal microscopy (LCSM) results indicated that, under in vitro conditions, ZnO NPs could significantly mitigate the negative effects of salt stress on leaf samples. These findings support the results from SEM and LSCM analyses. Our study suggests that ZnO NPs may provide a simple and effective method to protect O. viciifolia from severe NaCl stress.