NanoZnO foliar application confers salinity tolerance in alfalfa: integrative physiological and transcriptomic analysis

Salt stress severely limits alfalfa ( Medicago sativa L.) growth and productivity. Although zinc oxide nanoparticles (ZnO NPs) may improve plant stress tolerance, the physiological and transcriptional responses of alfalfa to ZnO NP treatment under salinity remain insufficiently understood. Here, we evaluated foliar-applied ZnO NPs using physiological and comparative transcriptomic analyses. Under salt stress, ZnO NP treatment increased plant height by 13.83%, total antioxidant capacity by 30.69%, and DPPH radical-scavenging activity by 21.54%, while reducing malondialdehyde accumulation by 30.11%. Transcriptome analysis identified 1,526 differentially expressed genes in the SZ vs. S comparison. These genes were enriched in pathways related to calcium signaling, phytohormone signaling, photosynthesis, and reactive oxygen species metabolism. Selected members of the WRKY, ERF, bHLH, and NAC transcription factor families were also upregulated following ZnO NP treatment under salinity. These findings indicate that foliar ZnO NP treatment was associated with improved salinity tolerance and coordinated physiological and transcriptional changes in alfalfa. However, because equivalent soluble-Zn and bulk ZnO controls were not included, the observed responses cannot be attributed exclusively to nanoparticle-specific effects.