Nano-priming modulates antioxidant enzymes and NHX/SOS-mediated ion homeostasis to improve salinity tolerance in barley genotypes

KEY MESSAGE: By controlling biochemical function and stress-related gene expression, ZnO nano-priming improved barley salinity tolerance beyond hydro-priming, demonstrating its coordinated biochemical and molecular effects. Salinity stress is a significant abiotic constraint that disrupts redox homeostasis and ionic equilibrium of barley. Nano-priming has emerged as an effective tool to mitigate these impacts by improving stress-responsive pathways. To investigate the biochemical and molecular responses to salt stress, 10 contrasting genotypes were selected from 170 genotypes screened under control conditions and at 200 mM NaCl using unprimed, hydro-primed, and nano-primed seeds. Biochemical traits, including protein, carbohydrates, proline, mineral absorption, and antioxidant activity, were assessed. QPCR was used to quantify the expression of 10 stress-related genes (CAT1, APX, SODA, GPX, GR, NHX1, NHX2, NHX3, SOS1, SOS3). Compared to unprimed and hydro-primed seeds, nano-priming significantly increased osmolyte accumulation, mineral absorption, and antioxidant enzyme activity. Principal component analysis and hierarchical clustering confirmed significant genotype-specific responses, with ionic traits (K⁺, K⁺/Na⁺) and antioxidant enzymes (CAT, GST, POD) driving separation under salinity. Under salinity, CAT1 was strongly expressed in the tolerant genotype HOR11370 (79.02-fold), while NHX3 was highly expressed in the tolerant genotype BCC1398 (208.16-fold). Nano-priming also significantly upregulates CAT1 in the sensitive genotype BCC532. These findings provide molecular evidence that nano-priming enhances barley resilience by reprogramming gene expression, ion transport, and antioxidant pathways. The differential regulations within and among tolerant and sensitive genotypes indicate that the integration of multiple mechanisms confers salinity tolerance in barley. These findings provide valuable insights into breeding strategies to improve crop performance under saline conditions.