Nitrate reductase-mediated nitric oxide production is involved in copper tolerance in shoots of hulless barley.

KEY MESSAGE: An NR-mediated early NO production in the shoots of hulless barley plays an important role in protecting hulless barley from Cu toxicity through enhanced antioxidant enzyme activities and antioxidant pools. Nitric oxide (NO) has been identified as an important signaling molecule that is involved in multiple plant physiological responses, especially under some abiotic stress. Here, we investigated NO production and its effects on copper (Cu) excess in hulless barley shoots. An early NO burst at 24 h was observed in shoots of hulless barley, and the synthesis of early NO was mediated through nitrate reductase (NR), but not through nitric oxide synthase (NOS). Application of the NO donor sodium nitroprusside (SNP) efficiently alleviated Cu-induced shoot inhibition and decrease in chlorophyll content, as well as oxidative damage and reactive oxygen species (ROS) accumulation, while inhibiting NO accumulation by a specific NO scavenger or a NR inhibitor aggravated shoot inhibition as well as the increase of hydrogen peroxide (H2O2) content, supporting the role of an NR-mediated early NO production in hulless barley responses to Cu toxicity. Furthermore, elevated antioxidant enzyme activities were induced by Cu stress in the shoots of hulless barley and further significantly enhanced by NO donor, whereas suppressed by NO scavenger or NR inhibitor. On the other hand, the application of NO scavenger significantly reduced Cu-induced accumulation of glutathione (GSH) and ascorbate (Asc) in the shoots of hulless barley. Taken together, our results indicate that NO may induce hulless barley seedling tolerance to Cu toxicity through modulating antioxidant enzyme activity and antioxidants accumulation.