Osmopriming-induced salt tolerance during seed germination of alfalfa most likely mediates through HO signaling and upregulation of heme oxygenase.

The present study showed that osmopriming or pretreatment with low HO doses (2 mM) for 6 h alleviated salt-reduced seed germination. The NADPH oxidase activity was the main source, and superoxide dismutase (SOD) activity might be a secondary source of HO generation during osmopriming or HO pretreatment. Hematin pretreatment similar to osmopriming improved salt-reduced seed germination that was coincident with the enhancement of heme oxygenase (HO) activity. The semi-quantitative RT-PCR confirmed that osmopriming or HO pretreatment was able to upregulate heme oxygenase HO-1 transcription, while the application of N,N-dimethyl thiourea (DMTU as trap of endogenous HO) and diphenyleneiodonium (DPI as inhibitor of NADPHox) not only blocked the upregulation of HO but also reversed the osmopriming-induced salt attenuation. The addition of CO-saturated aqueous rescued the inhibitory effect of DMTU and DPI on seed germination and α-amylase activity during osmopriming or HO pretreatment, but HO could not reverse the inhibitory effect of ZnPPIX (as HO inhibitor) or Hb (as CO scavenger) that indicates that the CO acts downstream of HO in priming-driven salt acclimation. The antioxidant enzymes and proline synthesis were upregulated in roots of seedlings grown from primed seeds, and these responses were reversed by adding DMTU, ZnPPIX, and Hb during osmopriming. These findings for the first time suggest that HO signaling and upregulation of heme oxygenase play a crucial role in priming-driven salt tolerance.