Hydrogen-rich water regulates cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner

Hydrogen gas (H₂) is an endogenous gaseous molecule in plants. Although its reputation is as a “biologically inert gas”, recent results suggested that H₂ has therapeutic antioxidant properties in animals and plays fundamental roles in plant responses to environmental stresses. However, whether H₂ regulates root morphological patterns is largely unknown. In this report, hydrogen-rich water (HRW) was used to characterize H₂ physiological roles and possible signaling transduction pathways in the promotion of adventitious root (AR) formation in cucumber explants. Our results showed that a 50% concentration of HRW was able to mimic the effect of hemin, an inducer of a carbon monoxide (CO) synthetic enzyme, and heme oxygenase-1 (HO-1), in restoring AR formation in comparison with the inhibition effect conferred by auxin-depletion treatment alone. It was further shown that the inducible effect of HRW could be further blocked by the co-treatment with N-1-naphthylphtalamic acid (NPA; an auxin transport inhibitor). The HRW-induced response, at least partially, was HO-1-dependent. This conclusion was supported by the fact that the exposure of cucumber explants to HRW up-regulates cucumber HO-1 gene expression and its protein levels. HRW-mediated induction of representative target genes related to auxin signaling and AR formation, such as CsDNAJ-1, CsCDPK1/5, CsCDC6, CsAUX22B-like, and CsAUX22D-like, and thereafter AR formation (particularly in the AR length) was differentially sensitive to the HO-1 inhibitor zinc protoporphyrin IX (ZnPP). Above blocking actions were clearly reversed by CO, further confirming that the above response was HO-1/CO-specific. However, the addition of a well-known antioxidant, ascorbic acid (AsA), failed to influence AR formation triggered by HRW, thus ruling out the involvement of redox homeostasis in this process. Together, these results indicated that HRW-induced adventitious rooting is, at least partially, correlated with the HO-1/CO-mediated responses. We also suggested that exogenous HRW treatment on plants might be a good option to induce root organogenesis.