Nitric Oxide Is Involved In Hydrogen Gas-induced Adventitious Root Development In Cucumber And Marigold

Adventitious rooting is a key step in the vegetative propagation of plants. Adventitious root development is a complex process regulated by several lines of environmental and endogenous factors. In recent years, many signaling molecules have been identified in the field of signal transduction during adventitious rooting. However, the complex network of signaling molecules associated with adventitious rooting remains unclear. A better understanding of the regulation of initial adventitious rooting by signaling molecules will advance our understanding of the molecular mechanisms regulating adventitious root development. NO is a redox-active small signaling molecule which may regulate various physiological processes and almost all stress responses in plants. NO is also required for root organogenesis, including lateral root formation, root hair formation, and adventitious rooting. Although numerous studies have been demonstrated the involvement of NO in adventitious roots formation; there is currently little information on its mechanism. More recently, there have been some reports indicating that H2 may play critical roles in plant development and stress response. Notably, it was reported that H2 might regulate cucumber adventitious root development in a heme oxygenase-1/carbon monoxide-dependent manner was reported.H2 and NO have been considered as signaling modulators with multiple biological functions in plants. Little information is available, however, about the role and relaitionship between H2 and NO in promoting adventitious root development. The current study presents the role and relationship of H2 and NO on the adventitious root development of cucumber(Cucumis sativus ‘Xinchun 4’) and marigold(Tagetes erecta L. ‘Marvel’) explants. The followings are the main results of this study:1. HRW and NO donor sodium nitroprusside(SNP) promoted the formation of adventitious root in cucumber and marigold with a maximal biological response at 50% HRW and 50 μM SNP. Moreover, NO specific scavenger c PTIO, NOS inhibitor L-NAME, NR inhibitor Na N3 reversed H2-induced adventitious root development in cucumber and in marigold, which indicates that endogenous NO is involved H2-induced adventiotious root development.2. H2 and NO treatments activated cell cycle with the transition of cell from G1 to S phrase. At the same time, H2 and NO also induced cell cycle related genes Cyc A, Cyc B, CDKA and CDKB expression. Thus, H2 and NO promoted adventitious root development via regulating cell cycle related genes and activating cell cycle for cell division rapidly in cucumber. While, c PTIO, L-NAME and Na N3 depressed the induction of H2 on cell cycle phrase and cell cycle related genes. Know then, that H2 induces adventitious root development by regulating of NO on cell cycle acvtivation in cucumber.3. H2 and NO increased the transcript levels of target genes related rooting Cs CDNAJ-1, Cs CDPK1 and Cs CDPK5, but the increase of these genes induced by H2 was reversed in c PTIO, L-NAME and Na N3 treatments. It is suggested that endogenous NO is involved in H2-mediated the expression of target genes related rooting.4. H2 induced NO production with dose- and time-dependent during adventitious root development in cucumber. NO content reached maximal response in 50% HRW treatments after 24 h. However, c PTIO, L-NAME and Na N3 decreased the effect of H2 on induction of NO production. So, the formation of adventitious root of cucumber promoted by H2 is induction NO production of explants.5. H2 induced the expression of NOS-like and NR activity with dose- and time-dependent. 50% HRW treatments significantly increased NOS-like and NR activity after 24 h. While, L-NAME inhibited the increase of NOS-like activity, Na N3 inhibited the increase of NR. Thus, H2 induced adventitious root development of cucumber via two pathways NOS-like and NR as well as regulating their activity.6. H2 increased the expression of NOS-like and NR, which indicates that H2 regulates NOS-like and NR genes in transcript levels, and then induces adventitious root development.7. H2 decreased electrical conductivity(EC) and stomatal opening and increased relative water content in marigold explants. Thus, H2 provides enough water for the formation of adventitious root by protecting cell membrane from damages and decreasing stomatal opening for increase of water in explants.8. H2 regulated water soluble carbohydrate, starch and soluble protein content and provided enough materials and energy supply for the formation of adventitious root in marigold. And H2 also increased POD, PPO and IAAO activity in induction of adventitious root of marigold.