The Roles Of H₂O₂ And Its Mechanism Mediate The Formation And Development Of Adventitious Roots In Mung Bean

Hydrogen peroxide (H₂O₂) is a moveable signaling molecule in plants. H₂O₂ is continually generated from various sources during normal metabolism. It is also produced in plants in response to a wide variety of abiotic and biotic stresses. It mediates some physiological and biochemical process, including systemic acquired resistance (SAR) and hypersensitive resistance (HR), senescence and programmed cell death (PCD), stomatal closure, root gravitropism and root development, formation of adventitious root, cell wall development, pollen-stigma interactions, et al., and gene expression in plants. Calcium fluxes and reversible protein phosphorylation are downstream components of H₂O₂ signaling cascade. Calcium and MAPKs cascade regulate the activity of other signaling proteins, further, expression of genes. H₂O₂ modulates the expression of various genes, including those encoding antioxidant enzymes, modulators of H₂O₂ production and peroxisome biogenesis genes. The intricate pathway of H₂O₂ signaling and its novel roles need to be revealed further. To date, we have not known whether H₂O₂ mediates the formation and development of adventitious roots.Adventitious roots arise from the stem and leaves and from non-pericycle tissues in old roots. It is one of the most important ways of vegetative propagation of plants. Many environmental and endogenous factors regulate adventitious rooting. Some of the endogenous factors have been identified including mineral nutrients, sugar, phenolics, ethylene, poryamines, nitric oxide, hydrogen peroxide, carbon monoxide, cGMP, MAPKs, phytohormone and peroxidase, function as messengers, and mediate auxin-induced adventitious rooting and auxin response gene expression. In recent years, some important progresses have made on auxin and adventitious roots response signaling pathway, for example, NO acts as signaling in auxin-induced adventitious rooting, and the IAA induces adventitious rooting via a pathway involving NO. cGMP and MAPK cascades are downstream signals of NO signaling pathway during adventitious rooting. Therefore, it is the question worthy to be investigated whether H₂O₂ which is similar to NO in chemical properties acting as a signaling in adventitious rooting.In this paper, the roles of H₂O₂ and its signal transduction and mechanisms in the formation and development of adventitious roots in mung bean (Mucuna pruriens (Linn.) DC.var. utilis) were investigated and reported, with the methods of morphology, physiology and biochemistry. The main results and conclusions obtained are as follows.1. Exogenous hydrogen peroxide (H₂O₂) has the effects on promoting the formation and development of adventitious roots in mung bean seedling explants. The treatment with 1-100 mM H₂O₂ for 8-18 h significantly promotes the formation and development of adventitious root. Catalase (CAT) and ascorbic acid (ASA), which are H₂O₂ scavenger or inhibitor, can eliminate the effects of exogenous H₂O₂ on promoting the adventitious rooting.2. H₂O₂ may be a messenger involving in IAA induced the adventitious rooting of seedling explants in mung bean. 2,3,5-Triiodobenzoic acid (TIBA) strongly inhibits adventitious rooting via blocking polar auxin transport in the stem. The formation of adventitious roots in mung bean was markedly inhibited as the concentration of TIBA used in treatment was high than 1μM, however, the inhibiting effects of TIBA on adventitious rooting can be partly reversed by IAA, also by H₂O₂.3. H₂O₂ may function as a downstream messenger in auxin signaling pathway and involve in auxin signal transduction induced adventitious rooting. DPI strongly inhibits the activity of NADPH oxidase which is one of the main sources of H₂O₂ formation in plant cells. The treatment with 10μM DPI for 48 h strongly inhibited the formation of adventitious roots in mung bean, and this inhibitory effects can be partly reversed by H₂O₂ or IBA. It indicated that as the generation of H₂O₂ through NADPH way was inhibited, the formation of adventitious roots was also inhibited, H₂O₂ mediated the IBA-induced the formation of adventitious roots.4. H₂O₂ may be only one of the downstream messengers involving auxin induced adventitious rooting. When CAT was applied with IBA together, CAT did not eliminate the effect of IBA on promoting adventitious rooting due to it is not a cell-permeable molecule. It is know that ASA is a cell permeable molecule. ASA used in the treatments can eliminate H₂O₂-induced increase in the number of adventitious roots, but can not eliminate IBA-induced increase in the number. The results suggested that H₂O₂ may be one of the signaling in the signal network of IAA in response to adventitious rooting. 5. IBA can promote the generation of H₂O₂ in seedling explants of mung bean. After 3 h the primary roots removal, IBA-treated markedly induced the generation of endogenous H₂O₂ in comparison with the treatment with water. This implies that the over-production of H₂O₂ was induced by IBA, and IBA promotes the formation of adventitious roots via a pathway involving H₂O₂.6. There was a crosstalk between H₂O₂ and NO signaling during the formation of adventitious roots, both may be the parallel downstream messengers in auxin signaling pathway, H₂O₂ may also be a downstream wignaling of NO. It is known that NO is a signaling in IAA-induced adventitious rooting, the treatment with DPI not only inhibite the H₂O₂-induced adventitious rooting but also inhibite the effect of NO.7. The formation of adventitious roots was not directly affected by EGTA, a chelator of extracellular calcium, but completely inhibited by Ruthenium red, a calcium channel blocker. It suggested that the calcium flow in the cytosol was needed during the induction of adventitious roots, and calcium was the downstream messenger in H₂O₂ signaling pathway.8. The formation and growth of adventitious roots was strongly inhibited by LY83583, a special inhibitor of GMP cyclase, the inhibitory effects of LY83583 can partly reversed by 8-Br-cGMP, H₂O₂ or IBA. It may indicate that H₂O₂ and IBA can promote the synthesis of cGMP in the cells, further, promote the adventitious rooting, and cGMP was downslream member in H₂O₂ signaling pathway.9. The formation and growth of adventitious roots was completely inhibited by 30μM PD98059, a special inhibitor of MAPKK. When the seedling explants were treated with 30μM PD98059 plus H₂O₂ or IBA, the effects of H₂O₂ and IBA on promoting the adventitious rooting were completely inhibited by PD98059. The results suggested that MAPK cascades mediated auxin response adventitious rooting. MAPKs are the downstream messengers in H₂O₂ signaling pathway.10. It was similar to the mechanism of IBA-induced adventitious rooting. H₂O₂-treated also markedly reduce the peroxidase (POD) activity in the induction phase of adventitious roots, in this phase, a high concentration of endogenous IAA was essential for the induction of adventitious roots, and the generation of H₂O₂ was induced by IAA, so the H₂O₂ signal transduction was started. Within 0 h-48 h after the primary roots removal in mung bean seedling explants, POD activity follow a rule of decrease—increase—decrease—increase. POD activity was significantly reduced at 3 h after the primary roots removal in the treatments with water or IBA. POD activity increased after 36 h after the primary roots removal, and the increase in POD activity was a prerequisite for the growth of adventitious root primordial because high POD activity can degrade the endogenous IAA, and low IAA concentration was needed in this development phase.11. Within 0 h-48 h after the primary roots removal in mung bean seedling explants, catalase (CAT) activity maintained a relative steadily level. Lesser changes in CAT activity took place during the formation of adventitious roots in the treatments used in experiments, but it has no statistical significance. The results may imply that CAT did not involve in the formation of adventitious roots.12. Both H₂O₂-treated and IBA-treated can significantly reduce the ascorbate peroxidase (APX) activity in the induction phase of adventitious roots. The change of APX activity was similar to that of POD during the formation of adventitious roots. Within 0 h-48 h after the primary roots removal in mung bean seedling explants, APX activity markedly reduced in treatments with water within 16 h-24 h and in treatments with IBA or H₂O₂ within 0 h-3 h after the primary roots removal, it increased within 36 h-48 h and reached a peak at 48 h.In conclusion, H₂O₂ may function as a signal molecule and promote the formation and growth of adventitious roots, and auxin induces adventitious rooting via a pathway involving H₂O₂. Both H₂O₂ and NO may be two parallel downstream signal molecules of auxin signaling cascade. Ca²⁺, cGMP and MAPK cascade are downstream signal molecules of H₂O₂ signaling pathway, and the components of auxin signaling network in response to adventitious rooting. The similar changs of activity of POD, CAT and APX induced by H₂O₂ and IBA indicate that the mechnisms of H₂O₂ on promoting adventitious rooting is same as that of IBA.