| It is reported that, in plants, the production of hydrogen peroxide (H2O2) deriving from polyamine oxidation has been correlated with cell wall maturation and lignification during development as well as with wound-healing and cell wall reinforcement during pathogen invasion. As a signal molecule, H2O2 derived from polyamine oxidation mediates cell death, the hypersensitive response and the expression of defence genes. Rencently, it has been reported that, hydrogen peroxide generated by polyamine oxidative degradation is involved in the development of lateral roots in soybean, hydrogen peroxide generated by copper amine oxidase plays an important role in abscisic acid-induced stomatal closure in Vicia faba, moreover, the work in our lab revealed that CuAO and its catalysate H2O2 were involved in light/dark-regulated stomatal movement. However, the physiological roles of CuAO and its catalysate H2O2 in formation of adventitious root were still unclear. In the present study, using mung bean(Phaseolus radiatus L.) as materials, by means of pharmacology analysis, the laser scanning confocal microscopy (LSCM) and enzyme activity analysis, the roles of CuAO and its catalysate H2O2 in formation of hypocotyl adventitious root in mung bean were investigated. The present results had an important theoretical significance for further understanding the roles of polyamine oxidative degradation during root development and the formation mechanisms of adventitious root.The results are as follows:1. CuAO catalyse putrescine (Put) oxidative degradation, AG, which is a specific irreversible inhibitor of CuAO, reduced the number of adventitious root significantly. The effect of AG was dose and time dependent. Decrease of CuAO activity resulted in reduction of adventitious roots number suggested that, CuAO was probably involved in the adventitious root formation of mung bean hypocotyl cuttings.2. The products from putrescine oxidation by CuAO include H2O2, ammonia (NH3) and 4-aminobutanal.4-aminobutanal can be easily catalyzed toγ-aminobutyric acid (GABA), which is subsequently transaminated and oxidized to succinic acid (Succ). The present results suggested that only H2O2 had significant effects on promoting rooting and could reverse AG-induced inhibition of rooting. These results indicated that H2O2 generated by CuAO play an important role in the adventitious root formation of mung bean hypocotyl cuttings.3. Put is a substrate of CuAO, so the inhibition effect of AG on rooting might be related to the accumulation of Put. The present results showed that exogenous Put had no significant effect on rooting, showing that the inhibitory effect of AG on rooting was not related to accumulation of Put.4. By means of LSCM based on H2DCF-DA, a specific molecular probe of H2O2, the dynamic change of endogenous H2O2 fluorescence was detected in transverse sections of basal 0-2mm rooting area of the control and AG treated cuttings. It showed that the green fluorescence of H2O2 was mainly located in the rooting area between the vascular bundles in all cuttings. During 0-60h, the density of green fluorescence of H2O2 increased as the time going, the H2DCF-DA-positive cells increased gradually, the area of H2O2 fluorescence expanded, the increase of H2O2 fluorescence was preceded to the generation of root primordia. Compared with control cuttings contemporaneously, the density of H2O2 fluorescence in AG treated cuttings was less, the extent of H2O2 fluorescence was smaller, and the development of root primordium was posterior. The results suggested that AG did inhibit rooting through arousing the decrease of endogenous H2O2 content, H2O2 generated by CuAO was involved in adventitious root formation.5. The dynamic change of CuAO activity was detected by histochemical mean in transverse sections of basal 0-2mm rooting area of the control and AG treated cuttings.3,3-diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H2O2 in the presence of peroxidase, and to produce a reddish-brown conjugate, which is stable in most solvents, thus when enough substrate Put was provided, the difference of DAB staining between the control and AG treated cuttings could reflect the dynamic distribution and change of CuAO activity indirectly. It showed that the reddish-brown DAB staining was mainly located in the rooting area between the vascular bundles in all cuttings. During 0-60h, the density of DAB staining increased as the time going in the rooting area, cells stained by DAB increased gradually, the area of DAB staining expanded, the increase of CuAO activity was preceded to the generation of root primordia, which is parallel to the distribution and change of H2O2 fluorescence. Compared with control cuttings contemporaneously, the density of DAB staining in AG treated cuttings was less, also, the extent of DAB staining was smaller, the activity of CuAO was weaker and the development of root primordium was posterior. Compared with AG treated cuttings, the net increment of DAB staining in control suggested that CuAO activity showing increasing trend. These results suggested that CuAO indeed was involved in formation of adventitious root.In summary, the present results showed that CuAO and its catalysate H2O2 were involved in adventitious root formation of mung bean hypocotyl cuttings. Diamine oxidative degradation play an important role in formation of adventitious root. |
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