CuAO及其催化产物H₂O₂在绿豆下胚轴不定根发生过程中的作用研究

【作者】 任筠；

【导师】 佘小平；

【作者基本信息】 陕西师范大学 ， 植物学， 2010， 硕士

【摘要】 植物多胺氧化降解产物过氧化氢(hydrogen peroxide, H2O2)参与发育过程中的细胞壁成熟和木质化以及病原体感染过程中的创伤愈合、细胞壁加厚过程。作为一种信号分子,多胺氧化降解产物H2O2介导细胞死亡、超敏反应以及防御基因表达。最近的研究表明,多胺氧化降解产物H2O2参与大豆侧根发育,铜胺氧化酶(copper amine oxidases, CuAO, EC 1.4.3.6)催化产物H2O2在ABA诱导的蚕豆气孔关闭过程中发挥了重要作用,本实验室的研究也证实CuAO及其催化产物H2O2参与光/暗调控蚕豆气孔运动,但至今尚未见CuAO及其催化产物H2O2参与不定根发生的报道。本实验以绿豆(mung bean)为材料,借助药理学分析、激光扫描共聚焦显微镜技术及酶活性分析方法,研究了CuAO及其催化产物H2O2在绿豆下胚轴不定根发生过程中的作用。本研究对于进一步了解多胺氧化降解在根发育过程中的作用以及不定根发生机理有重要理论意义。主要实验结果如下：1. CuAO催化腐胺(putrescine, Put)氧化降解,所试浓度CuAO专一性抑制剂氨基胍(Aminoguanidine, AG)显著抑制不定根发生,且呈明显剂量和时间依赖效应。CuAO活性降低抑制生根,表明CuAO可能参与绿豆下胚轴插条不定根发生。2. CuAO催化Put氧化降解的主要产物是H2O2, NH3和4-氨基丁醛,4-氨基丁醛再经几步反应可生成γ-氨基丁酸(GABA)和琥珀酸(Succ)。本实验结果表明,H2O2、NH3、GABA和Succ四种催化产物中只有H2O2显著促进不定根发生,而NH3、GABA和Succ无显著促进作用。另外,四种催化产物中只有H2O2能够显著逆转AG对不定根发生的抑制效应。这些结果表明,CuAO的催化产物H2O2在绿豆下胚轴不定根发生中起作用。3.Put为CuAO催化底物,AG抑制不定根生成的效应也可能与Put增加有关。我们的结果证实,外源Put并不影响生根,可见CuAO抑制剂抑制不定根生成与Put增加无关。4.利用H2O2特异荧光探针H2DCF-DA和激光扫描共聚焦显微镜(LSCM)检测插条基部0-2mm生根区域横切片内源H2O2水平及其分布的结果表明,对照和AG处理插条H2O2荧光主要分布在维管组织之间的区域及正在形成的根原基中,0-60h时间范围内上述区域H2O2荧光逐渐增强,呈现H2O2荧光的细胞逐渐增多,荧光面积逐渐扩大,且H2O2荧光的增强先于不定根的形成。与对照同期相比,AG处理H2O2荧光较弱,荧光范围较小,根原基发育相对延迟。此结果表明,AG处理的确通过引起内源H2O2含量的降低从而抑制生根,CuAO催化产物H2O2确实参与了不定根发生。5.本实验以组织化学原位检测法对不定根发生过程中插条基部0-2mm生根区域横切片CuAO活性水平进行了检测。当过氧化物酶存在时,H2O2与3,3-二氨基联苯胺(Diaminobenzidine, DAB)结合随即原位生成稳定的红棕色聚合物,当提供足够底物Put时,对照和AG处理DAB染色的差异可间接反映出H2O2生成酶CuAO活性的大小及变化。结果表明,对照和AG处理插条基部生根区域DAB染色所得红棕色沉淀主要分布在维管组织之间的区域及正在形成的根原基中,0-60h时间范围内上述区域红棕色沉淀强度逐渐增加,呈现红棕色染色印迹的细胞逐渐增多,面积扩大,红棕色沉淀增加先于不定根形成,这些与H2O2荧光的分布及变化一致。与对照同期相比,AG处理红棕色沉淀较少,沉淀分布范围较小,根原基发育也相对延迟。对照与处理相比,DAB染色的净增量表明CuAO活性呈现逐渐增加的趋势。此结果表明,CuAO确实参与不定根发生。综上所述,本研究的结果表明CuAO及其催化产物H2O2确实参与绿豆幼苗下胚轴不定根发生,二胺氧化降解在不定根发生过程中起着重要作用。更多还原

【Abstract】 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.更多还原