【作者】 李师翁；

【导师】 王勋陵； 安黎哲；

【作者基本信息】 兰州大学 ， 植物学， 2007， 博士

【摘要】 H₂O₂是植物细胞的可移动的信号分子，是植物细胞代谢的正常产物，各种生物和非生物胁迫能促使植物细胞产生过量H₂O₂，并通过H₂O₂信号转导响应环境胁迫。H₂O₂信号调控一系列重要的植物生理生化过程和基因表达，如系统获得抗性（SAR）和高度敏感抗性（HR），细胞衰老与程序化死亡（PCD），气孔关闭，根的向地性、根和侧根的生长，细胞壁的发育，柱头与花粉粒间的关系等。Ca²⁺流动和可逆蛋白磷酸化作用是H₂O₂信号途径的下游信号，通过MAPK级联作用于转录因子，最终调控基因的表达。已经发现H₂O₂调控多种基因的表达，包括编码抗氧化酶基因和调控H₂O₂产生的蛋白，以及过氧物酶体生物发生所需要的蛋白。虽然近年来对H₂O₂信号及其功能的研究取得了一系列重要进展，但关于H₂O₂在植物不定根形成与发育过程中的信号作用的研究还未见文献报道。不定根是指从茎、叶和老根的非中柱鞘组织产生的根，是植物无性繁殖的重要途径之一，普遍存在于被子植物和裸子植物界。各种外部环境的和内部的因子调控不定根的发生，已经发现的内部调控因子有矿质营养元素、糖、酚类、乙烯、多胺、NO、H₂O₂、CO、cGMP、MAPKs、植物激素和过氧化物酶等，其中生长素信号对不定根的形成起着关键作用，Ca²⁺、乙烯、NO、H₂O₂、CO、cGMP、MAPK等可能都是生长素信号途径中的信使分子，参与了生长素诱导的不定根形成的信号途径，并最终调控生长素响应基因和不定根响应基因的表达。近年来，对生长素和不定根响应信号的研究受到广泛关注，并取得了许多重要成果，如发现NO是生长素诱导的不定根形成的信号分子，在不定根的诱导中IAA可能是通过NO而起作用的，cGMP和MAPK级联是NO信号途径的下游分子。那么，在不定根的诱导中，与NO性质相似的H₂O₂是否也具有与NO相同的信号作用，正是本文需要研究的问题。本文基于当前的研究进展，运用形态学、生理学和生物化学的研究方法，以绿豆（Mucuna pruriens（Linn．）DC．var．utilis）为研究材料，研究了H₂O₂在不定根形成与发育中的作用、信号转导及其可能的生理生化机理。本文取得的主要结果与结论如下。1．外源H₂O₂具有促进绿豆幼苗外植体不定根形成与生长的作用。用1-100mMH₂O₂处理8-18h能显著促进不定根的形成与生长，H₂O₂清除剂CAT和ASA能去除外源H₂O₂对不定根形成和生长的促进作用。CAT是H₂O₂的敏感的清除剂，外源CAT对不定根的形成和生长没有显著影响，但外源100 U CAT和H₂O₂一同处理时，外源H₂O₂对不定根的促进作用被CAT去除；H₂O₂是ASA最重要的降解底物，4 mM ASA与H₂O₂一同处理时，外源H₂O₂对不定根的促进作用被ASA去除。2．H₂O₂是生长素诱导的绿豆幼苗外植体不定根形成过程中的信号分子。TIBA通过抑制IAA向茎基部的极性运输而强烈抑制不定根的形成，大于1μM的TIBA强烈抑制绿豆幼苗外植体不定根的形成，而TIBA对不定根形成的抑制作用能被IAA部分地逆转，同时也能被H₂O₂部分地逆转，这说明IAA诱导绿豆不定根的形成可能是通过H₂O₂而起作用。3．H₂O₂可能作为生长素的下游信号，参与生长素诱导的不定根形成的信号转导。DPI专一性抑制细胞内NADPH途径的H₂O₂的产生，发现10μM DPI处理48 h能强烈抑制绿豆不定根的形成，而H₂O₂和IBA能够部分地逆转DPI的抑制作用，这说明当NADPH氧化酶途径的内源H₂O₂的产生被抑制后，不定根的形成同时被抑制，证明H₂O₂可能参与了IBA诱导的不定根形成的信号过程。4．H₂O₂可能只是生长素诱导的不定根形成过程中的下游信号之一。CAT与IBA一同处理时，CAT不能去除IBA对不定根的促进作用，是由于CAT是非细胞透过的，所以对内源H₂O₂没有清除作用；由于ASA是细胞透过的，外源ASA能够清除细胞内的H₂O₂，ASA能够去除外源H₂O₂诱导的不定根数量的增加，但对IBA诱导的不定根形成没有明显去除作用，这可能说明，H₂O₂只是生长素响应的不定根形成信号网络中的成员之一。5．IBA处理能够显著促进绿豆幼苗外植体细胞内H₂O₂的产生。在初生根切除后3 h时，与H₂O处理相比较，IBA处理显著增加H₂O₂的浓度，这说明IBA诱导H₂O₂的产生，暗示IBA对不定根形成的诱导作用可能是通过H₂O₂实现的。6．H₂O₂和NO可能是生长素信号途径中两个平行的下游信号分子。已知NO是IAA诱导的不定根形成过程中的信号分子，但当用DPI抑制H₂O₂的产生，不仅抑制H₂O₂对不定根的诱导作用，而且NO诱导的不定根的形成作用也被抑制。这可能暗示H₂O₂和NO都是生长素信号途径的下游分子，而且H₂O₂可能位于NO的下游。7．Ca²⁺可能是H₂O₂下游信号分子。胞外Ca²⁺螯合剂EDTA（1 mM）对绿豆幼苗外植体不定根的形成没有显著地抑制作用，而胞内Ca²⁺释放抑制剂钌红（10μM）则完全抑制了不定根的形成，且这种抑制作用不能被H₂O₂和IBA去除，钌红（10μM）也完全抑制H₂O₂和IBA对不定根形成的诱导作用，这说明Ca²⁺的流动是不定根形成所需要的，且Ca²⁺可能是H₂O₂下游信号分子。8．cGMP可能是H₂O₂信号途径的下游成员。GMP环化酶专一性抑制剂LY83583（20μM）对绿豆幼苗外植体不定根的形成与生长有显著抑制作用，LY83583与cGMP类似物8-Br-cGMP、H₂O₂或IBA共同处理时，8-Br-cGMP、H₂O₂和IBA能部分逆转LY83583的抑制作用，这说明H₂O₂和IBA可能促进cGMP的合成，进而促进不定根的形成，cGMP可能是H₂O₂信号途径的下游成员。9．MAPK级联参与了生长素响应的植物不定根信号途径，且MAPK信号级联位于H₂O₂信号的下游。MAPKK专一性抑制剂PD98059（30μM）完全抑制绿豆幼苗外植体不定根的形成，PD98059也完全抑制H₂O₂和IBA诱导的不定根的形成，表明，MAPK级联参与了生长素响应的植物不定根信号途径，且MAPK信号级联位于H₂O₂信号的下游。10．与IBA的作用机理相似，H₂O₂和IBA处理都能显著降低不定根诱导阶段POD的活性，与这一时段需要较高的IAA浓度相关，而较高的IAA浓度可能在于促进H₂O₂的产生，启动H₂O₂信号转导。在绿豆幼苗外植体不定根形成的0 h-48 h内，POD活性的变化基本遵循降—升—降—升的规律，当用H₂O₂和IBA处理后3 h时，POD活性显著降低，在3 h-36 h之间，POD活性发生升降的变化，而在36 h后，POD活性升高，这一时段要求较低的IAA浓度以保证不定根原基的长出，较高的POD活性促进内源IAA的分解，降低了内源IAA的浓度，可能也在于降低H₂O₂的浓度。11．在绿豆幼苗外植体不定根形成的0 h-48 h阶段，CAT活性保持在一个相对稳定的水平，仅有比较小的变化，而这些变化基本没有统计学意义，这可能暗示，在不定根的诱导与根原基的形成阶段，CAT对细胞内H₂O₂浓度的变化不如POD敏感。12．H₂O₂和IBA处理都能显著降低不定根诱导阶段APX的活性，APX活性的变化规律与POD活性的变化极其相似，在不定根的形成过程中，APX与POD活性的变化具有同等意义，进一步说明H₂O₂与IBA的作用机理相同。IBA和H₂O₂处理，在0 h-3 h时间段降低APX活性，在36 h-48 h时间段升高APX活性，APX活性在48 h时均达到峰值。综上，我们认为H₂O₂在绿豆幼苗外植体不定根形成过程中，起信号分子的作用，能够促进不定根形成与生长，生长素对不定根形成的诱导作用可能是通过H₂O₂而起作用的，H₂O₂和NO可能是生长素信号途径中的两个平行的下游分子，且H₂O₂可能位于NO的下游，Ca²⁺、cGMP和MAPK级联都是H₂O₂信号途径的下游分子，共同组成生长素信号网络。H₂O₂和IBA对POD、CAT和APX活性的影响，反映出它们在不定根形成过程中具有相同的作用机理。更多还原

【Abstract】 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.更多还原