【作者】 郭艳玲；

【导师】 陈靠山；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2014， 博士

【摘要】 激发子(elicitor)是一类能激活寄主植物产生抗病防卫反应的生物或非生物因子。牛蒡低聚果糖(Burdock fructooligosaccharide, BFO)是从牛蒡(Arcitum lappa L.)根中分离出来的储存型低聚糖。前期研究表明,BFO是一种有效的激发子,能够增强植物对多种病原真菌和病毒的抗性。细菌是仅次于真菌和病毒的第三大类植物病原物,BFO是否能够增强植物对病原细菌的抗性还没有研究。引发(priming),是指被激发子诱导后的细胞对随后的侵染发生更强烈的防卫反应的现象。引发是植物诱导抗性的重要机制；气孔关闭可以作为抵御细菌入侵的屏障,也是植物抗病反应之一；这两个机制都可能影响植物对病原细菌的抗性。本文以模式植物拟南芥(Arabidopsis thaliana)为材料,研究了BFO处理对拟南芥防御Pseudomonas syringae pv. tomato DC3000(Pst DC3000)的影响及其引发效应。研究发现,5.0g/L BFO预处理能够显著抑制Pst DC3000在拟南芥叶片组织中增殖,减轻发病症状。BFO处理后接种Pst DC3000,引发拟南芥发生更强更快的细胞和分子防卫反应,拟南芥叶片组织出现过氧化氢(hydrogen peroxide, H2O2)积累、胼胝质沉积和过敏性细胞死亡；Pst DC3000单独接种,拟南芥没有发生显著的H202积累、胼胝质沉积和过敏性细胞死亡。BFO处理3d后接种Pst DC30006h,比单独接种Pst DC3000,拟南芥PRl基因的表达量升高27.5倍,PAL1基因的表达量升高2.7倍。在接种PstDC3000时,添加过氧化氢清除剂过氧化氢酶(catalase, CAT), BFO的引发效应被抑制。BFO诱导的引发效应在不积累水杨酸(salicylic acid, SA)的转基因植株NahG、SA缺陷突变体sid2和SA不敏感突变体nprl-1中被抑制,但在脱落酸(abscisic acid, ABA)缺陷突变体aba3-1中,BFO的引发效应不受影响。这些研究结果表明,BFO处理增强了拟南芥对Pst DC3000的抗性；BFO诱导拟南芥进入引发状态,再接种病原菌,拟南芥表现出更强的细胞和分子防卫反应,即引发效应；BFO的引发效应依赖H2O2、SA信号通路和NPR1基因,但可能不依赖ABA信号通路。研究了BFO处理3d内的拟南芥组织内活性氧相关变化及其对引发的作用。结果显示,BFO处理引起拟南芥组织内H2O2积累,并在6h达到高峰,此时H2O2含量是对照的2.4倍；24h后H2O2含量基本恢复到原来水平并保持稳定。GST1基因是植物抗膜脂过氧化的关键基因,其表达受氧化应激调控,BFO处理引起GST1基因表达上调；活性氧清除酶相关基因FSD1, CAT1、CAT2、APX1和ZAT10基因表达也上调。活性氧清除酶如超氧化物歧化酶(SOD)、过氧化物酶(POD),过氧化氢酶(CAT)的酶活性升高。进一步研究发现,BFO单独处理能够诱导拟南芥积累H2O2,但不引起胼胝质沉积和过敏性细胞死亡。BFO处理3d后接种Pst DC3000,不仅激发拟南芥组织内H2O2积累,还引起了过敏性细胞死亡和胼胝质沉积。在BFO处理时添加CAT,外源CAT抑制了BFO诱导的H2O2积累；再接种Pst DC3000后,拟南芥组织不发生显著的H2O2积累、胼胝质沉积和过敏性细胞死亡,同时拟南芥对PSt DC3000的抗性也被抑制。这些结果表明BFO处理可以诱导拟南芥活性氧积累,影响拟南芥的氧化-还原平衡,使氧化态升高；BFO诱导的活性氧积累和氧化还原状态的改变使植物进入引发状态(priming state),对病原菌的刺激表现出更强的防卫反应。气孔运动与植物防御反应关系密切。研究了BFO对气孔运动的影响,结果显示,BFO处理可以诱导拟南芥气孔关闭,并诱导拟南芥保卫细胞内积累活性氧(reactive oxygen species, ROS)。我们选用研究气孔运动的常用植物豌豆进一步研究了BFO诱导气孔关闭的作用机制。研究发现,BFO处理可以诱导豌豆气孔关闭,200μg/mL BFO使豌豆气孔开度减小54.5%,并且在20-200μg/mL范围内,与BFO的剂量呈正相关。BFO处理20min时,豌豆保卫细胞中ROS和一氧化氮(nitric oxide,NO)含量显著增加,ROS和NO的荧光强度分别是对照的2.8倍和1.64倍。ROS抑制剂过氧化氢酶同时抑制了BFO诱导的ROS积累和NO积累；而NO抑制剂L-NAME抑制了BFO诱导的NO积累,部分抑制了BFO诱导的ROS积累。过氧化氢酶和L-NAME都抑制了BFO诱导的气孔关闭。这些研究结果表明,BFO可以诱导豌豆气孔关闭；BFO诱导的气孔关闭依赖ROS和NO信号；BFO诱导的ROS可能位于NO上游,是其诱导NO所必需的NO位于ROS下游,并可能反作用于ROS。BFO是植物自身产生的一种菊糖型低聚糖,本研究证实BFO能够增强植物对病原细菌的抗性,揭示了BFO的引发效应,发现BFO可以诱导气孔关闭,对理解BFO诱导植物抗病的作用机制有一定理论价值,也为BFO在农业生产上的应用进一步提供了理论依据。更多还原

【Abstract】 Plants can acquire strengthened resistance to pathogens after treatment with incompatible pathogens or elicitors. Elicitors are chemicals or biofactors that can induce plant defense resonses. Burdock fructooligosaccharide (BFO) isolated from the root tissue of Arctium lappa is a reserve carbohydrate. BFO alone has no anti-microbial activities in vitro, but instead, it induces defensive responses in plants as a potential elicitor. BFO can induce resistance against a number of plant diseases, including fungal diseases and virus diseases.This study shows that BFO could increase resistance against Pseudomonas syringae pv. tomato DC3000(Pst DC3000) in Arabidopsis. The growth rate of Pst DC3000declined in Arabidopsis leaves and disease symptoms were suppressed in BFO-pretreated plants. Burdock fructooligosaccharide (BFO) induces plant defense responses through priming, leading to rapid counterattack against pathogen. To investigate the effects of priming by BFO on defense-related responses, Arabidopsis was treated with BFO and the effects of pathogen challenge on cellular and molecular defense responses were analyzed. BFO treatment and subsequent Pseudomonas syringae pv. tomato DC3000challenge triggered earlier expression of defense response genes and pronounced cellular defense events, including a hydrogen peroxide (H2O2) burst, hypersensitive cell death (HCD), and callose deposition. BFO-induced priming was absent in NahG [a transgenic plant that does not accumulate salicylic acid (SA)], sid2(a SA-deficient mutant), and nprl-1(a mutant that carries a deficient NPR1gene), but not in aba3-1[an abscisic acid (ABA)-deficient mutant]. Removal of H2O2by catalase almost completely nullified the cellular and molecular defense responses. Our results indicated that BFO-induced priming is dependent on H2O2, SA and intact NPR1, but is not affected by the ABA signal transduction pathway.To explore whether the disturbed redox homeostasis also functions in BFO-induced priming, we detected the H2O2accumulation, the activities of reactive oxygen species scavengers and the transcription of their encoding genes while reducing reactive oxygen species levels and GST1. The results showed that BFO triggered H2O2accumulation in Arabidopsis during the period tested and the level of H2O2reach the highest at6hours post treatment, but restore to the original level at24hours post treatment. Meanwhile, BFO treatment improved the activities of reactive oxygen species scavengers, the transcription of their encoding genes while reducing reactive oxygen species levels and GST1. These results suggested that the plants have escalated oxidative state. Combined with catalase and Pst DC3000inoculation, we analyzed the cellular defense responses. Catalase, which infiltrated with BFO treatment, nullifies the escalated oxidative state and the augmented ROS accumulation when infected by Pst DC3000. Moreover, the disease resistance was also abolished in BFO pretreated but catalase treated plants, which suggests that the disrupted redox homeostasis is required in BFO-induced priming.Stomatal closure is a part of plant innate immune response to restrict bacterial invasion. In this study, the effects of BFO on stomata movement in Pisum sativum and the possible mechanisms were studied. The results showed that BFO could induce stomatal closure accompanied by ROS and NO production, as is the case with ABA. BFO-induced stomatal closure was inhibited by pre-treatment with L-NAME (NG-nitro-L-arginine methyl ester, hydrochloride; nitric oxide synthase inhibitor) and catalase (hydrogen peroxide scavenger). Exogenous catalase completely restricted BFO-induced production of ROS and NO in guard cells. In contrast, L-NAME prevented the rise in NO levels but only partially restricted the ROS production. These results indicate that BFO-induced stomatal closure is mediated by ROS and ROS-dependent NO production. Another, BFO could also induce stomatal closure in Arabidopsis in this study.更多还原