【作者】 张鹏英；

【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， 2008， 博士

【摘要】 植物合成并释放一定量的挥发性物质（Volatile organic compound,VOCs）,这些挥发性物质包括烷烃、烯烃、醇、醛、醚、酯和羧酸,是植物次生代谢产物中的一个重要类群。挥发性物质的大部分组分具有抑菌活性,是植物抗性的物质基础和抵御病原侵染的化学防御因素;其中一些组分是防御基因表达的调节因子,是病原侵染过程中植株组织间或相邻植株间传递信息的信号分子。因此,挥发性物质在植物防御、通讯和/或抵制极端环境中发挥着重要的作用。发育进程、诱导子、环境因素及各种胁迫均影响植物挥发性物质的释放。不同病害侵染不同发育时期的植物,植物在不同发育期对同一病害的敏感性不同,这种差异和植物挥发性物质之间的关系是本文研究的主要内容。本文用C-C/MS法测定了番茄叶片在不同发育时期（发育阶段与叶龄）挥发性物质释放变化特性,探讨了其抑菌活性和植物系统抗性的关系;研究了壳寡糖对不同发育时期番茄叶片挥发性物质释放的影响特性;进一步全面的确定了牛蒡低聚果糖对植物系统抗性中重要事件的效应,证实该低聚果糖为高效的系统抗性调节子;本文还研究了稀土元素钕和镧提高植物抗病性的机理。主要结果如下:1.以不同发育阶段植株上部相同叶龄番茄叶片为材料,动态顶空吸附法收集叶片释放的VOCs,GC/MS法对其进行定性和定量分析;SQ-PCR法研究VOCs合成途径中关键酶脂氧合酶、苯丙氨酸转氨酶和4-香豆酸CoA连接酶基因的表达;测定了番茄叶片脂氧合酶和苯丙氨酸转氨酶活性;悬滴法检测了番茄叶片释放的VOCs的抑菌活性;离体挑战接种法调查了番茄叶片对灰葡萄孢的敏感性。结果发现:随着植株的生长,同叶龄番茄叶片释放的VOCs质和量均显著增加;二叶期、十叶期和开花期植株释放的VOCs中分别鉴定出13、19和29种组分;开花期和十叶期植株VOCs总量分别是二叶期植株释放量的5.36和11.3倍。合成酶基因在不同发育阶段中的表达量与VOCs的释放具有一定的正相关性。脂氧合酶和苯丙氨酸转氨酶活性随着植株的生长而逐渐增加,分别于十叶期和开花期达到最高。VOCs的抑菌能力,随着植株生长而逐渐增强;不同发育时期番茄叶片对灰葡萄孢的敏感性随着植株的生长而显著降低,开花期植株比二叶期植株对灰葡萄的抗性高66.7%。该研究表明,同叶龄番茄叶片VOCs的释放量及其抑菌活性和叶片对病原真菌抗性随着植株生长显著增加;番茄叶片VOCs合成途径中关键酶基因表达量及酶活性与VOCs的释放呈一定的正相关。2.以开花期番茄为材料,研究了不同叶龄（按叶位划分）番茄叶片VOCs释放及其抑菌活性,和叶片对灰葡萄孢敏感性的差异。结果发现:番茄叶片释放VOCs的种类和量随着叶龄的增加而减少,幼叶、成熟叶和衰老叶释放的VOCs中分别鉴定出20、18和11种组分,幼叶VOCs的释放量分别是成熟叶和衰老叶释放量的1.42和3.09倍。番茄叶片VOCs的抑菌活性及叶片对灰葡萄孢敏感性随着叶龄的增加而降低,与VOCs的质和量成正相关。该研究表明,随着叶龄的增加,番茄叶片VOCs的释放量及其抑菌活性和叶片对病原真菌的抗性显著降低。3.以不同发育阶段及开花期番茄植株为材料,研究了不同发育时期番茄防御酶同工酶酶谱及防御酶活性的差异。结果发现,同叶龄番茄叶片过氧化物酶、过氧化物歧化酶和酯酶同工酶随植株生长均显著增加;叶龄对三种同工酶的影响显著低于植株发育阶段对这三种酶的影响。同叶龄番茄叶片的β-1,3-葡聚糖酶和几丁质酶在不同发育阶段有显著差异,β-1,3-葡聚糖酶和几丁质酶活性分别在开花初期和六叶期植株中活性最高;β-1,3-葡聚糖酶活性随着叶龄的增加而升高,几丁质酶活性变化不大。该研究表明,植物的一些抗病防御相关因子与植物发育时期密切相关,植株发育阶段对叶片防御相关因子的影响显著高于叶龄对它的影响。4.以5.0 mg/ml壳寡糖（CO）处理不同发育阶段番茄植株,研究了不同发育阶段植株番茄叶片VOCs的释放及防御酶对CO的响应。结果发现:同叶龄番茄叶片VOCs的释放对CO的响应依赖于植株的发育阶段;CO处理后,二叶期植株的响应最强,VOCs的总量及主要组分在48 h内有所下降,随后显著升高;其次为六叶期植株,VOCs释放量降低,但组分种类显著增加;开花期植株VOCs的释放量显著降低,组分变化不大。CO诱导后,二叶期植株番茄叶片内脂氧合酶和β-1,3-葡聚糖酶活性的增加幅度最大,其次为六叶期,开花期植株这两种酶活性也显著增加,但增加幅度较平缓;六叶期和开花期植株番茄叶片内几丁质酶活性显著增加,二叶期植株中该酶活性没有显著变化。该研究表明,同叶龄番茄叶片VOCs的释放及防御相关酶对CO诱导的响应与植物的发育阶段密切相关。5.以壳寡糖处理开花期植株,研究了不同叶龄番茄叶片VOCs释放及病原真菌敏感性对CO的响应。结果发现:不同叶龄番茄叶片VOCs释放对CO的响应有显著差异;经CO诱导后,VOCs总量及四类化合物总量的变化相似,幼叶VOCs的释放量在96 h前显著下降,随后有所回升;成熟叶和衰老叶VOCs的释放量显著升高,成熟叶的响应显著高于衰老叶。CO诱导后,幼叶、成熟叶和衰老叶对灰葡萄孢的抗性分别提高了33.1%、39.4%和34.2%。该研究表明,植物叶片VOCs的释放对CO的响应依赖于叶龄;CO诱导后不同叶龄番茄叶片VOCs释放量与叶片对病原真菌抗性的提高呈一定相关性。6.以5.0 mg/ml牛蒡低聚果糖（BFO）处理二叶期、六叶期和开花期植株,研究BFO对不同发育阶段植株番茄VOCs释放及防御酶活性的影响。结果发现:同叶龄番茄叶片VOCs的释放对BFO的响应依赖于植株的发育阶段;BFO诱导后,六叶期植株的响应最强,新检出15种组分,VOCs总量、芳香族、单萜类和倍半萜类化合物量均显著增加;其次为二叶期植株,120 h时总量增加为对照组的238.8%,增加了5种组分;开花期植株VOCs的变化主要在量上,其总量、脂肪酸衍生物和倍半萜类组分的释放量显著降低,芳香族和单萜类组分含量均显著增加。BFO处理后,β-1,3-葡聚糖酶的活性在二叶期植株中响应最强,其次为六叶期;同叶龄番茄叶片几丁质酶对BFO的响应随着植株的生长而增强。该研究表明,番茄叶片对BFO的响应与植物发育阶段有关,开花前植株番茄VOCs的释放对该果糖响应较强,不同发育阶段防御酶对该果糖的响应有差异。7.以牛蒡低聚果糖处理开花期植株,研究不同叶龄番茄叶片VOCs的释放及其抑菌活性、防御相关因子及叶片的病原敏感性对BFO的响应。结果发现:BFO诱导后,幼叶VOCs的释放量显著降低,脂肪酸衍生物在24 h内显著增加,芳香族化合物先略有下降后显著增加,萜类化合物显著下降;成熟叶VOCs总量、脂肪酸衍生物和芳香族化合物释放量都显著增加,单萜类化合物变化不明显,倍半萜类化合物略有增加;衰老叶四类挥发性化合物含量均显著增加,其中单萜类组分增加最为显著。BFO处理后,成熟叶和衰老叶VOCs的抑菌活性显著增加,幼叶VOCs的抑菌活性基本没有变化。BFO处理后,不同叶龄番茄叶片内源自由基产生速率随着叶龄增加而增加;β-1,3-葡聚糖酶在不同叶龄番茄叶片中的变化趋势相似,其中幼叶响应较强;几丁质酶活性在成熟叶中响应最高,其次为幼叶;BFO处理后,幼叶防御酶同工酶的响应最高,其次为成熟叶。BFO处理后,三个叶龄番茄叶片对灰葡萄孢的抗性均显著增强;其中衰老叶的增加幅度最大,其次为成熟叶,幼叶略低。该研究表明,BFO处理显著提高不同叶龄番茄叶片对病原真菌的抗性,这种抗性的提高除与VOCs的释放有关外,也与氧爆发和防御酶活性密切相关。8.以八叶期番茄为材料,研究牛蒡低聚果糖对植物水杨酸及水杨酸信号转导途径相关因子的影响。结果发现:BFO诱导后,局部叶和系统叶中的SA和SAG水平显著升高,系统叶的增加幅度低于局部叶。BFO处理后,局部叶内PR2和Chit、LoxD和Pall基因表达量显著增加;系统叶内PR2、Chit和Pall的表达量也显著升高,LoxD表达量有所下降。BFO处理后,局部叶和系统叶自由基产生速率、β-1,3-葡聚糖酶和几丁质酶活性显著增加,系统叶较局部叶迟缓。该研究表明,BFO能够通过依赖于水杨酸的信号转导途径诱导植物系统抗性。9.以黄瓜子叶为材料,研究稀土元素钕对氧胁迫下的植物保护作用,及钕和镧对黄瓜子叶抗性相关生理指标的影响。结果发现:Nd³⁺预处理及Nd³⁺与H₂O₂同时处理都能显著降低H₂O₂对植物造成的伤害,缓解氧胁迫引起的细胞膜相对透性增加、自由基产生速率和MDA含量升高,光化学效率降低。Nd³⁺和La³⁺处理后,黄瓜子叶内源SA及SAG的含量迅速升高;内源O₂^-·产生速率在12 h内逐渐升高,随后降低;β-1,3-葡聚糖酶和几丁质酶活性显著增加。该研究表明,稀土元素钕可以通过直接降低自由基的水平而保护细胞膜,减轻植物所受伤害;稀土元素钕和镧通过植物水杨酸信号转导途径提高植物抗性。综上所述,番茄叶片挥发性物质的释放随着发育时期的变化有显著不同,与植物对病原真菌敏感性的差异相一致,挥发性物质中的大部分组分具有抑菌活性,是植物抵御病原侵染的化学防御因素。因此,在植物与病原微生物相互作用中,挥发性物质的释放与植物在不同发育阶段的抗性差异有关（植物阶段性抗性）。不同发育时期番茄叶片挥发性物质的释放对寡糖诱导子的响应有较大差异。植物中天然存在的储存型糖——牛蒡低聚果糖促进了植物系统抗性中的主要事件,其作用与壳寡糖相似,可能是一种新型诱导子,在开发无公害植物病害防治剂具有重要应用前景。本文还发现可以提高植物抗病性的稀土元素可能是通过水杨酸信号转导途径发挥作用的。更多还原

【Abstract】 A large variety of volatile organic compounds（VOCs）are synthesized by a range of physiological processes in many different plant tissues and themselves also extremely diverse（30 000 compounds）,and include alkanes,alkenes,alcohols, aldehydes,ethers,esters and carboxylic acids.Most of them can be assigned to the following classes（in order of decreasing size）:terpenoids,fatty acid derivatives including lipoxygenase pathway products,benzenoids and phenylpropanoids,and various nitrogen and sulfur containing compounds.Those low-molecular-weight volatiles that have different functional groups play a vital role in the plant life cycle by providing a way for plants to interact with the surrounding environment.Many factors could effects the quantity and quality of VOCs emitted from plants,including developmental stage,growth condition,and all sorts of stresses.Most of VOCs have the inhibited activity on microbe growth and are a part of plant chemical defense. Some of VOCs have the activity to enhance defense genes expression,and are an intercommunion signal molecular between plants themself or neighbor.In plant-microbe interaction,when older leaves/plants display increased resistance or reduced susceptibility to pathogens,this form of resistance often is referred to as age-related resistance（ARR）.The onset of some ARR forms correlates with flowering,while others correlate with plant age,leaf size,or the synthesis of secondary metabolites or defense proteins.In this paper,we studied developmental-dependent variations of VOCs emission and their inhibitory activity to Botrytis cinerea and Fusarium oxysporum,and systemic acquire resistance（SAR）in tomato leaves in order to investigate the role of VOCs in plant ARR.The effects of crop development on the emission of volatile in tomato leaves and their antifungal activity towards chitosan oligosaccharide（CO）and Burdock fructooligosaccharide（BFO）were investigated.The ability of BFO to induce systemic acquire resistance（SAR）in tomato was studied.The cotyledons of cucumber were used to investigate the protection of Nd³⁺on plant against the oxidative stress,and the effects of Nd³⁺and La³⁺on physiological characters in respect of plant resistance.The mainly results are as followed:1.VOCs emitted from the leaves of tomato at different developmental stages（the two-,ten-leaf and anthesis periods）were collected by gas absorbing method and analyzed by GC/MS.The expressions of several key genes of the VOCs synthesis ways including lipoxygenase D（Lox D）,phenylalanine transaminase 1（Pall）and 4-coumarate CoA ligase（4-CL）were investigated by SQ-PCR.The effects of VOCs emitted from tomato on B.cinerea and F.oxysporum were examined by a modified hanging drop methods.The resistances of tomato leaves at three developmental stages to B.cinerea in vitro were also studied.The results demonstrated that plant developmental stage variations in the levels and composition of VOCs emitted from leaves were notably.With plant developing,VOCs emitted from the leaves of tomato were significantly increased.In total 13,19,and 33 constituents were identified in VOCs emitted from leaves of tomatoes at two-,ten-leaf and anthesis periods, respectively.The amounts of VOCs emitted from ten-leaf and anthesis plants were 5.36 and 11.3 folds of those emitted from two-leaf plant,respectively.The expressions of LoxD,Pal1 and 4-CL,and the activities of LOX and PAL have a positive correlation with VOCs emitted from leaves of tomato at different developmental stages.The antifungal activities of VOCs emitted from leaves and the resistances of leaves to B.cinerea were enhanced with plant growth.2.Age-dependent variations of VOCs emission,its inhibitory activity,and resistance to B.cinerea in tomato leaves were investigated.The results demonstrated that volatile emitted from tomato decreased with leaf age（based on the leaf positions on the plant）.Contents of VOCs decreased steadily with leaf age,from 452.4μg/g·Fw in young leaf to 146.6μg/g·Fw in old leaf.VOCs emitted from young leaf demonstrated the best antifungal activity on spores germination and hyphal growth against B.cinerea and F.oxysporum.The leaves of tomato become more susceptible to B.cinerea with age increasing.3.Several resistance related factors including isozyme,β-1,3-glucanase and chitinase activities in leaf of tomatoes at different development stages were analyzed. The results demonstrated that the isozymes of POD,SOD and EST in the same age leaves were enhanced with plant growth.Effects of leaf age on these isozymes were much lower than plant developmental stage.The activities ofβ-1,3-glucanase were enhanced significantly in anthesis plant,while chitinase were the highest of all in six-leaf plant.β-1,3-glucanase activities were enhaced with leaf age increasing,while chitinase activities were not significantly different in three age leaf.4.Developmental change in leaf volatile emission and several resistance factors in response to CO were studied.The results demonstrated that the leaves of tomato become less susceptible to oligosaccharide inducement with plant growth.The amount of VOCs and several mainly compounds emitted from leaves of two-leaf tomato decreased a little in 48 h,and then significantly increased after CO treatment. Although the total amount of VOCs emitted from the leaf of six-leaf tomato decreased a little,the kinds of VOCs were markedly increased by CO elicited.The amount of VOCs emitted from anthesis plant decreased significantly.After CO treatment.LOX andβ-1,3-glucanase activities in two-leaf plant were significantly enhanced,and become more susceptible than six-leaf and anthesis plants.Chitinase activities were markedly increased in leaves of six-leaf and anthesis plants at 72 h after CO treatment. There were no significantly changes in leaf of two-leaf plant after CO treatment.5.To investigate the influence of leaf age（based on the leaf positions on the plant） on volatile emissions and leaf resistance response to elicitation,the anthesis tomatoes were treated with CO.The results demonstrated that the leaves of tomato become more susceptible to CO with age increasing.The responses of young leaf to CO treatment that were attenuated or delayed included the quantity and quality of VOCs. VOCs released from old and adult leaves were dramatically increased after treatment with CO.In contrast,VOCs emitted from young leaf was decreased to 88.4%of the control at 72 h after CO treated.Resistances of leaves at three ages to B.cinerea were all enhanced by CO treated.There were an increase of 33.1%,39.4%and 34.2%in the resistance of young,adult and old leaf compared to the control group.6.The effects of plant development on the VOCs emission and defense-related enzymes response to BFO were studied.The results demonstrated that the response of tomato leaf to BFO depended on the plant developmental stages.VOCs emitted from six-leaf plant response to BFO were increased,and detected fifteen new compounds. The amounts of VOCs,aromatic compounds,monoterpene and sesquiterpenoids emitted from leaf of six-leaf plant were enhanced markedly after BFO elicited.For BFO treatment,VOCs emitted from leaf of two-leaf plant were significantly enhanced and 138.8%higher than the control.After BFO treatment,VOCs emitted from anthesis plant changes greatly in quantity and total amount of VOCs decreased sharply.The amount of oxygenated aliphatic compounds and sesquiterpenoids emitted from anthesis plant were significantly decreased,but the amounts of aromatic compounds and monoterpene were markedly enhanced after BFO treated.β-1,3-glucanase activity in two-leaf plant was notbly enhanced by BFO treatment. Chitinase activity response to BFO was enhanced with plant growth. 7.To investigate the influence of leaf age on volatile emissions and their antifungal activity of VOCs,defense-related factors and leaf eliciting resistance,the anthesis plant leaf were treated with Burdock fructooligosaccharide.The results demonstrated that the leaf of tomato become more susceptible to BFO with leaf age increasing.After BFO treatment,the synthesis and release were attenuated or delayed in young leaf in terms of the quality and quantity of VOCs.VOCs emitted from young leaf were significantly reduced after BFO treated.After BFO treatment,the amount of oxygenated aliphatic compounds and aromatic compounds were significantly enhanced,but there were no significantly different in the amount of monoterpene and sesquiterpenoids different in adult leaf.VOCs emitted from old leaf were markedly enhanced after BFO elicited,especially in the content of monoterpene.The inhibitory activity of volatile emitted from adult and old leaves increased significantly against two fungi after BFO,while VOCs emitted from young leaf were not changes.After BFO treatment the generation rate of O₂^-·become more susceptible in tomato leaf with leaf age increasing.The changes ofβ-1,3-glucanase activity were similar in three leaf age,while the increase in young leaf were the most in three leaf ages after BFO treatment.For BFO treatment,chitinase activity was enhanced significantly in adult leaf,and a little in old leaf.The isozymes of POD and SOD were markedly enhanced by BFO eliciting in young leaf,and were increased in adult leaf after BFO treated.For BFO elicited,resistances of leaves at three ages to B.cinerea were all enhanced.An increase of resistance in old leaf was higher to BFO than adult and young leaves.8.We studied the ability of BFO to induce SAR in tomato.It is revealed that after BFO eliciting,level of SA and SAG in the first leaf sprayed with BFO and the untreated leaf of the same seedling increased sharply.The changes of SA and SAG in untreated leaf were lower than this change in local leaf after BFO treatment.BFO treatment enhanced markedly the expressions of PR2,Chit,LoxD,and Pal1 in local leaf,and PR2,Chit,and in systemic leaf Pal1 expression also increased significantly. In addition,the generation rate of O₂^-·reached the highest at 3h in local leaf and 12h in systemic leaf,and the extent of oxygen burst in systemic leaf was much lower than this in local leaf.β-1,3-glucanase and chitinase activities significantly increased both in the first leaf treated with BFO and in systemic leaf.9.The protection of Nd³⁺on the cotyledons of cucumber against the oxidative stress by H₂O₂ was studied.The result revealed that both Nd³⁺pretreatments and Nd³⁺treated with H₂O₂ synchronous decreased the injury of H₂O₂ effectively.The permeability of cell membrane,the generation rate of O₂^-·and the content of MDA were obviously lower than that of H₂O₂ treatment.The cucumber cotyledons were sprayed with Nd³⁺ and La³⁺,and the changes on SA and SAG contents,the generation rate of O₂^-·,and the activities ofβ-1,3-glucanase and chitinase were measured.The results demonstrated that the yields of endogenous SA and SAG in cucumber cotyledons were enhanced significantly in a short time in response to Nd³⁺and La³⁺treatments. The generation rate of O₂^-·increased gradually after Nd³⁺or La³⁺treatments,and then decreased in cucumber at 12 h.β-1,3-glucanase and chitinase activities were significantly enhaced by Nd³⁺and La³⁺treatments.In summary,the emission of VOCs,some defense related factors,and the resistance to pathogen of tomato leaf were affected by plant developmental stage and leaf age. Most compounds of VOCs have the inhibitory microbe growth activity and play a key role in plant chemical defense.Thus,VOCs emitted from tomato leaf play a role in plant age-related resistance.VOCs emission,several PR genes expression and PR activities of tomato leaf in response to oligosaccharide elicit varied with plant developmental stage and leaf age.Studies of these forms of resistance may help us to evaluate more exhaustively the plethora of levels of regulation during development, the variability of the defense potential of developing hosts and may have practical application,making it possible to reduce pesticide applications.Furthmore,we also confirmed that BFO may induce the SAR response through an SA dependent signal pathway to enhance defense related genes expression and improve PR enzymes activities.Due to the low cost and abundant supply of substandard Arctium lappa roots,we believed that the activation of plant defenses using the BFO elicitor could be a valuable tool that would contribute to a new alternative strategy for developing plant protection.Rare earth（RE）,a kind of natural mineral,has been used as the beneficial elements to enhance crops resistance to abiotic and biotic stress for quite a long time. The mechanism about RE biological activities is not clear yet.In this paper,these results suggest that RE can enhance the resistance of plant to stress factors and diseases due to directly decrease the injury of free radical to plant and through the signal pathway of salicylic acid.更多还原