蓟马取食、机械损伤以及外源水杨酸甲酯和茉莉酸对菜豆叶片防御酶活性的影响

【目的】探讨菜豆对昆虫取食防御反应的生化机制。【方法】研究了西花蓟马Frankliniella occidentalis取食、机械损伤以及外源水杨酸甲酯（MeSA）和茉莉酸（JA）处理后菜豆叶片防御酶活性的变化。【结果】西花蓟马取食、机械损伤及MeSA和JA处理均能明显提高过氧化物酶（POD）的活性,前2种处理POD活性在72 h上升到最高峰,而后2种处理则在48 h达到最高峰。蛋白酶抑制剂（PI）活性在西花蓟马取食后升高最明显。JA途径关键酶脂氧合酶（LOX）和多酚氧化酶（PPO）的活性在西花蓟马取食、机械损伤和JA诱导处理均升高,但外源MeSA诱导处理则不能诱导它们的活性(P>0.05)。SA途径的关键酶苯丙氨酸解氨酶（PAL）在西花蓟马取食和机械损伤后均有一个先升高后下降的过程,外源MeSA诱导只在24 h引起PAL活性升高,其余时间下和对照没有明显的区别,外源JA诱导未能引起PAL活性的显著变化(P>0.05)。西花蓟马取食、JA和MeSA诱导以及机械损伤均能诱导β-1,3 葡聚糖酶（PR-2）活性上升(P<0.05)。【结论】结果说明,不同处理可诱导菜豆植株产生明显的防御反应,但酶活性的变化与处理方式和处理时间有关。     

不同诱导处理对番茄叶片营养物质含量 及防御酶活性的影响

本文研究了番茄在不同时间下受西花蓟马Frankliniella occidentalis危害(DTF)、机械损伤(MW)、茉莉酸(JA)和水杨酸甲酯(MeSA)外源诱导后,叶片营养物质含量和防御酶活性的变化。结果表明:各种诱导处理24 h和36 h时,番茄叶片可溶性蛋白和可溶性糖含量不同程度的下降,其中虫害处理36 h时,叶片可溶性蛋白和可溶性糖含量下降最明显;在48 h时除水杨酸甲酯处理外,番茄营养物质含量均显著升高。β-1,3葡聚糖酶(PR-2)活性在虫害、机械损伤和茉莉酸处理24 h和36 h后均升高,其中虫害处理的PR-2活性最高。各种处理均能诱导番茄叶片苯丙氨酸解氨酶(PAL)活性明显升高,且均随时间的延长持续升高。所有处理24 h时的番茄叶片多酚氧化酶(PPO)均被激发。各种处理均能导致植株的脂氧合酶(LOX)活性升高,但不同处理诱导的LOX活性升高的时间不同。结果表明,番茄能通过改变营养物质含量和防御酶活性对不同诱导处理作出生理应激反应,但反应程度与诱导方式和时间有关。     

外源钙和茉莉酸甲酯诱导番茄植株抗灰霉病研究

以‘辽园多丽’番茄幼苗为材料,研究了经钙(Ca)、钙螯合剂(EGTA)和茉莉酸甲酯(MeJA)处理后接种番茄灰霉病幼苗叶片的病情指数、活性氧(H2O2、O2.-)含量和过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、过氧化物酶(POD)活性的变化。结果显示:(1)Ca、MeJA、MeJA+Ca处理番茄幼苗的灰霉病发病率分别比对照显著降低32.5%、38.0%和54.5%,而MeJA+Ca处理又显著低于Ca、MeJA处理32.6%和15.3%;MeJA+EGTA处理高于MeJA处理30.3%,但低于EGTA处理13.1%;Ca处理低于EGTA处理34.2%。(2)Ca、MeJA及MeJA+Ca处理番茄幼苗叶片中活性氧积累量高于对照,MeJA+Ca处理又高于Ca、MeJA处理;但MeJA+EGTA处理活性氧积累量低于MeJA处理,而高于EGTA处理;Ca处理的活性氧含量高于EGTA处理。(3)Ca、MeJA及MeJA+Ca处理幼苗叶片的SOD、CAT、POD的活性均比对照提高,且以MeJA+Ca处理最高;而MeJA+EGTA处理抗氧化酶活性低于MeJA处理,但高于EGTA处理;Ca处理抗氧化酶活性高于EGTA处理。研究表明,钙在茉莉酸甲酯诱导番茄抗灰霉病过程中具有重要调节作用,这种作用与钙促进茉莉酸甲酯诱导番茄活性氧积累和抗氧化酶活性有关。     

茉莉酸甲酯诱导邻近番茄植株抗斜纹夜蛾的浓度和距离效应

茉莉酸甲酯(Methyl jasmonate, MeJA)与植物间的防御通讯密切相关,其挥发性强,可以在空气中传播而诱导邻近植物产生防御反应,为探明MeJA诱导邻近植株抗虫性的浓度和距离效应,本研究在大棚(长4 m×宽1 m×高1 m)进口处喷以不同浓度MeJA,检测离气味源60 cm、120 cm、180 cm、240 cm和300 cm远的番茄植株叶片多酚氧化酶(Polyphenol oxidase, PPO),过氧化物酶(Peroxidase, POD)和脂氧合酶(Lipoxygenase, LOX)的活性以及斜纹夜蛾幼虫取食不同处理番茄植株叶片后的体重增长率,研究结果表明1 mmol/L和10 mmol/L MeJA挥发物可以显著诱导增强邻近番茄植株叶片PPO,POD和LOX酶的活性,以离10 mmol/L MeJA气味源60 cm处植株叶片的酶活性最高,并随着距离的增加而呈降低的趋势;取食60 cm和300 cm对照组植株叶片的斜纹夜蛾幼虫其体重增长率没有显著差异,然而1 mmol/L和10 mmol/L MeJA处理下,取食300 cm处植株叶片的斜纹夜蛾幼虫其体重增长率显著高于取食60 cm处的,相同时间下以取食10 mmol/L MeJA处理组60 cm处植株叶片的幼虫体重增长率最低,以上研究结果表明MeJA对邻近番茄植株抗虫性的诱导有明显的浓度和距离效应。     

不同方式诱导植物抗性对甜菜夜蛾幼虫及其寄生蜂斑痣悬茧蜂发育表现的影响

【目的】明确诱导植物抗性对植食性昆虫甜菜夜蛾 Spodoptera exigua (Hübner) 及内寄生蜂斑痣悬茧蜂 Meteorus pulchricornis (Wesmael) 生长发育的影响。【方法】采用取食损伤、机械损伤及外源喷施3种浓度 (0.01, 0.1和1 mmol/L) 茉莉酸甲酯 (MeJA) 3种方式来诱导菜薹抗性,观察甜菜夜蛾初孵幼虫及其寄生蜂斑痣悬茧蜂的发育表现。【结果】取食取食损伤和茉莉酸甲酯处理的菜薹后,甜菜夜蛾幼虫存活率显著降低,茉莉酸甲酯浓度与幼虫存活呈负相关; 取食0.1 mmol/L MeJA处理的菜薹后,甜菜夜蛾幼虫体重显著低于清水对照。甜菜夜蛾取食取食损伤和0.1 mmol/L MeJA处理的菜薹,其寄生蜂斑痣悬茧蜂幼虫的死亡率显著增加,同时结茧率显著降低;0.1 mmol/L MeJA处理的菜薹使成蜂体重显著低于对照。不同处理间斑痣悬茧蜂的卵巢发育无明显差异,卵巢由8~10根卵巢管组成,羽化24 h后成熟卵数量为7~10个,成熟卵的长度和宽度分别为350和78 μm。【结论】结果说明,取食损伤和茉莉酸甲酯处理诱导的菜薹反应会影响甜菜夜蛾的存活和大小,进而抑制斑痣悬茧蜂生长。     

虫咬及挥发物诱导后合作杨叶片中多酚氧化酶活性的变化

经杨扇舟蛾(Clostera anachoreta)幼虫取食,杨扇舟蛾幼虫诱导的挥发物熏蒸,及茉莉酸甲酯(MeJA)、水杨酸甲酯(MeSA)、顺式己烯醛(cis-hexenal)、反式己烯醛(trans-hexenal)及苯骈噻唑(benzothiazole)熏蒸后,分析了合作杨(Populus simonii×P. pyramidalis 'Opera 8277')叶片中多酚氧化酶(PPO)活性的变化.虫咬叶及其上部叶片中PPO活性增加了2～4倍,表明合作杨产生了系统抗性.经虫咬诱导的挥发物熏蒸后,合作杨叶片中的PPO活性增加了1倍,推测是合作杨受伤后释放出了具有信号作用的挥发物,诱导邻近植株产生了防御反应.5种外源挥发物都引起了叶片PPO活性升高,因此均可能是合作杨间潜在的气体信号,并且混合挥发物的诱导效果显著高于单一挥发物.EGTA(钙离子螯合剂)和氯化镧(钙通道抑制剂)明显抑制了合作杨虫咬叶片、虫咬上部叶片及虫咬诱导挥发物熏蒸叶中PPO活性的升高,说明钙信号在虫咬及挥发物诱导的PPO活性升高中起着重要作用.     

3种不同类型口器的昆虫取食对菜豆叶片PPO和PAL活性的影响

为研究不同口器昆虫取食对菜豆植株系统防御的影响,以斜纹夜蛾(Spodoptera litura)咀嚼取食、二斑叶螨(Tetranychus urticae)刺吸取食和西花蓟马(Frankliniella occidentalis)锉吸取食分别处理菜豆中部叶片6、24、48、72和96 h后,测定菜豆受处理的中部叶片及未处理的上部及下部位叶片多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)的活性。结果表明:危害方式、处理时间和叶片部位对菜豆叶片PPO和PAL活性有极显著影响(P0.01),且三者间有极显著的交互作用。不同危害方式诱导后中部处理叶片的PPO活性均是在6 h明显升高(P0.05),但随时间的延长PPO活性在不同处理下变化不同;中部叶片PAL活性在机械损伤处理48 h后才被显著激活,而斜纹夜蛾、二斑叶螨和西花蓟马取食后,PAL活性分别在6、6和24 h明显升高,且达到最大值,分别是对照的2.66、1.75和2.79倍;处理菜豆的上部和下部未受害叶片的PPO和PAL活性对虫害的响应明显比机械损伤迅速;二斑叶螨取食后PAL活性响应最快,上部和下部叶片均在6 h达到最大值,分别为对照的2.72和5.07倍。以上结果说明,害虫取食能引起菜豆植株产生系统防御反应,并且防御的时空效应与害虫的为害方式有关。     

茉莉酸对棉花单宁含量和抗虫相关酶活性的诱导效应

以植物生长调节物茉莉酸(Jasmonic acid,JA)为诱导子,以常规棉为研究对象,探讨了外源茉莉酸对棉花幼苗单宁和蛋白酶抑制素以及其它抗虫相关酶活性诱导的浓度依赖性和持久性,讨论了棉花抗虫相关物质的抗虫效果.结果表明,0.01、0.1和1.0 mmol/L茉莉酸都能在2周内诱导棉花单宁和胰蛋白酶抑制素(Proteinase inhibitors,PIs)含量增加,诱导多酚氧化酶(Polyphenol oxidase,PPO)、苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)、过氧化物酶(Peroxidase,POD)和过氧化氢酶(Catalase,CAT)活性升高.对3种浓度茉莉酸的诱导效应进行分析表明,0.1 mmol/L茉莉酸对于诱导PIs、PPO、POD和CAT最有效,0.1和1.0 mmol/L茉莉酸对于诱导棉花单宁和苯丙氨酸解氨酶等效,二者的诱导效应均高于0.01 mmol/L.对茉莉酸诱导抗性的持久性进行分析表明,最佳诱导效应发生的时间各不相同:POD活性在JA处理后第1天最高,随后呈下降趋势,PIs和单宁含量分别在JA处理后第7天和第14天达最大值;JA处理后第1天和第7天的PPO活性无明显差异,但明显高于第14天;JA处理后第7天和第14天的PAL活性无明显差异,但明显高于第1天;JA处理后第1、7和14天棉花叶片的CAT活性均无明显差异.以上结果表明,茉莉酸可通过增加棉叶单宁和PIs含量、提高棉叶PAL、PPO、POD和CAT活性等增强棉花幼苗的抗虫性.     

茉莉酸在植物诱导防御中的作用

茉莉酸(JA)和茉莉酸甲酯(MeJA)作为与损伤相关的植物激素和信号分子,广泛地存在于植物体中,外源应用能够激发防御植物基因的表达,诱导植物的化学防御,产生与机械损伤和昆虫取食相似的效果。大量研究表明,用茉莉酸类化合物处理植物可系统诱导蛋白酶抑制剂(PI)和多酚氧化酶(PPO),从而影响植食动物对营养物质的吸收,还能增加过氧化物酶、壳聚糖酶和脂氧合酶等防御蛋白的活性水平,导致生物碱和酚酸类次生物质的积累,增加并改变挥发性信号化合物的释放,甚至形成防御结构,如毛状体和树脂导管。经茉莉酸处理的植物提高了植食动物的死亡率,变得更加吸引捕食性和寄生性天敌。挥发性化合物——茉莉酸甲酯可以从植物的气孔进入植物体内,在细胞质中被酯酶水解为茉莉酸,实现长距离的信号传导和植物间的交流,诱导邻近植物产生诱导防御反应。茉莉酸和茉莉酸甲酯分别具有4种立体异构,其中具有活性的是顺式结构,但顺式结构不稳定,会差向异构化为反式结构。茉莉酸的代谢物(Z)-茉莉酮(cis-Jasmone)具电生理活性,在植物诱导防御中起作用,并且在防御信号的作用上不同于茉莉酸和茉莉酸甲酯。     

茉莉酸介导丛枝菌根真菌诱导番茄抗早疫病的机制

丛枝菌根真菌的定殖可以提高寄主植物的抗病性,但机制并不十分清楚。利用番茄茉莉酸信号转导途径前系统素过表达材料35S::PS、茉莉酸合成突变体spr2、茉莉酸信号识别突变体jai1及其野生型CM 4个不同基因型材料,分别在根系接种丛枝菌根真菌摩西斗管囊霉(Funneliformis mosseae, Fm),待菌根形成后,在叶片外源喷施10 mL 0.5μmol/L茉莉酸甲酯(MeJA)和接种番茄早疫病病原菌(Alternaria solani, As),比较不同基因型抗病防御反应以及对早疫病抗性的差异。结果表明:预先接种菌根真菌的CM和35S::PS番茄,在叶片接种病菌5 d和10 d后,其叶片中过氧化物酶(POD)、多酚氧化酶(PPO)和脂氧合酶(LOX)活性以及丙二烯氧化物环化酶基因(AOC)和茉莉酸信号受体基因(COI1)的转录水平显著高于只接种早疫病菌的处理、只接种菌根菌的处理以及未进行任何处理的健康植株,其早疫病发病率和病情指数也显著降低;外源喷施MeJA可增强预先接种菌根菌的CM和35S::PS番茄植抵抗早疫病的能力。与此同时,对预先接种菌根菌的spr2番茄外源喷施MeJA后接种早疫病菌,其酶活性和基因转录水平显著高于其他4个处理,发病率和病情指数也明显降低。然而,对jai1番茄进行MeJA处理不能增强其防御反应和抗病性。可见,丛枝菌根真菌侵染诱导番茄抗早疫病是与茉莉酸信号途径密切相关的。     

绿盲蝽取食与机械损伤对棉花叶片内防御性酶活性的影响

为探明绿盲蝽Apolygus lucorum (Mayer-Dür)取食和机械损伤对不同抗性棉花叶片内主要防御酶活性的影响以及防御酶与棉花抗绿盲蝽性的关系,以棉花3个不同抗性品系为材料,室内条件下测定绿盲蝽取食和机械损伤处理后棉叶中苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)和多酚氧化酶(PPO)的活性.结果表明:对...     

机械损伤对重瓣玫瑰防御酶的诱导

为研究重瓣玫瑰（Rosa rugosa ‘plena’）的诱导抗虫性,采用机械损伤方式诱导处理重瓣玫瑰,研究机械损伤对重瓣玫瑰叶片防御酶活性的影响。结果表明,机械损伤可诱导重瓣玫瑰叶片苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)活性的增加,PPO、POD活性先升高后下降,PAL活性的升高持续时间较长。T3（打孔损伤,3孔/叶）处理为最适的损伤程度,诱导的PAL、PPO、POD活性分别在第7、3、5天达到最高值。因此认为,采用适度地损伤能够诱导重瓣玫瑰较高的防御酶活性,提高植株的防御能力。     

Enzymatic browning reaction of apple juices prepared using a blender and a low-speed masticating household juicer

ABSTRACT

The aim of this study was to investigate the effect of juicer type (blender or LSM household juicer) on the browning reaction of apple juice and evaluate the remaining antioxidant activity in the juice. The blender apple juice showed a darker brown color and 4.5 times higher PPO activity than LSM apple juice. This result suggested that the blender caused severer damage to plastids in cells leading to leakage of PPO into the juice than the LSM juicer. The total polyphenol and flavonoid content of LSM apple juice was approximately 2 times higher than that of blender apple juice because polyphenols and flavonoids can be used as substrates by PPO. The antioxidant activity of LSM juice was higher than that of blender juice. Together, these results suggested that the LSM juicer is superior to the blender for preparation of fresh apple juices due to the minimization of enzymatic oxidation reactions.

Abbreviations: LSM: low-speed masticating; PPO: polyphenol oxidase; ABTS: 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl     

Import of polyphenol oxidase by chloroplasts is enhanced by methyl jasmonate

Polyphenol oxidase (PPO; EC 1.10.3.2 or EC 1.14.18.1) takes part in the response of tomato plants (Lycopersicon esculentum Mill.) to wounding and herbivore attack, mediated by the octadecanoid wound-signaling pathway. Wounding and methyl jasmonate (MeJA) induce expression of ppo genes and markedly increase the level of the enzyme. We report that pretreatment with MeJA also markedly increased the ability of isolated tomato chloroplasts to import and process PPO precursors (pPPO). Pea (Pisum sativum L.) chloroplasts showed no such response. Wounding or ethylene alone was ineffective but ethylene was synergistic with MeJA. Treatment with MeJA conferred a strong binding of pPPO, or its processing intermediate, to thylakoids and subsequent translocation into the lumen and processing to the mature protein. The effect on PPO import and translocation was evident after 8–16 h exposure to MeJA. Membrane-bound pPPO was cross-linked to a proteinaceous component of the thylakoid translocation apparatus, apparently induced by MeJA. The import and processing of other nuclear-encoded thylakoid proteins were not affected by MeJA in tomato. A 90-kDa protein that co-fractionated with thylakoids was induced along with the increase in competence for PPO import, and was identified as the proteinase-inhibitor multicystatin. It is concluded that the 90-kDa protein observed is part of the MeJA-induced defense response of tomato, not a component of the thylakoid translocation apparatus.Abbreviations Chl Chlorophyll - i and p Prefixes used to denote the intermediate and precursor forms of a protein, respectively - JA Jasmonic acid - LSU Large subunit of Rubisco - MeJA Methyl jasmonate - OE23 and OE33 23- and 33-kDa subunits of the oxygen-evolving complex of PSII - PC Plastocyanin - pPPO (iPPO, PPO) Precursor (intermediate, mature) form of polyphenol oxidase     

Kinetic characterization of the oxidation of esculetin by polyphenol oxidase and peroxidase

Esculetin has been described as an inhibitor of tyrosinase and polyphenol oxidase and, therefore, of melanogenesis. In this work, we demonstrate that esculetin is not an inhibitor but a substrate of mushroom polyphenol oxidase (PPO) and horseradish peroxidase (POD), enzymes which oxidize esculetin, generating its o-quinone. Since o-quinones are very unstable, the usual way of determining the enzymatic activity (slope of recordings) is difficult. For this reason, we developed a chronometric method to characterize the kinetics of this substrate, based on measurements of the lag period in the presence of micromolar concentrations of ascorbic acid. The catalytic constant determined was of the same order for both enzymes. However, polyphenol oxidase showed greater affinity (a lower Michaelis constant) than peroxidase for esculetin. The affinity of PPO and POD towards oxygen and hydrogen peroxide was very high, suggesting the possible catalysis of both enzymes in the presence of low physiological concentrations of these oxidizing substrates. Taking into consideration optimum pHs of 4.5 and 7 for POD and PPO respectively, and the acidic pHs of melanosomes, the studies were carried out at pH 4.5 and 7. The in vivo pH might be responsible for the stronger effect of these enzymes on L-tyrosine and L-3,4-dihydroxyphenylanaline (L-DOPA) (towards melanogenesis) and on cumarins such as esculetin towards an alternative oxidative pathway.     

枇杷果实多酚氧化酶活性影响因素的研究

研究了温度、pH、几种还原剂对枇杷果实多酚氧化酶活性的影响。结果表明:以邻苯二酚为作用底物,该酶的最适温度为25℃、最适pH为6.3,抗坏血酸、L-半胱氨酸、亚硫酸钠等还原剂对该酶活性有明显的抑制作用。     

Tentoxin stops the processing of polyphenol oxidase into an active protein

Previous studies in our laboratory have indicated that polyphenol oxidase (PPO), as measured by its activity, is not present in tentoxin-treated plants. In the present study, immunochemical techniques were used as a sensitive probe of tentoxin effects on PPO. Immunocytochemistry of PPO with peroxidase-antiperoxidase labelling techniques, revealed that in control Vicia faba L. chloroplasts, PPO was associated mainly with the thylakoids. Cytochemical staining of PPO activity using DL-dihydroxyphenylalanine (DOPA) as substrate was also localized only on the thylakoids in the control chloroplasts. In tentoxin-treated plants all of the immunologically-recognizable PPO accumulated at the plastid envelope although no PPO was detected by cytochemical methods. SDS polyacrylamide gels of extracts from control and tentoxin-treated Vicia leaves were blotted onto nitrocellulose and reacted with rabbit anti-PPO. Secondary labelling of the blots with goat-antirabbit IgG labelled with peroxidase revealed a 40 kdalton protein in both extracts. However, only the PPO from the control extract had DOPA oxidase activity. These data suggest that PPO accumulates in the plastid envelope membranes in tentoxin-affected cells and that PPO in these treated plants is not processed to an active protein.     

Genetic mapping of the gene affecting polyphenol oxidase activity in tetraploid durum wheat

The quality of durum wheat (Triticum turgidum ssp. durum) is influenced by polyphenol oxidase(PPO) activity and its corresponding substrates. A saturated molecular-marker linkage map was constructed previously by using a set of recombinant inbred (RI) lines, derived from a cross between durum wheat cultivars Jennah Khetifa and Cham 1. Quantitative trait loci (QTL) for PPO activity in seeds were mapped in this population. PPO activity in seeds of the parents and 110 RI lines was measured spectrophotometrically. The PPO activity of Cham 1 was significantly lower than that of Jennah Khetifa. QTL analysis of these data indicated that most of PPO activity was associated with major loci on the long arm of chromosome 2A. The trait was found to be strongly associated with the SSR marker Xgwm312@2A. With this knowledge, marker-assisted selection can be used to select genotypes with lower PPO activity in durum wheat populations.