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A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.)
一种简单快速测定豌豆(Pisum sativum L.)中植物防御反应指标-植物抗毒素豌豆素的方法   

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Abstract

Phytoalexins are antimicrobial substance synthesized in plants upon pathogen infection. Pisatin (Pisum sativum phytoalexin) is the major phytoalexin in pea, while it is also a valuable indicator of plant defense response. Pisatin can be quantitated in various methods from classical organic chemistry to Mass-spectrometry analysis. Here we describe a procedure with high reproducibility and simplicity that can easily handle large numbers of treatments. The method only requires a spectrophotometer as laboratory equipment, does not require any special analytical instruments (e.g., HPLC, mass spectrometers, etc.) to measure the phytoalexin molecule quantitatively, i.e., most scientific laboratories can perform the experiment.

Keywords: Pisatin(豌豆素), Phytoalexin(植物抗毒素), Nonhost resistance(非宿主抗病性), Plant defense response(植物防御反应), Pea(豌豆)

Background

Plants have host resistance and nonhost resistance depending upon the nature of plant-pathogen interactions. Host resistance is mostly controlled by R genes and less durable, whereas nonhost resistance is generally a multi-gene trait and more durable in comparison with host resistance (Gill et al., 2015; Lee et al., 2017). The pea plant has served as a model system for research on the signals that trigger the nonhost defense response when challenged by incompatible pathogens that fall outside that species’ host range (Hadwiger, 2008). An indicator of this response in peas is the induction of a secondary metabolism to the isoflavonoid, pisatin. Pisatin has strong antifungal properties but its presence is a valuable indicator of plant defense response. Pisatin can be quantitated in various ways from classical organic chemistry procedures (Schwochau and Hadwiger, 1969) to Mass-spec analysis (Seneviratne et al., 2015). However, a procedure with high reproducibility and simplicity is described herein that can easily handle large numbers of treatments. The targeted tissue is the inside layer of an immature pea pod, called endocarp. This pristine cuticle-free tissue is capable of responding rapidly to candidate microbes or elicitor compounds to generate the pea defense response. The exposed epidermal layer of cells can be monitored for light microscope-visible or stained cellular component changes. The overall changes that culminate in pisatin accumulations can be determined by immersing the pod half in 5 ml of hexane for 4 h in the dark and subsequently allowing the decanted hexane to evaporate in the air flow of a hood. The residue remaining is dissolved in 1 ml of 95% alcohol and read at OD309 using a spectrophotometer: 1 OD309 = 43.8 µg pisatin/ml in 1 cm pathlength (Cruickshank and Perrin, 1961; Perrin and Cruickshank, 1965; Teasdale et al., 1974). This reading minus the background control tissue and the characteristic UV spectrum are essentially free from other hexane soluble components of the pea tissue. This protocol was used in our recent publications (Hadwiger and Tanaka, 2014 and 2017; Tanaka and Hadwiger, 2017).

Materials and Reagents

  1. Spatula–smooth narrow tip and smooth glass rod
  2. Plastic Petri dishes (60 x 15 mm) (Corning, catalog number: 351007 )
  3. Plastic container with wet Kimwipes inside for humidity
  4. Paper towel or Kimwipe
  5. Immature pea pods (1.5-2.0 cm in length) grown in sand and clay pots at 65-70 F under greenhouse conditions and freshly harvested (use within 3 h of applying a treatment). Remove calyx and retain briefly in sterile water (Figure 1). Endocarp will be used for the assay (see Note 1 in detail)
  6. Glass vials 30 ml
  7. Candidate elicitor solutions best dissolved in deionized water (For exceptions see Procedure 1)
  8. DMSO
  9. Hexane
  10. 95% ethanol

Equipment

  1. Adjustable pipettes (P-200 and P-1000 and corresponding tips)
  2. Flask 500 ml with 5 ml dispenser top or 5 ml pipet for dispensing hexane
  3. Glass beakers, 30 ml
  4. Room temperature dark cabinet space for pathogen or elicitor treatments (as described in step 3b)
  5. UV spectrometer (Shimadzu, model: UV160 )
  6. 1 cm Pathlength quartz cuvettes (Sigma-Aldrich, catalog number: C5178 )
    Note: This product has been discontinued.

Procedure

  1. Preparation of elicitors
    The selection of elicitors is by design open to innovation. Follow the directions of manufactures for solubility procedures. Water soluble compounds dissolved at near neutral pH are preferred. When solubility depends on ethanol, DMSO etc., there must be suitable control applications with only the respective solvent. Incompatible pathogen can be a positive control for an inducer of nonhost resistance. See Note 2 in detail.
  2. Preparation of pod halves
    1. Harvest pods and remove calyx as shown in Figure 1. Hold these pods in a sterile deionized water reservoir to keep the tissue moist.
    2. Select uniform sized and conditioned pea pods from their water reservoir.
    3. Separate the pod halves with a smooth spatula avoiding wounding as much as possible.
    4. Fresh weight of pod halves is determined.
    5. Lay endocarp (inner) surface layer up in a Petri dish (Figure 1).


      Figure 1. Pea endocarp. The inside layer (endocarp) of an immature pea pod is cuticle-free tissue capable of responding rapidly to microbes or elicitor compounds. Right picture shows endocarp tissues treated in an elicitor solution in Petri dishes. Incubation is performed in a plastic container with wet paper towels to maintain humidity.

  3. Application of elicitors
    1. Apply 25 µl of elicitor candidate solution and lightly distribute over the entire surface with a glass rod. For the control, apply the same solvent used for dissolving the elicitor.
    2. Treated pods are retained in a plastic container with wet paper towels (Figure 1) to maintain humidity and then incubate in the dark or moderate light for up to 24 h.
  4. Extraction and measurement of pisatin
    1. Pods are transferred to 30 ml glass vials using forceps and immersed in 5 ml of hexane for 4 h in the dark. Typically, 400 mg fresh weight per 5 ml of hexane.
    2. The hexane is decanted off into 30 ml beakers and the hexane evaporated in the air stream of a hood in low light because pisatin is not stable in bright light (typically light strength in the lab is not incandescent).
    3. One milliliter of 95% ethanol is added to the residue and read at 309 nm in a cuvette using spectrophotometer.
    4. To insure purity, a UV spectrum is measured in the range of 220-320 nm to verify the characteristic pisatin spectrum (Figure 2). See Note 3 in detail.


      Figure 2. Pisatin spectrum. UV spectrum in the range of 220-320 nm was run to verify the purity of pisatin.

Data analysis

After subtracting the OD309 value of non-treated control, the numbers are converted based on the equation: 1.0 OD309 unit = 43.8 µg/ml pisatin in 1 cm pathlength (see Note 4 in detail). Data should be shown with pisatin quantity (µg) per fresh weight of tissues (g). Data obtained should be analyzed using ANOVA followed by Student’s t-test. Difference with P < 0.05 is considered significant.

Notes

  1. The pea endocarp tissue has some potential to condition or partially take up small percentages of insoluble materials in suspension, such as the cell wall fragments released by fungal spores, detected in electron microscope view (Hadwiger et al., 1981).
  2. Effect of elicitors can be compared with that of an incompatible fungal pathogen, e.g., Fusarium solani f.sp. phaseoli (Fsph), which is a pathogen for bean (not for pea). An example result was shown in Figure 3, in which the data show that no detectable pisatin above background in water-treated tissue, and accumulation of pisatin induced by a fungal wall carbohydrate (i.e., chitosan) and by an authentic inducer of nonhost resistance in pea, a fungal pathogen of bean (i.e., Fsph).
  3. Pisatin has been purified from the final step with both thin layer chromatography and mass spectrometry (Teasdale et al., 1974; Seneviratne et al., 2015). The assay is accurate because pisatin only absorbs at 309 nm.
  4. Pisatin in ethanol has a characteristic UV absorption spectrum with two peaks at 286 nm and 309 nm (Figure 2). When pisatin is the only light-absorbing compound in the solution, the ratio OD309 to OD286 is 1.47 (Cruickshank and Perrin, 1961).


    Figure 3. An example result of pisatin measurement. Pisatin was extracted and measured from endocarps (n = 3) after 24 h incubation with water (control), 1 mg/ml chitosan (elicitor), or 4 x 106 spores of Fsph (Fusarium solani f.sp. phaseoli). Data was modified from our previous publication (Hartney et al., 2006). There were statistically significant differences between group means as determined by one-way ANOVA (P < 0.05).

Acknowledgments

This work was partly supported by Biologically-Intensive Agriculture and Organic Farming (BIOAg) grant from the Center for Sustaining Agriculture and Natural Resources (CSANR) at Washington State University. PPNS No. 0740, Department of Plant Pathology, College of Agricultural, Human, and Natural Resource Sciences, Agricultural Research Center, Hatch Project No. WNP01844, Washington State University, Pullman, 99164-6430, USA.

References

  1. Cruickshank, I. A. M. and Perrin D. R. (1961). Studies on phytoalexins III. The isolation, assay, and general properties of a phytoalexin from Pisum sativum L. Aust J Biol Sci 14: 336-348.
  2. Gill, U. S., Lee, S. and Mysore, K. S. (2015). Host versus nonhost resistance: distinct wars with similar arsenals. Phytopathology 105: 580-87.
  3. Hadwiger, L. A. (2008). Pea-Fusarium solani interactions contributions of a system toward understanding disease resistance. Phytopathology 98: 372-379.
  4. Hadwiger, L. A., Beckman, J. M. and Adams, M. J. (1981). Localization of Fungal components in the Pea-Fusarium interaction detected immunochemically with anti-chitosan and anti-fungal cell wall antisera. Plant Physiol 67: 170-175.
  5. Hadwiger, L. A. and Tanaka, K. (2014). EDTA a novel inducer of pisatin, a phytoalexin indicator of the non-host resistance in peas. Molecules 20: 24-34.
  6. Hadwiger, L. A. and Tanaka, K (2017). Non-host resistance: DNA damage is associated with SA signaling for induction of PR genes and contributes to the growth suppression of a pea pathogen on pea endocarp tissue. Front Plant Sci 8: 446.
  7. Hartney, S., Carson, J. and Hadwiger, L. A. (2006). The use of chemical genomics to detect functional systems affecting the non-host disease resistance of pea to Fusarium solani f.sp. phaseori. Plant Sci 172: 45-56.
  8. Lee, H-A., Lee, H-Y., Seo, E., Lee, J., Kim, S-B., Oh, S., Choi, E., Choi, e., Lee, s. E. and Choi, D. (2017). Current understandings of plant nonhost resistance. Mol Plant-Microbe Interact 30: 5–15
  9. Perrin, D. R. and Cruickshank, I. A. M. (1965). Studies on phytoalexins VII. Chemical stimulation of pisatin formation in Pisum sativum L. Aust J Biol Sci 18: 803-816.
  10. Schwochau, M. E. and Hadwiger, L. A. (1969). Regulation of gene expression by actinomycin D and other compounds which change the conformation of DNA. Arch Biochem Biophys 134: 34-41.
  11. Seneviratne, H. K., Dalisay, D. S., Kim, K. W., Moinuddin, S. G., Yang, H., Hartshorn, C. M., Davin, L. B. and Lewis, N. G. (2015). Non-host disease resistance response in pea (Pisum sativum) pods: Biochemical function of DRR206 and phytoalexin pathway localization. Phytochemistry 113: 140-148.
  12. Tanaka, K. and Hadwiger, L. A. (2017). Nonhost resistance: reactive oxygen species (ROS) signal causes DNA damage prior to the induction of PR genes and disease resistance in pea tissue. Physiol Mol Plant Pathol 98: 18-24.
  13. Teasdale, J., Daniels, G., Davis, W. C., Eddy, R., Hadwiger, L. A. (1974). Physiological and cytological similarities between disease resistance and cellular incompatibility responses. Plant Physiol 54: 690-695.

简介

植物毒素是在病原体感染后在植物中合成的抗微生物物质。 豌豆中的主要植物抗毒素豌豆中的豌豆豌豆(Pisatin)是豌豆中的主要植物抗坏血酸,而它也是有价值的 植物防御反应指标。 茜素可以从经典有机化学到质谱分析的各种方法进行定量。 在这里我们描述了一个具有高重现性和简单性的程序,可以轻松处理大量的治疗。 该方法仅需要使用分光光度计作为实验室设备,不需要任何特殊的分析仪器(例如,如HPLC,质谱仪,等等)来定量测量植物抗毒素分子, ,大多数科学实验室都可以进行实验。
【背景】植物具有宿主抗性和非宿主抗性,取决于植物 - 病原体相互作用的性质。宿主抗性主要由R基因控制,耐久性较差,而非宿主抗性通常是多基因性状,与宿主抗性相比更耐用(Gill等人,2015; Lee et al。等等,2017)。豌豆植物作为一个模型系统,用于研究在不属于该物种主体范围的不相容病原体挑战时触发非宿主防御反应的信号(Hadwiger,2008)。豌豆中这种反应的一个指标是诱导异黄酮(pifatin)的次级代谢。靛蓝具有很强的抗真菌性能,但其存在是植物防御反应的有价值的指标。茜素可以从古典有机化学方法(Schwochau和Hadwiger,1969)到质谱分析(Seneviratne等人,2015)以各种方式进行定量。然而,本文描述了具有高重现性和简单性的方法,其可以容易地处理大量的治疗。目标组织是未成熟豌豆荚的内层,称为内果皮。这种原始无角质层的组织能够迅速响应候选微生物或诱发剂化合物,以产生豌豆防御反应。暴露的细胞表皮层可以监测光学显微镜 - 可见或染色的细胞组分变化。通过将荚果半部浸入5ml己烷中在黑暗中浸泡4小时,随后允许倾析的己烷在发动机空气流中蒸发,可以确定最终达到抑制素积聚的整体变化。残留的残余物溶解在1ml 95%乙醇中,并使用分光光度计在OD 309中读取:1OD 309%=43.8μg靛红色素/ ml,1cm光程(Cruickshank和Perrin,1961; Perrin和Cruickshank,1965; Teasdale等人,1974)。该读数减去背景控制组织,特征UV光谱基本上不含豌豆组织的其他己烷可溶组分。我们最近的出版物(Hadwiger和Tanaka,2014和2017; Tanaka和Hadwiger,2017)都使用了这个协议。

关键字:豌豆素, 植物抗毒素, 非宿主抗病性, 植物防御反应, 豌豆

材料和试剂

  1. Spatula光滑的窄头和光滑的玻璃棒
  2. 塑料培养皿(60 x 15毫米)(康宁,目录号:351007)
  3. 塑料容器用湿Kimwipes内部湿度
  4. 纸巾或Kimwipe
  5. 在温室条件下,65-70°F生长在砂土和陶壶中的新生豌豆荚(长1.5-2.0厘米),新鲜收获(施用处理3小时后使用)。取出花萼并短暂保持在无菌水中(图1)。内果皮将用于测定(见详细说明1)
  6. 玻璃小瓶30 ml
  7. 候选激发剂溶液最好溶于去离子水中(例如见程序1)
  8. DMSO
  9. 己烷
  10. 95%乙醇

设备

  1. 可调式移液器(P-200和P-1000及相应的提示)
  2. 烧瓶500毫升与5ml分配器顶部或5ml移液管用于分配己烷
  3. 玻璃烧杯,30 ml
  4. 用于病原体或诱导剂处理的室温暗柜空间(如步骤3b所述)
  5. 紫外光谱仪(Shimadzu,型号:UV160)
  6. 1厘米路径石英比色皿(Sigma-Aldrich,目录号:C5178)
    注意:本产品已停产。

程序

  1. 诱导器的准备
    激发器的选择是通过设计开放创新的。按照制造商的说明进行溶解度程序。在近中性pH下溶解的水溶性化合物是优选的。当溶解度取决于乙醇,DMSO等时,必须有合适的控制应用,只有相应的溶剂。不相容的病原体可以是非宿主抗性诱导物的阳性对照。详见附注2.
  2. 准备荚半部
    1. 收获荚果并清除花萼,如图1所示。将这些荚放在无菌去离子水储存器中,以保持组织湿润。
    2. 从水库选择一般尺寸和调理的豌豆荚。
    3. 用平滑的铲子分离荚半部,尽可能避免受伤。
    4. 确定荚半重量的重量。
    5. 在培养皿中放置内果皮(内)表面层(图1)

      图1.豌豆内果皮未成熟豌豆荚的内层(内皮)是能够快速响应微生物或诱发剂化合物的无角质层组织。右图显示在培养皿中用诱导剂溶液处理的内皮组织。孵育在带有湿纸巾的塑料容器中进行,以保持湿度
  3. 诱发器的应用
    1. 应用25μl引发剂候选溶液,并用玻璃棒轻轻分布在整个表面上。为了控制,应用与溶解激发剂相同的溶剂。
    2. 处理的荚被保留在带湿纸巾的塑料容器中(图1)以保持湿度,然后在黑暗或中等光照下孵育长达24小时。
  4. 提取和测量靛红
    1. 使用镊子将荚子转移到30ml玻璃小瓶中,并在黑暗中浸入5ml己烷中4小时。通常,每5ml己烷具有400mg鲜重
    2. 将己烷倒入30ml烧杯中,并在低光下在发动机罩的气流中蒸发己烷,因为在明亮的光线中,因为靛红不稳定(通常实验室中的光强度不是白炽灯)。
    3. 将1毫升95%乙醇加入到残留物中,并用分光光度计在比色杯中以309nm读数。
    4. 为了确保纯度,在220-320nm的范围内测量UV光谱以验证特征性的美沙酮谱(图2)。详见附注3.


      图2.靛红色谱。测试在220-320nm范围内的紫外光谱,以验证皮沙汀的纯度。

数据分析

在减去未处理对照的OD 309值之后,根据以下等式转换数:1.0 OD 309单位=43.8μg/ ml在1cm光程中的美沙酮详见附注4)。数据应以每个鲜重的组织(g)的豌豆素量(μg)显示。所获得的数据应使用ANOVA进行分析,然后是Student's 测试。与 P 的区别0.05被认为是重要的。

笔记

  1. 豌豆内果皮组织具有一些潜力,可以在电子显微镜观察中检测到(例如,Hadwiger等人)检测到悬浮液中不溶性物质的小百分比,例如由真菌孢子释放的细胞壁碎片, 1981)
  2. 诱导子的作用可以与不相容的真菌病原体(例如,镰孢镰刀菌fssp. (Fsph)(Fsph)进行比较,豆的病原体(不是豌豆)。在图3中显示了一个示例性结果,其中数据显示在水处理的组织中没有可检测到的高于背景的皮屑素,以及由真菌壁碳水化合物(即,壳聚糖)诱导的美沙酮的积累以及由真菌壁碳水化合物豌豆中的非宿主抗性的真正诱导物,豆的真菌病原体(,Fsph)。
  3. 使用薄层色谱和质谱法(Teasdale等人,1974; Seneviratne等人,2015),从最终步骤中纯化靛蓝。该测定是准确的,因为只有在309nm处才能吸收靛红
  4. 乙酸中的靛红具有特征性紫外吸收光谱,在286 nm和309 nm处有两个峰(图2)。当靛红是溶液中唯一的光吸收化合物时,OD 309与OD 286之比为1.47(Cruickshank和Perrin,1961)。


    图3.靛红测定的实例结果在与水(对照),1mg / ml壳聚糖(激发剂)或4个培养基孵育24小时后,从内切片(n = 3)中提取和测量茜素Fsph(镰孢镰刀菌fssp。相位柱)的孢子的X 10 。数据是从我们以前的出版物(Hartney等人,2006)修改的。通过单因素方差分析(p <0.05)确定组间平均值差异有统计学意义。

致谢

华盛顿州立大学维持农业和自然资源中心(CSANR)的生物强化农业和有机农业(BIOAg)补助金部分得到了这项工作的支持。 PPNS第0740号,农业,人力和自然资源科学学院农业研究中心植物病理学系,华盛顿州立大学哈尔滨工程WNP01844,铂尔曼,99164-6430,美国。

参考

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  3. Hadwiger,LA(2008)。 Pea- em> Fusarium solani 系统相互作用对理解疾病抵抗的贡献。 植物病理学 98:372-379。
  4. Hadwiger,LA,Beckman,JM和Adams,MJ(1981)。
  5. Hadwiger,LA和Tanaka,K.(2014)。 EDTA一种新型诱导剂豌豆中的非宿主抗性的植物依赖蛋白指示剂。分子 20:24-34。
  6. Hadwiger,LA和Tanaka,K(2017)。&nbsp; 非宿主抗性:DNA损伤与SA信号传导相关,用于诱导PR基因,并有助于豌豆内果皮组织对豌豆病原体的生长抑制。前植物科学8:446 。
  7. Hartney,S.,Carson,J.和Hadwiger,LA(2006)。使用化学基因组学检测影响豌豆对镰刀菌的非宿主病抗性的功能系统。 植物科学 172:45-56。
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  9. Perrin,D.R。和Cruickshank,I.A.M.(1965)。植物抗毒素研究VII。化学刺激豌豆豌豆中的蛋白酶形成.Li。J.Journal J Biol Sci 18:803-816。
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  11. Seneviratne,H.K.,Dalisay,D.S.,Kim,K.W.,Moinuddin,S.G.,Yang,H.,Hartshorn,C.M.Davin,L.B.and Lewis,N.G。(2015)。 豌豆中的非宿主病抗性反应(豌豆豌豆荚果:DRR206的生物化学功能和植物抗毒素途径定位。 植物化学 113:140-148。
  12. Tanaka,K.和Hadwiger,LA(2017)。非寄生虫抗性:活性氧(ROS)信号在诱导PR基因之前引起DNA损伤,并在豌豆组织中引起疾病抗性。“生理分子植物Pathol”98:18-24。 />
  13. Teasdale,J.,Daniels,G.,Davis,WC,Eddy,R.,Hadwiger,LA(1974)。&lt; a class =“ke-insertfile”href =“http://www.plantphysiol.org/内容/ 54/5/690“target =”_ blank“>疾病抗性与细胞不相容性反应之间的生理和细胞学相似性植物生理54:690-695。
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引用:Hadwiger, L. A. and Tanaka, K. (2017). A Simple and Rapid Assay for Measuring Phytoalexin Pisatin, an Indicator of Plant Defense Response in Pea (Pisum sativum L.). Bio-protocol 7(13): e2362. DOI: 10.21769/BioProtoc.2362.
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