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Lignin Extraction and Quantification, a Tool to Monitor Defense Reaction at the Plant Cell Wall Level
木质素的提取和定量测定是检测植物在细胞壁水平的防御反应的一种工具

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Abstract

Lignin is a complex polymer of phenolic compounds (monolignins), which contributes to the rigidity of the plant cell wall. Lignification is essential for plant development, however it is also one of the mechanisms of plant defense. Accumulation of lignin and the polymerization of monolignins at sides of pathogen attack protect the cell wall against cell wall-degrading enzymes and prevent therefore the pathogen’s penetration. In addition to cross-linkage of phenolic compounds, this resistance mechanism includes also callose and cellulose appositions on the cell wall. This results in structures called papillae, which provide the necessary resistance to the mechanical pressure exercised by fungal appressorium. Lignin accumulation in cell walls is therefore a part of plant defense responses. Here we describe a quantification method for lignin and cell wall phenolic compounds, which is based on an acid-catalyzed reaction resulting in a colored and soluble lignin-thioglycolate complex suitable for photometric measurements.

Keywords: Plant Defense(植物防御), Cell Wall(细胞壁), Priming(启动), Lignin(木质素)

Materials and Reagents

  1. Plant material
  2. Flg22 (a flagellin-derived, 22 amino acid-long peptide) (QRLSTGSRINSAKDDAAGLQIA)
  3. 80% aqueous methanol (Carl Roth, catalog number: 4627.6 )
  4. Distilled water
  5. Acetone (VWR International, catalog number: UN1090 )
  6. NaOH (Carl Roth, catalog number: 6771.1 )
  7. 86% H3PO4 (J.T.Baker®, catalog number: 6024 )
  8. Ethyl acetate (Carl Roth, catalog number: 6784.4 )
  9. Thioglycolic acid (Sigma-Aldrich, catalog number: T3758 )
  10. 32% HCl (Carl Roth, catalog number: P074.3 )
  11. Lignin (alkali) (Sigma-Aldrich, catalog number: 370959 )
  12. 1 M NaOH (see Recipes)
  13. 2 M HCl (see Recipes)

Equipment

  1. Plastic equipment (Grainer Falcon 50 ml tubes, catalog number: 227261 ; Grainer 6-well culture plates, catalog number: 657160 )
  2. Freeze drying machine
  3. Tissue Lyser2 (manufactured by Retsch, provided by QIAGEN) and stainless steel beads (QIAGEN, catalog number: 69989 )
  4. Shaker for tube rotation (with variable temperature)
  5. Centrifuge (with variable temperature) (Eppendorf, centrifuge number: 5417R )
  6. Vacuum pump (Savant Systems LLC, catalog number: SC110 : 25-30 Hg)
  7. Rotary shaker for tube rotation
  8. Photometer (Eppendorf, BioSpectrometer basic)

Note: The extraction procedure includes a separation of different fractions of phenolic compounds depending on their chemical properties. The soluble phenolic fraction is extracted with 80% methanol (Day 1). The cell wall-bound phenolic fraction is hydrolyzed (alkaline hydrolysis) and solved in ethyl acetate. The lignin fractions are extracted in the later steps of the procedure by binding the to the lingo-thioglycolic acid complex (Days 2 and 3). The soluble and cell wall-bound phenols fractions can be measured according the Folin-Ciocalteau method after Strack et al. (1988), or used for further HPLC analyses. The lignin complex can be measured directly after resolving in NaOH.

Procedure

  1. Plant material: Arabidopsis seeds were surface-disinfected, germinated, and grown for 2 weeks on ½ MS plant growth media (Murashige and Skoog, 1962). Thereafter, seedlings were transferred into liquid plant growth media for 3 days in 6-well plates for the appropriate treatment. The growth chamber was set to 22 °C with 150 µmol/m2/s light and 8/16 h day/night photoperiod.
  2. To induce the defense reaction(s), plants were challenged with flg22 for 24 h. Flg22 was added directly to the Arabidopsis seedlings floated on liquid plant growth media, at a final concentration of 100 nM.
  3. Four grams of Arabidopsis leaves was harvested in 15 ml Falcon tubes and freeze dried (lyophilized) for 3 days.
  4. Two technical replicates (2 x 40 mg) of Arabidopsis dry leaves were filed into 2 ml tube and homogenized with metal beads in the Tissue Lyser (frequency: 30/sec for 30 sec).

    Day 1: Start of extraction
  5. Before the phenolic extraction, samples were protected against light with aluminum foil.
  6. One ml of 80% aqueous methanol was added to the samples (plant powder) and incubated for 1 h at room temperature with continuous shaking (120 rpm) to extract the first fraction of soluble phenolic compounds.
  7. Next, the samples were centrifuged at 13,000 x g for 10 min at 4 °C.
  8. The supernatants were collected.
  9. The remaining pellets were again incubated with 1 ml of 80% aqueous methanol for 1 h at room temperature.
  10. Samples were re-centrifuged at 13,000 x g for 10 min at 4 °C.
  11. Supernatants were merged and the pellets were used for further extraction.
  12. Pellets were washed with 1 ml of 80% aqueous methanol, distilled water, and acetone, subsequently.
  13. The washing steps were performed in each case for 15 min (incubation) followed by centrifugation (13,000 x g for 10 min).
  14. After the wash with acetone, the pellets were dried in the SpeedVac for 10 min.
  15. For alkaline hydrolysis, dried pellets were incubated with 1 ml of 1 M NaOH for 1 h at 80 °C and subsequently overnight at room temperature.

    Day 2
  16. The pH was lowered by adding 100 µl of 86% phosphoric acid (H3PO4) to the samples.
  17. To solve the cell wall-bound phenolic compounds, 500 µl of ethyl acetate was added to the samples and samples were incubated for 30 min at room temperature on a rotary shaker.
  18. After centrifugation at 13,000 x g for 5 min at room temperature the upper phase (around 500 µl) was collected in new tubes. This supernatant contained the cell wall-bound phenolic compounds.
  19. Additional 500 µl of ethyl acetate was added to the remaining samples and incubated for 30 min at rotary shaker.
  20. After centrifugation at 13,000 x g for 5 min at room temperature the upper phase (around 500 µl) was collected and combined with the previous. Both were the cell wall-bound phenolics fraction (about 1 ml).
  21. For lignin extraction, 500 µl of 80% aqueous methanol was added to the remaining samples (lower phases), and samples were precipitated by centrifugation (13,000 x g for 10 min).
  22. Resulting pellets were washed with 1 ml 80% methanol, distilled water, and acetone, like in point 13.
  23. After the wash with acetone, pellets were dried in the SpeedVac for 10 min.
  24. 1.5 ml of 2 M HCl and 0.3 ml of thioglycolic acid were added to the dry pellets and incubated for 4 h at 95 °C with regular shaking.
  25. Samples were shortly cooled on ice and centrifuged at 13,000 x g for 5 min at room temperature.
  26. Supernatants were removed.
  27. Pellets were washed twice with distilled water and centrifuged at 13,000 x g for 10 min at room temperature.
  28. The residues were incubated with 1 ml of 0.5 M NaOH overnight on a shaker.

    Day 3
  29. After the overnight incubation samples were centrifuged at 13,000 x g for 5 min at room temperature.
  30. The supernatants were collected and stored in new 2 ml tubes.
  31. 0.5 ml NaOH was again added to the remaining pellets and centrifuged at 13,000 x g for 5 min at room temperature.
  32. The supernatants were combined and acidified with 300 µl of 32% HCl, for the ligno-thioglycolic acid complex precipitation.
  33. Samples were incubated for 4 h at 4 °C with continuous shaking.
  34. Thereafter, samples were centrifuged at 13,000 x g for 5 min at room temperature.
  35. The supernatant was discarded and the lignin pellet was solved in 600 µl of 0.5 M NaOH.
  36. The quantification of lignin was performed after appropriate dilution (if necessary) by measuring the absorbance at 340 nm. Calibration was performed using a standard curve made of lignin, alkali (Figure 1).


    Figure 1. Standard curve of lignin. The colorimetric visualization of lignin was performed in a concentration range of 1 to 0.0001 mg/ml. Lignin was solved in 0.5 M NaOH. The absorbance at 340 nm was measured with a bio-spectrometer and the regression line was calculated accordingly.

Representative data

  1. For representative data see Schenk et al. (2014).
  2. For reproducibility see Notes.

Notes

Working under a laboratory fume hood during the whole extraction procedure is recommended. The metal beads used to crush the plant material should be removed in the first steps of extraction procedure. For reproducibility dry plant material should be weighed for adequate calculation, the content of roots and leave tissue should be the same in all samples, and the dry plant material should be homogenized via TissueLyser to an appropriated powder.

Recipes

  1. 1 M NaOH
    2 g of NaOH in 50 ml H2O
  2. 2 M HCl
    1.964 ml of 32% HCl in 10 ml H2O

Acknowledgments

The protocol was modified after Bruce and West (1989), Eynck et al. (2009) and Strack et al. (1988). This work was supported by the Federal Office for Agriculture and Food (BLE), Germany.

References

  1. Bruce, R. J. and West, C. A. (1989). Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean. Plant Physiol 91(3): 889-897.
  2. Eynck, C., Koopmann, B., Karlovsky, P. and von Tiedemann, A. (2009). Internal resistance in winter oilseed rape inhibits systemic spread of the vascular pathogen Verticillium longisporum. Phytopathology 99(7): 802-811.
  3. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plantarum 15, 473-497.
  4. Schenk, S. T., Hernandez-Reyes, C., Samans, B., Stein, E., Neumann, C., Schikora, M., Reichelt, M., Mithofer, A., Becker, A., Kogel, K. H. and Schikora, A. (2014). N-Acyl-homoserine lactone primes plants for cell wall reinforcement and induces resistance to bacterial pathogens via the salicylic acid/oxylipin pathway. Plant Cell 26(6): 2708-2723.
  5. Strack, D., Heilemann, J., Momken, M., and Wray, V. (1988). Cell wall-conjugated phenolics from coniferae leaves. Phytochemistry 27, 3517-3521.

简介

木质素是酚类化合物(木质素)的复杂聚合物,其有助于植物细胞壁的刚性。木质素是植物发育的关键,但它也是植物防御的机制之一。木质素的积累和木质素在病原体攻击侧的聚合保护细胞壁免受细胞壁降解酶的影响,从而防止病原体的渗透。除了酚类化合物的交联之外,该阻力机制还包括在细胞壁上的胼cal质和纤维素结合。这导致称为乳头的结构,其提供对由真菌附属物施加的机械压力的必要的抵抗力。因此,细胞壁中的木质素积累是植物防御反应的一部分。在这里我们描述了木质素和细胞壁酚类化合物的量化方法,其基于酸催化反应,产生适合于光度测量的有色和可溶性木质素 - 巯基乙酸酯复合物。

关键字:植物防御, 细胞壁, 启动, 木质素

材料和试剂

  1. 植物材料
  2. Flg22(鞭毛蛋白衍生的,22个氨基酸长的肽)(QRLSTGSRINSAKDDAAGLQIA)
  3. 80%甲醇水溶液(Carl Roth,目录号:4627.6)
  4. 蒸馏水
  5. 丙酮(VWR International,目录号:UN1090)
  6. NaOH(Carl Roth,目录号:6771.1)
  7. 86%H 3 PO 4(J.T.Baker ,目录号:6024)
  8. 乙酸乙酯(Carl Roth,目录号:6784.4)
  9. 硫代乙醇酸(Sigma-Aldrich,目录号:T3758)
  10. 32%HCl(Carl Roth,目录号:P074.3)
  11. 木质素(碱)(Sigma-Aldrich,目录号:370959)
  12. 1 M NaOH(见配方)
  13. 2 M HCl(见配方)

设备

  1. 塑料设备(Grainer Falcon 50ml管,目录号:227261; Grainer 6孔培养板,目录号:657160)
  2. 冷冻干燥机
  3. Tissue Lyser2(由Retsch制造,由QIAGEN提供)和不锈钢珠(QIAGEN,目录号:69989)
  4. 用于管旋转的振动器(具有可变温度)
  5. 离心机(可变温度)(Eppendorf,离心机编号:5417R)
  6. 真空泵(Savant Systems LLC,目录号:SC110:25-30Hg)
  7. 用于管旋转的旋转振动器
  8. 光度计(Eppendorf,BioSpectrometer basic)

注意:提取程序包括根据酚类化合物的化学性质分离不同的酚类化合物。 用80%甲醇萃取可溶性酚馏分(第1天)。 将细胞壁结合的酚部分水解(碱性水解)并溶解在乙酸乙酯中。 木质素级分在程序的后续步骤中通过结合至隐式巯基乙酸复合物(第2天和第3天)来提取。 的 可以在Strack等人(1988)之后根据Folin-Ciocalteau方法测量可溶性和细胞壁结合的酚部分,或用于进一步的HPLC分析。在NaOH中解析后可以直接测量木质素络合物。

程序

  1. 植物材料:将拟南芥种子表面消毒,发芽,并在1/2MS植物生长培养基(Murashige和Skoog,1962)上生长2周。此后,将幼苗在6孔板中转移到液体植物生长培养基中3天,以进行适当的处​​理。生长室设定为22℃,具有150μmol/m 2光照和8/16小时日/夜光周期。
  2. 为了诱导防御反应,用flg22攻击植物24小时。将Flg22直接加入浮在液体植物生长培养基上的拟南芥幼苗,最终浓度为100nM。
  3. 在15ml Falcon管中收获4g拟南芥叶并冷冻干燥(冻干)3天。
  4. 将两个技术重复(2×40mg)的拟南芥干叶置于2ml管中,并用组织裂解器中的金属珠匀浆(频率:30 /秒,30秒)。

    第1天:开始提取
  5. 酚醛树脂萃取前,样品用铝箔防光
  6. 向样品(植物粉末)中加入1ml 80%甲醇水溶液,并在室温下连续振荡(120rpm)温育1小时以提取可溶性酚类化合物的第一部分。
  7. 接下来,将样品在4℃以13,000×g离心10分钟。
  8. 收集上清液。
  9. 将剩余的沉淀再次与1ml 80%甲醇水溶液在室温下孵育1小时
  10. 将样品在4℃下以13,000×g离心10分钟
  11. 将上清液合并,将沉淀物用于进一步提取
  12. 然后用1ml 80%甲醇水溶液,蒸馏水和丙酮洗涤沉淀
  13. 在每种情况下进行洗涤步骤15分钟(温育),然后离心(13,000×10分钟)。
  14. 用丙酮洗涤后,将粒料在SpeedVac中干燥10分钟
  15. 对于碱性水解,将干燥的沉淀物与1ml 1M NaOH在80℃下温育1小时,随后在室温下过夜。

    第2天
  16. 通过向样品中加入100μl的86%磷酸(H 3 PO 4)来降低pH。
  17. 为了解决细胞壁结合酚类化合物,向样品中加入500μl乙酸乙酯,并将样品在室温下在旋转振荡器上温育30分钟。
  18. 在室温下以13,000xg离心5分钟后,将上层相(约500μl)收集在新管中。这种上清液含有细胞壁结合的酚类化合物
  19. 向剩余的样品中加入另外500μl的乙酸乙酯,并在旋转振荡器上孵育30分钟。
  20. 在室温下以13,000×g离心5分钟后,收集上层相(约500μl)并与前一步合并。两者都是细胞壁结合的酚类级分(约1ml)
  21. 对于木质素提取,向剩余样品(下相)中加入500μl80%甲醇水溶液,并通过离心(13,000×g 10分钟)沉淀样品。
  22. 所得颗粒用1ml 80%甲醇,蒸馏水和丙酮洗涤,如点13
  23. 用丙酮洗涤后,将粒料在SpeedVac中干燥10分钟。
  24. 将1.5ml 2M HCl和0.3ml巯基乙酸加入到干燥的沉淀中,并在95℃下定期摇动孵育4小时。
  25. 将样品在冰上短暂冷却,并在室温下以13,000xg离心5分钟。
  26. 除去上清液。
  27. 将沉淀用蒸馏水洗涤两次,并在室温下以13,000×g离心10分钟。
  28. 将残余物与1ml 0.5M NaOH在振荡器上孵育过夜
    第3天
  29. 过夜温育后,将样品在室温下以13,000×g离心5分钟。
  30. 收集上清液并储存在新的2ml管中
  31. 再次向剩余的丸粒中加入0.5ml NaOH并在室温下以13,000×g离心5分钟。
  32. 将上清液合并,用300μl32%HCl酸化,用于木质 - 巯基乙酸复合物沉淀。
  33. 将样品在4℃下连续振荡孵育4小时
  34. 此后,将样品在室温下以13,000×g离心5分钟
  35. 弃去上清液,将木质素沉淀溶于600μl0.5M NaOH中
  36. 木质素的定量在适当稀释(如果需要)后通过测量340nm处的吸光度进行。使用由木质素,碱制成的标准曲线进行校准(图1)

    图1.木质素的标准曲线。木质素的比色显现在1至0.0001mg/ml的浓度范围内进行。将木质素溶解在0.5M NaOH中。用生物分光光度计测量340nm处的吸光度,并相应地计算回归线

代表数据

  1. 有代表性的数据参见Schenk等人。(2014)。
  2. 重复性见注释。

笔记

建议在整个萃取过程中在实验室通风橱下工作。 用于粉碎植物材料的金属珠应在提取步骤的第一步中除去。 为了重复性,应对干燥植物材料进行称重,以便进行充分计算,根和残留组织的含量在所有样品中应相同,并且干燥植物材料应通过TissueLyser均化至适当的粉末。

食谱

  1. 1 M NaOH
    2g NaOH在50ml H 2 O中的溶液
  2. 2 M HCl
    1.964ml在10ml H 2 O中的32%HCl

致谢

在Bruce和West(1989),Eynck等人(2009)和Strack等人(1988)后修改了该方案。 这项工作得到了德国联邦农业和食品局(BLE)的支持。

参考文献

  1. Bruce,R.J。和West,C.A。(1989)。 蓖麻籽悬浮培养物中果胶片段对木质素生物合成和异过氧化物酶活性的诱导。 Plant Physiol 91(3):889-897。
  2. Eynck,C.,Koopmann,B.,Karlovsky,P。和von Tiedemann,A。(2009)。 冬季油籽油菜的内部阻力抑制血管病原体长链口孢霉的全身传播。 Phytopathology 99(7):802-811。
  3. Murashige,T。和Skoog,F。(1962)。 用烟草组织培养快速生长和生物测定的修订培养基。 Physiol Plantarum 15,473-497。
  4. Schenk,ST,Hernandez-Reyes,C.,Samans,B.,Stein,E.,Neumann,C.,Schikora,M.,Reichelt,M.,Mithofer,A.,Becker,A.,Kogel,KH和Schikora,A。(2014)。 N-酰基 - 高丝氨酸内酯引物植物用于细胞壁增强,并通过水杨酸诱导对细菌病原体的抗性酸/oxylipin途径。植物细胞26(6):2708-2723。
  5. Strack,D.,Heilemann,J.,Momken,M。,和Wray,V。(1988)。 来自coniferae叶的细胞壁缀合酚类。植物化学 27,3517-3521。

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引用:Schenk, S. T. and Schikora, A. (2015). Lignin Extraction and Quantification, a Tool to Monitor Defense Reaction at the Plant Cell Wall Level. Bio-protocol 5(6): e1430. DOI: 10.21769/BioProtoc.1430.
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