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Quantification of Anthocyanin Content
花青素含量的定量测定   

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Abstract

Anthocyanins are a class of flavonoids and important plant pigments. They attract insects to pollinate flowers, protect plants from UV irradiation, and act as antimicrobial agents against herbivores and pathogens. Biosynthesis of anthocyanin is stimulated by diverse developmental signals and environmental stresses including drought, wounding, pathogen infection and insect attack. Plant hormones such as jasmonates, a stress-related plant hormone, also induce accumulation of anthocyanins. Sensitivity of plants to these stress stimuli can be measured by accumulation of anthocyanins. Here we describe a simple method for measurement of anthocyanins in Arabidopsis thaliana seedlings. Amount of anthocyanins are calculated only from absorbances at 530 and 657 nm of crude extract.

Materials and Reagents

  1. Arabidopsis thaliana seedlings (~10 days after germination)
    Note: Amount of anthocyanin per seedling weight is higher in young seedlings.
  2. Bleach solution
  3. Sterile dH2O
  4. Methanol
  5. Acetic acid
  6. Murashige and Skoog medium salt (Wako Pure Chemical Industries, catalog number: 392-00591 )
  7. Sucrose
  8. 2-Morpholinoethanesulfonic acid (MES)
  9. Agar (for plant culture)
  10. Modified Murashige and Skoog medium (see Recipes)
  11. Extraction buffer (see Recipes)

Equipment

  1. Paper towel
  2. Spectrophotometer
  3. Microcentrifuge
  4. Microcentrifuge tubes
  5. Mortar and pestle
  6. Liquid nitrogen
  7. Electric balance

Procedure

  1. Add bleach solution to Arabidopsis thaliana seeds and shake gently for 7 min.
  2. Rinse three to five times with sterile dH2O.
  3. Sow surface-sterilized seeds on modified MS medium.
  4. Grow the plants under long day (16 h light/8 h dark) or continuous light condition (50~70 µmol/m2/s) at 22 °C for 7~10 days.
  5. Measure fresh weight of 10~15 seedlings.
    Note: Remove surface moisture with paper towel before measurement.
  6. Freeze seedlings with liquid nitrogen and grind in a mortar and pestle.
  7. Add 5 volumes (based on fresh weight) of extraction buffer and mix thoroughly.
  8. Centrifuge at 12,000 x g for 5 min at room temperature.
  9. Transfer supernatant to new tube.
  10. Centrifuge at 12,000 x g for 5 min at room temperature.
  11. Transfer supernatant to new tube.
  12. Measure absorbances at 530 and 637 nm.
  13. Calculate anthocyanin content (Abs530/g F.W.) by [Abs530 - (0.25 x Abs657)] x 5.
    Note: To correct contribution of chlorophyll to the absorbances at 530 nm, the formula Abs530 - (0.25 x Abs657) was used. Multiplication of “5” can be changed depending on volumes of added extraction buffer in step 5. For example, if 10 volumes of extraction buffer was added, multiply “10”.

Recipes

  1. Modified Murashige and Skoog medium
    1x Murashige and Skoog medium salt
    0.5% Sucrose
    0.05% MES
    Adjust pH to 5.7 with KOH
    0.8% agar
  2. Extraction buffer
    45% methanol
    5% acetic acid

Acknowledgments

This protocol is adapted from Nakata et al. (2013).

References

  1. Nakata, M., Mitsuda, N., Herde, M., Koo, A. J., Moreno, J. E., Suzuki, K., Howe, G. A. and Ohme-Takagi, M. (2013). A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis. Plant Cell 25(5): 1641-1656.

简介

花青素是一类黄酮类化合物和重要的植物色素。 它们吸引昆虫授粉花,保护植物免受紫外线辐射,并作为抗食草动物和病原体的抗微生物剂。 花青素的生物合成由多种发育信号和环境胁迫(包括干旱,伤害,病原体感染和昆虫攻击)刺激。 植物激素例如茉莉酮酸酯(一种应激相关的植物激素)也诱导花青素的积累。 植物对这些胁迫刺激的敏感性可以通过花色素苷的积累来测量。 在这里,我们描述了一种测量拟南芥幼苗中花青素的简单方法。 花青素的量仅由粗提取物的530和657nm处的吸光度计算

材料和试剂

  1. 拟南芥幼苗(发芽后约10天)
    注意:幼苗中每株幼苗的花青素含量较高。
  2. 漂白溶液
  3. 无菌dH 2 O 2/b
  4. 甲醇
  5. 乙酸
  6. Murashige和Skoog中盐(Wako Pure Chemical Industries,目录号:392-00591)
  7. 蔗糖
  8. 2-吗啉乙磺酸(MES)
  9. 琼脂(植物培养)
  10. 修改Murashige和Skoog介质(见配方)
  11. 提取缓冲液(参见配方)

设备

  1. 毛巾
  2. 分光光度计
  3. 微量离心机
  4. 微量离心管
  5. 砂浆和杵
  6. 液氮
  7. 电子天平

程序

  1. 向拟南芥种子中加入漂白溶液并轻轻摇动7分钟。
  2. 用无菌dH 2 O冲洗三至五次。
  3. 在改良的MS培养基上播种表面灭菌的种子
  4. 在长日照(16小时光照/8小时黑暗)或连续光照条件(50〜70μmol/m 2/s)下在22℃下生长植物7〜10天。 >
  5. 测量10〜15棵幼苗的鲜重 注意:在测量之前用纸巾除去表面水分。
  6. 用液氮冷冻幼苗,用研钵和杵研磨
  7. 加入5倍体积(基于鲜重)的提取缓冲液,并彻底混合
  8. 在室温下以12,000×g离心5分钟
  9. 将上清液转移到新试管中
  10. 在室温下以12,000×g离心5分钟
  11. 将上清液转移到新试管中
  12. 测量530和637nm处的吸光度。
  13. 通过[Abs 530 - (0.25×Abs 657)]×5计算花色素苷含量(Abs 530/g F.W.)。
    注意:为了校正叶绿素对530nm处吸光度的贡献,使用式Abs530 - (0.25×Abs657)。 "5"的乘法可以根据步骤5中添加的提取缓冲液的体积而改变。例如,如果添加了10体积的提取缓冲液,则乘以"10"。

食谱

  1. 修改Murashige和Skoog介质
    1x Murashige和Skoog中盐
    0.5%蔗糖 0.05%MES
    用KOH调节pH至5.7 0.8%琼脂
  2. 提取缓冲区
    45%甲醇
    5%乙酸

致谢

该协议改编自Nakata等人(2013)。

参考文献

  1. Nakata,M.,Mitsuda,N.,Herde,M.,Koo,A.J.,Moreno,J.E.,Suzuki,K.,Howe,G.A。和Ohme-Takagi, bHLH型转录因子,ABA诱导BHLH型转录因子/JA相关MYC2- LIKE1,作为在拟南芥中负调节茉莉酮酸信号的阻遏物。 植物细胞25(5):1641-1656。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nakata, M. and Ohme-Takagi, M. (2014). Quantification of Anthocyanin Content. Bio-protocol 4(7): e1098. DOI: 10.21769/BioProtoc.1098.
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mohamad padri
Naresuan University
dear authors,

I would like to ask about what kind of Volume do we use that you mention in the 7th step of procedure ?
secondly, based on the calculation, the result will be in Abs530/g F.W. would you mind to exemplify this ?

many thanks

best regards
2/15/2016 10:42:38 PM Reply