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Jun 2016

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A Protocol for Flavonols, Kaempferol and Quercetin, Staining in Plant Root Tips
植物根尖中黄酮醇、山奈酚和槲皮素的染色方法   

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Abstract

Flavonols are a subclass of flavonoids of the group of plant secondary metabolites. In planta, flavonols play various functions such as antioxidant and natural regulator of auxin polar transport. Many lines of evidence have shown that flavonols also contribute to human health in anti-oxidation, anti-inflammation, and even prevention some types of cancer. Several methods have been utilized to measure flavonols such as high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and diphenylboric acid-2-aminoethyl ester (DPBA) staining. While HPLC or LC-MS can quantitatively determine the level of flavonols, DPBA staining can provide an in-situ view of flavonols accumulation in the plants. In this protocol, a detailed procedure for staining the flavonols in Arabidopsis root tips is described. Five-day-old Arabidopsis seedlings are soaked in a solution containing DPBA and latterly the flavonols (kaempferol and quercetin) can be observed under a confocal microscope.

Keywords: DPBA staining ( DPBA 染色), Kaempferol (山奈酚), Flavonoids (黄酮), Flavonols (黄酮醇), Quercetin (槲皮素)

Background

In Arabidopsis, flavonols are biosynthesized from a condensation reaction between one molecule of p-coumaroyl-CoA and three molecules of malonyl-CoA. Lewis et al. (2011) found that quercetin, but not kaempferol, is an inhibitor of root basipetal auxin transport. In addition, flavonols also function as an efficient antioxidant for plants and humans as well. A prior study investigated that DPBA can fluoresce when it interacts with flavonols (Sheahan and Rechnitz, 1992). Based on this, DPBA has been widely applied to detect the flavonols accumulation in the plants (Nguyen et al., 2013, 2015 and 2016; Vu et al., 2015). Here, a detailed protocol for DPBA-based detection of flavonols is described. Apart from Arabidopsis, this protocol can be also used for other plants such as Brassica napus L. (Vu et al., 2015). Since other plant species may have a thicker and larger tissue than Arabidopsis, a vacuum can be applied to facilitate the penetration of DPBA into the plant tissues.

Materials and Reagents

  1. Square dishes for tissue culture [External dimension (mm): 126.40 x 126.40 x 20.00] (SPL Life Sciences, catalog number: 10125)
  2. 1.5 ml micro-tubes (Eppendorf, catalog number: 0030121589)
  3. Microscope slides (dimensions: 76 x 26 mm; thickness: 1 mm) (Marienfeld, catalog number: 1000200)
  4. Transparent slides coverslips (dimensions: 60 x 24 mm; thickness: 0.170 mm ± 0.005 mm) (Marienfeld, catalog number: 0107242)
  5. Arabidopsis thaliana
  6. Diphenylboric acid-2-aminoethyl ester (DPBA) (Sigma-Aldrich, catalog number: D9754 )
  7. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  8. Murashige & Skoog medium including B5 vitamins (Duchefa Biochemie, catalog number: M0231 )
  9. Phyto Agar (Duchefa Biochemie, catalog number: P1003 )
  10. Sucrose (Duchefa Biochemie, catalog number: S0809 )
  11. DPBA staining solution (see Recipes)

Equipment

  1. Tweezers
  2. A plant growth chamber
  3. Micro-tubes rotator
  4. Pipette
  5. Confocal microscopy system (Leica, model: SP8 )

Procedure

  1. Plant growth and DPBA staining procedure
    1. Here, Arabidopsis thaliana is used. However, this protocol can be applied for other plants as well. Grow plants on MS medium supplemented with 2% sucrose and 1.2% phyto-agar for 5 days (Figure 1). The growth chamber conditions were 22 ± 1 °C, long-day (16 h light/8 h dark), and light intensity 100 μmol m−2 s−1.
      Note: After seeding, remember to place the medium plates vertically in the growth chamber. Thereby, the roots grow on the surface of the medium and intact root tip samples can be obtained.


      Figure 1. Five-day-old Arabidopsis seedlings. Scale bar = 5 mm.

    2. Add 1 ml of DPBA staining solution (Recipe 1) to each micro-tube.
    3. Use a clean tweezers to carefully transfer the 5-day-old Arabidopsis seedlings to the micro-tube containing DPBA staining solution (5 seedlings/micro-tube).
    4. Place the micro-tube in the rotator and rotate for 5 min at room temperature.
    5. Stop the rotator, transfer the micro-tube to a rack and remove the DPBA staining solution.
      Note: At this step, carefully use a pipette to remove the DPBA staining solution and try to not damage the plants, especially the root tips.
    6. For washing, add 1.5 ml of distilled water to each micro-tube. Place the micro-tube in the rotator and rotate for 2 min at room temperature.
    7. Stop the rotator, transfer the micro-tube to a rack and remove water.
    8. Repeat Steps A6 and A7 two more times.
      Note: After washing, can keep the plants in water and try to detect immediately. Do not leave the samples staying in water for longer than 30 min.
    9. Transfer the seedlings to a microscope slide, cover it and detect the flavonols accumulation in the root tips by a confocal microscope.

  2. Confocal microscopy
    1. For DPBA-kaempferol, apply the emission spectrum (475-500 nm).
    2. For DPBA-quercetin, apply the emission spectrum (585-619 nm).
    3. Remember to take an additional bright field picture for control.

Data analysis

Arrange the data as following order: (1) Bright field; (2) Kaempferol; and (3) Quercetin. Please check some previous publications for details (Nguyen et al., 2013, 2015 and 2016; Vu et al., 2015).


Figure 2. Accumulation of flavonols (kaempferol and quercetin) in the root tips of 5-day-old Arabidopsis seedlings (wild-type). Scale bar = 100 μm.

Recipes

  1. DPBA staining solution
    Reagent
    Amount for 100 ml
    DPBA
    0.25 g
    Triton X-100
    20 μl
    Milli-Q water
    up to 100 ml
    Notes:
    1. Some crystals of DPBA can be retained and seen after mixing.
    2. This staining solution can be stored at -20 °C for further uses.

Acknowledgments

I would like to appreciate Dr. Cuong Thach Nguyen (Nguyen Tat Thanh University, Vietnam) and Dr. Minh Tan Nguyen (UCLA School of Dentistry, US) for their critical reading this protocol. This protocol was derived from previous publications (Nguyen et al., 2013, 2015 and 2016). The authors declare that they have no conflict of interest.

References

  1. Lewis, D. R., Ramirez, M. V., Miller, N. D., Vallabhaneni, P., Ray, W. K., Helm, R. F., Winkel, B. S. and Muday, G. K. (2011). Auxin and ethylene induce flavonol accumulation through distinct transcriptional networks. Plant Physiol 156(1): 144-164.
  2. Nguyen, H. N., Kim, J. H., Hyun, W. Y., Nguyen, N. T., Hong, S. W. and Lee, H. (2013). TTG1-mediated flavonols biosynthesis alleviates root growth inhibition in response to ABA. Plant Cell Rep 32(4): 503-514. 
  3. Nguyen, N. H., Jeong, C. Y., Kang, G. H., Yoo, S. D., Hong, S. W. and Lee, H. (2015). MYBD employed by HY5 increases anthocyanin accumulation via repression of MYBL2 in Arabidopsis. Plant J 84(6): 1192-1205. 
  4. Nguyen, N. H., Kim, J. H., Kwon, J., Jeong, C. Y., Lee, W., Lee, D., Hong, S. W. and Lee, H. (2016). Characterization of Arabidopsis thaliana FLAVONOL SYNTHASE 1 (FLS1) -overexpression plants in response to abiotic stress. Plant Physiol Biochem 103: 133-142. 
  5. Sheahan, J. J. and Rechnitz, G. A. (1992). Flavonoid-specific staining of Arabidopsis thaliana. Biotechniques 13(6): 880-883.
  6. Vu, T. T., Jeong, C. Y., Nguyen, H. N., Lee, D., Lee, S. A., Kim, J. H., Hong, S. W. and Lee, H. (2015). Characterization of Brassica napus Flavonol Synthase Involved in Flavonol Biosynthesis in Brassica napus L. J Agric Food Chem 63(35): 7819-7829.

简介

[摘要]黄酮醇是植物次生代谢物中黄酮类的一个亚类。在植物中,黄酮醇具有多种功能,例如抗氧化剂和生长素极性转运的天然调节剂。许多证据表明,黄酮醇还有助于抗氧化,抗炎症甚至预防某些类型的癌症,从而对人类健康做出贡献。几种方法已用于测量黄酮醇,例如高效液相色谱(HPLC),液相色谱-质谱(LC-MS)和二苯硼酸-2-氨基乙基酯(DPBA)染色。HPLC或LC-MS可以定量测定黄酮醇的含量 DPBA染色可以提供植物中黄酮醇积累的原位视图。在该协议中,描述了对拟南芥根尖中的黄酮醇进行染色的详细步骤。将五天大的拟南芥幼苗浸入含有DPBA的溶液中,然后在共聚焦显微镜下观察黄酮醇(山ka酚和槲皮素)。

[背景]在拟南芥,黄酮醇是从缩合反应对香豆酰基辅酶A的一个分子和丙二酰-CoA的三个分子之间生物合成。Lewis等。(2011年)发现槲皮素而不是山奈酚是根型根茎植物生长素转运的抑制剂。此外,黄酮醇也可作为植物和人类的有效抗氧化剂。先前的研究调查了DPBA与黄酮醇相互作用时可以发出荧光(Sheahan和Rechnitz,1992)。基于此,DPBA已被广泛应用于检测植物中黄酮醇的积累(Nguyen等,2013、2015和2016 ; Vu等,2015)。在此,描述了用于基于DPBA的黄酮醇检测的详细协议。除拟南芥外,该方案还可用于其他植物,例如甘蓝型油菜(Vu等人,2015)。由于其他植物物种可能比拟南芥更厚和更大的组织,因此可以施加真空以促进DPBA渗透到植物组织中。

关键字:DPBA 染色, 山奈酚, 黄酮, 黄酮醇, 槲皮素

材料和试剂
 
用于组织培养的方盘[外部尺寸(mm):126.40 x 126.40 x 20.00](SPL Life Sciences,目录号:10125)
1.5 ml微型管(Eppendorf,目录号:0030121589)
显微镜载玻片(尺寸:76 x 26毫米;厚度:1毫米)(Marienfeld ,目录号:1000200)
透明幻灯片盖玻片(尺寸:60 x 24 mm;厚度:0.170 mm±0.005 mm)(Marienfeld ,目录号:0107242)
拟南芥
二苯硼酸-2-氨基乙酯(DPBA)(Sigma-Aldrich,目录号:D9754)
海卫一X-100 (Sigma-Aldrich,目录号:T8787)
含B5维生素的Murashige &Skoog培养基(Duchefa Biochemie ,目录号:M0231)
Phyto Agar (Duchefa Biochemie ,目录号:P1003)
蔗糖(Duchefa Biochemie ,目录号:S0809)
DPBA染色液(请参阅配方)
 
设备
 
镊子
植物生长室
微管旋转器
吸管
共聚焦显微镜系统(Leica,型号:SP8)
 
程序
 
植物生长和DPBA染色程序
在这里,阿拉比dopsis拟南芥使用。但是,该协议也可以应用于其他工厂。在补充2%蔗糖和1.2%植物琼脂的MS培养基上培养植物5天(图1)。生长室条件为22±1℃,长日照(16小时光照/ 8小时黑暗),和光强度100微摩尔米-2小号-1 。
注意:播种后,切记将中板垂直放置在生长室中。从而,根在培养基的表面上生长,并且可以获得完整的根尖样品。




图1.五天大的拟南芥幼苗。标尺a a r = 5毫米。
 
向每个微管中加入1 ml DPBA染色溶液(配方1)。
用干净的镊子将5天大的拟南芥幼苗小心地转移到装有DPBA染色液的微管中(5苗/微管)。
将微管放在旋转器中,在室温下旋转5分钟。
停止旋转器,将微管转移到机架上,然后去除DPBA染色溶液。
注意:在此步骤中,请小心使用移液器除去DPBA染色溶液,并尽量不要损坏植物,尤其是根尖。
洗涤时,向每个微管中加入1.5 ml蒸馏水。将微管放在旋转器中,在室温下旋转2分钟。
停止旋转器,将微管转移到机架上并除去水。
再重复两次步骤A6和A7 。
注:一压脚提升冲洗,能保持植物在水中,并尝试立即检测。请勿将样品在水中停留超过30分钟。
将幼苗转移到显微镜载玻片上,盖上并用共聚焦显微镜检测根尖中的黄酮类化合物。
 
共聚焦显微镜
对于DPBA-山emp酚,应用发射光谱(475-500 nm)。
对于DPBA-槲皮素,应用发射光谱(585-619 nm)。
请记住要拍摄其他明场照片以进行控制。
 
数据分析
 
按以下顺序排列数据:(1)明场;(2)山萘酚;(3)槲皮素。请查看以前的出版物以获取详细信息(Nguyen等人,2013、2015和2016 ; Vu等人,2015)。
 


图2. 5天大拟南芥幼苗(野生型)根尖中的黄酮醇(山ka酚和槲皮素)的积累。刻度尺b AR = 100 μ米。
 
菜谱
 
DPBA染色液
100 ml的试剂量             
DPBA 0.25克             
海卫一X-100 20              μ升
Milli-Q水高达100毫升              
注意事项:
混合后可以保留并看到一些DPBA晶体。
该染色液可在-20℃保存 °C进一步使用。
 
致谢
 
我要感谢Cuong Thach Nguyen博士(越南阮达城大学)和Minh Tan Nguyen博士(美国加州大学洛杉矶分校牙科学院)对本协议的批判性阅读。该协议源自先前的出版物(Nguyen等,2013、2015和2016)。作者宣称他们没有利益冲突。
 
参考文献
 
Lewis,DR,Ramirez,MV,Miller,ND,Vallabhaneni ,P.,Ray,WK,Helm,RF,Winkel,BS和Muday ,GK(2011)。生长素和乙烯通过不同的转录网络诱导黄酮醇积累。植物生理学156(1):144-164。
Nguyen,HN,Kim,JH,Hyun,WY,Nguyen,NT,Hong,SW and Lee,H.(2013年)。TTG1介导的黄酮醇的生物合成减轻了对ABA响应的根系生长抑制。植物细胞Rep 32(4):503-514。              
Nguyen,NH,Jeong ,CY,Kang,GH,Yoo ,SD,Hong,SW和Lee,H.(2015)。通过采用HY5增加MYBD通过压制花青素积累MYBL2在拟南芥。植物J 84(6):1192-1205。              
Nguyen,NH,Kim,JH,Kwon,J.,Jeong ,CY,Lee,W.,Lee,D.,Hong,SW和Lee,H.(2016年)。表征拟南芥黄酮醇合成酶1(FLS1)-overexpression植物响应于非生物胁迫。植物生理学生物化学103:133-142。              
Sheahan,JJ和Rechnitz,GA(1992)。拟南芥的类黄酮特异性染色。生物技术13(6):880-883。
Vu,TT,Jeong ,CY,Nguyen,HN,Lee,D.,Lee,SA,Kim,JH,Hong,SW和Lee,H.(2015年)。表征油菜黄酮醇合成酶在黄酮生物合成相关油菜L. ĴAGRIC食品化学63(35):7819-7829。
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引用:Nguyen, N. H. (2020). A Protocol for Flavonols, Kaempferol and Quercetin, Staining in Plant Root Tips. Bio-protocol 10(19): e3781. DOI: 10.21769/BioProtoc.3781.
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