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The primary aerial surfaces of all land plants are coated by a lipidic cuticle, which restricts non-stomatal water loss and protects the plant from pathogens, herbivores, and ultraviolet radiation. The cuticle is made up of two components: cutin, a polymer of hydroxy- and epoxy- long-chain fatty acid derivatives and glycerol, and cuticular waxes, which are derivatives of very-long-chain fatty acids. While chemical analysis of cutin can be a lengthy and technically challenging task, analysis of cuticular waxes is relatively simple, and can be routinely used to characterize different plant species, adaptations of a given species to environmental conditions, or mutant phenotypes. Here, we present a protocol tailored for the analysis of cuticular waxes on the surface of the model organism Arabidopsis thaliana. Because cuticular waxes are found on the outermost surface of the plant, the wax extraction process is very simple, and sample processing can be completed in less than one day. Chemical analysis involves quantitation of wax monomers by gas chromatography coupled with flame ionization detection (GC/FID), and identification of wax monomers by either mass spectrometry or comparison of retention times of individual wax components to those of known standards.

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Wax Analysis of Stem and Rosette Leaves in Arabidopsis thaliana
拟南芥茎和莲座叶的蜡质层分析

植物科学 > 植物生物化学 > 脂质
作者: Tegan M Haslam
Tegan M HaslamAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
For correspondence: tegan.haslam@botany.ubc.ca
Bio-protocol author page: a612
 and Ljerka Kunst
Ljerka KunstAffiliation: Department of Botany, University of British Columbia, Vancouver, Canada
For correspondence: ljerka.kunst@ubc.ca
Bio-protocol author page: a611
Vol 3, Iss 11, 6/5/2013, 4874 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.782

[Abstract] The primary aerial surfaces of all land plants are coated by a lipidic cuticle, which restricts non-stomatal water loss and protects the plant from pathogens, herbivores, and ultraviolet radiation. The cuticle is made up of two components: cutin, a polymer of hydroxy- and epoxy- long-chain fatty acid derivatives and glycerol, and cuticular waxes, which are derivatives of very-long-chain fatty acids. While chemical analysis of cutin can be a lengthy and technically challenging task, analysis of cuticular waxes is relatively simple, and can be routinely used to characterize different plant species, adaptations of a given species to environmental conditions, or mutant phenotypes. Here, we present a protocol tailored for the analysis of cuticular waxes on the surface of the model organism Arabidopsis thaliana. Because cuticular waxes are found on the outermost surface of the plant, the wax extraction process is very simple, and sample processing can be completed in less than one day. Chemical analysis involves quantitation of wax monomers by gas chromatography coupled with flame ionization detection (GC/FID), and identification of wax monomers by either mass spectrometry or comparison of retention times of individual wax components to those of known standards.
Keywords: Arabidopsis(拟南芥), Cuticle(角质层), Wax(蜡), Lipid(脂质), Gas chromatography(气相色谱法)

[Abstract]

Materials and Reagents

  1. Chloroform, ACS grade (Sigma-Aldrich, catalog number: 319988-4L )
  2. N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane (99:1 BSTFA+TMCS) (Sigma-Aldrich, catalog number: 33148 )
  3. Pyridine, ACS grade (Thermo Fisher Scientific, catalog number: P368-500 )
  4. Tetracosane (solid, for use as an internal standard) (Sigma-Aldrich, catalog number: 87089-1G )

Equipment

  1. Ruler or sticker of known dimensions
  2. Overhead transparent sheets (only needed for leaf wax analysis)
  3. Camera, ideally with a small tripod
  4. Scissors
  5. Forceps
  6. 11 ml glass tubes (Wheaton, catalog number: 358606 )
  7. 1.5 ml GC vials (Agilent, catalog number: 5182-0715 )
  8. 250 μl GC vial inserts (Agilent, catalog number: 5183-2085 )
  9. GC vial rack
  10. 100 μl glass syringe (Hamilton, catalog number: 80630 )
  11. 500 μl glass syringe (Hamilton, catalog number: 80865 )
  12. Oven
  13. Reacti-Vap evaporator (Thermo Fisher Scientific, catalog number: TS-18825 ) with Reacti-Therm heating module (Thermo Fisher Scientific, catalog number: TS-18822 )
  14. Nitrogen gas (Praxair NI M-T)
  15. Adobe Photoshop
  16. Microsoft Excel
  17. Gas chromatography system
    1. Agilent 7890A GC system with FID
    2. HP1 methyl siloxane column (Agilent, 19091Z-313 )
    3. hydrogen (GC carrier gas and FID fuel) and compressed air (for FID)

Procedure

Note: A few steps of the protocol vary between leaves and stems. Chloroform, pyridine, and BSTFA should all be handled in a fume hood.

  1. Prepare a 1 μg μl-1 internal standard by dissolving 500 μg tetracosane in 500 μl chloroform. If you expect to be analyzing waxes routinely, scale this up to make a large stock, and aliquot known amounts of the stock in 1.5 ml GC vials. Allow the chloroform to evaporate off, seal the vials, and store at 4 °C. Resuspend the aliquots in chloroform to their initial volume as required for immediate use.
  2. Prepare one 11 ml glass tube for each sample you will collect. We recommend using 3-6 technical replicates for each genotype you are investigating. Rinse all glass tubes with chloroform three times before you begin. Then, fill the tubes with approximately 10 ml chloroform, and add 10 μl internal standard to each sample. It is critical to remember to include a set of wild-type samples as a positive control every time you do a wax analysis experiment. Wax load and composition can vary greatly depending on the conditions your plants are grown in, so this control is necessary to distinguish between changes caused by different genotypes vs. changes in the environmental conditions or developmental stage of the plants at the time of harvest. If you wish to check the GC trace of the solvent and standard alone, you can prepare an extra tube at this step, which will not have a tissue sample dipped in it.
  3. STEMS: Stem wax extraction and image capture for surface area calculation. Take care to minimize sample handling, as handling will remove wax from your sample. Use forceps, not your hands, whenever possible.
    1. Cut stems approximately 10cm from the apical meristem, remove leaves and branches, and lie flat on a light, even background with a ruler or item of known dimensions for size comparison. Label each sample by writing on the background. Only one stem for each replicate is needed.
    2. Photograph the samples so that you have all stems, their labels, as well as the ruler or size marker in view (Figure 1). Photographs will be used later to calculate stem surface area.
    3. Dip stems in solvent tubes for 30 s to extract wax. Stems can be thrown out after dipping.

      Figure 1. How to collect stem area data. Stems are trimmed and photographed for measurement before dipping in chloroform. The round green sticker was used as a reference for size.

    ROSETTE LEAVES: Rosette leaf wax extraction and image capture for area calculation. As with stems, take care to minimize sample handling, and use forceps, not your hands, whenever possible.

    1. Arabidopsis rosette leaves carry approximately 10% of the wax load found on inflorescence stems. Therefore, you will need to collect more leaf tissue to complete your analysis. Aim to collect at least 6 leaves for each replicate. Also, you will likely find more variability in leaf wax composition and load than you will for stem, so using more technical replicates for each genotype (5-8) is advisable.
    2. As rosette leaves of Arabidopsis are often curved and warped, it is difficult to calculate their area based on a 2D image. Our solution is to first dip leaves in chloroform for 30 s, then, after the wax has been extracted and the leaf is limp and coated with solvent, spread the leaf flat on a plastic transparency sheet. The transparency sheet will melt very slightly with the chloroform, helping to hold the leaf flat. Label each sample by writing on the transparency sheet (Figure 2).
    3. Repeat for all leaves for each replicate, and all replicates for each genotype. Photograph the samples after wax extraction, with the labels and ruler or size marker in view. Photographs will be used later to calculate stem surface area.
    4. Try to work quickly, as the leaves will eventually dry out after wax extraction, and your area calculation will be inaccurate if you do not photograph the samples quickly enough.

      Figure 2. How to collect leaf area data. Leaves are laid flat (which is sometimes easier if small notches are cut in the leaf blade) and photographed for measurement after dipping in chloroform.

  4. Evaporate off all of the solvent by passing a gentle stream of nitrogen gas from an evaporator manifold over each sample. Clean the steel needles of the manifold in chloroform before and after each use.
    Note: A greater flow of nitrogen will not necessarily reduce the time required to evaporate your sample. Use a low flow and position the needles so that there is a shallow dimple in the top of your sample from the outflow of gas. If your sample is splashing or bubbling, you are wasting nitrogen, and risk losing your sample.
  5. After the solvent has dried off, you should be able to see a white film of wax at the bottom of each tube. Resuspend this wax residue in 100-200 μl fresh chloroform using a glass syringe, and transfer to a GC vial insert in a GC vial. Evaporate solvent off as above. Be very careful with the outflow of nitrogen this time; because the vial inserts are narrow, it is very easy to apply too much nitrogen and lose your concentrated sample.
  6. After the solvent has dried off completely, add 10 μl pyridine and 10 μl BSTFA+TMCS to each sample using a glass syringe. Seal the GC vials tightly, and incubate for 1 h in an oven set to 80 °C.
  7. Remove the vials from the oven and allow them to cool to room temperature for 1-2 min. Evaporate off the solvent again as above.
    Note: Pyridine, BSTFA, and TMCS will evaporate much more slowly than chloroform.
  8. Resuspend the final derivatized residues in 40 μl chloroform, and seal the GC vials tightly.
  9. Inject samples on the GC column. We use a 2.7:1 split for stem wax analysis, and run leaf waxes in splitless mode. For both, we inject 1 μl sample. The program we use is as follows:
        50 °C for 2 min
        ramp 40 °C/min to 200 °C
        hold 1 min
        ramp 3 °C/min to 320 °C
        hold 15 min
  10. Calculate the area of your samples. Upload your photos to Adobe Photoshop, and use the magic wand tool to select and record the area of each of your samples and the size marker in pixels. As the size of the marker is known, you can use a simple ratio to determine the 2D areas of your samples in centimeters or inches. For leaves, 2D area is used as is. For stems, the 2D area represents the stem diameter multiplied by length. Therefore, you will need to multiply the 2D area by pi to obtain the surface area for the entire stem.
  11. After the GC has finished running your samples, download your data. Typically, a wild-type sample will be run through GC/MS using the same program as for GC/FID so that you can determine the chemical identities of each of the peaks in your trace from GC/FID. Alternatively, you can compare the peaks in the GC/FID chromatogram from your wild-type sample to a chromatogram where the peaks have been identified, and determine the exact retention times that you expect for each monomer on your system. Identify the peaks for the wax monomers you are interested in, and the internal standard, and copy their retention times and peak areas into a Microsoft Excel spreadsheet. Repeat this for all of your replicates and different genotypes.
  12. 10 μg tetracosane standard was added to each sample. Therefore, you can use a ratio with the tetracosane standard mass and peak area and the peak area of each of your wax monomers to determine the mass of each monomer for every sample. Finally, the amount of each monomer is divided by the sample area, so that you express wax monomer amount in μg/cm2 for stems, or μg/dm2 for leaves. If you include all wax monomers in your analysis, you can tally these values to determine the total wax load for each sample.

References

  1. Haslam, T. M., Manas-Fernandez, A., Zhao, L. and Kunst, L. (2012). Arabidopsis ECERIFERUM2 is a component of the fatty acid elongation machinery required for fatty acid extension to exceptional lengths. Plant Physiol 160(3): 1164-1174.

材料和试剂

  1. 氯仿,ACS级(Sigma-Aldrich,目录号:319988-4L)
  2. N,O-双(三甲基甲硅烷基)三氟乙酰胺与三甲基氯硅烷(99:1BSTFA + TMCS)(Sigma-Aldrich,目录号:33148)
  3. 吡啶,ACS级(Thermo Fisher Scientific,目录号:P368-500)
  4. 二十四烷(固体,用作内标)(Sigma-Aldrich,目录号:87089-1G)

设备

  1. 尺寸或已知尺寸的标签
  2. 顶部透明片(仅用于叶蜡分析)
  3. 相机,理想情况下用小三脚架
  4. 剪刀
  5. 镊子
  6. 11ml玻璃管(Wheaton,目录号:358606)
  7. 1.5ml GC小瓶(Agilent,目录号:5182-0715)
  8. 250μlGC小瓶插入物(Agilent,目录号:5183-2085)
  9. GC小瓶架
  10. 100μl玻璃注射器(Hamilton,目录号:80630)
  11. 500μl玻璃注射器(Hamilton,目录号:80865)
  12. 烤箱
  13. 使用Reacti-Therm加热模块(Thermo Fisher Scientific,目录号:TS-18822)的Reacti-Vap蒸发器(Thermo Fisher Scientific,目录号:TS-18825)
  14. 氮气(Praxair NI M-T)
  15. Adobe Photoshop
  16. Microsoft Excel
  17. 气相色谱系统
    1. 带有FID的Agilent 7890A GC系统
    2. HP1甲基硅氧烷柱(Agilent,19091Z-313)
    3. 氢气(GC载气和FID燃料)和压缩空气(用于FID)

程序

注意:协议的几个步骤在叶和茎之间有所不同。 氯仿,吡啶和BSTFA都应在通风橱中处理。

  1. 通过将500μg二十四烷溶解在500μl氯仿中制备1μgμl -1 内标。 如果你希望经常分析蜡,扩大规模以制造大量库存,并在1.5 ml GC小瓶中等分已知量的原料。 让氯仿蒸发掉,密封小瓶,并在4°C下保存。 将氯仿中的等分试样重悬至其初始体积,以便立即使用。
  2. 为您将收集的每个样品准备一个11毫升玻璃管。我们建议对您正在研究的每个基因型使用3-6个技术重复。开始之前,用氯仿冲洗所有玻璃管三次。然后,用约10ml氯仿填充管,并向每个样品加入10μl内标。至关重要的是,每次进行蜡分析实验时,记住包括一组野生型样品作为阳性对照。蜡的负荷和组成可以根据您的植物生长的条件大不相同,因此这种控制是必要的,以区分由不同基因型引起的变化与环境条件或收获时植物发育阶段的变化。如果您想单独检查溶剂和标准品的GC谱图,您可以在此步骤准备额外的试管,这样不会有组织样品浸入其中。
  3. STEMS:用于表面积计算的吸蜡和图像捕获。注意尽量减少样品处理,因为处理会从样品中除去蜡。尽可能使用镊子,而不是您的手。
    1. 切茎距离顶端分生组织约10cm,去除叶和枝,并平放在光,甚至背景与尺寸或已知尺寸的项目进行大小比较。通过在背景上书写标记每个样品。每个重复只需要一个茎。
    2. 拍摄样品,使您拥有所有茎,其标签,以及视图中的标尺或尺寸标记(图1)。照片将用于以后计算茎表面积。
    3. 浸泡在溶剂管中30秒以提取蜡。浸泡后可能会丢弃茎。

      图1.如何收集茎区数据。在浸入氯仿之前,对茎进行修剪和拍照以进行测量。圆形绿色贴纸用作尺寸的参考

    ROSETTE LEAVES:玫瑰叶蜡提取和区域图像捕获 计算。与茎一样,注意尽量减少样品处理,和 使用镊子,而不是你的手,尽可能。

    1. 拟南芥玫瑰花叶携带大约10%的花序茎上发现的蜡负载。因此,您将需要收集更多的叶组织以完成您的分析。目标是为每个重复收集至少 6个叶子。此外,您可能会发现叶蜡组成和负载比您对茎更多的变异性,因此使用更多的技术重复每个基因型(5-8)是可取的。
    2. 由于拟南芥的莲座叶通常是弯曲的和弯曲的,所以难以基于2D图像计算它们的面积。我们的解决方案是首先将叶子浸在氯仿中30秒,然后,在蜡提取并且叶子柔软并用溶剂涂覆之后,将叶片平铺在塑料透明片上。透明片将与氯仿非常轻微地融化,有助于保持叶平。通过在透明片上书写标记每个样品(图2)。
    3. 对每个复制的所有树叶重复此操作,为每个复制复制所有复制 基因型。蜡提取后,用标签拍摄样品 和标尺或尺寸标记。照片将在以后使用 计算茎表面积。
    4. 尝试快速工作,因为叶子将最终干后蜡 提取,你的面积计算将不准确,如果你不 以足够快的速度拍摄样品
      图2.如何收集叶面积数据。将叶子平放(如果在叶片中切割小切口,有时更容易),并在浸入氯仿后拍照进行测量。

  4. 通过将温和的氮气流从蒸发器歧管通过每个样品蒸发掉所有溶剂。在每次使用之前和之后,在氯仿中清洁歧管的钢针。
    注意:较大的氮气流量不一定会减少蒸发样品所需的时间。使用低流量并定位针头,以便样品顶部有一个浅的凹坑,从气体的流出。如果您的样品是飞溅或冒泡,您正在浪费氮气,可能会失去您的样品。
  5. 溶剂干燥后,您应该能够在每个管的底部看到白色的蜡膜。使用玻璃注射器将此蜡残留物重新悬浮在100-200μl新鲜氯仿中,并转移到GC小瓶中的GC小瓶插入物。如上所述蒸发掉溶剂。这一次要注意氮的流出;因为小瓶插件很窄,很容易施加太多的氮气并损失浓缩的样品
  6. 溶剂完全干燥后,使用玻璃注射器向每个样品中加入10μl吡啶和10μlBSTFA + TMCS。紧紧密封GC小瓶,并在设置为80℃的烘箱中孵育1小时
  7. 从烘箱中取出小瓶,让它们冷却至室温1-2分钟。如上所述再次蒸发掉溶剂。
    注意:吡啶,BSTFA和TMCS将比氯仿蒸发得慢得多。
  8. 将最终的衍生化残留物重悬在40μl氯仿中,并密封GC小瓶
  9. 在GC柱上注入样品。我们使用2.7:1分割用于茎蜡分析,并且以不分流模式运行叶蜡。对于两者,我们注射1μl样品。我们使用的程序如下:
         50°C 2分钟
        以40°C/min升至200°C
        持续1分钟
        以3°C/min升至320°C
        持续15分钟
  10. 计算样品的面积。将照片上传到Adobe Photoshop,然后使用魔术棒工具选择并记录每个样本的区域和以像素为单位的大小标记。由于标记的大小是已知的,您可以使用简单的比率来确定样本的2D区域(以厘米或英寸为单位)。对于叶,2D区域被原样使用。对于茎,2D区域表示茎直径乘以长度。因此,您将需要将2D面积乘以pi以获得整个茎的表面积。
  11. GC运行完样品后,下载数据。通常,野生型样品将通过GC/MS使用与GC/FID相同的程序运行,以便可以从GC/FID确定谱图中每个峰的化学特性。或者,您可以将来自野生型样品的GC/FID色谱图中的峰与已经确定峰的色谱图进行比较,并确定系统中每个单体的预期保留时间。确定您感兴趣的蜡单体的峰和内标,并将其保留时间和峰面积复制到Microsoft Excel电子表格中。对所有重复和不同基因型重复此操作。
  12. 向每个样品中加入10μg二十四烷标准品。因此,您可以使用与二十四烷标准质量和峰面积的比率以及每种蜡单体的峰面积来确定每种单体的质量 。 最后,将每种单体的量除以样品面积,以便表示茎的单位μg/cm 2 的蜡单体量,或叶的μg/dm 2 。 如果在分析中包括所有蜡单体,则可以计算这些值以确定每个样品的总蜡负载。

参考文献

  1. Haslam,T. M.,Manas-Fernandez,A.,Zhao,L.and Kunst,L。(2012)。 拟南芥 ECERIFERUM2是脂肪酸需要脂肪酸延长机制的一个组成部分 酸延伸到特殊长度。植物生理 160(3):1164-1174。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Haslam, T. M. and Kunst, L. (2013). Wax Analysis of Stem and Rosette Leaves in Arabidopsis thaliana. Bio-protocol 3(11): e782. DOI: 10.21769/BioProtoc.782; Full Text
  2. Haslam, T. M., Manas-Fernandez, A., Zhao,L. and Kunst, L. (2012). Arabidopsis ECERIFERUM2 is a component of the fatty acid elongationmachinery required for fatty acid extension to exceptional lengths. Plant Physiol 160(3): 1164-1174.




  3. 可重复性反馈:

    • 添加图片
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    我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

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    登陆 | 注册
    11/13/2013 12:25:39 AM  

    girija chalumuru
    university of horticultural sciences, karnataka, india

    ITS A VERY WORTH-FULL PROTOCOL ABOUT LEAF CUTICULAR WAX. SIR CAN I HAVE A STANDARD PROTOCOL FOR ESTIMATION OF EPICUTICULAR WAX THROUGH CALORIMETRIC METHOD WITH STANDARD CURE OF ANY NATURAL WAX, AS I AM A RESEARCH SCHOLAR CONDUCTING MY RESEARCH ON TUBEROSE WAX ESTIMATION .

    PLEASE KINDLY ATTACH THE PROTOCOL TO MY MAIL ID FOR THE SAME IF AVAILABLE.


    THANK YOU SIR

    Reply

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