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Analysis of Flower Cuticular Waxes and Cutin Monomers
花表面角质层蜡份和角质单体的分析

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Abstract

Here we describe procedures for the flower cuticular waxes extraction, modification and subsequent qualitative and quantitative analysis by gas-chromotography-mass spectrometry (GC-MS) and gas-chromotography with flame ionization detector (GC-FID), accordingly. To characterize flower cutin monomers two experimental setup are described: (i) analysis of enzymatically isolated cuticles in order to determine the relative proportions of cutin monomers; (ii) analysis of freeze-dried material for quantitative estimation of the cutin content. This report is an adaptation of the earlier published protocols developed for the chemical analysis of the cuticles in vegetative organs (Leide et al., 2007).

Keywords: Cuticular waxes(表皮蜡), Cutin(角质), Flower(花), GC-MS(气相色谱-质谱联用), GC-FID(气相色谱法)

Materials and Reagents

  1. Chloroform (Carl Roth, catalog number: 7331.2 )
  2. Heptatriacontane (Fluka, catalog number: 51848 )
  3. Dotriacontane (Fluka, catalog number: 44253 )
  4. N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) (Macherey-Nagel, catalog number: 701220.201 )
  5. Pyridine (Merck, catalog number: 1074630500 )
  6. 1.25 M methanol-HCl (Fluka, catalog number: 17935 )
  7. Sodium chloride-saturated aqueous solution (Applichem, catalog number: A2824 )
  8. Anhydrous sodium sulfate salt (Applichem, catalog number: A1048 )
  9. Pectinase (Trenolin Super D) (Erbsloh, catalog number: 20312 )
  10. Cellulase (Cellulast) (Novo Nordisk AIS, catalog number: CA451088 )
  11. Citric acid monohydrate (Sigma-Aldrich, catalog number: C1909 )
  12. Liquid nitrogen
  13. 20 mM citrate buffer, pH 3.0 supplemented with 1 mM sodium azide (see Recipes)

Equipment

  1. Freeze-drier
  2. Nitrogen cylinder and blow-down system
  3. Block heater with supports for glass scintillation vials
  4. Glass scintillation vials (for volume 15-20 ml)
  5. Glass vials with conic bottom (for volume 1-1.5 ml)
  6. Glass vials (with volume 1.5 ml) and glass inserts for them (with volume 0.2 ml)
  7. Glass funnels and glass Pasteur pipette
  8. Screw caps with Teflon or PTFE liners for the glass vials
  9. Glass syringes (for volumes 1,000, 100, 50 and 10 μl)
  10. Paper filters
  11. Nylon filters (with pores 41 μm) (EMD Millipore, model: NY4102500 )
  12. Glass filtration apparatus
  13. GC-MS: Temperature controlled capillary gas chromatograph (Agilent Technologies, model: 6890N ) with on-column injection (J&W Scientific; 30 m DB-1, 320 μm i.d., df = 1 mm) and a mass spectrometric detector (Agilent Technologies, model: 5973N ; 70 eV; m/z 50–750)
  14. GC-FID: Capillary gas chromatography (Hewlett-Packard, model: 5890 II ) and flame ionization detection

Procedure

  1. Wax analysis
    1. Flowers of Solanum lycopersicum L. cv. MicroTom were collected in anthesis and frozen in liquid nitrogen. Plant material was freeze-dried overnight.
    2. About 10 to 30 mg of freeze-dried material was submersed in 10 ml chloroform containing 3 mg of heptatriacontane (internal standard) in 15 ml vessels for 1 min and then filtered through a paper filter.
    3. The filtrate was evaporated under a flow of nitrogen to volume 1 ml and transferred into glass-vials with conic bottom and volume 1-1.5 ml. The solvent was totally evaporated under a flow of nitrogen.
    4. Hydroxyl-containing compounds were transformed into the corresponding trimethylsilyl derivatives using 10 μl BSTFA and 10 μl pyridine. The mixtures were incubated at 70 °C for 30 min and then dissolved in 50-100 μl of chloroform.
    5. Solution was transferred into the glass inserts, which were placed in glass vials with volume 1.5 ml.
    6. The qualitative composition was identified with temperature controlled capillary gas chromatography and on-column injection with helium carrier gas inlet pressure programmed at 50 kPa for 5 min, 3.0 kPa min-1 to 150 kPa, and at 150 kPa for 40 min. Separation of the wax mixtures was achieved using an initial temperature of 50 °C for 2 min, raised by 40 °C min-1 to 200 °C, held at 200 °C for 2 min, and then raised by 3 °C min-1 to 320 °C and held at 320 °C for 30 min. These parameters are universal for analysis of the cuticular waxes and allow good peak resolution for samples derived from diverse plant species. Individual compounds were identified according to their retention times and mass-spectra obtained from commercially available and domestic libraries.
    7. Quantitative composition of the mixtures was studied using capillary gas chromatography and flame ionization detection under the same gas chromatographic conditions as above, but with hydrogen as carrier gas. Single compounds were quantified against the heptatriacontane. Wax load was calculated to dry weight of freeze-dried flowers.

  2. Cutin analysis
    1. For the cutin analysis freeze-dried material (a) or enzymatically isolated cuticles (b) were used.
      1. About 25 mg of freeze-dried flowers were briefly washed with chloroform at room temperature and then with a new chloroform portion at 50 °C for 30 min. Afterwards samples were incubated for one week in chloroform changed daily (at room temperature). Wax-free material was air dried and stored on silica.
      2. Cuticles from fresh flowers were isolated enzymatically with pectinase and cellulose in the citrate buffer supplemented with sodium azide. Material was incubated for 4 weeks in the enzymatic solution with occasional shaking. Every week material was collected on the nylon filter and then replaced into fresh portion of enzymatic solution. Isolated cuticles were washed with water and dried out under an air stream. Then cuticles were delipidated by chloroform and again air dried.
    2. Subsequent isolation of cutin monomers and their analysis did not differ for two types of samples. Dried samples were trans-esterified with 1 ml of 1.25 M methanol/HCl at 80 °C overnight to release methyl esters of cutin acid monomers and phenolics.
    3. Afterwards 1 ml of sodium chloride-saturated aqueous solution, 2 ml of chloroform spiked with 20 μg of dotriacontane (internal standard) were added to the reaction mixture. All components were intermingled by shaking and then allowed to segregate into two phase.
    4. Lower phase represented by chloroform with depolymerized transmethylated cutin components was collected with the glass syringe and transferred into a glass vial.
    5. New 2 ml portion of chloroform was added to the reaction mixture. All components were intermingled by shaking and then allowed to segregate into two phase.
    6. Lower phase was again collected with the glass syringe and pooled with the extract from step 11.
    7. New 2 ml portion of chloroform was added to the reaction mixture. All components were intermingled by shaking and then allowed to segregate into two phase.
    8. Lower phase was again collected with the glass syringe and pooled with the extract from step 11. Thus, the extraction was performed thrice.
    9. The combined organic phases were dried over anhydrous salt of sodium sulfate. For this the salt was added to the solution in small portions till the salt stopped conglomerate. The amount required depends on the amount of water in the solvent solution, and it varies from experiment to experiment. The solution should be dried out until salt crystals float free.
    10. The solution was filtered via paper filters to get rid of the salt and the organic solvent was evaporated under a continuous flow of nitrogen.
    11. Hydroxyl-containing compounds were transformed into the corresponding trimethylsilyl derivatives like waxes and then dissolved in 250 μl of chloroform.
    12. Solution was transferred to into the glass inserts, which were placed in glass vials with volume 1.5 ml.
    13. GC-MS and GC-FID of the cutin components was conducted with the use of the same equipment, but different conditions. Inlet pressure programmed at 50 kPa for 60 min, 10.0 kPa min-1 to 150 kPa. Initial temperature of 50 °C for 2 min, raised by 10 °C min-1 to 150 °C, held at 150 °C for 2 min, and then raised by 3 °C min-1 to 320 °C and held at 320 °C for 30 min. Single compounds were quantified against the dotriacontane. Relative proportion of the cutine components were calculated to dry weight of isolated wax-free cuticles. Quantitative cutine composition was calculated was calculated to the dry weight of delipidated freeze-dried flowers.

Recipes

  1. 20 mM citrate buffer, pH 3.0 supplemented with 1 mM sodium azide
    2,000 ml distilled water
    20 ml Cellulast (Cellulase)
    20 ml Trenolin Super D (Pectinase)
    0.13 g sodium azide
    Store at room temperature

Acknowledgments

This report is an adaptation of earlier published protocols developed for the chemical analysis of cuticles in vegetative organs (Leide et al., 2007).

References

  1. Leide, J., Hildebrandt, U., Reussing, K., Riederer, M. and Vogg, G. (2007). The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6). Plant Physiol 144(3): 1667-1679.
  2. Smirnova, A., Leide, J. and Riederer, M. (2013). Deficiency in a very-long-chain fatty acid beta-ketoacyl-coenzyme a synthase of tomato impairs microgametogenesis and causes floral organ fusion. Plant Physiol 161(1): 196-209. 

简介

在这里我们介绍花角质蜡提取,修改和随后的气相色谱 - 质谱(GC-MS)和气相色谱与火焰离子化检测器(GC-FID)的定性和定量分析的程序。 为了表征花角质单体,描述了两个实验装置:(i)分析酶分离的角质层,以确定角质单体的相对比例; (ii)冷冻干燥材料的分析,用于定量估计角质含量。 该报告是对早期公开的用于营养器官中角质层的化学分析的协议的改编(Leide等人,2007)。

关键字:表皮蜡, 角质, 花, 气相色谱-质谱联用, 气相色谱法

材料和试剂

  1. 氯仿(Carl Roth,目录号:7331.2)
  2. 七七烷(Fluka,目录号:51848)
  3. Dotriacontane(Fluka,目录号:44253)
  4. N,O-双 - 三甲基甲硅烷基 - 三氟乙酰胺(BSTFA)(Macherey-Nagel,目录号:701220.201)
  5. 吡啶(Merck,目录号:1074630500)
  6. 1.25M甲醇-HCl(Fluka,目录号:17935)
  7. 氯化钠饱和水溶液(Applichem,目录号:A2824)
  8. 无水硫酸钠盐(Applichem,目录号:A1048)
  9. 果胶酶(Trenolin Super D)(Erbsloh,目录号:20312)
  10. 纤维素酶(Cellulast)(Novo Nordisk AIS,目录号:CA451088)
  11. 柠檬酸一水合物(Sigma-Aldrich,目录号:C1909)
  12. 液氮
  13. 20mM柠檬酸盐缓冲液,pH 3.0,补充有1mM叠氮化钠(参见Recipes)

设备

  1. 冷冻干燥器
  2. 氮气瓶和排污系统
  3. 带玻璃闪烁瓶支架的块式加热器
  4. 玻璃闪烁瓶(体积15-20ml)
  5. 具有锥形底部(体积为1-1.5ml)的玻璃小瓶
  6. 玻璃小瓶(体积1.5ml)和用于它们的玻璃插入物(体积0.2ml)
  7. 玻璃漏斗和玻璃巴斯德吸管
  8. 带玻璃瓶的聚四氟乙烯或PTFE衬垫的螺旋盖
  9. 玻璃注射器(体积为1,000,100,50和10μl)
  10. 滤纸
  11. 尼龙过滤器(孔41μm)(EMD Millipore,型号:NY4102500)
  12. 玻璃过滤装置
  13. GC-MS:具有柱上进样(J& W Scientific; 30m DB-1,320μmid,d f = 1mm)的温控毛细管气相色谱仪(Agilent Technologies,型号:6890N) )和质谱检测器(Agilent Technologies,型号:5973N; 70eV; m/z 50-750)。
  14. GC-FID:毛细管气相色谱(Hewlett-Packard,型号:5890 II)和火焰离子化检测

程序

  1. 蜡分析
    1. 花的叶子。 MicroTom在开花期收集并在液氮中冷冻。 将植物材料冷冻干燥过夜。
    2. 将约10至30mg冷冻干燥材料浸没在含有3mg三十七烷(内标物)的10ml氯仿的15ml容器中1分钟,然后通过滤纸过滤。
    3. 将滤液在氮气流下蒸发至体积1ml,并转移至具有圆锥形底部和1-1.5ml体积的玻璃小瓶中。 溶剂在氮气流下完全蒸发
    4. 使用10μlBSTFA和10μl吡啶将含羟基的化合物转化成相应的三甲基甲硅烷基衍生物。 将混合物在70℃温育30分钟,然后溶于50-100μl氯仿中
    5. 将溶液转移到玻璃插入物中,将玻璃插入物放置在体积为1.5ml的玻璃小瓶中
    6. 通过温度控制的毛细管气相色谱和柱上注射,用氦载气入口压力在50kPa下编程5分钟,3.0kPa min -1 -1至150kPa和150kPa 40分钟。蜡混合物的分离使用50℃的初始温度2分钟,以40℃min升高至200℃,在200℃保持2分钟,然后升高3℃min -1至320℃并在320℃保持30min。这些参数对于表皮蜡的分析是通用的,并且允许来自不同植物物种的样品的良好峰分辨率。根据其从市售和国内图书馆获得的保留时间和质谱来鉴定单个化合物
    7. 在与上述相同的气相色谱条件下,用氢气作为载气,使用毛细管气相色谱和火焰离子化检测来研究混合物的定量组成。单一化合物针对庚三十烷定量。蜡装载量计算为冻干花的干重
  2. Cutin分析
    1. 对于角质分析,使用冷冻干燥的材料(a)或酶分离的角质层(b)。
      1. 在室温下用氯仿短暂洗涤约25mg冷冻干燥的花,然后在50℃下用新的氯仿部分洗涤30分钟。然后将样品在氯仿中每天更换(在室温下)孵育一周。将无蜡材料风干并储存在二氧化硅上
      2. 用补充有叠氮化钠的柠檬酸盐缓冲液中的果胶酶和纤维素酶促分离来自鲜花的角质层。材料在酶溶液中孵育4周,偶尔摇动。每周材料在尼龙过滤器上收集,然后更换为新鲜部分的酶溶液。用水洗涤分离的角质层,并在空气流下干燥。然后用氯仿将角质层脱脂,再次风干
    2. 随后的角质单体的分离和它们的分析对于两种类型的样品没有区别。干燥的样品用1ml的1.25M甲醇/HCl在80℃下酯交换过夜,以释放角质酸单体和酚类的甲酯。
    3. 然后,向反应混合物中加入1ml氯化钠饱和水溶液,2ml氯仿和20μg三十二烷(内标)。所有组分通过摇动混合,然后允许分离成两相
    4. 用氯仿和解聚的转甲基化角质组分代表的下相用玻璃注射器收集并转移到玻璃小瓶中。
    5. 将新的2ml氯仿加入到反应混合物中。所有组分通过摇动混合,然后允许分离成两相
    6. 用玻璃注射器再次收集下相,并与步骤11的提取物合并
    7. 将新的2ml氯仿加入到反应混合物中。所有组分通过摇动混合,然后允许分离成两相
    8. 用玻璃注射器再次收集下相,并与步骤11的提取物合并。因此,提取进行三次。
    9. 合并的有机相用无水硫酸钠干燥。为此,将盐以小份加入溶液中,直到盐停止聚集。所需的量取决于溶剂溶液中的水的量,并且其根据实验变化。溶液应该干燥,直到盐晶体游离为止。
    10. 通过滤纸过滤该溶液以除去盐,并在连续氮气流下蒸发有机溶剂
    11. 将含羟基的化合物转化成相应的三甲基甲硅烷基衍生物如蜡,然后溶于250μl氯仿中
    12. 将溶液转移到玻璃插入物中,将玻璃插入物放置在体积为1.5ml的玻璃小瓶中
    13. 使用相同的设备,但是不同的条件进行角质组分的GC-MS和GC-FID。在50kPa下编程60分钟,10.0kPa min -1至150kPa的入口压力。初始温度为50℃,持续2分钟,以10℃/min升至150℃,在150℃下保持2分钟,然后升高3℃min。 -1至320℃,并在320℃保持30分钟。单一化合物针对三十二烷定量。计算所述角质组分的相对比例,作为分离的无蜡角质层的干重。计算定量的角质组成,计算为脱脂冻干花的干重

食谱

  1. 20mM柠檬酸盐缓冲液,pH 3.0,补充有1mM叠氮化钠 2,000毫升蒸馏水
    20ml Cellulast(纤维素酶)
    20ml Trenolin Super D(果胶酶)
    0.13克叠氮化钠 在室温下贮存

致谢

该报告是对早期公开的用于营养器官中角质层的化学分析开发的方案的改编(Leide等人,2007)。

参考文献

  1. Leide,J.,Hildebrandt,U.,Reussing,K.,Riederer,M。和Vogg,G。(2007)。 番茄果实蜡堆积的发育模式及其对表皮蒸腾屏障特性的影响:缺陷的影响 在β-酮脂酰辅酶A合酶(LeCER6)中。植物生理学144(3):1667-1679。
  2. Smirnova,A.,Leide,J.和Riederer,M。(2013)。 在非常长链脂肪酸β-酮脂酰辅酶缺乏番茄合成酶损害微粒形成 并导致花器官融合。植物生理 161(1):196-209。
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Smirnova, A., Leide, J. and Riederer, M. (2013). Analysis of Flower Cuticular Waxes and Cutin Monomers. Bio-protocol 3(18): e899. DOI: 10.21769/BioProtoc.899.
  2. Leide, J., Hildebrandt, U., Reussing, K., Riederer, M. and Vogg, G. (2007). The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration barrier properties: effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6). Plant Physiol 144(3): 1667-1679.
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