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GC-MS-Based Analysis of Chloroform Extracted Suberin-Associated Root Waxes from Arabidopsis and Other Plant Species
采用GC-MS分析氯仿提取的拟南芥和其它植物物种中的木栓质根蜡   

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Abstract

The periderm and exodermis of taproots and tuberous taproots contain an extracellular lipid polymer, suberin, deposited in their cell walls. This polymer is intractable in organic solvents, and is co-deposited with chloroform-extractable waxes. These suberin-associated root waxes are typically composed of alkanes, primary alcohols, fatty acids, alkyl ferulates, alkyl caffeates, and alkyl coumarates (Espelie et al., 1980; Li et al., 2007; Kosma et al., 2015). They are believed to contribute to the diffusion barrier properties of suberized cell walls (Soliday et al., 1979), and possibly have other roles yet to be discovered. Here we describe a protocol to extract and analyze waxes associated with root suberin. This fraction of aliphatic components is extracted by whole root immersion in chloroform, and is then chemically modified to prepare samples that are more suitable to gas-chromatography analysis. This protocol is optimized for Arabidopsis thaliana, but can be used with roots of other plants as described herein.

Materials and Reagents

  1. 3.5 inch pots for Arabidopsis, bigger pots if necessary for other plants
  2. 9 ml glass tubes with polytetrafluoroethylene (PFTE)-lined caps (Corning, catalog number: 9826-13 )
  3. Glass Pasteur pipets
  4. Glass wool (Sigma-Aldrich, catalog number: 18421 )
  5. 2 ml GC vials and caps (Agilent Technologies, catalog number: 5190-2240 ) with 250 µl glass inserts (Agilent Technologies, catalog number: 5181-1270 )
  6. 10 µl and 500 µl Hamilton syringes (Hamilton Company, catalog number: 80075 and 81216 , respectively)
    Note: It is advisable to rinse the glass Hamilton syringes with acetone then chloroform before each use and chloroform then acetone after each use. This will help to prevent clogging of the syringes.
  7. Arabidopsis thaliana or other plant seeds [e.g., Brassica napus, Raphanus sativus, Beta vulgaris, see Kosma et al., (2015) for a comprehensive list of species that have been analyzed with this protocol]
  8. Soil-less growing medium
    1. 2:1 (v/v) mixture of potting mix (Pro-Mix BX, Premier Tech Horticulture, Rivière-du-Loup)
    2. calcined clay granules (PPC Greens Grade, Profile, Buffalo Grove)
      Note: For images of these growing media please refer to https://ag.purdue.edu/hla/Hort/greenhouse/pages/101-ways-to-grow-arabidopsis.aspx
  9. Distilled water
  10. Chloroform (CHCl3) (≥ 99.5% purity) (Sigma-Aldrich, catalog number: C2432 )
  11. 95% Ethanol (e.g., Corning, Koptec, catalog number: V1105 )
  12. Suggested internal standards
    1. Pentadecanoic acid (15:0) (Nu-Check Prep, catalog number: N-15-A ) or heptadecanoic acid (17:0) (Nu-Check Prep, catalog number: N-17-A )
    2. Tricosan-1-ol (23:0-OH) (Nu-Check Prep, catalog number: A-624 )
    3. Monoheptadecanoin (17:0 monoacylglycerol) (Nu-Check Prep, catalog number: M-159 )
    4. Octacosane (28:0) (Sigma-Aldrich, catalog number: O504 )
    5. Tridecyl (13:0) ferulate, if used need to synthesize according to Kosma et al., (2012)
    6. Heptadecyl (17:0) coumarate, if used need to synthesize according to Kosma et al., (2012)
    7. Nonadecyl (19:0) caffeate, if used need to synthesize according to Razeq et al., (2014)
  13. Nitrogen gas (> 99% Purity)
  14. Pyridine, anhydrous (C5H5N) (Sigma-Aldrich, catalog number: 270970 )
  15. N, O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA) (Sigma-Aldrich, catalog number: 15222 )
  16. n-heptane, anhydrous (Sigma-Aldrich, catalog number: 246654 )
  17. Toluene, anhydrous (Sigma-Aldrich, catalog number: 244511 )

Equipment

  1. Controlled-environment plant growth chamber (e.g., Percival Scientific, Conviron, etc.)
  2. Small pair of scissors or a razor blade
  3. Temperature-controlled evaporator connected to nitrogen tank (e.g., Organomation Associates, catalog number: 11155 )
  4. Dry heating block capable of reaching a temperature of 110 ºC and with a metal block containing 13 mm orifices
  5. Gas chromatograph-mass spectrometer (e.g., Agilent Technologies, model: 6850/5975 GC-MS ) equipped with a HP-5MS capillary column (length 30 m, id 0.25 mm, film thickness 0.25 µm) (Agilent Technologies, J&W Scientific)

Software

  1. ImageJ software (National Institute of Health)
  2. Excel software (Microsoft office)

Procedure

  1. Grow plants to maturity in a controlled-environment plant growth chamber. Typical growth parameters are 21 °C to 22 °C, 40% to 60% humidity, a 16/8-h light/dark cycle, and a fluorescent light intensity of 80 to 100 μmol m-2 s-1. For Arabidopsis, seven-week-old plants should be used to allow development of mature periderm enriched in root waxes. For Arabidopsis, a density of up to four to five evenly spaced plants per pot is optimal with each replicate consisting of roots pooled from all the plants in three to four pots (12-20 plants per replicate). Four to five replicates are recommended. If multiple genotypes are employed, then a randomized complete block design should be employed to account for environmental gradients and other sources of variation. For plants with large taproots, like rapeseed (Brassica napus), each replicate can comprise one taproot from a single plant.
  2. The root waxes accumulate in the periderm of mature taproots (Kosma et al., 2012) and so it is convenient to cut away non-taproot tissues including secondary roots (taproots are up to 5 cm from the base of the Arabidopsis rosette; see Figure 1). For plants like rapeseed, the taproot will be longer.


    Figure 1. Arabidopsis taproot typical of what would be used for root wax extraction. A. Although lateral roots are pictured here, these should be removed prior to extraction. Scale bar = 2 cm (Kosma et al., 2015). B. Illustration of root measurements required to calculate root surface area.

  3. Five cm of Arabidopsis taproot can be readily removed from growing media by turning the pot upside-down into your hand and removing the bulk of potting medium with a gentle stream of water or by submersion into a tub of distilled water. Lateral roots are excised with a small pair of scissors or razor blade. Root mass below 5 cm is also removed by excision with a small pair of scissors or razor blade.
  4. Rinse roots well with distilled water to remove remaining potting medium and debris. Gently blot dry with a paper towel. Measure and record the fresh weight.
  5. For Arabidopsis, place roots into a 9 ml glass tube that employs a PTFE-lined screw cap. Use a long pair of forceps to gently place roots into the glass tube. For plant species with a larger taproot like rapeseed, a glass beaker or larger glass tube should be used. All tubes or beakers must be rinsed with organic solvents to remove residual detergents and/or other sources of lipid contamination. For this, rinse tubes or beakers once with ethanol and twice with chloroform prior to use for extractions.
  6. Fill glass tube or beaker (containing roots) with volume of chloroform that allows complete submersion of the root(s). For Arabidopsis, between 5 and 7 ml of chloroform is sufficient (see Figure 2). Larger taproots will require a larger volume of solvent.


    Figure 2. Example of a periderm wax extraction from Arabidopsis taproots. Image of a typical extraction for 1 replicate; 16 taproots submerged in 5 ml of chloroform in a 9 ml glass tube with a PTFE-lined screw cap.

  7. Gently rotate the tube for 1.5 min with Arabidopsis or 2 min with larger taproots. If using a beaker for larger taproots, then solvent-rinsed forceps may be used to completely submerge the roots; gently agitate these roots in the beaker of chloroform.
  8. For Arabidopsis, decant chloroform, being careful not to remove taproots, into a solvent-rinsed 9 ml glass tube (with PTFE-lined cap). For larger taproots, the chloroform can be decanted into a large volume, solvent-rinsed tube (e.g., 25 ml). For very large volumes of chloroform (> 25 ml), solvent can be decanted into a solvent-rinsed round bottom flask (50 - 250 ml) with a ground glass neck to facilitate removal of chloroform using a rotary.
  9. Keep roots for surface area determination (step 17 below).
  10. If many particulates are visible in the extracts, extracts should be filtered through glass wool. For small volumes of extract, a small plug of glass wool can be packed into a Pasteur pipet. For larger volumes, pack glass wool into a solvent-rinsed glass funnel. Pre-rinse the glass wool with chloroform, then pour or pipet the chloroform extract through glass wool into a new, solvent-rinsed glass tube or round bottom flask.
  11. Add internal standards to chloroform extract using a chloroform-rinsed 10 µl Hamilton syringe. For the parameters described herein for Arabidopsis, a single, fixed concentration in the range of 5 to 10 µg of each internal standard is recommended. Larger quantities of internal standards may be required for plants like rapeseed that possess large taproots.
  12. For small volumes of extract (<25 ml), evaporate samples to dryness under a gentle stream of nitrogen with the bath heated to a temperature of 30 - 40 °C. For small volumes, samples will evaporate in approximately 15-20 min. For larger volumes of extract (>25 ml), round bottom flasks should be connected to a rotary evaporator under reduced pressure and with a water bath temperature of 30 - 40 °C. Once most solvent has been removed by rotary evaporation, transfer the remaining extract to a solvent-rinsed 9 ml tube and evaporate to dryness under a gentle stream of nitrogen.
  13. To each tube containing dry extract, add 0.5 ml of pyridine and 0.5 ml of BSTFA using a chloroform-rinsed 500 µl Hamilton syringe (Note: Volumes here can be approximate and so a Pasteur pipet can be used instead.). Cap tubes under nitrogen gas and incubate at 110 °C for 10 min using a dry heating block. This step silylates hydroxyl and carboxylate groups (derivatization) making compounds containing these groups more amenable to separation by gas chromatography.
  14. Evaporate derivatized samples to dryness under a gentle stream of nitrogen (no heating).
  15. Dissolve samples in a 1:1 mixture of n-heptane:toluene. Add 100 μl for Arabidopsis extracts. For extracts from larger taproots, greater volumes will be required.
  16. Transfer each sample to a GC vial containing a fresh glass insert and place on GC autosampler.
  17. For GC-MS analysis, the helium flow rate is 1.5 ml per minute and samples usually injected in splitless mode. For typical root wax samples, the temperature settings are: inlet at 350 °C; detector at 320 °C; oven program at 130 °C for 2 min, increased to 325 °C at 5 °C per min, and then held at 325 °C for 10 min.
  18. To calculate root surface area (SA), each whole root is scanned along its longitudinal axis using a desktop scanner. Image files should be scanned at a resolution of 200 dpi (78.74 dots per cm) and saved as TIFF files. The digital TIFF files are opened in ImageJ and the built-in software tools used to determine radius (r) of the cut base and length of the side of the root (s) (Figure 1B). s is taken as the mean length of two sides from the digitized two-dimensional image and r calculated as ½ the measured width of the cut base. The surface areas of the taproots are then calculated using the formula SA = π * r * s (a conical geometry is assumed).
  19. A typical chromatogram of an Arabidopsis taproot wax extract is shown in Figure 3. The amount (μg) of each constituent is calculated from its peak area relative to the peak area of the respective internal standard. Fatty alcohol peaks should be compared to the tricosan-1-ol internal standard, alkyl coumarate peaks to the heptadecyl coumarate internal standard, etc. Constituent amounts (χ μg) are calculated by multiplying the internal standard (IS) amount (μg) with the peak area (PA) of the constituent of interest (COI) and dividing by the peak area of the internal standard. In the following formula, 10 μg of IS is used: χ μg = 10 μg * PACOI/PAIS. The following is an example using 10 micrograms of IS, PACOI of 25,000 (arbitrary units), and a PAIS of 12,000: χ μg = 10 μg * 25,000 area units/12,000 area units, χ μg = 10 μg * 2.0833, χ = 20.833 μg. The wax constituent amounts are then divided by total surface area or fresh weight (FW) of root(s) used for extraction (e.g., µg cm-2 or µg g-1 FW). Continuing with the above example, if one replicate consists of 100 mg of FW and our peak amount is 20.833 μg then 20.833 μg/100 mg FW = 0.20833 μg mg-1 FW. However, surface area is generally preferred because the root waxes are being extracted from surface tissue layer (periderm), thereby allowing for better comparisons between species of differing root size (surface area to fresh weight ratios vary dramatically).

Representative data


Figure 3. Example chromatogram of Arabidopsis root waxes. Peak IDs are as follows: 1) 17:0 FFA*; 2) 18:0-OH; 3) 18:0 FFA; 4) 20:0-OH; 5) 20:0 FFA; 6) 22:0-OH; 7) 22:0 FFA; 8) 23:0-OH*; 9) β-17:0 MAG*; 10) α-17:0 MAG*; 11) 28:0 Alkane*; 12) cis-13:0 ferulate*; 13) β-20:0 MAG; 14) trans-13:0 ferulate*; 15) α-20:MAG; 16) cis-17:0 coumarate*; 17) 28:1 sterol; 18) 29:2 sterol; 19) 29:1 sterol; 20) trans-17:0 coumarate*; 21) cis-18:0 caffeate; 22) trans-18:0 coumarate; 23) trans-18:0 ferulate; 24) trans-18:0 caffeate; 25) cis-20:0 caffeate; 26) trans-20:0 coumarate; 27) trans-20:0; ferulate; 28) trans-20:0 caffeate; 29) cis-22:0 caffeate; 30) trans-22:0 coumarate; 31) trans-22:0 ferulate; 32) trans-22:0 caffeate. Abbreviations are as follows: FFA = free fatty acid, -OH = primary fatty alcohol, MAG = monoacylglycerol, * indicates an internal standard (used for quantification).

Notes

  1. The soil-less medium used here leaves little debris on the roots and thus the roots are easily cleaned prior to chloroform extraction. Other growing media, including soil, can be used instead but then it may be more difficult to wash material away from the roots.
  2. It is important that no plasticware, such as plastic pipettemen tips, are used during the extraction and processing of root waxes or else there is a good chance of plastic contamination in the GC traces. Use solvent rinsed glassware and Hamilton syringes throughout the procedure. New glass Pasteur pipets do not need to be rinsed with solvent.
  3. The optimum mix of internal standards depends on the chemical composition of root waxes. The mix recommended herein is ideal for Arabidopsis root waxes. For example, not all species contain alkyl caffeates. As such, the nonadecyl caffeate internal standard could then be excluded. It is important that the internal standards used do not co-elute with any of the native root wax components. Each root wax component should be quantified relative to an internal standard of like chemical structure (i.e., they only differ in chain length).
  4. Root waxes are influenced by stage of development (e.g., may accumulate only in mature periderm) and by environment (e.g., may be stress induced). Therefore, one needs to ensure that control and sample plants (e.g., a mutant) are grown together and sampled exactly the same way.
  5. Although the protocol described here is optimized for Arabidopsis root waxes, it can be modified for any other plant by taking into account differing root sizes and root wax compositions, as noted above and, for example, as reported in Razeq et al. (2014) and Kosma et al. (2015).

Acknowledgments

The original version of this protocol was reported in Li et al. (2007) with adjustments made in subsequent publications (Molina et al., 2009; Kosma et al., 2012; Vishwanath et al., 2013) to produce the protocol reported here.

References

  1. Espelie, K. E., Sadek, N. Z. and Kolattukudy, P. E. (1980). Composition of suberin-associated waxes from the subterranean storage organs of seven plants: Parsnip, carrot, rutabaga, turnip, red beet, sweet potato and potato. Planta 148(5): 468-476.
  2. Kosma, D. K., Molina, I., Ohlrogge, J. B. and Pollard, M. (2012). Identification of an Arabidopsis fatty alcohol:caffeoyl-Coenzyme A acyltransferase required for the synthesis of alkyl hydroxycinnamates in root waxes. Plant Physiol 160(1): 237-248.
  3. Kosma, D. K, Rice, A. and Pollard M. (2015). Analysis of aliphatic waxes associated with root periderm or exodermis from eleven plant species. Phytochemistry 117: 351-362.
  4. Li, Y., Beisson, F., Ohlrogge, J. and Pollard, M. (2007). Monoacylglycerols are components of root waxes and can be produced in the aerial cuticle by ectopic expression of a suberin-associated acyltransferase. Plant Physiol 144(3): 1267-1277.
  5. Molina, I., Li-Beisson, Y., Beisson, F., Ohlrogge, J. B. and Pollard, M. (2009). Identification of an Arabidopsis feruloyl-coenzyme A transferase required for suberin synthesis. Plant Physiol 151(3): 1317-1328.
  6. Razeq, F. M., Kosma, D. K., Rowland, O. and Molina, I. (2014). Extracellular lipids of Camelina sativa: characterization of chloroform-extractable waxes from aerial and subterranean surfaces. Phytochemistry 106: 188-196.
  7. Soliday, C. L., Kolattukudy, P. E., and Davis, R. W. (1979). Chemical and ultrastructural evidence that waxes associated with the suberin polymer constitute the major diffusion barrier to water vapor in potato tuber (Solanum tuberosum L.). Planta 146(5): 607-614.
  8. Vishwanath, S. J., Kosma, D. K., Pulsifer, I. P., Scandola, S., Pascal, S., Joubes, J., Dittrich-Domergue, F., Lessire, R., Rowland, O. and Domergue, F. (2013). Suberin-associated fatty alcohols in Arabidopsis: distributions in roots and contributions to seed coat barrier properties. Plant Physiol 163(3): 1118-1132.

简介

子叶和块根状茎的外胚层和外皮含有沉积在其细胞壁中的细胞外脂质聚合物,软骨素。该聚合物在有机溶剂中难以处理,并与氯仿萃取蜡共沉积。这些软骨素相关的根蜡通常由烷烃,伯醇,脂肪酸,烷基阿魏酸酯,烷基咖啡酸酯和烷基香豆酸酯组成(Espelie等人,1980; Li等人,/em>,2007; Kosma ,,2015)。它们被认为有助于增加蜂窝细胞壁的扩散阻挡性能(Soliday等人,1979),并且可能具有尚待发现的其它作用。在这里我们描述一个协议,以提取和分析与根软骨素相关的蜡。该部分脂族组分通过在氯仿中全根浸提提取,然后化学改性以制备更适合于气相色谱分析的样品。该方案针对拟南芥进行优化,但可以与本文所述的其他植物的根一起使用。

材料和试剂

  1. 用于拟南芥的3.5英寸盆,如果其他植物需要,可以使用更大的盆
  2. 9ml带有聚四氟乙烯(PFTE)衬里帽(Corning,目录号:9826-13)的玻璃管
  3. 玻璃巴斯德吸管
  4. 玻璃棉(Sigma-Aldrich,目录号:18421)
  5. 带有250μl玻璃插入物(Agilent Technologies,目录号:5181-1270)的2ml GC小瓶和盖(Agilent Technologies,目录号:5190-2240)
  6. 10μl和500μlHamilton注射器(Hamilton Company,目录号:分别为80075和81216) 注意:建议在每次使用前用丙酮和氯仿冲洗Hamilton注射器,每次使用后用氯仿和丙酮清洗。这将有助于防止注射器堵塞。
  7. 拟南芥或其他植物种子[例如 Brassica napus ,Raphanus sativus ,Beta vulgaris >,见Kosma等人,,(2015)有关此协议分析的物种的全面列表]
  8. 无土生长介质
    1. (Pro-Mix BX,Premier Tech Horticulture,Rivière-du-Loup)的2:1(v/v)混合物
    2. 煅烧粘土颗粒(PPC Greens Grade,Profile,Buffalo Grove)
      注意:有关这些不断增长的媒体的图片,请参阅 https://ag.purdue.edu/hla/Hort/greenhouse/pages/101-ways-to-grow-arabidopsis.aspx
  9. 蒸馏水
  10. 氯仿(CHCl 3)(纯度≥99.5%)(Sigma-Aldrich,目录号:C2432)
  11. 95%乙醇(例如Corning,Koptec,目录号:V1105)
  12. 建议的内部标准
    1. 十五烷酸(15:0)(Nu-Check Prep,目录号:N-15-A)或 十七烷酸(17:0)(Nu-Check Prep,目录号:N-17-A)
    2. (23:0-OH)(Nu-Check Prep,目录号:A-624)
    3. 单十七烷酸(17:0单酰基甘油)(Nu-Check Prep,目录号:M-159)
    4. 二十八烷(28:0)(Sigma-Aldrich,目录号:O504)
    5. 十三烷基(13:0)阿魏酸酯,如果使用需要根据Kosma等人(2012)合成。
    6. 十七烷基(17:0)香豆酸盐,如果使用,需要根据Kosma等人的综述,(2012)
    7. 如果使用十七烷基(19:0)咖啡酸,需要根据Razeq等人的综述,(2014)
  13. 氮气(> 99%纯度)
  14. 吡啶,无水(C 5 H 5 N)(Sigma-Aldrich,目录号:270970)
  15. (三甲基甲硅烷基) - 三氟乙酰胺(BSTFA)(Sigma-Aldrich,目录号:15222)的二甲基甲酰胺
  16. 无水(Sigma-Aldrich,目录号:246654)的无水庚烷
  17. 无水甲苯(Sigma-Aldrich,目录号:244511)

设备

  1. 控制环境植物生长室(例如Percival Scientific,Conviron等)。
  2. 小剪刀或剃刀刀片
  3. 连接到氮气罐的温度控制蒸发器(例如,Organomation Associates,目录号:11155)
  4. 干燥加热块能够达到110℃的温度和含有13毫米孔的金属块
  5. 装备有HP-5MS毛细管柱(长度30m,内径0.25mm,膜厚度0.25μm)(Agilent公司)的气相色谱 - 质谱仪(例如Agilent Technologies,型号:6850/5975 GC-MS) Technologies,J& W Scientific)

软件

  1. ImageJ软件(国立卫生研究所)
  2. Excel软件(Microsoft office)

程序

  1. 在受控环境的植物生长室中使植物生长至成熟。典型的生长参数是21℃至22℃,40%至60%的湿度,16/8-h光/暗循环,以及80至100μmol/m 2的荧光强度。 s -1 。对于拟南芥,应使用七周龄植物以允许富含根蜡的成熟外胚层的发育。对于拟南芥,每盆最多四至五个均匀间隔的植物的密度是最佳的,每个重复由从三到四个盆(每个重复12-20个植物)中的所有植物收集的根组成。建议重复四至五次。如果使用多种基因型,则应采用随机完全区组设计来考虑环境梯度和其他变异来源。对于具有大的根,例如油菜籽(欧洲油菜)的植物,每个重复可以包含来自单个植物的一个根。
  2. 根蜡累积在成熟根的周皮中(Kosma等人,2012),因此方便地切除非根部组织,包括次生根(根部距离基部至多5cm)的拟南芥花瓣;参见图1)。对于像油菜籽这样的植物,树根将更长

    图1. 拟南芥 taproot是用于根蜡提取的典型。 A.虽然在这里描述了侧根,但在提取之前应该将其去除。比例尺= 2cm(Kosma等人,2015)。 B.计算根表面面积所需的根测量图示。

  3. 通过将罐翻转到你的手中并用温和的水流除去大量的盆栽培养基,或者通过浸没到蒸馏水桶中,可以容易地从生长培养基中除去5cm的拟南芥[水。用小剪刀或剃刀刀片切除侧根。通过用小剪刀或剃刀刀片切除也除去5cm以下的根质量
  4. 用蒸馏水冲洗根以除去剩余的灌封介质和碎片。用纸巾轻轻擦干。测量并记录鲜重。
  5. 对于拟南芥,将根部放入9ml玻璃管中,该玻璃管采用PTFE内衬螺旋盖。使用一双长的镊子轻轻地把根插入玻璃管。对于具有较大的根茎如菜籽的植物物种,应使用玻璃烧杯或更大的玻璃管。所有管或烧杯必须用有机溶剂冲洗,以去除残留的清洁剂和/或其他脂质污染源。为此,在用于萃取之前,用乙醇冲洗管或烧杯一次,用氯仿冲洗两次
  6. 填充玻璃管或烧杯(含根)与体积的氯仿,允许根的完全浸没。对于拟南芥,在5和7ml之间的氯仿是足够的(参见图2)。较大的原料需要较大体积的溶剂。


    图2.从拟南芥 taproot中提取胚毛蜡的实施例。 1个重复的典型提取的图像;将16根根淹没在5ml氯仿中的9ml带有PTFE衬里螺旋盖的玻璃管中。

  7. 用拟南芥轻轻旋转管子1.5分钟,或用较大的根茎轻轻旋转管子2分钟。如果使用烧杯用于较大的根,则可以使用溶剂冲洗的镊子来完全浸没根;轻轻搅拌这些根在氯仿的烧杯。
  8. 对于拟南芥,倾析氯仿,小心不要除去根,进入溶剂冲洗的9毫升玻璃管(用PTFE内衬帽)。对于较大的根,可以将氯仿倾析入大体积,溶剂冲洗的管(例如25ml)中。对于非常大体积的氯仿(> 25ml),可以将溶剂倾析到具有研磨的玻璃颈的溶剂冲洗的圆底烧瓶(50-250ml)中,以利于使用旋转器除去氯仿。
  9. 保持根表面积测定(下面的步骤17)。
  10. 如果提取物中有许多颗粒,则应通过玻璃棉过滤提取物。对于小体积的提取物,可以将一小块玻璃棉填充到巴斯德吸管中。对于更大的体积,将玻璃棉包装在溶剂冲洗的玻璃漏斗中。用氯仿预冲洗玻璃棉,然后将氯仿提取物通过玻璃棉倒入或移液到新的,溶剂冲洗的玻璃管或圆底烧瓶中。
  11. 使用氯仿 - 冲洗的10μlHamilton注射器将内标添加到氯仿提取物中。对于本文中对于拟南芥所述的参数,推荐在5至10μg每种内标的范围内的单一固定浓度。对于具有大根子的油菜籽等植物,可能需要更大量的内标
  12. 对于小体积的提取物(<25ml),在温和的氮气流下将样品蒸发至干,将浴加热至30-40℃的温度。对于小体积,样品将在约15-20分钟内蒸发。对于较大体积的提取物(> 25ml),应将圆底烧瓶在减压下和30-40℃的水浴温度下连接到旋转蒸发器。一旦通过旋转蒸发除去大多数溶剂,将剩余的提取物转移至溶剂冲洗的9ml管中,并在温和的氮气流下蒸发至干。
  13. 向每个含有干提取物的试管中加入0.5ml吡啶和0.5ml BSTFA,使用氯仿冲洗的500μlHamilton注射器(注意:这里的体积可以是近似值,因此可以使用巴斯德吸量管。) >)。帽管在氮气下,并使用干燥加热块在110℃孵育10分钟。该步骤使羟基和羧酸酯基团(衍生化)甲硅烷基化,使含有这些基团的化合物更易于通过气相色谱分离。
  14. 在温和的氮气流下(不加热)将衍生的样品蒸发至干
  15. 将样品溶解在正庚烷:甲苯的1:1混合物中。为拟南芥提取物加入100μl。对于较大的根的提取物,需要更大的体积
  16. 将每个样品转移到含有新鲜玻璃插入物的GC小瓶中,并置于GC自动进样器上。
  17. 对于GC-MS分析,氦气流速为1.5ml /分钟,样品通常以不分流模式注射。对于典型的根蜡样品,温度设置为:入口在350℃;检测器在320℃;烘箱程序在130℃下2分钟,以5℃/分钟升至325℃,然后在325℃下保持10分钟。
  18. 为了计算根表面积(SA),使用台式扫描仪沿着其全部纵向轴扫描每个整根。图像文件应以200 dpi(78.74点/厘米)的分辨率扫描,并另存为TIFF文件。在ImageJ中打开数字TIFF文件,并且使用内置的软件工具来确定根切的半径( r )和根的边长( s )(图1B)。将 s 作为从数字化二维图像的两侧的平均长度,并将 r 计算为切割基底的测量宽度的1/2。然后使用公式SA =π* em(假设锥形几何形状)计算基干的表面积。
  19. 拟南芥皂草提取物的典型色谱图显示在图3中。各组分的量(μg)由其峰面积相对于相应内标的峰面积计算。脂肪醇峰应与三聚糖-1-醇内标物,十九烷基香豆酸盐内标物的烷基香豆酸盐峰等比较。组成量(χμg)通过内标(< (μg)与感兴趣的组分的峰面积(PA)(COI )之比,并除以内标的峰面积。在下式中,使用10μg的IS:χμg=10μg* PA 子/PA 子。以下是使用10微克IS,25,000(任意单位)的PA COI 和12,000的PA sub:IS的例子:χμg=10μg×25,000面积单位/12,000面积单位,χμg=10μg* 2.0833,χ=20.833μg。然后将蜡组分量除以用于提取的根的总表面积或鲜重(FW)(例如μgcm -2 -2/s)或μgg -1 FW)。继续上述实施例,如果一个重复由100mg FW组成并且我们的峰量为20.833μg,则20.833μg/100mg FW =0.20833μgmg -1 FW。然而,表面积通常是优选的,因为根蜡是从表面组织层(外胚层)提取的,从而允许在不同根大小的物种(表面积与鲜重比率显着变化)之间更好地比较。

代表数据


图3.拟南芥根蜡的示例色谱图。峰ID如下:1)17:0 FFA *; 2)18:0-OH; 3)18:0 FFA; 4)20:0-OH; 5)20:0 FFA; 6)22:0-OH; 7)22:0 FFA; 8)23:0-OH *; 9)β-17:0 MAG *; 10)α-17:0 MAG *; 11)28:0烷烃*; 12) cis -13:0 ferulate *; 13)β-20:0 MAG; 14) trans -13:0 ferulate *; 15)α-20:MAG; 16)顺式-17:0香豆酸酯*; 17)28:1甾醇; 18)29:2甾醇; 19)29:1固醇; 20) trans -17:0 coumarate *; 21) cis -18:0 caffeate; 22) trans -18:0 coumarate; 23) trans -18:0 ferulate; 24) trans -18:0 caffeate; 25) cis -20:0咖啡因; 26) trans -20:0 coumarate; 27) trans -20:0;阿魏尔28) trans -20:0 caffeate; 29)顺式-22:0咖啡酸盐; 30) trans -22:0 coumarate; 31) trans -22:0 ferulate; 32) trans -22:0 caffeate。缩写如下:FFA =游离脂肪酸,-OH =主要脂肪醇,MAG =单酰基甘油,*表示内标(用于定量??)。

笔记

  1. 这里使用的无土介质在根上留下少量碎屑,因此在氯仿提取之前根容易清洁。可以使用其他生长介质,包括土壤,但是然后可能更难以从根除去材料。
  2. 重要的是,在根蜡的提取和加工期间不使用塑料器皿,例如塑料移液管吸头,否则GC痕迹中有很大的塑料污染的机会。在整个程序中使用溶剂冲洗的玻璃器皿和Hamilton注射器。新的玻璃巴斯德吸管不需要用溶剂冲洗。
  3. 内标的最佳混合取决于根蜡的化学组成。本文推荐的混合物是拟南芥根根蜡的理想选择。例如,不是所有的物种都含有烷基咖啡因。因此,可以排除十九烷基咖啡酸内标。重要的是,所使用的内标不与任何天然根蜡组分共洗脱。每个根蜡组分应当相对于具有相似化学结构的内标物定量(即,它们仅在链长度上不同)。
  4. 根蜡受发育阶段(例如,仅在成熟的周皮中积累)和环境(例如可以是胁迫诱导)的影响。因此,需要确保对照植物和样品植物(例如突变体)一起生长并以完全相同的方式进行取样。
  5. 尽管本文所述的方案针对拟南芥根蜡进行优化,但是如上所述,可以通过考虑不同的根大小和根蜡组成对任何其它植物进行修饰,例如, Razeq等人(2014年)和Kosma等人(2015年)。

致谢

该方案的原始版本在Li等人(2007)中报道,并在随后的出版物中进行了调整(Molina等人,2009; Kosma等人。,2012; Vishwanath ,2013)以产生此处报告的方案。

参考文献

  1. Espelie,K.E.,Sadek,N.Z.and Kolattukudy,P.E。(1980)。 来自七种植物的地下储存器官的软木素相关蜡的组成:欧洲防风草,胡萝卜,芜菁,萝卜,红甜菜,甘薯和马铃薯。 148(5):468-476。
  2. Kosma,D.K.,Molina,I.,Ohlrogge,J.B.and Pollard,M。(2012)。 鉴定拟南芥脂肪醇:咖啡酰辅酶A酰基转移酶所需的在根蜡中合成羟基肉桂酸烷基酯。植物生理学 160(1):237-248。
  3. Kosma,D.K,Rice,A.and Pollard M.(2015)。 分析来自十一种植物物种的根部皮层或外皮的脂肪蜡。 Phytochemistry 117:351-362。
  4. Li,Y.,Beisson,F.,Ohlrogge,J。和Pollard,M。(2007)。 单酰基甘油是根蜡的组分,并且可以通过异位表达suberin-相关酰基转移酶。 植物生理学 144(3):1267-1277。
  5. Molina,I.,Li-Beisson,Y.,Beisson,F.,Ohlrogge,J.B。和Pollard,M。(2009)。 鉴定拟南芥阿魏酸辅酶A转移酶,是suberin合成所必需的。 Plant Physiol 151(3):1317-1328。
  6. Razeq,F.M.,Kosma,D.K.,Rowland,O。和Molina,I。(2014)。 Camelina sativa的细胞外脂质::天线中氯仿萃取蜡的表征和地下表面。 Phytochemistry 106:188-196。
  7. Soliday,C.L.,Kolattukudy,P.E.,and Davis,R.W。(1979)。 化学和超微结构证据表明与软木脂聚合物相关的蜡是马铃薯水蒸汽的主要扩散障碍块茎( Solanum tuberosum L)。 146(5):607-614。
  8. Vishwanath,SJ,Kosma,DK,Pulsifer,IP,Scandola,S.,Pascal,S.,Joubes,J.,Dittrich-Domergue,F.,Lessire,R.,Rowland,O。和Domergue, )。 拟南芥中的Suberin相关脂肪醇:根系分布和贡献种子外壳屏障性质。植物生理学 163(3):1118-1132。
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引用:Kosma, D. K., Molina, I. and Rowland, O. (2015). GC-MS-Based Analysis of Chloroform Extracted Suberin-Associated Root Waxes from Arabidopsis and Other Plant Species. Bio-protocol 5(24): e1679. DOI: 10.21769/BioProtoc.1679.
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