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Histochemical Preparations to Depict the Structure of Cauliflower Leaf Hydathodes
用于研究花椰菜叶泌水孔结构的组织化学制备   

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Abstract

Hydathodes are plant organs present on leaf margins of a wide range of vascular plants and are the sites of guttation. Both anatomy and physiology of hydathodes are poorly documented. We have recently reported on the anatomy of cauliflower and Arabidopsis thaliana hydathodes and on their infection by the vascular pathogenic bacterium Xanthomonas campestris pv. campestris (Xcc) (Cerutti et al., 2017). Because hydathodes are natural infection routes for several pathogens, it is necessary to have a deep knowledge of their anatomy to further better interpret images of infected hydathodes. Here, we described different detailed protocols for gaining information on hydathode anatomy which are applicable to a wide range of plants (including monocots like barley and rice). Nomarsky and confocal microscopy were used to observe clarified thick samples. Optical microscopy in transmitted light and transmission electron microscopy were used to observed thin and ultrathin sections.

Keywords: Cauliflower(花椰菜), Hydathode(泌水孔), Xanthomonas(黄单胞菌)

Background

In literature, different techniques were used to study hydathodes (Perrin, 1972; Chen and Chen, 2007; Wang et al., 2011; Singh, 2014). From light microscopy (on entire tissues or on section of resin-embedded samples) to scanning or transmission electron microscopy, a large panel of protocols and techniques was available. To our knowledge, these techniques were not used in combination and laser confocal microscopy was never used to depict hydathode structures. Moreover, we noticed variations from protocols to protocols. We presented here different techniques used in combination. They are well-adapted to cauliflower and Arabidopsis thaliana. They have been successfully applied to other plants like monocotyledons (barley and rice) and should be likely used to a larger variety of plant species. We encourage the users to apply these protocols to gain complementary information on the hydathode at different scales during infection.

Materials and Reagents

  1. Microscope glass slides (Thermo Fisher Scientific, Superfrost, catalog number: 10143560WCUT )
  2. Cover slip 24 x 60 mm (Thermo Fisher Scientific, catalog number: 15747592 )
  3. Razor blades
  4. Cauliflower (Brassica oleracea var. botrytis, cultivar Clovis)
    Notes:
    1. Cauliflower plants were grown in a controlled greenhouse.
    2. All the experiments used the second true leaf from four-weeks-old plants.
  5. Chloral hydrate (Sigma-Aldrich, catalog number: 23100 )
  6. Glycerol (C3H8O3) (VWR, catalog number: 24387.326 )
  7. Calcofluor (Fluorescent Brightener 28) (Sigma-Aldrich, catalog number: F3543 )
  8. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  9. Sodium cacodylate (Sigma-Aldrich, catalog number: C4945 )
  10. Glutaraldehyde EM grade (Electron Microscopy Sciences, catalog number: 16214 )
  11. Osmium tetroxide (OsO4) (Electron Microscopy Sciences, catalog number: 19150 )
  12. Ethanol (C2H5OH) (Sigma-Aldrich, catalog number: 32221 )
  13. Epon (Electron Microscopy Sciences, catalog number: 14120 )
    Note: Composition of the resin: Embed-812 (45 g), DDSA (36 g), NMA (18 g) and BDMA (1.35 ml). Embedding kit in which accelerator DMP30 is replaced by BDMA (Electron Microscopy Sciences, catalog number: 11400 ).
  14. Borax (Sigma-Aldrich, catalog number: B3545 )
  15. Toluidine blue (RAL Diagnostics, catalog number: 361590 )
  16. Methylene blue (Merck, catalog number: 159270 )
  17. Basic fuchsin (Sigma-Aldrich, catalog number: 857343 )
  18. Periodic acid (VWR, Prolabo, catalog number: 20.593.151 )
  19. Acetic acid (CH3COOH) (CARLO ERBA Reagents, catalog number: 302016 )
  20. Thiocarbohydrazide (Sigma-Aldrich, catalog number: 223220 )
  21. Silver proteinate (Roques for histology) (Sigma-Aldrich, catalog number: 05495 )
  22. Aceton (CH3COCH3) (Fisher Scientific, catalog number: 10395640 )
  23. Clarification solution (see Recipes)
  24. Fixation solution (see Recipes)
  25. Sodium cacodylate buffer (see Recipes)
  26. Borax solution with toluidine blue and methylene blue (see Recipes)
  27. Basic fuchsin solution (see Recipes)
  28. Periodic acid solution (see Recipes)
  29. Silver proteinate solution (see Recipes)

Equipment

  1. Hollow punch (Harris, Uni-Core) (Electron Microscopy Sciences, catalog number: 69039-70 )
  2. Ultra-microtome (Leica Microsystems, Reichert-Jung, model: UltraCut E )
  3. Optical microscope equipped for Nomarski (Leica Microsystems, model: DM IRB-E )
  4. Confocal microscope (Leica Microsystems, model: Leica TCS SP2 ) equipped with a diode laser at 405 nm
  5. Diaphragm pump vacuum or other vacuum sources
  6. Stirrer hotplate (IKA)
  7. Microwave apparatus (Leica Microsystems, model: EM AMW )
  8. Flat bottom embedding capsule (Electron Microscopy Sciences, catalog number: 70021 )
  9. Oven (Memmert, model: UFE500BO )
  10. Histo diamond knife (Diatome Histo) for semi-thin sections (0.5 µm) to optical observations
  11. Ultra diamond knife (Diatome Ultra 45) for ultra-thin sections (70-80 nm) to transmission electron microscopy
  12. Hot plate (slides warmer) (C & A Scientific, Premiere, model: XH-2002 )
  13. Optical microscope (ZEISS, model: Axioplan 2 )
  14. CCD camera (ZEISS, model: AxioCam MRc )
  15. Transmission electron microscope (Hitachi, model: HT7700 )
  16. Gold grid (Electron Microscopy Sciences, catalog number: FCF200-Au )

Software

  1. Pro Plus 4.0 Imaging software (Media Cybernetics, Silver Spring, MD, USA)

Procedure

Nomarski and confocal microscopy were used to observe clarified thick samples to depict the complex vascularization and the epithem, respectively. Optical microscopy in transmitted light (on thin sections) and transmission electron microscopy (on ultrathin sections) were used to localize both bacteria within the hydathode and changes in cell or tissue structure during infection.

  1. Clarification of the samples for Nomarski and laser confocal microscopy
    1. Use the hollow punch or razor blades to take pieces of leaf margins.
    2. Immerse the samples in the chloral hydrate-based clarification solution (see Recipe 1) for several weeks (usually 1-3 weeks) at 4 °C in the dark (in a fridge).
    3. Mount samples in the same solution and observe by Nomarski microscopy. Acquire images (see Figure 1).


      Figure 1. Typical images of cauliflower (A) and Arabidopsis (B) acquired in Nomarsky. Scale bars = 100 µm.

    4. For confocal microscopy of the same samples, rinse the samples overnight in distilled water at 4 °C.
    5. Stain the samples in an aqueous solution of 0.01% calcofluor (for cellulose) for 1 h at room temperature.
    6. Rinse in distilled water and mount in water on a glass slide.
    7. Acquire images using the 405 nm ray line of laser diode for excitation and collect the emitted fluorescence between 410 and 500 nm. The samples being thick, we use objective lens with long working distance to avoid too much pressure on the sample during observation and acquisition. Thus, we favor water immersion objective lenses (working distance of 2 to 3 mm) to perform observations in depth (see Figure 2).


      Figure 2. Confocal plane (A-B) of paradermal optical sectioning of cauliflower hydathode. A. View of the edge of the hydathode with two pores (arrows) and a large chamber below the pore (open arrow). B. Image of the epithem tissue of the hydathode with numerous meatuses. e: epidermal cell; p: parenchyma cell. C. Maximal projection of 20 confocal planes (z-stack) within a hydathode to depict the organization of the tracheids and their ornamentation. Scale bars = 25 µm.

  2. Sample preparation for electron and optical microscopy
    1. With a razor blade, take pieces (2-3 mm2) of leaf margins encompassing hydathodes.
    2. Fix the samples under vacuum for 10 min with fixation solution (see Recipe 2). Release the vacuum slowly and repeat (2-3 times) until the samples sink.
    3. Fix the samples for 1 h, at the atmospheric pressure, in the fixation solution without Triton X-100.
    4. Rinse the samples three times in 0.2 M sodium cacodylate buffer (15 min each time, see Recipe 3).
    5. Post-fix the samples with 2% osmium tetroxide in 0.2 M sodium cacodylate buffer for 1 h at room temperature.
    6. Rinse the samples three times in 0.2 M sodium cacodylate buffer (15 min each time).
    7. Dehydrate the samples in a graded series of aqueous solution of ethanol concentrations (25%, 50%, 1 h for each treatment).
    8. Conserve the samples in ethanol 70% (minimum 1 h) until the use of Leica microwave apparatus (AMW) to dehydrate and infiltrate the Epon resin (The infiltration is done according to the following program, see Table 1).

      Table 1. Procedure of resin infiltration

      Notes:
      1. *Slope: means that the temperature raised progressively to reach the indicated value at the end of the time.
      2. **Continuous: means that the temperature reached as soon as possible the indicated value and was then maintained.

    9. Place the samples in flat bottom embedding capsule filled with pure resin for 48 h of polymerization at 60 °C in an oven.

    For optical microscopy
    1. Cut the resin-embedded sample at a thickness of 0.5 µm with a Histo-diamond knife. The sections are placed on a drop of water on a glass slide and warmed to 50 °C on a hot plate to dry and stick on the glass.
    2. Stain the sections by covering the slide in a 1% borax solution (see Recipe 4) containing 0.1% toluidine blue and 0.2% methylene blue (these two stains were used to contrast thin sections from resin embedded samples). Rinse in water and then cover the slide with an aqueous solution of 0.07% of basic fuchsin (use to stain acidic compounds, see Recipe 5).
    3. Acquire images (see Figure 3) using an optical microscope (ZEISS, Axioplan 2 imaging) equipped with a CCD color camera (ZEISS, AxioCam MRc)


      Figure 3. Paradermal sections (0.5 µm in thickness) of resin embedded hydathode (A-B) from cauliflower leaf (3 dpi) infected by Xcc strain 8004::GUS-GFP (Cerutti et al., 2017). e: epidermal cell; p: parenchyma cell of the epithem tissue; xv: xylem vessel. A. Note a pore (arrow), a large chamber below the pore (open arrow); B. The presence of bacteria in the meatuses (arrowhead). Scale bars = 25 µm.

    For transmission electron microscopy
    1. Cut the resin-embedded sample at a thickness of 70-80 nm and collect the sections on gold grids and keep to dry overnight before the periodic acid-thiocarbohydrazide-silver (Ag) proteinate (PATAg) treatment.
    2. Float the sections (on the grids) for 30 min at room temperature on an aqueous solution of 1% (w/v) of periodic acid (see Recipe 6).
    3. Rinse twice the sections on distilled water for 2 h (1 h for each).
    4. Treat the sections overnight at 4 °C with a 20% acetic acid solution containing 0.2% thiocarbohydrazide.
    5. Wash the sections in solutions of decreasing concentrations of acetic acid (20%, 10%, and 5% acetic acid solutions to distilled water) for 30 min each at room temperature.
    6. Float the sections in a 1% (w/v) silver proteinate (see Recipe 7) for 30 min in the dark at the room temperature.
    7. Wash the sections in water (2-3 times, 20 min each time) and air-dry.
    8. Observe the sections under a transmission electron microscope operating at 80 kV and acquire images (see Figure 4).


      Figure 4. Images of a non-infected hydathode (A) and infected hydathode (B at 3 dpi, and C at 6 dpi) from cauliflower leaf infected by Xcc strain 8004::GUS-GFP (Cerutti et al., 2017). xv: xylem vessel. The arrows indicate the secondary cell wall ornamentation of the xylem vessels. The arrowheads indicate the epithem meatuses colonized by bacteria. Note in (C) the vessel invaded by bacteria. Scale bars = 3 µm.

Data analysis

Images of hydathodes from different clarified plant samples can be further analyzed to count the xylem terminations, to determine the size of the different cell types, the number of pores and their size per hydathode. Through analysis of the captured images, we characterized the hydathodes of cauliflower and Arabidopsis (Cerutti et al., 2017). All the measurements were done using Image-Pro Plus 4.0 Imaging software (Media Cybernetics, Silver Spring, MD, USA). It is also possible to use other imaging software like ImageJ:

  1. To count the xylem terminations (or the pores) of a hydathode, it is most often necessary to acquire images at different focus to visualize all the terminations. Use a measurement tool which saves the positioning of the point feature to avoid overcounting of the xylem terminations within a hydathode. At least 30 to 40 hydathodes were analyzed (Cerutti et al., 2017).
  2. To determine the size (area) of the cells (epidermal or epithemal cells), it is crucial to obtain well -contrasted images. To solve this problem, stain the cell wall with calcofluor and rinse the samples extensively with water before image acquisition.

Recipes

  1. Clarification solution
    45 g chloral hydrate
    7.6 ml distilled water
    9.3 ml 60% glycerol
  2. Fixation solution
    2.5% glutaraldehyde
    0.2 M sodium cacodylate buffer (pH 7.2)
    0.1% Triton X-100
  3. Sodium cacodylate buffer
    0.2 M solution of cacodylate (4.28 g in100 ml) in distilled water at pH 7.2
    Adjust the pH with several drops of HCl (1 N)
  4. Borax solution with toluidine blue and methylene blue
    In an aqueous solution of 1% borax add 0.1% (w/v) of toluidine blue and then 0.2% (w/v) methylene blue
  5. Basic fuchsin solution
    0.07% (w/v) of basic fuchsin in distilled water
  6. Periodic acid solution
    1% (w/v) of periodic acid in distilled water
  7. Silver proteinate solution
    Prepare a fresh solution of 1% (w/v) of silver proteinate in distilled water. In a fume bottle glass, add progressively the silver proteinate under stirring. Filter the solution before use and store it in the dark

Acknowledgments

This work was supported by a PhD grant from the French Ministry of National Education and Research to AC. LIPM is part of the French Laboratory of Excellence project (TULIP ANR-10-LABX-41; ANR-11-IDEX-0002-02). We also thank the Région Occitanie for the financial support of the microscopy platform. Thanks to the CMEAB (Centre de Microscopie Electronique Appliquée à la Biologie) staff for their invaluable technical assistance. The procedures for fixation and embedding were adapted from: a) Glauert, A. M. (1975). Fixation, Dehydration and embedding of biological specimens in Practical Methods in Electron Microscopy. North-Holland Publishing Compagny-Amsterdam. b) Hawes, C. and Satiat-Jeunemaitre, B. (2001). Plant Cell Biology: A Practical Approach. Oxford University Press. The PATAg treatment was adapted from Thiery, J. P. (1967). Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. J; Microsc. 6: 927-1018.

References

  1. Chen C. C. and Chen Y. R. (2007). Study on laminar hydathodes of Ficus formosana (Moraceae) III. Salt injury of guttation on hydathodes. Bot Stud 48: 215-226.
  2. Cerutti, A., Jauneau, A., Auriac, M. C., Lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Berthomé, R. and Noël, L. D. (2017). Immunity at cauliflower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiol 174(2): 700-716.
  3. Glauert, A. M. (1975). Fixation, Dehydration and embedding of biological specimens in Practical Methods in Electron Microscopy. North-Holland Publishing Compagny-Amsterdam.
  4. Hawes, C. and Satiat-Jeunemaitre, B. (2001). Plant Cell Biology: A Practical Approach. Oxford University Press.
  5. Thiery, J. P. (1967). Mise en évidence des polysaccharides sur coupes fines en microscopie électronique. J Microsc 6: 927-1018
  6. Perrin, A. (1972). Contribution à l’étude de l’organisation et du fonctionnement des hydathodes: recherches anatomiques ultrastructurales et physiologiques. Université Claude-Bernard–Lyon.
  7. Singh, S. (2014). Guttation: quantification, microbiology and implications for phytopathology. In: Lüttg, U., Beyschlag, W., Cushman, J. (Eds). Prog. Bot. Vol. 75. Springer pp 187-214.
  8. Wang, B., Xu, B., Wang, H., Li, J., Huang, H. and Xu L. (2011). YUCCA genes are expressed in response to leaf adaxial-abaxial juxtaposition and are required for leaf margin development. Plant Physiol 157: 1805-1819.

简介

水溶性植物是存在于各种维管植物的叶缘上的植物器官,是排列的位置。水解的解剖学和生理学都很少被证明。我们最近报道了花椰菜和拟南芥水解的解剖结构及其血管病原菌黄单胞菌(Xanthomonas campestris)pv的感染。 Cerutti等人,2017)。由于水阴是多种病原体的自然感染途径,因此需要对其解剖学有深入的了解,以进一步更好地解释受感染水合物的图像。在这里,我们描述了不同的详细方案,以获得适用于广泛植物(包括单子叶植物如大麦和水稻)的水溶性解剖学信息。用Nomarsky和共焦显微镜观察澄清的厚样品。透射光和透射电子显微镜中的光学显微镜用于观察薄和超薄切片。
【背景】在文献中,使用不同的技术来研究水溶性(Perrin,1972; Chen and Chen,2007; Wang等人,2011; Singh,2014)。 从光学显微镜(在整个组织或树脂嵌入样品的部分)到扫描或透射电子显微镜,可以使用大型方案和技术。 据我们所知,这些技术并不被组合使用,激光共焦显微镜从未用于描绘水溶性结构。 此外,我们注意到从协议到协议的变化。 我们在这里介绍了组合使用的不同技术。 它们适合花椰菜和拟南芥(Arabidopsis thaliana)。 它们已成功应用于其他植物,如单子叶植物(大麦和大米),应适用于各种植物物种。 我们鼓励用户应用这些协议,在感染过程中获得不同规模的水溶性辅助信息。

关键字:花椰菜, 泌水孔, 黄单胞菌

材料和试剂

  1. 显微镜玻片(Thermo Fisher Scientific,Superfrost,目录号:10143560WCUT)
  2. 封面24 x 60 mm(Thermo Fisher Scientific,目录号:15747592)
  3. 剃刀刀片
  4. 花椰菜(Brassica oleracea) var。,葡萄品种Clovis)
    注意:
    1. 花椰菜植物在受控的温室中生长。
    2. 所有的实验都使用了四周龄植物的第二个真叶。
  5. 水合氯仿(Sigma-Aldrich,目录号:23100)
  6. 甘油(C 3 H 8 O 3)(VWR,目录号:24387.326)
  7. Calcofluor(荧光增白剂28)(Sigma-Aldrich,目录号:F3543)
  8. Triton X-100(Sigma-Aldrich,目录号:T8787)
  9. 二甲胂酸钠(Sigma-Aldrich,目录号:C4945)
  10. 戊二醛EM级(电子显微镜科学,目录号:16214)
  11. 四氧化锇(OsO 4)(Electron Microscopy Sciences,目录号:19150)
  12. 乙醇(C 2 H 5 OH)(Sigma-Aldrich,目录号:32221)
  13. Epon(Electron Microscopy Sciences,目录号:14120)
    注意:树脂组合物:Embed-812(45g),DDSA(36g),NMA(18g)和BDMA(1.35ml)。加速器DMP30被BDMA替代的嵌入试剂盒(Electron Microscopy Sciences,catalog number:11400)。
  14. 硼砂(Sigma-Aldrich,目录号:B3545)
  15. 甲苯胺蓝(RAL Diagnostics,目录号:361590)
  16. 亚甲基蓝(Merck,目录号:159270)
  17. 基本品红(Sigma-Aldrich,目录号:857343)
  18. 周期酸(VWR,Prolabo,目录号:20.593.151)
  19. 乙酸(CH 3 COOH)(CARLO ERBA试剂,目录号:302016)
  20. 硫代碳酰肼(Sigma-Aldrich,目录号:223220)
  21. 蛋白质银(用于组织学的Roques)(Sigma-Aldrich,目录号:05495)
  22. Aceton(CH 3 COCH 3)(Fisher Scientific,目录号:10395640)
  23. 澄清解决方案(见配方)
  24. 固定液(参见食谱)
  25. 二甲胂酸钠缓冲液(见食谱)
  26. 硼烷溶液与甲苯胺蓝和亚甲蓝(见食谱)
  27. 基本品红解决方案(请参阅食谱)
  28. 周期酸溶液(见配方)
  29. 银蛋白溶液(参见食谱)

设备

  1. 空心冲孔机(Harris,Uni-Core)(Electron Microscopy Sciences,目录号:69039-70)
  2. 超切片机(Leica Microsystems,Reichert-Jung,型号:UltraCut E)
  3. 用于Nomarski的光学显微镜(Leica Microsystems,型号:DM IRB-E)
  4. 配有405nm二极管激光器的共焦显微镜(Leica Microsystems,型号:Leica TCS SP2)
  5. 隔膜泵真空或其他真空源
  6. 搅拌器电热板(IKA)
  7. 微波设备(Leica Microsystems,型号:EM AMW)
  8. 平底嵌入胶囊(电子显微镜科学,目录号:70021)
  9. 烤箱(Memmert,型号:UFE500BO)
  10. Histo金刚石刀(Diatome Histo)用于半薄片(0.5μm)至光学观察
  11. 超级金刚石刀(Diatome Ultra 45)用于超薄切片(70-80nm)至透射电子显微镜
  12. 热板(幻灯片加热器)(C& A Scientific,Premiere,型号:XH-2002)
  13. 光学显微镜(ZEISS,型号:Axioplan 2)
  14. CCD相机(ZEISS,型号:AxioCam MRc)
  15. 透射电子显微镜(日立,型号:HT7700)
  16. 金网格(电子显微镜科学,目录号:FCF200-Au)

软件

  1. Pro Plus 4.0成像软件(Media Cybernetics,Silver Spring,MD,USA)

程序

Nomarski和共聚焦显微镜用于观察澄清的厚样本,分别描绘复杂的血管形成和外膜。透射光(薄切片)和透射电子显微镜(超薄切片)的光学显微镜用于定位阴茎内的两种细菌,以及感染期间细胞或组织结构的变化。

  1. 澄清Nomarski和激光共焦显微镜的样品
    1. 使用空心打孔刀或刀片刮刀片。
    2. 将样品浸入基于水合氯醛的澄清溶液(见配方1)中,在4℃(黑夜)(冰箱)中浸泡数周(通常为1-3周)。
    3. 将样品装入相同的溶液中,由Nomarski显微镜观察。获取图像(见图1)。


      图1.在Nomarsky获得的花椰菜(A)和拟南芥(B)的典型图像()。比例尺= 100μm。

    4. 对于同一样品的共焦显微镜,在4℃的蒸馏水中冲洗样品过夜。
    5. 将样品在0.01%的calcofluor(用于纤维素)的水溶液中在室温下染色1小时。
    6. 用蒸馏水冲洗并放在玻璃载玻片上的水中。
    7. 使用激光二极管的405nm射线捕获图像,并收集410和500 nm之间的发射荧光。样品很厚,我们使用长工作距离的物镜,以避免在观察和采集过程中对样品施加过大的压力。因此,我们倾向于使用水浸式物镜(工作距离2至3 mm)进行深度观察(见图2)。


      图2.花椰菜水合物的真皮光学切片的共焦平面(A-B)。A.具有两个孔(箭头)和孔下方的大室(开口箭头)的水阴极边缘的视图。 B.具有多种口腔的水溶性阴道的上皮组织的图像。 e:表皮细胞; p:薄壁组织细胞C.在阴茎内的20个共焦平面(z-叠层)的最大投影,以描绘组织的气管及其装饰。比例尺= 25μm
  2. 电子和光学显微镜样品制备
    1. 用剃刀刀片,取包括水合物的叶片边缘(2-3毫米 2 )。
    2. 用固定溶液将样品固定10分钟(参见方案2)。缓慢释放真空并重复(2-3次),直到样品沉没。
    3. 在没有Triton X-100的固定溶液中,在大气压下固定样品1小时。
    4. 在0.2M二甲胂酸钠缓冲液中冲洗样品三次(每次15分钟,参见方法3)。
    5. 在0.2M二甲胂酸钠缓冲液中用2%四氧化锇将样品固定1小时。
    6. 在0.2M二甲胂酸钠缓冲液中冲洗样品三次(每次15分钟)
    7. 以等级系列的乙醇浓度水溶液(25%,50%,每次处理1小时)使样品脱水。
    8. 保存乙醇中的样品70%(最少1 h),直到使用徕卡微波设备(AMW)脱水并渗入Epon树脂(渗透按照以下程序进行,见表1)。

      表1.树脂渗透程序

      注意:
      1. *斜率:表示温度逐渐升高以在时间结束时达到指定值。
      2. 连续:表示温度尽快达到指定值,然后维护 d。

    9. 将样品放在装有纯树脂的平底嵌入胶囊中,在60℃下在烘箱中聚合48小时。

    对于光学显微镜
    1. 用Histo-diamond刀切成厚度为0.5μm的树脂嵌入样品。将这些部分放置在玻璃载玻片上的一滴水上,并在热板上加热至50℃以干燥并粘在玻璃上。
    2. 通过在含有0.1%甲苯胺蓝和0.2%亚甲蓝的1%硼砂溶液(参见方案4)中覆盖载玻片来污染切片(使用这两种污渍对比来自树脂嵌入样品的薄片)。在水中冲洗,然后用0.07%碱性品红的水溶液覆盖载玻片(用于染色酸性化合物,参见配方5)。
    3. 使用配有CCD彩色相机(ZEISS,AxioCam MRc)的光学显微镜(ZEISS,Axioplan 2成像)获取图像(参见图3)


      图3.来自由XCC 菌株8004 :: 感染的花椰菜叶(3dpi)的树脂嵌入的水合物(AB)的树皮剖面(0.5μm厚) em> GUS-GFP (Cerutti等人,2017)。 e:表皮细胞; p:外膜组织的薄壁组织细胞; xv:木质部容器A.注意孔(箭头),孔下方的大室(开口箭头); B.细菌在口腔中的存在(箭头)。比例尺= 25μm

    对于透射电子显微镜
    1. 切割厚度为70-80nm的树脂嵌入样品,并收集金网上的切片,并在高碘酸 - 硫代碳酰肼 - 银(Ag)蛋白质(PATAg)处理之前将其干燥过夜。
    2. 在1%(w / v)的高碘酸水溶液中,将部分(网格)在室温下漂浮30分钟(参见方案6)。
    3. 在蒸馏水上冲洗两次两小时(每次1小时)。
    4. 在4℃下用含有0.2%硫代碳酰肼的20%乙酸溶液处理切片过夜
    5. 在室温下将各浓度乙酸(20%,10%,5%醋酸溶液至蒸馏水)溶液中的切片洗涤30分钟。
    6. 在室温下,在黑暗中将1%(w / v)蛋白质蛋白酶(参见食谱7)的部分漂浮30分钟。
    7. 在水中洗涤切片(2-3次,每次20分钟)并风干。
    8. 观察在80kV下运行的透射电子显微镜下的部分,并获取图像(见图4)

      图4.未感染的阴茎(A)和感染的水阴(来自3dpi的B,6dpi的C)的图像,其由感染了Xcc 菌株8004 :: em的花椰菜叶> GUS-GFP (Cerutti等,2017)。 xv:木质部容器。箭头表示木质部血管的二次细胞壁装饰。箭头表示由细菌定殖的上皮细胞。注意(C)细菌侵入的血管。比例尺= 3μm

数据分析

可以进一步分析来自不同澄清植物样品的水合物的图像以计数木质部终端,以确定不同细胞类型的大小,孔的数量及其每个阴茎的大小。通过对捕获图像的分析,我们将花椰菜和拟南芥的水合物(Cerutti等人,2017年)进行了表征。所有测量均使用Image-Pro Plus 4.0成像软件(Media Cybernetics,Silver Spring,MD,USA)完成。也可以使用其他成像软件,如ImageJ:

  1. 为了计算水合物的木质部终端(或孔),最常见的是需要以不同的焦点获取图像,以便可视化所有的终止。使用一种测量工具,可以节省点特征的定位,以避免在阴极内的木质部终端超量计数。分析至少30至40个水阴极(Cerutti等人,2017)。
  2. 为了确定细胞(表皮或表皮细胞)的大小(面积),获得良好的图像至关重要。为了解决这个问题,用calcofluor染色细胞壁,并在图像采集之前用水充分冲洗样品。

食谱

  1. 澄清解决方案
    45克水合氯醛
    7.6毫升蒸馏水
    9.3 ml 60%甘油
  2. 固定解决方案
    2.5%戊二醛
    0.2 M二甲胂酸钠缓冲液(pH 7.2)
    0.1%Triton X-100
  3. 二甲胂酸钠缓冲液
    0.2M柠檬酸钠溶液(4.28g,100ml)在蒸馏水中,pH7.2 用几滴HCl(1N)调节pH值
  4. 硼砂溶液与甲苯胺蓝和亚甲蓝
    在1%硼砂的水溶液中加入0.1%(w / v)甲苯胺蓝,然后加入0.2%(w / v)亚甲基蓝色
  5. 基本品红解决方案
    碱性品红色在蒸馏水中的0.07%(w / v)
  6. 周期酸溶液
    1%(w / v)的高碘酸在蒸馏水中
  7. 银蛋白溶液
    准备1%(w / v)蛋白质银在蒸馏水中的新鲜溶液。在烟雾瓶玻璃中,在搅拌下逐渐加入蛋白质银。使用前过滤溶液并将其存储在黑暗中

致谢

这项工作得到法国国家教育和研究部授予AC的博士资助。 LIPM是法国卓越实验室项目(TULIP ANR-10-LABX-41; ANR-11-IDEX-0002-02)的一部分。我们也感谢RégionOccitanie对显微镜平台的财务支持。感谢CMEAB(中心微电子技术公司)的工作人员提供了宝贵的技术援助。固定和嵌入的程序改编自:a)Glauert,A.M。(1975)。固体,脱水和嵌入生物标本在电子显微镜实践方法。北荷兰出版公司阿姆斯特丹。 b)Hawes,C.和Satiat-Jeunemaitre,B.(2001)。植物细胞生物学:实践方法。牛津大学出版社。 PATAg治疗改良自Thiery,J.P。(1967)。 Mise enévidencedes polysaccharides sur coupes fines en microopieélectronique。焦耳; Microsc。 6:927-1018。

参考

  1. Chen C. C.和Chen Y. R.(2007)。 榕树的层状水合物研究(Moraceae )III 。盐水上的盐水损伤。 Bot Stud 48:215-226。
  2. Cerutti,A.,Jauneau,A.,Auriac,M.C.,Lauber,E.,Martinez,Y.,Chiarenza,S.,Leonhardt,N.,Berthomé,R.andNoël,L.D。(2017)。 花椰菜水合物的免疫力通过黄单胞菌(Xanthomonas campestris)控制全身感染 pv campestris 。植物生理学 174(2):700-716。
  3. Glauert,A.M。(1975)。 固定,生物标本的脱水和嵌入在电子显微镜的实践方法中。 北荷兰出版公司阿姆斯特丹。
  4. Hawes,C.和Satiat-Jeunemaitre,B.(2001)。 植物细胞生物学:实践方法。牛津大学出版社。
  5. Thiery,J.P。(1967)。 Mise enévidencedes polysaccharides sur coupes fines en microopieélectronique。 J Microsc 6:927-1018
  6. Perrin,A.(1972)。 贡献àl'étudede l'organization et du fonctionnement des hydathodes:recherches anatomiques ultrastructurales et physiologiques。大学克劳德 - 伯纳德里昂。
  7. Singh,S。(2014)。 喷雾:量化,微生物学和植物病理学的影响。 :Lüttg,U.,Beyschlag,W.,Cushman,J.(Eds)。 PROG。博特。卷。 75. 第187-214页。
  8. Wang,B.,Xu,B.,Wang,H.,Li,J.,Huang,H.,Xu L.(2011)。基因以叶片近轴 - 背面并列的形式表达,并且分别是叶片边缘发育需要。植物生理 157:1805-1819。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cerutti, A., Auriac, M., Noël, L. D. and Jauneau, A. (2017). Histochemical Preparations to Depict the Structure of Cauliflower Leaf Hydathodes. Bio-protocol 7(20): e2452. DOI: 10.21769/BioProtoc.2452.
  2. Cerutti, A., Jauneau, A., Auriac, M. C., Lauber, E., Martinez, Y., Chiarenza, S., Leonhardt, N., Berthomé, R. and Noël, L. D. (2017). Immunity at cauliflower hydathodes controls systemic infection by Xanthomonas campestris pv campestris. Plant Physiol 174(2): 700-716.
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