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Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells
拟南芥表皮、叶肉和血管实质细胞的激光扫描共聚焦显微镜检查   

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Abstract

Investigation of protein targeting to plastids in plants by confocal laser scanning microscopy (CLSM) can be complicated by numerous sources of artifact, ranging from misinterpretations from in vivo protein over-expression, false fluorescence in cells under stress, and organellar mis-identification. Our studies have focused on the plant-specific gene MSH1, which encodes a dual targeting protein that is regulated in its expression and resides within the nucleoid of a specialized plastid type (Virdi et al., 2016). Therefore, our methods have been optimized to study protein dual targeting to mitochondria and plastids, spatial and temporal regulation of protein expression, and sub-organellar localization, producing a protocol and set of experimental standards that others may find useful for such studies.

Keywords: Confocal(共聚焦), Plastid(质体), Chloroplast(叶绿体), GFP(GFP), Localization(定位), Autofluorescence(自发荧光), Organellar(细胞器)

Background

Protein targeting behavior in plants is influenced by amino-terminal presequences as well as internal sequence features that can influence suborganellar localization behaviors (Baginsky and Gruissem, 2004). Combined with promoter-driven spatial and temporal regulation in expression, a protein’s activity can be extremely precise and specialized by virtue of timing and location. In the case of MSH1, this nuclear-encoded, plant-specific protein is dual targeted to mitochondria and plastids (Xu et al., 2011). Promoter features direct its expression to reproductive, epidermal and vascular parenchyma cells (Virdi et al., 2016). Internal protein features direct its localization to the mitochondrial and plastid nucleoid, as well as to the plastid thylakoid membrane. Discovery of these unusual protein features was greatly facilitated by laser scanning confocal microscopy using methodologies described here. Much of this detail would have been overlooked using more traditional organellar subfractionation methodologies.

Materials and Reagents

  1. 5 ml tube
  2. Needleless syringe
  3. Double edge razor blades (Electron Microscopy Sciences, PersonnaTM, catalog number: 72000 )
  4. Glass cover slips, No. 1.5 (VWR, catalog number: 16004-302 )
  5. Dissecting needle or probe pin
  6. Petri dish (VWR, catalog number: 25384-302 )
  7. Centrifuge tube (VWR, catalog number: 89039-668 )
  8. 0.45 μm filter (VWR, catalog number: 28145-481 )
  9. Disposable transfer pipette (Fisher Scientific, catalog number: 13-7117M )
  10. Glass slides (VWR, catalog number: 48300-048 )
  11. Arabidopsis leaf/flower/stem tissue (Ecotype Col-0, 6 weeks old plants)
  12. Lurie broth
  13. Tween 20 (Sigma-Aldrich, catalog number: P1379 )
  14. Incubation solution
  15. Potassium phosphate dibasic (K2HPO4) (Sigma-Aldrich, catalog number: P3786 )
  16. Potassium phosphate monobasic (KH2PO4) (Sigma-Aldrich, catalog number: P5655 )
  17. Ammonium sulfate, (NH4)2SO4 (Sigma-Aldrich, catalog number: A4418 )
  18. Sodium citrate dihydrate (Sigma-Aldrich, catalog number: C8532 )
  19. Magnesium sulfate (MgSO4) (1 M stock solution) (Sigma-Aldrich, catalog number: M2643 )
  20. Glucose (Sigma-Aldrich, catalog number: G8270 )
  21. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  22. MES (Sigma-Aldrich, catalog number: M2933 )
  23. KOH or NaOH
  24. Acetosyringone (3’,5’-dimethoxy-4’-hydroxyacetophenone) (Sigma-Aldrich, catalog number: D134406 )
  25. MS medium basal salts (Sigma-Aldrich, catalog number: M5519 )
  26. Cellulase ‘onozuka’ R-10 (Yakult Honsha, Tokyo, Japan)
  27. Macerozyme R-10 (Yakult Honsha, Tokyo, Japan)
  28. Mannitol (Sigma-Aldrich, catalog number: M1902 )
  29. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  30. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C7902 )
  31. BSA (optional) (Sigma-Aldrich, catalog number: A7906 )
  32. Induction medium (see Recipes)
  33. Infiltration medium (see Recipes)
  34. Cellulase/macerozyme solution (see Recipes)
  35. Washing and incubation solution (WI) (see Recipes)

Equipment

  1. 387/478/555 nm triple-bandpass filter excitation with a broad emission filter ~400-700 nm
  2. LED illumination, 488 nm, 543 nm (Lumencor AURA light engine)
  3. Incubator shaker
  4. Platform shaker
  5. Forceps (VWR, catalog number: 82027-440 ) or (Cole-Parmer Instrument, catalog number: EW-07287-09 )
  6. 90i upright compound microscope (Nikon Instruments)
  7. 10x Plan Apo 0.45NA (Nikon Instruments, model: OFN25 )
  8. 20x Plan Apo 0.75NA (Nikon Instruments, model: lambda )
  9. 60x Plan Apo VC water immersion lens 1.2NA (Nikon Instruments, model: MRD07602 )
  10. Confocal laser scanning microscope (CLSM) (Nikon Instruments, model: A1+ )
  11. Autoclave
  12. Water bath
  13. Vacuum desiccator

Software

  1. NIS-Elements software

Procedure

  1. Agrobacterium mediated transient transformation of tobacco leaves
    1. Culture Agrobacterium strain C58C1 carrying the target gene of interest in a 5 ml culture medium of Lurie broth overnight at 30 °C in an incubation shaker and spin down at 1,000 x g.
    2. Transfer the bacterial cells into 5 ml of induction medium (see Recipes) and grow at 30 °C for 16 h with shaking.
    3. Spin the cells down and re-suspended in 1 ml infiltration medium (see Recipes).
    4. Measure the bacterial concentration to an optical density of 0.8 at 600 nm in a fresh 5 ml tube of infiltration medium. Infiltrate young leaves of about 4 weeks old tobacco plants, Nicotiana tabacum cv. xanthi using a needleless syringe. Visualize plant expression after 48 h.
    Note: Protocol modified from Van den Ackerveken et al. (1996).

  2. Preparation of Arabidopsis plant samples
    1. GFP or YFP when viewed through the oculars with a broad emission filter coupled with a 488 nm LED will not only allow visualization of GFP or YFP (with less excitation efficiency) but also the plastids. This full spectrum visualization allows the user to differentiate damaged areas where plastids shift from red emission to orange. Furthermore in variegated tissues, ocular observation may be the only method of differentiating low GFP or YFP and damaged/stressed areas. Ocular observations have the benefit of much deeper optical sections, providing context for localization. In this case, 10x and 20x visualization provided clues to pursue vascular localization.
    2. RFP excited with 543 nm light visualized with a broad emission filter will appear a deep orange color in a ruby red background from the plastids. With low RFP expression, it’s difficult to differentiate the two, and the CSLM should be used to separate the two.
    3. The benefit of using LED over more common mercury vapor lamps is the reduction in photobleaching or degradation of the plastid pigments, which will shift emission causing erroneous analyses.
      1. Leaf/flower
        The surface tension of the water used for wet mounting under a larger cover slip will hold samples steady during imaging. The addition of a trace of Tween 20 (5-10 µl in 250 ml water) will assist in the wetting of flowers, which are far more hydrophobic than leaves.
      2. Stems
        A double-edged razor blade snapped in half longitudinally before unwrapping is used to make cross sections of stems. Snapping double-edged razor blades is hazardous (Figure 1). The stem sections should be taken in the middle of the internodes to avoid leaf vascular traces found near the nodes (Figure 2). The sections should be thin enough and consistent enough to fit under a coverslip (< 0.3 mm) while in water. Cut the stem in a forward and downward motion exposing the tissue to clean blade areas preventing further damage (Figure 3). The blade will dull within roughly 30 cross sections and should be rotated or disposed of. Dull blades will compress the stems and pull the helical thickenings of the secondary cell wall out of the metaxylem.
        Note: The cut and damaged cells and those intact but in contact with the water under the coverslip will begin to degrade immediately. It is necessary to image deeper into the tissue than these cells with CSLM. Familiarity with the phenomenon of emission spectra shifting from far red plastid autofluorescence to green is best characterized with wild type plant material. The degradation of chlorophylls from damage causes the spectra to move from far red to green, this may result in autofluorescence being mistaken for GFP.


        Figure 1. Method for using double edged razor blades. A. Grasp the blade so that the sharp edges are not pressured by the fingers. B. The index fingers are retracted while slow and constant pressure is used to bend the blade in half. C. As the two outer edges come closer, the index fingers and thumbs are placed near the folded side and pressure there will cause the blade to snap into two separate blades. D. Unwrapping the blade is done carefully again to prevent cuts through the paper. Each half of the blade may be used for dissection.


        Figure 2. Arabidopsis thaliana flowering stem used for analysis. The internode region is where cross sections should be made (bracket).


        Figure 3. Method for cutting and mounting Arabidopsis stem cross sections. A. The stem with the unneeded portions removed is laid flat on the slide with water. B. Sections are cut as thin as possible in a forward and downward motion. The blade may be rotated to expose a sharp edge, or the other half may be used. C. While cutting, the sections may stack, this is remedied by gentle manipulation with a dissecting needle or probe or insect pin. D. Once all sections are separated, a cover slip is placed on the samples. Uniform sections are important for level cover slip placement. Water may be added or wicked off to keep the sections in water, but without movement.  

  3. Preparation of enzymatically digested plant tissues and visualization
    Partial enzymatic digestion of A. thaliana leaves for visualization of fluorescence in plant vasculature. Enzymatic digestion is done by modifying the protocol of Yoo et al. (2007) used for Protoplast Isolation Procedure. 
    1. Cut the leaves so most of the lamina is trimmed off from the midrib and veins and subjected to enzyme digestion.
      Note: A six to seven weeks old plant is normally used for this experiment.
    2. Place the plant material to be digested in a Petri dish containing the enzyme solution and put it in a vacuum desiccator and vacuum infiltrate for 15 min.
      Note: A 5-10 ml cellulase/macerozyme solution (see Recipes) is enough for a flowering A. thaliana plant.
    3. Take out the Petri dish from vacuum and continue digestion at RT for 2 h with gentle shaking on a platform shaker, between 30-50 RPM.
    4. The proper digestion time is near when the epidermis is digested away, and mesophyll cells begin sloughing away. The slurry generated to visualize vascular parenchyma with intact leaf or is easily transferred with a transfer pipette or fine tip forceps as the samples are not allowed to fully digest to the protoplast stage. The sample can be transferred to a Petri dish containing 10 ml of the WI buffer. On a compound microscope, if the vasculature can be seen, the sample is ready for confocal analysis. Vasculature is easily identified by the helical thickenings in the xylem with transmitted light at 10x or 20x. Digestion time will vary with the hardiness of the plant material.
    5. Mount samples on slides with coverslips for confocal microscopy. The incubation solution is used to mount the sample, as it will support the cells during imaging.
      1. GFP or YFP can be visualized with RFP with relative ease with the correct filter sets on the CSLM, although not simultaneously. Sequential or simultaneous acquisitions is dictated by the fluorescent protein used and the relative strength compared to various sources of autofluorescence. It is crucial to compare CSLM settings to the wild type material at the same settings used to detect the fluorescent proteins. When expression of the fluorescent protein(s) is low, it is suggested to check and recheck against negative controls.
      2. Plastid suborganellar localization often requires practices outside of routine confocal imaging. Operators should be aware of data clipping from under and overexposure of the detectors. Underexposed pixels may imply lost data. Overexposed pixels are data lost, and there is concern regarding overloading of the detectors. NIS-Elements has an indicator display for under and overexposed pixels. When expression levels are low or near the same signal levels as autofluorescence, it is suggested that the look up table (LUT) be manipulated to increase the brightness on the display screen after image acquisition. Images acquired in the same way between the negative and putative positives should be viewed with these same artificial increases in signal. Increasing the signal to the detectors will only increase the artifacts and noise in the images. Chlorophyll autofluorescence, detected in the 650-720 nm range at 60x or higher will overlap autofluorescence in other detection channels. Signal not colocalized with the chlorophyll autofluorescence may indicate positive fluorophore signal. It is crucial to repeat the experiment and continue to compare the images between the negative and putative positives.
        Nikon A1 CSLM notes:

Data analysis

Factors that affect sample observations:

  1. Transgene construct features influence microscopy outcomes
    Promoter selection can influence signal strength significantly in our experiments. Consequently, we have combined both transient, high level expression experiments with native promoter, stable transformation experiments for any constructs to be tested (Figure 4). For proteins targeted to subcellular organelles, it appears that truncation of the full-length gene can sometimes produce unreliable results. Figure 5 shows that the MSH1 protein, which targets to the nucleoid subcompartment of the plastid, can be found localized to the nucleoid only when at least domains I–III of the protein coding region are included in the analysis (Virdi et al., 2016).


    Figure 4. Expression pattern in leaves and level of expression vary depending on the promoter used. A. pCAMBIA1302C::35S::MSH1::GFP transient expression in tobacco. There are more punctate GFP expression signals when the gene construct is driven by the CaMV 35S promoter. B. pCAMBIA1302C (-35S)::NativePro::MSH1::GFP stable expression in Arabidopsis cells. The native promoter resulted in fewer punctate GFP signals (as seen by number of green dots per plastid). All scale bars are 25 µm. GFP pseudocolored - green, chloroplast autofluorescence pseudocolored - red, maximum intensity projections. 


    Figure 5. Protein localization pattern can vary depending on truncations in Arabidopsis thaliana Col-0 leaf tissue. A. Native promoter::MSH1(ATG to Domain I)::GFP; domain I alone gives a diffused signal in the plastids. Single frame image. B. Native promoter::MSH1(ATG to Domain III)::GFP. Single frame image. C. Native promoter::MSH1(ATG to Domain V)::GFP. MSH1 ATG start to domain III is necessary to give the punctate localization. Maximum intensity projection. GFP pseudocolored green, chloroplast autofluorescence pseudocolored red for all images. All scale bars are 25 µm. D. Schematic diagram of the different domains of MSH1 from Arabidopsis thaliana used for the truncation studies (Virdi et al., 2016).

  2. Tissue preparation can also influence resolution in a confocal microscopy study
    Plants stably transformed with a full-length gene construct under control of its native promoter offer the opportunity to document spatial and temporal expression behaviors. Figure 6 shows the result of partial tissue digestion in revealing the localization of the MSH1 protein within specialized plastids of the vascular parenchyma tissues. These observations are confirmed, in Figure 7, by stem cross-sections that show MSH1-GFP signal within the vascular bundle tissues. However, it is essential that the tissues assayed are not derived from a stressed plant. Figure 8 shows that artifactual fluorescence in a plant under stress can be confused with RFP signal.


    Figure 6. Partial enzymatic digestion of A. thaliana leaf midrib and side veins to study sub-cellular localization of fluorescence. Native Pro::MSH1::GFP stable transformed A. thaliana Col-0 leave digested with cellulose and macerozyme as described above. MSH1::GFP expression in specialized plastids can be seen concentrated around phloem and xylem but not in the mesophylls. All scale bars are 25 µm. A. Minor leaf vein. GFP pseudocolored - green, chloroplast autofluorescence pseudocolored - red, xylem autofluorescence pseudocolored - blue. Maximum intensity projection. B. Midrib of leaf. GFP pseudocolored - green, chloroplast autofluorescence pseudocolored - red.


    Figure 7. Hand sectioning technique to visualize tissue specificity of proteins. A. Cross section of native promoter::MSH1::GFP stable transformed floral stems hand sectioned as described in Materials and Methods. MSH1::GFP expression localizes to the vascular system. GFP pseudocolored green, chloroplast autofluorescence pseudocolored red, xylem autofluorescence pseudocolored blue with crosstalk in the GFP channel. B. Cross section of a dually transformed A. thaliana with MSH1::GFP and PPD3::RFP (AT1G76450); co-localization of two proteins in the same tissue and plastid type. GFP pseudocolored - green, RFP pseudocolored - red, chloroplast autofluorescence pseudocolored - blue. Single frame images and all scale bars are 25 µm.


    Figure 8. Artifacts that users need to be aware of in Confocal Microscopy. A stressed A. thaliana Col-0 leaf vacuoles. Scale bar is 25 µm. Chloroplast autofluorescence pseudocolored - green, vacuole autofluorescence pseudocolored - red. Maximum intensity projection.

Recipes

  1. Induction medium (1 L)
    10.5 g K2HPO4
    4.5 g KH2PO4
    1 g (NH4)2SO4
    0.5 g sodium citrate dihydrate
    1 ml MgSO4 (1 M stock solution)
    2 g glucose
    5 ml glycerol
    10 mM MES
    Adjust pH to 5.6 with KOH or NaOH and autoclave at 115 °C for 20 min
    Cool down medium and add antibiotics and 50 μg/ml acetosyringone
  2. Infiltration medium
    Half strength MS medium basal salts
    10 mM MES
    Adjust pH to 5.6 with KOH
    Add 150 μg/ml acetosyringone before use
  3. Cellulase/macerozyme solution
    1.5% cellulase ‘onozuka’ R-10 (0.15 g/10 ml)
    0.4% macerozyme R-10 (Yakult Honsha, Tokyo, Japan) (0.04 g/10 ml)
    0.4 M mannitol (0.8 M mannitol stock)
    20 mM KCl (2 M KCl stock)
    20 mM MES, pH 5.7 (0.2 M MES stock)
    Heat the enzyme solution in a 55 °C water bath for 10 min in 15 ml centrifuge tube (stirring not needed)
    Cool it to room temperature
    Add 10 mM CaCl2 (1 M CaCl2 stock); 0.1% BSA (optional) (10% stock, sterile)
    Pass the enzyme through a 0.45 μm filter
  4. Washing and incubation solution (WI)
    0.5 M mannitol
    4 mM MES, pH 5.7
    20 mM KCl

Acknowledgments

We wish to thank Dr. Kamaldeep Virdi and Sunil Kumar for supplying images from their research in Arabidopsis for demonstration purposes. We also thank Cecil Renfro for her assistance with photography. We gratefully acknowledge support from the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of DOE (DE-FG02-10ER16189) to S.M. for this work.

References

  1. Baginsky, S. and Gruissem, W. (2004). Chloroplast proteomics: potentials and challenges. J Exp Bot 55(400): 1213–1220.
  2. Van den Ackerveken, G., Marois, E. and Bonas, U. (1996). Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell 87(7):1307–1316.
  3. Virdi, K. S, Wamboldt, Y., Yu, J., Laurie, J. D., Kumar, S., Kundariya, H., Xu, Y., Elowsky, C., Basset, G., Bricker, T., Leubker, S., Keren, I. and Mackenzie, S. A. (2016). MSH1 is a plant organellar DNA binding and thylakoid protein under precise spatial regulation to alter development. Mol Plant 9: 245–60.
  4. Xu, Y. Z., Arrieta-Montiel, M. P., Virdi, K. S., de Paula, W. B., Widhalm, J. R., Basset, G. J., Davila, J. I., Elthon, T. E., Elowsky, C. G., Sato, S. J., Clemente, T. E. and Mackenzie, S. A. (2011). MutS HOMOLOG1 is a nucleoid protein that alters mitochondrial and plastid properties and plant response to high light. Plant Cell 23: 3428–3441.
  5. Yoo, S. D., Cho, Y. H. and Sheen, J. (2007). Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat Protoc 2(7): 1565-1572.

简介

通过共焦激光扫描显微镜(CLSM)对植物中质体进行蛋白质靶向的研究可能由于许多来源的伪像而复杂化,其范围从体内蛋白质过度表达的误解,应激细胞中的假荧光,和细胞器错误识别。我们的研究集中在植物特异性基因MSH1上,其编码双重靶向蛋白,其在其表达中被调节并且位于特定质体类型的核内(Virdi等人,2016)。因此,我们的方法已被优化,以研究蛋白质双重靶向线粒体和质体,蛋白质表达的空间和时间调节和亚细胞定位,产生其他人可能对这些研究有用的方案和一组实验标准。

背景 植物中的蛋白质靶向行为受氨基末端前序列以及可影响亚组织定位行为的内部序列特征的影响(Baginsky和Gruissem,2004)。结合启动子驱动的表达空间和时间调节,蛋白质的活性可以通过时间和位置而非常精确和专一。在MSH1的情况下,这种核编码的植物特异性蛋白质是双重靶向线粒体和质体(Xu et al。,2011)。启动子特征将其表达指导到生殖,表皮和血管薄壁细胞(Virdi等人,2016)。内部蛋白质特征将其定位于线粒体和质体核,以及质体类囊体膜。使用这里描述的方法,通过激光扫描共聚焦显微镜大大促进了这些不寻常的蛋白质特征的发现。使用更传统的细分器分割方法,大部分细节将被忽略。

关键字:共聚焦, 质体, 叶绿体, GFP, 定位, 自发荧光, 细胞器

材料和试剂

  1. 5ml管
  2. 无针注射器
  3. 双刃刀片(Electron Microscopy Sciences,Personna TM ,目录号:72000)
  4. 玻璃盖玻片,1.5号(VWR,目录号:16004-302)
  5. 解剖针或探头针
  6. 培养皿(VWR,目录号:25384-302)
  7. 离心管(VWR,目录号:89039-668)
  8. 0.45μm过滤器(VWR,目录号:28145-481)
  9. 一次性转移移液器(Fisher Scientific,目录号:13-7117M)
  10. 玻璃滑梯(VWR,目录号:48300-048)
  11. 拟南芥叶/花/茎组织(Ecotype Col-0,6周龄植物)
  12. Lurie肉汤
  13. 吐温20(Sigma-Aldrich,目录号:P1379)
  14. 孵化解决方案
  15. 磷酸氢二钾(K 2/2 HPO 4)(Sigma-Aldrich,目录号:P3786)
  16. 磷酸二氢钾(KH 2 PO 4)(Sigma-Aldrich,目录号:P5655)
  17. 硫酸铵(NH 4)2 SO 4(Sigma-Aldrich,目录号:A4418)
  18. 柠檬酸钠二水合物(Sigma-Aldrich,目录号:C8532)
  19. 硫酸镁(MgSO 4)(1M储备溶液)(Sigma-Aldrich,目录号:M2643)
  20. 葡萄糖(Sigma-Aldrich,目录号:G8270)
  21. 甘油(Sigma-Aldrich,目录号:G5516)
  22. MES(Sigma-Aldrich,目录号:M2933)
  23. KOH或NaOH
  24. 氨基丁酮(3',5'-二甲氧基-4'-羟基苯乙酮)(Sigma-Aldrich,目录号:D134406)
  25. MS中等基础盐(Sigma-Aldrich,目录号:M5519)
  26. 纤维素酶'onozuka'R-10(Yakult Honsha,Tokyo,Japan)
  27. Macerozyme R-10(Yakult Honsha,Tokyo,Japan)
  28. 甘露醇(Sigma-Aldrich,目录号:M1902)
  29. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  30. 氯化钙滴液(CaCl 2·2H 2 O)(Sigma-Aldrich,目录号:C7902)
  31. BSA(可选)(Sigma-Aldrich,目录号:A7906)
  32. 感应介质(见配方)
  33. 渗透介质(见食谱)
  34. 纤维素酶/macerozyme溶液(参见食谱)
  35. 洗涤和孵化溶液(WI)(参见食谱)

设备

  1. 387/478/555 nm三通带滤波器激发与宽发射滤波器〜400-700 nm
  2. LED照明,488nm,543nm(Lumencor AURA光引擎)
  3. 孵化器摇床
  4. 平台摇床
  5. 镊子(VWR,目录号:82027-440)或(Cole-Parmer仪器,目录号:EW-07287-09)
  6. 90i立式复合显微镜(尼康仪器)
  7. 10x Plan Apo 0.45NA(Nikon Instruments,型号:OFN25)
  8. 20x Plan Apo 0.75NA(Nikon Instruments,型号:lambda)
  9. 60x Plan Apo VC水浸镜头1.2NA(尼康乐器,型号:MRD07602)
  10. 共焦激光扫描显微镜(CLSM)(尼康仪器,型号:A1 +
  11. 高压灭菌器
  12. 水浴
  13. 真空干燥器

软件

  1. NIS-Elements软件

程序

  1. 农杆菌介导烟草叶片的瞬时转化
    1. 培养在农杆菌菌株C58C1中携带目的基因的目标基因在5升Lurie肉汤培养基中在30℃下在孵育振荡器中过夜并以1,000×g旋转。
    2. 将细菌细胞转移到5ml的诱导培养基中(参见食谱),并在30℃振荡培养16小时。
    3. 旋转细胞并重新悬浮在1ml渗透培养基中(见食谱)。
    4. 在新鲜的5ml管渗透培养基中,在600nm处测量细菌浓度至0.8的光密度。渗透大约4周龄烟草植物的青叶,烟草烟草 cv。 xanthi使用无针注射器。 48小时后可视化植物表达。
    注意:修改自Van den Ackerveken等人的协议(1996)。

  2. 拟南芥植物样品的制备
    1. 当通过与488nm LED耦合的宽发射滤光片通过眼睛观察时,GFP或YFP将不仅允许GFP或YFP的可视化(具有较少的激发效率),而且还可以显示质体。这种全频谱可视化允许用户区分质量从红色发射到橙色的损坏区域。此外,在杂色组织中,眼睛观察可能是区分低GFP或YFP和损伤/应激区域的唯一方法。眼睛观察具有更深的光学部分的优点,为本地化提供了上下文。在这种情况下,10x和20x可视化提供了追踪血管定位的线索。
    2. 用广泛的发射滤光器显示的543nm光激发的RFP将在质体的红宝石红色背景中显现深橙色。由于RFP表达低,难以区分两者,CSLM应用于分离两者。
    3. 将LED用于更常见的汞蒸气灯的好处是减少了漂白或质子颜料的降解,这会导致发生错误分析的排放。
      1. 叶/花
        在较大的覆盖层下用于湿式安装的水的表面张力将在成像期间保持样品稳定。加入痕量的吐温20(5-10毫升在250毫升水中)将有助于润湿比叶更疏水的鲜花。

      2. 一个双刃剃刀刀片在展开前被纵向咬合一半,用于制作茎的横截面。咬合双刃剃刀刀片是危险的(图1)。茎节应在节间的中部,以避免在节点附近发现叶脉血管痕迹(图2)。这些部分应足够薄且足够一致,以适合在水中的盖玻片(<0.3mm)。切割杆向前和向下运动,使组织清洁,以防止进一步损坏(图3)。刀片将在大约30个横截面内变钝,并应旋转或处理。钝的叶片将压缩茎,并将次生细胞壁的螺旋状增厚物从甲状腺体外拉出。
        注意:切割和损坏的细胞以及那些完整但与盖玻片下的水接触的细胞将立即开始降解。有必要使用CSLM比这些细胞对组织进行更深的成像。熟悉发射光谱从远红色质感自发荧光转变为绿色的现象,最好以野生型植物材料为特征。叶绿素从损伤中的降解导致光谱从远红色移动到绿色,这可能导致自发荧光被误认为GFP。


        图1.使用双刃剃刀刀片的方法。 A.握住刀片,使锋利的边缘不被手指压。 B.食指缩回,而使用缓慢且恒定的压力将刀片弯曲成一半。 C.当两个外边缘靠近时,食指和拇指放置在靠近折叠的一侧,压力将导致刀片卡入两个单独的刀片。 D.再次仔细拆开刀片,以防止纸张断开。刀片的每一半可用于解剖。


        图2.拟南芥开花茎用于分析。 节间区域应该是横截面(括号)。


        图3.切割和固定方法切割茎横截面 A.将带有不需要部分的茎除去水放在载玻片上。 B.切片在向前和向下运动中切割尽可能薄。刀片可以旋转以暴露锋利的边缘,或者可以使用另一半。 C.切割时,切片可以堆叠,这通过用解剖针或探针或昆虫针进行温和操作来补救。 D.一旦所有部分分开,样品上都会放置盖子。均匀部分对于水平盖滑移位置很重要。水可能会被添加或恶化,以使部分保持在水中,但不会移动。  

  3. 酶消化的植物组织和可视化的准备
    部分酶促消化。 thaliana 留下植物脉管系统中荧光的可视化。酵素消化是通过修改Yoo等人的方案进行的。 (2007)用于原生质体隔离程序。
    1. 切开叶子,使大部分叶片从中脉和静脉中剪下来,并进行酶消化。
      注意:本实验通常使用6至7周龄的植物。
    2. 将待消化的植物材料放入含有酶溶液的培养皿中,并放入真空干燥器中,真空渗透15分钟。
      注意:5-10毫升的纤维素酶/巨噬菌体溶液(参见食谱)对于开花的拟南芥植物来说是足够的。
    3. 从真空中取出培养皿,并在平台摇床上,在30-50RPM之间,在室温下继续消化2小时。
    4. 当表皮被消化时,适当的消化时间接近,叶肉细胞开始脱落。产生的浆液用完整的叶观察血管薄壁,或者当样品不能完全消化到原生质体阶段时,可以用转移移液管或细尖镊子容易地转移。样品可以转移到含有10ml WI缓冲液的培养皿中。在复合显微镜上,如果可以看到脉管系统,样品就可以进行共聚焦分析了。脉管系统容易通过木质部的螺旋增厚识别,透射光为10x或20x。消化时间将随着植物材料的耐受性而变化。
    5. 载玻片上的样品用盖玻片共聚焦显微镜。孵育溶液用于安装样品,因为它将在成像期间支持细胞。
      1. GFP或YFP可以用RFP可视化,而CSLM上的正确滤波器组相对容易,尽管不是同时的。顺序或同时采集由所使用的荧光蛋白和与各种自发荧光源相比的相对强度决定。在用于检测荧光蛋白的相同设置下,将CSLM设置与野生型材料进行比较是至关重要的。当荧光蛋白的表达低时,建议检查和重新检查阴性对照。
      2. 质体亚组织定位通常需要常规共焦成像以外的实践。操作人员应注意检测器下方的数据剪切和过度曝光。曝光不足的像素可能意味着丢失的数据。曝光过度的像素是数据丢失,并且有关于检测器的超载问题。 NIS-Elements具有下曝光和曝光过度的像素的指示器显示。当表达水平低或与自发荧光相同的信号水平时,建议在图像采集之后操纵查找表(LUT)以增加显示屏上的亮度。应以同样的方式在负面和推定的正面之间获得的图像以同样的人为增加信号来观察。将信号增加到检测器将仅增加图像中的伪像和噪声。在60〜60nm范围内检测到的叶绿素自身荧光在其他检测通道中将重叠自发荧光。不与叶绿素自身荧光共定位的信号可能表明阳性荧光信号。重复实验至关重要,并继续比较阴性和推测阳性之间的图像。
        尼康A1 CSLM注意:

数据分析

影响样本观察的因素:

  1. 转基因结构特征影响显微镜结果
    启动子选择可以在我们的实验中显着影响信号强度。因此,我们将瞬态,高水平表达实验与天然启动子相结合,对待测试的任何构建体进行稳定转化实验(图4)。对于靶向亚细胞器的蛋白质,看来全长基因的截短有时会产生不可靠的结果。图5显示,仅当蛋白质编码区的至少区域I-III包含在分析中时,可以发现靶向质体的核苷酸亚室的MSH1蛋白定位于核型(Virdi等人。,2016)。


    图4.叶中的表达模式和表达水平根据所使用的启动子而变化。 A.烟草中的pCAMBIA1302C :: 35S :: MSH1 :: GFP瞬时表达。当基因构建体由CaMV 35S启动子驱动时,有更多的点状GFP表达信号。 B.pCAMBIA1302C(-35S):: NativePro :: MSH1 :: GFP稳定表达在拟南芥细胞中。天然启动子导致较少的点状GFP信号(如通过质体数量的绿点所见)。所有比例尺为25μm。 GFP伪着色 - 绿色,叶绿体自发荧光假染色 - 红色,最大强度预测。 


    图5.蛋白定位模式可以根据拟南芥Col-0叶组织中的截短而变化。 A。天然启动子:: MSH1(ATG到结构域I):: GFP;单独的域I在质体中产生扩散信号。单帧图像B.原生启动子:: MSH1(ATG到结构域III):: GFP。单帧图像C.原生启动子:: MSH1(ATG到域V):: GFP。 MSH1 ATG开始到域III是必要的,以进行点状定位。最大强度投影。 GFP假绿色,叶绿体自发荧光伪染红色为所有图像。所有比例尺为25μm。 D.用于截断研究(Virdi等人,2016)的拟南芥MSH1的不同结构域的示意图。

  2. 组织制备也可以影响共聚焦显微镜研究中的分辨率 在其天然启动子控制下用全长基因构建体稳定转化的植物提供记录空间和时间表达行为的机会。图6显示部分组织消化的结果,揭示MSH1蛋白在血管薄壁组织的特殊质体中的定位。在图7中,这些观察结果通过在血管束组织中显示MSH1-GFP信号的干细胞横截面得到证实。然而,测定的组织不是来源于应激植物是重要的。图8显示植物在胁迫下的人为荧光可能与RFP信号混淆

    图6.部分酶消化。拟南芥叶中脉和侧脉,研究荧光的亚细胞定位。 Native Pro :: MSH1 :: GFP稳定转化A。如上所述,用纤维素和macerozyme消化Col-0。 MSH1 :: GFP特异性质体中的表达可以看作是围绕韧皮部和木质部,而不是在叶肉中。所有比例尺为25μm。 A.小叶静脉GFP伪着色 - 绿色,叶绿体自发荧光假染色 - 红色,木质部自发荧光假染色 - 蓝色。最大强度投影。 B.叶中。 GFP伪着色 - 绿色,叶绿体自发荧光假染色 - 红色。


    图7.用于显示蛋白质组织特异性的手工分割技术。 A.天然启动子的横截面:: MSH1 :: GFP稳定转化的花茎手部分如材料和方法中所述。 MSH1 :: GFP表达定位于血管系统。 GFP假绿色,叶绿体自发荧光假染色红色,木质部自发荧光假绿色在GFP通道中具有串扰。 B.双重变换的横截面。具有MSH1 :: GFP和PPD3 :: RFP(AT1G76450)的拟南芥;两种蛋白质在同一组织和质体型中的共定位。 GFP伪着色 - 绿色,RFP伪着色 - 红色,叶绿体自发荧光假染色 - 蓝色。单幅图像和所有比例尺均为25μm。


    图8.用户需要在共聚焦显微镜中注意的人工制品。 A。拟南芥 Col-0叶空泡。刻度棒为25μm。叶绿体自发荧光假绿色,液泡自发荧光假绿色 - 红色。最大强度投影。

食谱

  1. 感应介质(1L)
    10.5g K 2 HPO 4
    4.5g KH 2 PO 4
    1g(NH 4)2 SO 4
    0.5克柠檬酸钠二水合物 1ml MgSO 4(1M储备溶液)
    2 g葡萄糖
    5毫升甘油
    10 mM MES
    用KOH或NaOH将pH调节至5.6,并在115℃下高压灭菌20分钟 冷却培养基并加入抗生素和50μg/ml乙酰丁香酮
  2. 渗透介质
    半强度MS中等基础盐
    10 mM MES
    用KOH调节pH至5.6 使用前加入150μg/ml乙酰丁香酮
  3. 纤维素酶/macerozyme溶液
    1.5%纤维素酶'onozuka'R-10(0.15g/10ml)
    0.4%macerozyme R-10(Yakult Honsha,Tokyo,Japan)(0.04g/10ml)
    0.4M甘露醇(0.8M甘露醇储备液)
    20mM KCl(2M KCl原料)
    20mM MES,pH 5.7(0.2M MES储备)
    将酶溶液在55℃的水浴中在15ml离心管中加热10分钟(不需要搅拌)
    将其冷却至室温
    加入10mM CaCl 2(1M CaCl 2)原料); 0.1%BSA(可选)(10%储备,无菌)
    将酶通过0.45μm过滤器
  4. 洗涤和孵化溶液(WI)
    0.5 M甘露醇
    4 mM MES,pH 5.7
    20 mM KCl

致谢

为了演示,我们要感谢Kamaldeep Virdi博士和Sunil Kumar博士提供他们在拟南芥研究中的图像。我们也感谢Cecil Renfro对摄影的帮助。我们非常感谢能源部基础能源科学办公室(DE-FG02-10ER16189)的化学科学,地球科学和生物科学部门向S.M.提供支持。为这项工作。

参考文献

  1. Baginsky,S.和Gruissem,W.(2004)。叶绿体蛋白质组学:潜力和挑战。 J Exp Bot 55(400):1213-1220。
  2. Van den Ackerveken,G.,Marois,E.and Bonas,U.(1996)。  识别细菌无毒素蛋白AvrBs3发生在宿主植物细胞内。细胞 87(7):1307-1316。
  3. Virdi,K.S,Wamboldt,Y.,Yu,J.,Laurie,JD,Kumar,S.,Kundariya,H.,Xu,Y.,Elowsky,C.,Basset,G.,Bricker, Leubker,S.,Keren,I。和Mackenzie,SA(2016)。 MSH1是精确空间调节的植物细胞器DNA结合和类囊体蛋白以改变发育。分子植物 9:245-60。
  4. Xu,YZ,Arrieta-Montiel,MP,Virdi,KS,de Paula,WB,Widhalm,JR,Basset,GJ,Davila,JI,Elthon,TE,Elowsky,CG,Sato,SJ,Clemente,TE and Mackenzie,SA (2011)。 MutS HOMOLOG1是一种核型蛋白,可以改变线粒体和质体属性和植物对高光的反应。植物细胞23:3428-3441。
  5. Yoo,SD,Cho,YH and Sheen,J.(2007)。  拟南芥叶肉原生质体:用于瞬时基因表达分析的多功能细胞系统。 Nat Protoc 2(7):1565-1572。 >
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引用:Elowsky, C., Wamboldt, Y. and Mackenzie, S. (2017). Laser Scanning Confocal Microcopy for Arabidopsis Epidermal, Mesophyll, and Vascular Parenchyma Cells. Bio-protocol 7(5): e2150. DOI: 10.21769/BioProtoc.2150.
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