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A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta
活体成像的热激诱导系统测定植株的Aux/IAA降解率   

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参见作者原研究论文

本实验方案简略版
Plant Physiology
Sep 2015

Abstract

An emerging theme in biology is the importance of cellular signaling dynamics. In addition to monitoring changes in absolute abundance of signaling molecules, many signal transduction pathways are sensitive to changes in temporal properties of signaling components (Purvis and Lahav, 2013). The phytohormone auxin regulates myriad processes in plant development. Many of these require the nuclear auxin signaling pathway, in which degradation of the Aux/IAA repressor proteins allows for transcription of auxin-responsive genes (Korasick et al., 2015). Using a heterologous yeast system, we found that Aux/IAAs exhibit a range of auxin-induced degradation rates when co-expressed in isolation with F-box proteins (Havens et al., 2012). Subsequent studies connecting signaling dynamics to plant growth and development confirmed that Aux/IAAs show similar differences in plants (Guseman et al., 2015; Moss et al., 2015). Here, we describe in detail the use of a heat-shock-inducible fluorescence degradation system to capture Aux/IAA degradation in real time in live plant roots. By employing this method, we were able to obtain high Aux/IAA expression and avoid the dampening long term effects of turnover, feedback and silencing. Degradation was dependent on the presence of an Aux/IAA degron and rates increased in response to exogenous auxin.

Keywords: Auxin (生长素), Fluorescence (荧光), Degradation (降解), Root (根), Aux/IAA (Aux/IAA)

Materials and Reagents

  1. 1,000 μl pipet tip (Thermo Fisher Scientific, FisherbrandTM, catalog number: 02-681-4 ) 

  2. 50 ml conical centrifuge tube (Thermo Fisher Scientific, FisherbrandTM, catalog number: 06-443-20 ) 

  3. 1.5 ml microcentrifuge tubes (Thermo Fisher Scientific, FisherbrandTM, catalog number: 05-408-129 ) 

  4. Square (100 x 100 x 15 mm) Petri plates (Thermo Fisher Scientific, FisherbrandTM, catalog number: 08-757-11A )
  5. Round (35 mm) Petri plates (VWR International, catalog number: 10799-192 ) 

  6. Micropore tape 1530-0 (3M, catalog number: 70200412230 ) 

  7. Aluminum foil
  8. Razor blades
  9. Rectangular (24 x 50 mm) microscopy coverslips (Thermo Fisher Scientific, catalog number: 12-544E )
  10. Labeling tape 

  11. > 4 ml glass perfume spray bottles (available in craft stores) 

  12. Arabidopsis thaliana Columbia-0 (Col) seeds transformed with heat-shock constructs (Guseman et al., 2015; Moss et al., 2015)

  13. Bacto Agar (BD, catalog number: 214010 )
  14. Linsmaier-Skoog Media (Caisson Laboratories, catalog number: LSP03-1LT ) (see Note 1)
  15. Triton X-100 (GE Healthcare, catalog number: US22686 ) 

  16. Indole-3-acetic acid (IAA) (bioWORLD, catalog number: 705490 )
  17. Kanamycin (Thermo Fisher Scientific, catalog number: BP906-5 )
  18. 95% ethanol
  19. Seed sterilization solution (see Recipes)
  20. 0.5x Linsmaier-Skoog (LS) liquid media (see Recipes)
  21. Sterile plating agar solution (see Recipes)
  22. 0.5x LS media + 0.8% agar (see Recipes)
  23. Cover slip media (0.5x LS Media + 1.2% agar) (see Recipes)
  24. IAA stock solution (5 mM) (see Recipes)

Equipment

  1. Slide warmer (Thermo Fisher Scientific, model: 11-474-470 )
  2. Microscope (Leica Biosystems, model: DMI 3000B ) fitted with a Lumencor SOLA light source and YFP filter cube
  3. Leica long working 40x HCX PL FLUOTAR objective (see Note 2)
  4. Leica camera (Leica Microsystems, model: DFC345 FX )

  5. Forceps (Electron Microscopy Sciences, model: Dumont Tweezers Style 2A )
  6. Timer

Software

  1. Leica LAS AF version 2.6.0 for image acquisition
  2. Fiji/ImageJ software for image analysis 

  3. GraphPad Prism6 software package for graphing and statistical analysis

Procedure

Note: Expression levels of Aux/IAA proteins are typically very low, so we have modified an existing heat-shock-inducible promoter system (Gray et al., 2001) to generate high levels of a fluorescently-tagged Aux/IAA for live cell imaging in root tissues prior to hormone treatment.

  1. Generation of transgenic Arabidopsis plants
    1. Heat-shock inducible VENUS-tagged Aux/IAA constructs (HS::VENUS-IAA-NLS) were cloned as previously described (Guseman et al., 2015; Moss et al., 2015) and based on pGREEN vectors containing the soybean heat shock promoter HS6871 (Gray et al., 2001).
    2. Arabidopsis plants were transformed with these constructs using the floral dip method (Clough and Bent, 1998). The plants were then selected for two generations on 0.5x LS agar plates containing 50 mg/ml kanamycin, to obtain T2 lines. For the experiments described below, 2-3 independent T2 lines for each construct should be used (see Notes 3-5).

  2. Plating and growing seedlings
    Note: steps B1-4 below should be carried out in aseptic conditions in a sterile hood.
    1. Pour 0.5x LS + 0.8% agar into square Petri plates in a sterile hood. Pour 1 plate for each independent plant line per experiment. 

    2. Aliquot ~30 seeds into 1.5 ml tubes and add 300 μl of seed sterilization solution. Set tubes on their sides to ensure all seeds are in contact with solution. Let sit for 15 min and then remove sterilization solution and replace with 95% ethanol. Invert several times to wash. Completely remove 95% ethanol using a pipette and replace with sterile plating agar solution. Be sure to also plate non-fluorescent siblings (or other non-fluorescent line) to use as controls.
    3. Use a 1,000 μl pipette tip to gently aspirate seeds and transfer onto 0.5x LS plates. For each independent line, plate into 2 rows of 12 plants per plate (Figure 1A). Seal plates with micropore tape or Parafilm. 

    4. Stratify seedlings vertically at 4 °C in the dark (cover with aluminum foil) for 2 days. 

    5. Grow vertically in a growth chamber set under 16L:8D light conditions and 20 °C for 7 days.


  3. Heat shock induction

    1. After 7 days of growth, place plates horizontally on a slide warmer set at 37 °C for 2 h to induce expression of the VENUS-IAA protein (Figures 1B and 2A).


      Figure 1. Experimental set-up and analysis for heat-shock degradation assay. A. 7-day old seedlings sown on square, vertically-grown 0.5x LS + 0.8% agar plates. B. Seedling plate undergoing heat shock on a slide warmer. C. Cover slip wrapped with lab tape to fit in a slide holder. D. Disc cut from coverslip media agar using a small Petri dish. E. Trimming edges off of disc so that it will fit evenly on the coverslip. F. Perfume bottle containing 0.5x LS media with ethanol or 5 μM IAA. G. Agar pad with seedlings arranged on top. H. Cover slip placed on top of agar pad and flipped over. I. Coverslip placed in the microscope slide holder. J. Close-up image of agar-plant-coverslip in slide holder.

  4. Auxin-induced VENUS-Aux/IAA degradation assay
    1. Prepare 0.5x LS Media + 1.2% agar coverslip media. This agar concentration is critical because it is rigid enough to handle, but low enough concentration to reduce background autofluorescence during fluorescence imaging. 

    2. Pour 4 plates in a sterile hood, adding IAA to 2 plates and ethanol (mock) to 2 plates. To do this, pour 50 ml of media into a sterile 50 ml tube, add 50 μl of 5 mM IAA or 95% ethanol. Mix by gentle inversions and pour into two square plates. 

    3. Prepare a rectangular 24 x 50 mm coverslip to fit within a microscope slide holder. Wrap lab tape
around each end ~3 times in order to add length so that the coverslip will fit snugly in the holder (Figure 1C). These thin coverslips break easily, so be sure to handle gently. 

    4. When coverslip media plates are solid, use a 35 mm round Petri dish to cut circles into the agar (Figure 1D). Use a razor blade to further cut the circle so that the agar slice will fit over the coverslip and plants (Figure 1E). While cutting a circle with the small Petri dish is not a necessity, we found the agar slices cut this way were easier to remove from the plate. Alternatively, use a razor blade to cut your slice and carefully lift it from the plate.
    5. Prepare 5 μM IAA or mock treatments to be sprayed onto seedlings. Pipet 4 ml liquid 0.5x LS
into two clean 5 ml perfume bottles (Figure 1F). Add 4 μl of either 95% ethanol (mock) or 5 mM IAA stock solution into the perfume bottle. Keep perfume bottles clean by washing with ethanol, and then rinsing with water and LS. Save separate, labeled bottles for mock and IAA solutions.
    6. Using forceps, transfer 5-6 seedlings from the plate warmer to a separate agar plate (use an extra non-supplemented plant-growth plate). Spray with liquid 0.5x LS containing either 5 μM IAA or vehicle (95% ethanol). Use enough liquid to fully cover the root tips. 

    7. Using forceps, quickly arrange these seedlings onto an agar block (prepared in step D4) containing appropriate
treatment (mock or 5 μM IAA) (Figure 1G). Place a coverslip over the agar block sandwiching the seedlings (Figure 1H). Be careful to avoid bubbles near the root tips. Flip the agar-plant-coverslip over and place in slide holder to image (Figure 1I-J).

    8. Image plant root tips at 0, 10, 20, 30, 40, and 60 min post-treatment (Figure 2B). It is important to minimize time between sample prep and imaging, as well as to maintain consistency between samples. We used a Leica DMI 3000B microscope fitted with a Leica long working 40x HCX PL FLUOTAR objective (see Note 2), illuminated with a Lumencor SOLA light source, and YFP filter cube. Images were captured using Leica LAS AF version 2.6.0 software and a Leica DFC345 FX camera. It is important to image all plants with the same intensity and gain settings. We used a 1 sec exposure and 3.6 gain. Based on the microscope system and particular fluorophore, it may be necessary for each lab to determine ideal exposure and gain settings. It is important to note that photobleaching may occur. In our experiments, we used controls lines with VENUS alone (i.e., with no fused IAA protein) and also mock-treated VENUS-IAA lines to test and document the extent of photobleaching that occurs over the imaging time course (Guseman et al., 2015; Moss et al., 2015).


      Figure 2. Schematic of auxin-induced VENUS-Aux/IAA degradation (A) and representative fluorescent root tip images following auxin or mock treatment (B)

  5. Fluorescence quantification and analysis
    1. Using Fiji software, open image files making sure that “autoscale” is not checked at the bottom left of the initial pop-up window. Images will all
initially appear black. The images for individual plants should be arranged into a stack of time points (Figure 3A-B). Set all images to the same brightness/contrast by using the Image > Adjust > Brightness/Contrast function for a single image, and then check the “Propagate to all other open images” box. We used a minimum and maximum of 500 and 2,200, respectively (Figure 3C). 

    2. Using the box tool, create a box the size of the root tip region of interest (Figure 3D). Rotate the box to fit by using the Edit > Selection > Rotate function, and then move the box over the region of interest (Figure 3D). Measure pixel intensity by pressing the “M” button. Be sure that “Mean gray value” is checked under Analyze > Set Measurements. Save the region of interest box by opening Analyze > Tools > ROI Manager, and repeat measurement with this identical box on all other images in the stack. For the next stack, you may need to rotate the box to fit the new root angle, and you can save this new angled box in the ROI manager as well.


      Figure 3. Analysis of heat-shock degradation assay using Fiji software. A. Image file stacks representing each individual plant from a single experiment. B. Each stack contains 6 images representing each of the time points. C. Dialog box showing Brightness/Contrast settings in Fiji. D. ROI box drawn to the width and length of the lateral root image, ROI box rotated to fit over fluorescent portion of root image, and rotated ROI box positioned of the root tip area to be measured.

    3. Use non-fluorescent siblings (or other non-fluorescent line) to calculate background levels.
    4. Copy pixel intensities from the Measurements box and paste into a spreadsheet file. Label each
line, treatment, and time point. This is the raw data. 

    5. For each line, compute the average of the background fluorescence from all non-fluorescent plants [there will likely be 2-3 (see Note 3)] at each time point. In a new column, subtract this background fluorescence value from the time point matched fluorescence value of each experimental plant (i.e., the averaged background fluorescence for time point T0 will be subtracted from all T0 values for fluorescent plants). Individual plants with T0 values lower than 350 AU were found to be within the range of noise and were removed from the dataset. Plotting the averaged background-subtracted data for each line and replicate (Figure 4A, left panel) illustrates the variability in basal fluorescence observed between lines and replicates.
    6. Next, normalize the fluorescence of each time point within each stack/plant by dividing by the initial fluorescence value (i.e., fold-initial normalization). Plotting the averaged normalized data for each line and replicate enables better comparison of lines and replicates (Figure 4A, right). A small number of lines behaved significantly differently from others with the same genotype (e.g., fluorescence was much higher and did not respond to auxin treatment), likely due to position effect or faulty integration of the T-DNA during transformation. These lines were removed from the analysis.
    7. Finally, plot the fold-initial fluorescence values averaged for all individuals from all lines and replicates. Example graphs (Figure 4B) show differences in auxin-induced degradation dynamics between VENUS-IAA1 and VENUS-IAA28.


      Figure 4. Representative quantification of VENUS-Aux/IAA degradation following auxin or mock treatment. A. Background-subtracted fluorescence measurements for three independent VENUS-IAA1 lines. The data is not normalized in the left panel, and is fold-initial normalized in the right panel. Each data point represents mean fluorescence from 5-6 plants carried out over replicate experiments with error bars representing standard error of the mean. B. Fold-initial normalized data for IAA1-VENUS and IAA28- VENUS, including multiple plants from at least two replicates from 2-3 independent lines. Error bars represent standard error of the mean.

Notes

  1. We used LS media for all of our experiments. We have not tried these experiments with MS media; however, we assume it is likely that MS can be used in place of LS.
  2. The microscope we used is an inverted scope with a 40x long working distance (LWD) objective. The LWD objective is not required, but a 40x objective will work best. Also, if the scope used is not inverted, the agar-plant-coverslip may be placed upright on a slide for imaging.
  3. We used segregating T2 HS::IAA-VENUS lines that included plants both hemi- and homozygous for the T-DNA, as well as non-fluorescing plants. Consistent with this mixed population of genotypes, we did observe some variability in absolute fluorescence intensity in our raw data. Fold-initial normalization corrected for this variability and revealed the same degradation trend within each line. We used the non-fluorescent siblings to calculate background fluorescence. If you would like to use homozygous T3 lines, you will also need to grow non-fluorescent plants in parallel and use 2-3 per experiment for background fluorescence measurements.
  4. To further confirm the genotype of the transgenic plants, one can isolate genomic DNA from each seedling (A small leaf or two from 2-3 week old seedlings should suffice.) and perform a genotyping PCR using primers to amplify the transgene. For example, we used a forward primer binding to the HS promoter region, and a reverse primer binding to the specific Aux/IAA sequence.
  5. The number of plants to test for each line will depend on the variability you see within the line. Some lines are far more variable. In general, we treated 5-6 plants per line per experiment and repeated the experiment at least twice.

Recipes

  1. Seed sterilization solution
    35 ml of 100% ethanol

    25 μl of 100% Triton X-100

    Fill to 50 ml with sterile water
    Invert several times to mix
  2. 0.5x Linsmaier-Skoog (LS) liquid media 

    Add 1,800 ml water to a beaker with stir bar. While stirring, add 1 LS packet to water. Once dissolved, bring volume up to 2 L with water. Pour 500 ml each into a separate 1 L glass bottle and autoclave (121 °C) for 20 min.

    Immediately use the remaining solution to prepare agar media (see below).
  3. Sterile plating agar solution
    0.1% Bacto-agar in sterile water
    Autoclave (121 °C) for 20 min 

  4. 0.5x LS media + 0.8% agar 

    From the remaining media above, pour 500 ml into each of two separate 1 L glass bottles. Add 4 g agar to each bottle (= 0.8% agar) and autoclave (121 °C) for 20 min.
  5. Cover slip media (0.5x LS Media + 1.2% agar) 

    Pour the remaining 500 ml of 0.5x LS liquid media into a 1 L glass bottle. Add 6 g of agar (=1.2% agar) and autoclave (121 °C) for 20 min. 

  6. IAA stock solution (5 mM) 

    First make a 100 mM stock solution:

    8.76 mg indole-3-acetic acid (IAA, MW: 175.18)
    500 μl 95% ethanol 

    Dilute 100 mM stock with 95% ethanol to make a 5 mM solution. These stocks should be stored in the dark at -20 °C. 


Acknowledgments

This protocol was adapted from Gray et al. (2001), and it was performed by Guseman et al. (2015) and Moss et al. (2015). This work was supported by the Paul G. Allen Family Foundation (J. L. N.), National Science Foundation (MCB-1411949 to J. L. N.) and National Institute of Health (R01-GM107084 to J. L. N.). B. L. M. received fellowship support from the National Cancer Institute of the National Institutes of Health (F32CA180514). J. M. G. was supported by the Developmental Biology Predoctoral Training Grant [T32HD007183] from the National Institute of Child Health and Human Development (NICHD). The authors thank Jodi LS Lilley for careful reading of this manuscript.

References

  1. Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16(6): 735-743.
  2. Gray, W. M., Kepinski, S., Rouse, D., Leyser, O. and Estelle, M. (2001). Auxin regulates SCF(TIR1)-dependent degradation of AUX/IAA proteins. Nature 414(6861): 271-276.
  3. Guseman, J. M., Hellmuth, A., Lanctot, A., Feldman, T. P., Moss, B. L., Klavins, E., Calderon Villalobos, L. I. and Nemhauser, J. L. (2015). Auxin-induced degradation dynamics set the pace for lateral root development. Development 142(5): 905-909.
  4. Havens, K. A., Guseman, J. M., Jang, S. S., Pierre-Jerome, E., Bolten, N., Klavins, E. and Nemhauser, J. L. (2012). A synthetic approach reveals extensive tunability of auxin signaling. Plant Physiol 160(1): 135-142.
  5. Korasick, D. A., Jez, J. M. and Strader, L. C. (2015). Refining the nuclear auxin response pathway through structural biology. Curr Opin Plant Biol 27: 22-28.
  6. Moss, B. L., Mao, H., Guseman, J. M., Hinds, T. R., Hellmuth, A., Kovenock, M., Noorassa, A., Lanctot, A., Villalobos, L. I., Zheng, N. and Nemhauser, J. L. (2015). Rate motifs tune auxin/indole-3-acetic acid degradation dynamics. Plant Physiol 169(1): 803-813.
  7. Purvis, J. E. and Lahav, G. (2013). Encoding and decoding cellular information through signaling dynamics. Cell 152(5): 945-956.

简介

生物学中一个新兴的主题是细胞信号传导动力学的重要性。除了监测信号分子的绝对丰度的变化,许多信号转导途径对信号传导组分的时间性质的变化敏感(Purvis和Lahav,2013)。植物激素生长素调节植物发育中的无数过程。这些中的许多需要核生长素信号通路,其中Aux/IAA阻抑蛋白的降解允许生长素应答基因的转录(Korasick等人,2015)。使用异源酵母系统,我们发现当与F-box蛋白分离共表达时,Aux/IAAs表现出一系列生长素诱导的降解速率(Havens等人,2012)。随后的连接信号动力学与植物生长和发育的研究证实Aux/IAAs在植物中显示相似的差异(Guseman等人,2015; Moss等人,2015)。在这里,我们详细描述热休克诱导荧光降解系统捕获Aux/IAA实时在活植物根中的降解的使用。通过采用这种方法,我们能够获得高的Aux/IAA表达,并避免抑制周转,反馈和沉默的长期影响。降解取决于Aux/IAA降解的存在,并且对外源生长素的反应速率增加。

关键字:生长素, 荧光, 降解, 根, Aux/IAA

材料和试剂

  1. 1000μl移液管吸头(Thermo Fisher Scientific,Fisherbrand TM ,目录号:02-681-4)
  2. 50ml的锥形离心管(Thermo Fisher Scientific,Fisherbrand ,目录号:06-443-20)
  3. 1.5ml微量离心管(Thermo Fisher Scientific,Fisherbrand TM ,目录号:05-408-129)
  4. 方形(100×100×15mm)培养皿(Thermo Fisher Scientific,Fisherbrand TM,目录号:08-757-11A)
  5. 圆形(35mm)培养皿(VWR International,目录号:10799-192)
  6. 微孔胶带1530-0(3M,目录号:70200412230)
  7. 铝箔
  8. 剃刀刀片
  9. 矩形(24×50mm)显微镜盖玻片(Thermo Fisher Scientific,目录号:12-544E)
  10. 贴上标签
  11. > 4毫升玻璃香水喷雾瓶(可在工艺品店购买)
  12. 用热休克构建体转化的拟南芥 Columbia-0(Col)种子(Guseman等人,2015; Moss等人,2015年, )
  13. Bacto琼脂(BD,目录号:214010)
  14. Linsmaier-Skoog Media(Caisson Laboratories,目录号:LSP03-1LT)(见注1)
  15. Triton X-100(GE Healthcare,目录号:US22686)
  16. 吲哚-3-乙酸(IAA)(bioWORLD,目录号:705490)
  17. 卡那霉素(Thermo Fisher Scientific,目录号:BP906-5)
  18. 95%乙醇
  19. 种子灭菌溶液(见配方)
  20. 0.5x Linsmaier-Skoog(LS)液体培养基(参见配方)
  21. 无菌电镀琼脂溶液(见配方)
  22. 0.5x LS培养基+ 0.8%琼脂(参见配方)
  23. 盖玻片介质(0.5x LS培养基+ 1.2%琼脂)(参见配方)
  24. IAA储备溶液(5mM)(参见配方)

设备

  1. 载玻片加热器(Thermo Fisher Scientific,型号:11-474-470)
  2. 配有Lumencor SOLA光源和YFP过滤器立方体的显微镜(Leica Biosystems,型号:DMI 3000B)
  3. Leica长工作40x HCX PL FLUOTAR物镜(见注2)
  4. Leica相机(Leica Microsystems,型号:DFC345 FX)
  5. 钳(电子显微镜科学,型号:Dumont Tweezers Style 2A)
  6. 定时器

软件

  1. Leica LAS AF版本2.6.0用于图像采集
  2. 用于图像分析的Fiji/ImageJ软件
  3. GraphPad Prism6软件包用于绘图和统计分析

程序

注意:Aux/IAA蛋白的表达水平通常非常低,因此我们已经修饰了现有的热休克诱导启动子系统(Gray等人,2001)以产生高水平的荧光标记的Aux/IAA用于在激素治疗之前的根组织中的活细胞成像

  1. 产生转基因拟南芥植物
    1. 如前所述克隆热激诱导的VENUS标记的Aux/IAA构建体(HS :: VENUS-IAA-NLS)(Guseman等人,2015; Moss等人,2015),并基于含有大豆热休克启动子HS6871的pGREEN载体(Gray等人,2001)。
    2. 使用花浸渍法(Clough和Bent,1998)用这些构建体转化拟南芥植物。然后将植物在含有50mg/ml卡那霉素的0.5x LS琼脂平板上选择两代,以获得T2系。对于下述实验,应使用每个构建体2-3个独立的T2系(见注释3-5)
  2. 电镀和生长苗
    注意:以下步骤B1-4应在无菌条件下在无菌罩中进行。
    1. 在无菌罩中将0.5x LS + 0.8%琼脂倒入方形培养皿中。每个实验每个独立植物系列倒1板。
    2. 等分约30种子到1.5ml管中,加入300μl种子灭菌溶液。在其侧面设置管,以确保所有种子都与溶液接触。让静置15分钟,然后取出灭菌溶液,并更换为95%乙醇。反转几次洗。使用移液管完全删除95%乙醇,并更换无菌电镀琼脂溶液。一定要将非荧光兄弟姐妹(或其他非荧光线)作为对照
    3. 使用1000微升移液器吸头轻轻地吸取种子,并转移到0.5x LS板。对于每个独立的系,将板平板成每板12个植物的2排(图1A)。用微孔带或石蜡膜密封板。
    4. 在4℃在黑暗中(用铝箔覆盖)垂直地将幼苗分层2天。
    5. 在设置在16L:8D光照条件下和20℃下的生长室中垂直生长7天。

  3. 热冲击感应
    1. 生长7天后,将板水平放置在37℃的载玻片加热器上2小时以诱导VENUS-IAA蛋白的表达(图1B和2A)。


      图1.热休克降解测定的实验设置和分析 A.将7天龄的幼苗播种在正方形的垂直生长的0.5x LS + 0.8%琼脂平板上。 B.苗木板在载玻片加热器上经受热休克。 C.用实验室胶带包裹的盖玻片以适合载玻片架。 D.使用小培养皿从盖玻片培养基琼脂上切下圆盘。 E.修剪光盘边缘,使其均匀地贴合在盖玻片上。 F.含有0.5x LS培养基与乙醇或5μMIAA的香水瓶。 G.琼脂垫与幼苗排列在上面。 H.将盖片置于琼脂垫顶部并翻转。 I.将盖片放置在显微镜载片架中。 J.载玻片架中琼脂植物盖玻片的特写图像
  4. 生长素诱导的VENUS-Aux/IAA降解测定
    1. 制备0.5x LS培养基+ 1.2%琼脂盖玻片培养基。这种琼脂浓度是关键的,因为它是刚性足以处理,但足够低的浓度,以减少荧光成像期间背景自身荧光。
    2. 在无菌罩中倒入4个板,将IAA加入2个板中,并将乙醇(模拟)加入2个板中。为此,将50ml培养基倒入无菌的50ml管中,加入50μl5mM IAA或95%乙醇。通过温和倒置混合并倒入两个正方形板中。
    3. 准备一个矩形24 x 50毫米盖玻片适合显微镜载玻片支架。每端缠绕实验室胶带约3次,以增加长度,以便盖玻片将紧贴在支架(图1C)。这些薄盖玻片容易破碎,所以一定要轻轻处理。
    4. 当盖玻片培养基板是固体时,使用35mm圆形培养皿将圆切成琼脂(图1D)。使用剃刀刀片进一步切割圆,使琼脂切片适合盖玻片和植物(图1E)。虽然用小培养皿切割圆不是必要的,但我们发现这种方式切割的琼脂切片更容易从板上去除。或者,使用剃刀刀片切割切片,并小心地将其从板上提起
    5. 准备5微米IAA或模拟处理,以喷洒到幼苗。吸管4毫升液体0.5x LSinto两个干净的5毫升香水瓶(图1F)。添加4微升的95%乙醇(模拟)或5毫米IAA储备液到香水瓶。保持香水瓶清洁,用乙醇洗涤,然后用水和LS冲洗。保存单独的,标记瓶的模拟和IAA解决方案。
    6. 使用镊子,将5-6个幼苗从板式加热器转移到单独的琼脂板(使用额外的非补充植物生长板)。用含有5μMIAA或载体(95%乙醇)的液体0.5x LS喷雾。使用足够的液体以完全覆盖根尖。
    7. 使用镊子,迅速安排这些幼苗到琼脂块(在步骤D4中制备)包含适当处理(模拟或5μMIAA)(图1G)。将盖玻片放在夹住幼苗的琼脂块上(图1H)。小心避免根尖附近的气泡。将琼脂 - 植物 - 盖玻片翻转并放置在载玻片保持器中以进行成像(图11I-J)。
    8. 在处理后0,10,20,30,40和60分钟的图像植物根尖(图2B)。重要的是尽量减少样品制备和成像之间的时间,以及保持样品之间的一致性。我们使用装有Leica longworking 40x HCX PL FLUOTAR物镜(见注2)的Leica DMI 3000B显微镜,用Lumencor SOLA光源和YFP滤光立方体照明。使用Leica LAS AF版本2.6.0软件和Leica DFC345 FX照相机捕获图像。重要的是对具有相同强度和增益设置的所有植物成像。我们使用1秒曝光和3.6增益。基于显微镜系统和特定荧光团,每个实验室可能需要确定理想的曝光和增益设置。重要的是注意可能发生光漂白。在我们的实验中,我们使用单独用VENUS(没有融合的IAA蛋白)的对照线以及模拟处理的VENUS-IAA细胞系来测试和记录在成像上发生的光漂白的程度时间过程(Guseman等人,2015; Moss等人,2015)。


      <图>图2.生长素诱导的VENUS-Aux/IAA降解(A)和生长素或模拟处理后的代表性荧光根尖图像(B)的示意图

  5. 荧光定量和分析
    1. 使用Fiji软件,打开图像文件,确保未在初始弹出窗口的左下角选中"自动缩放"。图像将初始显示为黑色。单个植物的图像应该排列成一叠时间点(图3A-B)。使用图像>将所有图像设置为相同的亮度/对比度。调整>亮度/对比度功能,然后检查"传播到所有其他打开的图像"框。我们分别使用最小和最大值500和2,200(图3C)。
    2. 使用框工具,创建感兴趣的根尖区域大小的框(图3D)。使用编辑>旋转框以适应。选择>旋转功能,然后将框移动到感兴趣的区域上(图3D)。按"M"按钮测量像素亮度。确保在分析>下选中"平均灰度值"。设置测量。通过打开Analyze>保存感兴趣区域框工具> ROI管理器,并使用此相同的框在堆叠中的所有其他图像上重复测量。对于下一个堆叠,您可能需要旋转框以适应新的根角,并且您可以将这个新的有角框保存在ROI管理器中。


      图3.使用斐济软件进行的热休克降解测定分析。A.来自单个实验的代表每个植物的图像文件堆栈。 B.每个堆栈包含表示每个时间点的6个图像。 C.对话框显示斐济的亮度/对比度设置。 D. ROI框绘制为横向根图像的宽度和长度,ROI框旋转以适合根图像的荧光部分,并旋转ROI框定位待测量的根尖区域。

    3. 使用非荧光兄弟姐妹(或其他非荧光线)来计算背景水平。
    4. 从"测量"框中复制像素亮度,并粘贴到电子表格文件中。标记每行,处理和时间点。这是原始数据。
    5. 对于每一行,在每个时间点计算来自所有非荧光植物的背景荧光的平均值(可能为2-3(参见注3))。在新柱中,从每个实验植物的时间点匹配荧光值中减去该背景荧光值(即,对于荧光植物,从时间点T0的平均背景荧光将从所有T0值中减去) )。发现T0值低于350AU的个体植物在噪声范围内,并从数据集中移除。绘制每条线和复制品的平均背景扣除数据(图4A,左图)说明在品系和重复品之间观察到的基础荧光的变异性。
    6. 接下来,通过除以初始荧光值(即,初始归一化倍数)来归一化每个堆叠/植物内的每个时间点的荧光。绘制每条线和重复的平均归一化数据使得能够更好地比较线和重复(图4A,右)。少数品系与具有相同基因型的其他品系的表现显着不同(例如,,荧光高得多,并且对生长素处理没有反应),可能是由于位置效应或T- DNA。这些行从分析中删除。
    7. 最后,绘制来自所有线和重复的所有个体的平均的折叠初始荧光值。实施例图(图4B)显示了VENUS-IAA1和VENUS-IAA28之间生长素诱导的降解动力学的差异。


      图4.在生长素或模拟处理后VENUS-Aux/IAA降解的代表性定量。A.三个独立的VENUS-IAA1品系的背景扣除荧光测量。数据未在左图中标准化,并且在右图中进行倍数初始归一化。每个数据点表示通过重复实验进行的5-6株植物的平均荧光,误差条表示平均值的标准误差。 B. IAA1-VENUS和IAA28-VENUS的折叠初始标准化数据,包括来自2-3个独立系的至少两个重复的多个植物。误差线表示平均值的标准误差。

笔记

  1. 我们使用LS培养基进行我们的所有实验。我们还没有尝试这些实验与MS媒体;然而,我们假设可能MS可以用来代替LS。
  2. 我们使用的显微镜??是一个40x长工作距离(LWD)物镜的倒置镜。 LWD目标不是必需的,但是40x的目标将工作最好。此外,如果使用的范围不是倒置的,琼脂 - 植物 - 盖玻片可以直立地放置在载玻片上用于成像。
  3. 我们使用分离的T2HS :: IAA-VENUS品系,其包括对于T-DNA是半和纯合的植物以及非发荧光的植物。与这种混合的基因型群体一致,我们确实观察到在我们的原始数据中绝对荧光强度的一些变异性。折叠初始归一化校正了这种变异性,并揭示了每行内同样的退化趋势。我们使用非荧光兄弟姐妹来计算背景荧光。如果您想使用纯合T3系,您还需要平行培养非荧光植物,每次实验使用2-3个背景荧光测量。
  4. 为了进一步确认转基因植物的基因型,可以从每个幼苗分离基因组DNA(来自2-3周龄幼苗的小叶或两个应足够),并使用引物扩增转基因进行基因分型PCR。例如,我们使用结合HS启动子区的正向引物和结合特异性Aux/IAA序列的反向引物。
  5. 要测试每条线的植物数量将取决于您在线内看到的变化。有些行变化更大。一般来说,我们每个实验处理5-6株植物/株,并重复实验至少两次。

食谱

  1. 种子灭菌溶液
    35ml的100%乙醇 25μl100%Triton X-100 用无菌水填充至50ml 反转几次混合
  2. 0.5x Linsmaier-Skoog(LS)液体培养基
    向带搅拌棒的烧杯中加入1,800ml水。在搅拌的同时,向水中加入1L LS包。一旦溶解,用水带来体积高达2升。将500ml每个倒入单独的1L玻璃瓶中并高压灭菌(121℃)20分钟。
    立即使用剩余的溶液制备琼脂培养基(见下文)。
  3. 无菌电镀琼脂溶液
    0.1%细菌琼脂在无菌水中的溶液 高压灭菌(121℃)20分钟
  4. 0.5x LS培养基+ 0.8%琼脂
    从上面剩余的培养基中,将500ml倒入两个单独的1L玻璃瓶中的每一个中。向每个瓶(= 0.8%琼脂)和高压灭菌(121℃)中加入4g琼脂20分钟
  5. 盖玻片介质(0.5×LS培养基+ 1.2%琼脂)
    将剩余的500ml 0.5×LS液体培养基倒入1L玻璃瓶中。加入6g琼脂(= 1.2%琼脂)和高压灭菌(121℃)20分钟。
  6. IAA储备溶液(5mM) 首先制备100mM储备溶液:
    8.76mg吲哚-3-乙酸(IAA,MW:175.18) 500μl95%乙醇
    用95%乙醇稀释100mM储备液以制备5mM溶液。这些储备应储存在-20℃的黑暗中。

致谢

此协议改编自Gray等人 。 (2001),并且由Guseman等人进行。 (2015)和Moss等人。 (2015)。这项工作得到了Paul G. Allen家庭基金会(J.L.N.),国家科学基金会(MCB-1411949至J.L.N.)和国立卫生研究所(R01-GM107084至J.L.N.)的支持。 B. L. M.从国立卫生研究院的国家癌症研究所(F32CA180514)获得了奖学金支持。 J.M.G.得到国家儿童健康和人类发展研究所(NICHD)的发育生物学预发性训练补助金[T32HD007183]的支持。作者感谢Jodi LS Lilley仔细阅读这份手稿。

参考文献

  1. Clough,SJ和Bent,AF(1998)。  Floral dip:农杆菌介导的拟南芥转化的简化方法。 植物J16(6):735-743。
  2. Gray,WM,Kepinski,S.,Rouse,D.,Leyser,O.and Estelle,M。(2001)。  生长素调节AUX/IAA蛋白的SCF(TIR1)依赖性降解。

    414(6861):271-276 。
  3. Gasseman,JM,Hellmuth,A.,Lanctot,A.,Feldman,TP,Moss,BL,Klavins,E.,Calderon Villalobos,LI and Nemhauser,JL(2015)。  生长素诱导的降解动力学设定侧根发育的速度。发育 em> 142(5):905-909。
  4. Havens,KA,Guseman,JM,Jang,SS,Pierre-Jerome,E.,Bolten,N.,Klavins,E.and Nemhauser,JL(2012)。  合成方法揭示了生长素信号的广泛可调性。植物生理学 160(1 ):135-142。
  5. Korasick,DA,Jez,JM和Strader,LC(2015)。  通过结构生物学优化核生长素应答途径。 Curr Opin Plant Biol 27:22-28。
  6. Moss,BL,Mao,H.,Guseman,JM,Hinds,TR,Hellmuth,A.,Kovenock,M.,Noorassa,A.,Lanctot,A.,Villalobos,LI,Zheng,N.and Nemhauser,JL 2015)。  Rate motifs tune auxin/indole-3-乙酸降解动力学。植物生理学 169(1):803-813
  7. Purvis,JE和Lahav,G。(2013)。  通过信号动力学对细胞信息进行编码和解码。 152(5):945-956。
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Copyright: © 2016 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. Guseman, J. M., Nemhauser, J. L. and Moss, B. L. (2016). A Live-imaging, Heat Shock-inducible System to Measure Aux/IAA Degradation Rates in Planta. Bio-protocol 6(15): e1881. DOI: 10.21769/BioProtoc.1881.
  2. Moss, B. L., Mao, H., Guseman, J. M., Hinds, T. R., Hellmuth, A., Kovenock, M., Noorassa, A., Lanctot, A., Villalobos, L. I., Zheng, N. and Nemhauser, J. L. (2015). Rate motifs tune auxin/indole-3-acetic acid degradation dynamics. Plant Physiol 169(1): 803-813.
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