搜索

Real-time Analysis of Auxin Response, Cell Wall pH and Elongation in Arabidopsis thaliana Hypocotyls
拟南芥下胚轴植物生长素响应、细胞壁pH和伸长的实时分析   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
eLIFE
Sep 2016

Abstract

The rapid auxin-triggered growth of the Arabidopsis hypocotyls involves the nuclear TIR1/AFB-Aux/IAA signaling and is accompanied by acidification of the apoplast and cell walls (Fendrych et al., 2016). Here, we describe in detail the method for analysis of the elongation and the TIR1/AFB-Aux/IAA-dependent auxin response in hypocotyl segments as well as the determination of relative values of the cell wall pH.

Keywords: Auxin signaling (植物生长素信号), Cell wall pH (细胞壁pH), Cell elongation (细胞伸长), Hypocotyl (下胚轴), Live-cell imaging (活细胞成像)

Background

Phytohormone auxin induces rapid growth in Arabidopsis thaliana hypocotyls. This process requires the TIR1/AFB-Aux/IAA auxin co-receptor. Auxin promotes the binding of TIR1/AFB and Aux/IAA, which leads to ubiquitination and degradation of the latter, and results in transcription of auxin-responsive genes. This protocol focuses on measuring the growth, auxin signaling and cell wall acidification in Arabidopsis thaliana etiolated hypocotyls. This protocol is based on the previous work of Schenck et al., 2010, Takahashi et al., 2012, Fraas et al., 2014 and Spartz et al., 2014; but unlike the published work, we describe the procedures that enable measuring a larger spectrum of processes occurring during growth of hypocotyls; from the macroscopically visible organ elongation, cell wall pH monitored by confocal microscopy to the real-time nuclear auxin signaling visualized by luciferase bioluminescence.

Materials and Reagents

  1. Aluminum foil
  2. Razor blades (Gillette WilkinsonTM Sword)
  3. Cellophane foil 80 mm diameter (AA Packaging, catalog number: 325 P cellulose film )
  4. Black filter paper 90 mm diameter (MACHEREY-NAGEL, catalog number: 409009 )
  5. Falcon 60 x 15 mm dishes (Corning, catalog number: 353004 )
  6. 12-well tissue culture plates (TPP Techno Plastic Products, catalog number: 92412 )
  7. 2-well Lab-TekTM chambered #1.0 borosilicate cover glass (Thermo Fisher Scientific, catalog number: 155380 )
  8. Arabidopsis thaliana seeds: Col-0, apo-pHusion apoplastic pH marker line (Gjetting et al., 2012), auxin responsive promoter driving the expression of the firefly luciferase enzyme marker DR5::LUC (Moreno-Risueno et al., 2010)
  9. Household bleach (sodium hypochlorite 4.7%)
  10. 37% hydrochloric acid (Sigma-Aldrich, catalog number: 435570 )
  11. MES (Duchefa Biochemie, catalog number: M1503.0100 )
  12. Sucrose (Sigma-Aldrich, catalog number: 84097-1KG )
  13. Potassium hydroxide (KOH) (Merck, catalog number: 105021 )
  14. Agar, plant cell culture tested (Alfa Aesar, catalog number: H26724 )
  15. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541-500G )
  16. Phytagel (Sigma-Aldrich, catalog number: P8169 )
  17. 10 mM 3-Indoleacetic acid (IAA) (Sigma-Aldrich, catalog number: I2886-5G ) dissolved in ethanol
  18. 1 mM D-luciferin (Duchefa Biochemie, catalog number: L1349.0100 ) dissolved in 1x PBS
  19. Chlorine gas (see Recipe 1)
  20. Half-strength MS agar media (AM+) (see Recipe 2)
  21. Depletion medium (DM) (see Recipe 3)

Equipment

  1. Forceps Dumont #5
  2. Binocular dissecting microscope Leica EZ4 (Leica Microsystems, model: Leica EZ4 )
  3. Flatbed scanner Epson Perfection V370 Photo (Epson, model: V370 Photo )
  4. For the bioluminescence dark box:
    Lumazone Manual Stage Dark Box (Photometric, model: LMZ-DRK-BOX )
    Evolve EMCCD camera (Photometric, model: Evolve® 512, catalog number: EVO‐512‐M‐FW-16-AC-RP )
    17 mm fixed lens/0.95 (Edmund Optics, model: 59-832 )
    125 mm lens (Thorlabs, model: LA1384-A )
  5. Zeiss 700 LSM confocal microscope (ZEISS, model: LSM 700 ) with a 20x/0.8 Plan-Apochromat M27 objective

Software

  1. Microsoft Excel program (https://products.office.com/en/excel)
  2. Fiji program (http://fiji.sc/)
  3. MATLAB program (https://www.mathworks.com/products/matlab.html)
  4. AutoIt program (https://www.autoitscript.com/site/autoit/)

Procedure

  1. Hypocotyl elongation measurement
    1. Surface sterilize Col-0 seeds (or any other genotype of your interest) by chlorine gas (Recipe 1) overnight. Plate the sterilized seeds on the AM+ medium (Recipe 2). Stratify for two days at 4 °C in the dark, then place vertically under light to cultivate for around 6 h in a growth room at 21 °C. Wrap with aluminum foil and grow for another 66 h vertically at 21 °C.
    2. Prepare a depletion plate with 5 ml depletion medium (DM, Recipe 3) in a Falcon 60 x 15 mm dish. After solidification, place cellophane foil onto the surface. Damp the cellophane foil with liquid depletion medium solution.
    3. Place a dissecting microscope in a dark room and cover the illumination with a green filter made of 8 layers of green office foil (Figure 1).


      Figure 1. Binocular dissecting microscope with a green filter

    4. Uncover the Petri dishes with seedlings and select the seedlings with similar hypocotyl length excluding the longest and shortest ones. Decapitate the seedlings right below the apical hook and before the shoot-root junction to get a hypocotyl segment by cutting them on the surface of the agar using a very sharp razor blade. Prepare 6-8 segments for each treatment. Using sharp forceps, transfer the segments onto the cellophane foil in the depletion plate without squeezing them, the sample preparation procedure is depicted in Figure 2. Keep in darkness for 30-60 min.


      Figure 2. The sample preparation procedure

    5. Afterwards, transfer the segments by flipping the cellophane foil onto a treatment plate with the depletion medium supplemented with the desired treatment (Figure 2), in our case 10 µM 3-Indoleacetic acid (IAA) and the mock control (Ethanol equivalent).
    6. Immediately place the treatment plates on a flatbed scanner, imaging through the layer of the phytagel. A wet black filter paper is placed into the lid of the dish to improve the contrast of the image. Scan the samples in the 8-bit grayscale and at 2,400 dpi every 10 min automatically using the AutoIt program (see Supplemental file 1).

  2. Measuring the TIR1/AFB-Aux/IAA dependent response using the DR5::LUC marker line
    1. Prepare the 6-10 decapitalized segments of DR5::LUC marker line for each treatment, as described before, on depletion medium. Add around 50 µl of 1 mM D-luciferin dissolved in 1x PBS and immerse the segments entirely for 30 min.
    2. Prepare the treatment solution (DM with desired drugs)–in our case, DM + mock or DM + 10 µM IAA. Pour 3 ml medium into each well of a 12 wells tissue culture plate and let the medium solidify; four wells can be imaged simultaneously.
      Note: There is no D-luciferin in the treatment solution, the substrate originates from Step B1. This is sufficient for approx. 5-h imaging. Alternatively, one can put D-luciferin in the treatment solution for a longer time of imaging.
    3. Using the cellophane foil, transfer the segments onto the surface of the treatment medium and remove the foil.
    4. Immediately image in a dark box (Figure 3) with a Photometric Evolve® EMCCD camera equipped with a 17 mm fixed lens/0.95 and an additional 125 mm lens. Set the multiplier EMCCD gain to 150 and the exposure time to 110 sec, and image every 2 min.


      Figure 3. Dark box with a Photometric Evolve EMCCD camera

  3. Imaging the apoplastic pH using apo-pHusion apoplastic pH marker line
    1. Prepare the decapitated hypocotyl of the apo-pHusion apoplastic pH marker line as above.
    2. Prepare 5 ml of DM medium with or without 10 µM IAA.
    3. Transfer 5 hypocotyl segments onto the surface of the agar with the treatment. Cut out a piece of the agar with the segments using a spatula. Place the agar with the segments into the Lab-TekTM chambered cover glass so that the segments are placed between the cover glass and the agar. When using the 2-well chambered glass, a treatment and a control sample can be imaged simultaneously.
    4. Alternatively, the treatment can be very carefully pipetted to the hypocotyl segments during imaging. Then ~50 μl DM with the treatment can be used, but one must be extremely careful not to move the sample during imaging.
    5. Using a confocal microscope with a 20x/0.8 Plan-Apochromat M27 objective, set the position of each segment using the position manager so that the apical region of the hypocotyl segment is imaged. Image 5 z-sections, z-thickness matched to the pinhole size, of each hypocotyl segment.
    6. Set the microscope for simultaneous imaging of GFP and RFP by exciting using 488 and 555 nm diode lasers, and splitting the emitted light with a short pass 550 nm and long pass 560 nm filters, 16 bits per pixel. Image all positions every 5 min.

Data analysis

  1. Hypocotyl elongation image analysis
    1. To achieve unbiased measurement, we created a Fiji macro (see Supplemental file 1) for analyzing the length of the segment at each time point. The macro firstly creates the time lapse of the image sequence captured from the scanner, then allows you to manually create a rectangle ROI for each segment, followed by automatically thresholding each ROI and measuring the Feret’s diameter, the maximum caliper, as the length of the segment. The macro eventually generates ‘.txt’ file for each ROI or hypocotyl, including the Feret’s diameter of that hypocotyl in each time point.
    2. Copy and paste the result into Excel, set the initial length of the segment as 100%, and calculate the length of the hypocotyl at each time point to obtain a growth curve (Figure 4C). Besides, growth can be visualized by creating a montage-kymograph of individual hypocotyl segment in Fiji (Figure 4A).

  2. Analysis of the bioluminescence intensity
    1. Analyze the image sequence in Fiji (Schindelin et al., 2012). Manually outline all the segments via Polygon selection, at their brightest time frame, and add them into the region of interest (ROI) manager, followed by multi-measuring the mean grey value. This gives the average intensity of each segment at each time point.
    2. Copy and paste the result into Excel. Take the initial intensity of the segment as 100%, and analyze the average of the luminescence intensity of each hypocotyl at each time point, to get an intensity curve in time. Additionally, one can visualize the growth and the luminescence intensity by creating a montage-kymograph in Fiji (Figures 4B and 4D).


      Figure 4. Hypocotyl segment growth and DR5::LUC intensity measurements. A. The kymograph of the hypocotyl segment of Col-0 under mock treatment (upper row) and 10 μM IAA treatment (lower row) from 0 to 460 min; time interval of 10 min. The kymograph was done by making montage of the growing part of one representative sample in Fiji. (Note that to visualize the growth better, the upper half part of the hypocotyl where growth takes place was used for making montage). Vertical and horizontal scale bars represent 1 mm and 20 min, respectively. B. The kymograph of the luminescence intensity in the mock-treated DR5::LUC hypocotyls (upper row) and 10 μM IAA-treated (lower row) from 0 to 172 min; time interval of 2 min. The ‘FIRE’ look-up table was applied in Fiji. Scale bar is 10 min. C. Quantification of the growth of Col-0 hypocotyls treated with mock or 10 μM IAA from 0 to 460 min. The growth is expressed as the percentage of the original segment length. D. Quantification of the luminescence intensity in the DR5::LUC hypocotyls treated with mock or 10 μM IAA. The Fold change is the average intensity of the hypocotyls normalized by the intensity at timepoint 0.

  3. Image analysis of the cell wall pH
    1. Analyze the apoplastic pH using Fiji. We use the SUM projections of the z-stacks (Figure 5A). Set the threshold of the apoplast region using the RFP channel so that only the cell wall signal is selected. Create the selection using the ‘create selection’ command and measure intensity in GFP and RFP channels. Analyze the intensity ratios in Excel program. We analyze the apoplastic pH change in relative values the lower the GFP intensity, the lower the apoplastic pH is (Gjetting et al., 2012).
    2. Alternatively to Step C1, the apoplastic pH can be visualized and measured using the AreaKymo MATLAB® script, Figure 5B (Supplemental file 1). The AreaKymo script essentially does the same procedure as described in Step C1, but does so automatically without the user input, allowing for rapid processing of several hypocotyls at a time. The user first merges several hypocotyl SUM projection time series into one ‘tif.’ series using the Fiji program (‘combine stacks’ command) and converts them into 16-bit ‘tif.’ images (MATLAB does not handle well the 32-bit images that the SUM projection command creates). Then the user should find a threshold of the RFP channel that is optimal for selecting just the apoplast signal. In MATLAB, the AreaKymo script is run, the combined time series is selected, and the user specifies the value of the threshold for the RFP channel and the desired width of the rectangle that will represent the individual timeframe. The script outputs the visual representation of apoplastic pH and also the values in the form of a series of boxplots.


      Figure 5. Analysis of the apoplast pH. A. The apoplast pH after application of auxin to a hypocotyl expressing the apo-pHusion sensor. Auxin application is indicated with an arrowhead, GFP is shown in green while RFP in magenta. B. The output of the AreaKymo MATLAB® script. Time is progressing from top to bottom; ratio of the two fluorophores is shown using the ‘FIRE’ look-up-table.

Notes

During all steps where the hypocotyls are manipulated (cut and transferred to new plates) it is crucial to be extremely gentle with the tissue, not squeeze it but rather scoop it using sharp forceps. The tissue needs to be protected from drying; plates must be kept closed whenever possible to prevent excessive evaporation.

Recipes

  1. Chlorine gas sterilization
    100 ml household bleach
    4.5 ml 37% HCl
  2. Half-strength MS agar media (AM+)
    Half Murashige and Skoog Basal Salts
    1% sucrose
    Adjust pH to 5.8 by KOH
    0.8% agar, plant cell tested
    MiliQ water as solvent
  3. Depletion medium (DM)
    10 mM KCl
    1 mM MES
    Adjust pH to 6 by KOH
    1.5% phytagel
    MiliQ water as solvent
    Note: The phytagel brings better transparency than normal agar, contributing to the better quality of the scanning.

Acknowledgments

This protocol was adapted from Fendrych et al., 2016. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie Grant Agreement No. 665385, and Austrian Science Fund (FWF) [M 2128-B21]. The authors declare no conflict of interests.

References

  1. Fendrych, M., Leung, J. and Friml, J. (2016). TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls. Elife 5.
  2. Fraas, S., Niehoff, V., and Lüthen, H. (2014). A high-throughput imaging auxanometer for roots and hypocotyls of Arabidopsis using a 2D skeletonizing algorithm. Physiol Plant 151: 112-118.
  3. Gjetting, K. S., Ytting, C. K., Schulz, A. and Fuglsang, A. T. (2012). Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor. J Exp Bot 63(8): 3207-3218.
  4. Moreno-Risueno, M. A., Van Norman, J. M., Moreno, A., Zhang, J., Ahnert, S. E. and Benfey, P. N. (2010). Oscillating gene expression determines competence for periodic Arabidopsis root branching. Science 329(5997): 1306-1311.
  5. Schenck, D., Christian, M., Jones, A., and Lüthen, H. (2010). Rapid auxin-induced cell expansion and gene expression: a four-decade-old question revisited. Plant Physiol 152: 1183-5.
  6. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  7. Spartz, A.K., Ren, H., Park, M.Y., Grandt, K.N., Lee, S.H., Murphy, A.S., Sussman, M.R., Overvoorde, P.J., and Gray, W.M. (2014). SAUR inhibition of PP2C-D phosphatases activates plasma membrane H+-ATPases to promote cell expansion in Arabidopsis. Plant Cell 26: 2129-2142.
  8. Takahashi, K., Hayashi, K., and Kinoshita, T. (2012). Auxin activates the plasma membrane H+-ATPase by phosphorylation during hypocotyl elongation in Arabidopsis. Plant Physiol 159: 632-41.

简介

拟南芥下胚轴的快速生长素触发的生长涉及核TIR1 / AFB-Aux / IAA信号传导并伴随质外体和细胞壁的酸化(Fendrych 等,2016)。 在这里,我们详细描述了在下胚轴节段中的延伸和TIR1 / AFB-Aux / IAA依赖性生长素响应的分析方法以及细胞壁pH的相对值的确定。


【背景】植物激素生长素诱导拟南芥下胚轴的快速生长。 这个过程需要TIR1 / AFB-Aux / IAA生长素共同受体。 生长素促进TIR1 / AFB和Aux / IAA的结合,导致后者的泛素化和降解,并导致生长素应答基因的转录。 这个协议的重点是测量拟南芥下胚轴的生长,生长素信号和细胞壁酸化。 该协议是基于以前Schenck等人的工作,2010年,Takahashi等人,2012年,Fraas 等人,2014年 和Spartz 等。,2014; 但与已发表的工作不同,我们描述了能够测量下胚轴生长期间发生的更大范围过程的程序; 从肉眼可见的器官伸长,通过共焦显微镜监测的细胞壁pH到通过荧光素酶生物发光显现的实时核生长素信号传导。

关键字:植物生长素信号, 细胞壁pH, 细胞伸长, 下胚轴, 活细胞成像

材料和试剂

  1. 铝箔
  2. 剃刀刀片(吉列威尔金森TM刀)
  3. 玻璃纸箔直径80毫米(AA包装,目录号:325 P纤维素薄膜)
  4. 直径90毫米的黑色滤纸(MACHEREY-NAGEL,目录号:409009)
  5. 猎鹰60 x 15毫米盘子(康宁,目录号:353004)
  6. 12孔组织培养板(TPP Techno Plastic Products,目录号:92412)
  7. 2孔Lab-Tek TM室#1.0硼硅酸盐盖玻片(Thermo Fisher Scientific,目录号:155380)
  8. 拟南芥种子:Col-0,apo-pHusion质外pH标记系(Gjetting等人,2012),驱动萤火虫萤光素酶酶标记物表达的生长素应答启动子DR5 :: LUC(Moreno-Risueno ,2010)
  9. 家用漂白剂(次氯酸钠4.7%)
  10. 37%盐酸(Sigma-Aldrich,目录号:435570)
  11. MES(Duchefa Biochemie,目录号:M1503.0100)
  12. 蔗糖(Sigma-Aldrich,目录号:84097-1KG)
  13. 氢氧化钾(KOH)(Merck,目录号:105021)
  14. 琼脂,测试植物细胞培养(阿法埃莎,目录号:H26724)
  15. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541-500G)
  16. Phytagel(Sigma-Aldrich,目录号:P8169)
  17. 溶于乙醇的10mM 3-吲哚乙酸(IAA)(Sigma-Aldrich,目录号:I2886-5G)
  18. 1mM D-luciferin(Duchefa Biochemie,目录号:L1349.0100)溶于1x PBS中
  19. 氯气(见配方1)
  20. 半强度MS琼脂培养基(AM +)(见方法2)
  21. 消耗介质(DM)(见方法3)

设备

  1. 杜普斯杜蒙#5
  2. 双目解剖显微镜徕卡EZ4(徕卡显微系统,型号:徕卡EZ4)
  3. 平板扫描仪爱普生完美V370照片(爱普生,型号:V370照片)
  4. 对于生物发光暗箱:
    Lumazone手动舞台暗箱(光度,型号:LMZ-DRK-BOX)
    进化EMCCD相机(光度计,型号:Evolve 512,目录号:EVO-512-M-FW-16-AC-RP)
    17毫米固定镜头/0.95(埃德蒙光学,型号:59-832)
    125毫米镜头(Thorlabs,型号:LA1384-A)
  5. Zeiss 700 LSM共聚焦显微镜(ZEISS,型号:LSM 700),带有20x / 0.8 Plan-Apochromat M27物镜

软件

  1. Microsoft Excel程序( https://products.office.com/en/excel ) >
  2. 斐济计划( http://fiji.sc/
  3. MATLAB程序( https://www.mathworks.com/products/matlab.html
  4. AutoIt程序( https://www.autoitscript.com/site/autoit/ ) />

程序

  1. 下胚轴伸长测量
    1. 使用氯气(配方1)将Col-0种子(或您感兴趣的任何其他基因型)过夜灭菌。在AM +培养基上消毒灭菌的种子(方案2)。在4℃的黑暗中分层两天,然后垂直放置在光照下,在21℃的培养室中培养约6小时。
      用铝箔包裹,在21°C垂直生长66 h
    2. 用Falcon 60 x 15 mm培养皿准备5 ml消耗培养基(DM,Recipe 3)的消耗板。凝固后,将玻璃纸贴在表面上。用液体消耗介质溶液来润湿玻璃纸箔。
    3. 在黑暗的房间放置一个解剖显微镜,并用8层绿色办公室箔(图1)制成的绿色过滤器覆盖照明。


      图1.具有绿色过滤器的双目解剖显微镜

    4. 用幼苗揭开培养皿并选择具有相似的下胚轴长度(不包括最长和最短的幼苗)的幼苗。在尖端钩子下方和芽根连接处将幼苗取出,使用非常锋利的剃刀刀片在琼脂表面上切下下胚轴片段。准备6-8段每个治疗。使用锋利的镊子,将片段转移到耗尽板中的玻璃纸箔上而不挤压它们,样品制备程序如图2所示。保持在黑暗中30-60分钟。


      图2.样品制备程序

    5. 之后,通过将玻璃纸箔翻转到处理板上并用补充有所需处理的耗尽培养基(图2)(在本例中为10μM3-吲哚乙酸(IAA)和模拟对照(乙醇等同物))转移片段。 br />
    6. 立即将处理板放在平板扫描仪上,通过植物层的成像。将黑色的湿滤纸放在碟子的盖子上以改善图像的对比度。使用AutoIt程序自动扫描8位灰度样本和每10分钟2400 dpi的样本(参见补充文件1 )。

  2. 使用DR5 :: LUC标记线测量TIR1 / AFB-Aux / IAA依赖性应答
    1. 如前所述,在耗尽培养基上,为每次处理准备6-10份不饱和的DR5 :: LUC标记系的片段。加入溶解在1x PBS中的约50μl的1mM D-荧光素并将片段完全浸没30分钟。
    2. 准备治疗解决方案(DM与所需药物) - 在我们的情况下,DM +模拟或DM +10μMIAA。将3ml培养基倒入12孔组织培养板的每个孔中,让培养基固化;四口井可以同时成像。
      注:处理溶液中没有D-荧光素,底物来源于步骤B1。这对于大约是足够的。 5小时成像。或者,可以将D-荧光素放入处理溶液中进行较长时间的成像。
    3. 使用玻璃纸箔,将这些部分转移到处理介质的表面上,并去除箔。
    4. 立即用配备有17mm固定镜头/0.95和另外的125mm镜头的Photometric Evolve EMCCD相机在黑盒子(图3)中成像。将乘数EMCCD增益设置为150,曝光时间设置为110秒,每2分钟拍摄一次。


      图3.使用EMCCD照相机的暗盒

  3. 使用apo-pHusion质外pH标记物线对质外pH进行成像
    1. 准备apo-pHusion apoplastic pH标记线如上所述的断头的下胚轴。
    2. 准备5毫升含有或不含10微米IAA的DM培养基。
    3. 用处理将5个下胚轴段转移到琼脂表面上。用抹刀切出一段琼脂。将带有片段的琼脂放入Lab-Tek TM室盖玻璃中,使片段置于盖玻片和琼脂之间。当使用2孔玻璃时,可以同时对处理和对照样品进行成像。
    4. 或者,在成像过程中,可以非常小心地将处理移植到下胚轴部分。然后可以使用约50μlDM的治疗,但是在成像过程中必须非常小心,不要移动样本。
    5. 使用具有20x / 0.8 Plan-Apochromat M27物镜的共聚焦显微镜,使用位置管理器设置每个片段的位置,使得下胚轴片段的顶端区域成像。图片5每个胚轴下段的z-切片,与针孔大小相匹配的z-厚度。
    6. 通过使用488和555nm二极管激光器激发,设置显微镜以同时成像GFP和RFP,并且以短通道550nm和长通560nm滤波器(每像素16位)分离发射的光。每5分钟拍摄一次所有姿势。

数据分析

  1. 下胚轴伸长图像分析
    1. 为了实现无偏差的测量,我们创建了一个斐济宏(参见补充文件1 )来分析每个时间点的细分的长度。该宏首先创建从扫描仪捕获的图像序列的时间间隔,然后允许您为每个分段手动创建一个矩形ROI,然后自动阈值化每个ROI并测量Feret直径,最大卡尺,作为分割。该宏最终会为每个ROI或下胚轴产生“.txt”文件,包括每个时间点的该胚轴的费雷特直径。
    2. 将结果复制并粘贴到Excel中,将片段的初始长度设置为100%,并计算每个时间点下胚轴的长度以获得生长曲线(图4C)。此外,通过在斐济建立一个单独的下胚轴段蒙太奇-kypograph(图4A),可以看到增长。

  2. 生物发光强度的分析
    1. 分析斐济的图像序列(Schindelin等人,2012年)。在最亮的时间框架中,通过多边形选择手动勾画所有的段,然后将其添加到感兴趣区域(ROI)管理器中,然后多次测量平均灰度值。这给出了每个时间点上每个片段的平均强度。
    2. 将结果复制并粘贴到Excel中。以该段的初始强度为100%,分析各时间点下胚轴发光强度的平均值,及时得到强度曲线。此外,人们可以通过在斐济创建一个蒙太奇-kymograph(图4B和4D)来观察生长和发光强度。


      图4.下胚轴段生长和DR5 :: LUC强度测量。 :一种。 Col-0的下胚轴片段在模拟处理(上排)和10μMIAA处理(下排)下从0到460分钟的kymograph;时间间隔10分钟。 kymograph是通过在斐济的一个代表性样本的成长部分蒙太奇。 (请注意,为了更好地观察生长情况,生长发生的下胚轴的上半部分被用于制作蒙太奇)。垂直和水平比例尺分别代表1毫米和20分钟。 B.从0到172分钟,模拟处理的DR5 :: LUC下胚轴(上排)和10μMIAA处理的(下排)发光强度的比较图;时间间隔2分钟。 “FIRE”查询表在斐济应用。比例尺是10分钟。 C.用模拟物或10μMIAA处理0至460分钟的Col-0胚轴的生长量化。增长表示为原始分段长度的百分比。 D.用模拟物或10μMIAA处理的DR5 :: LUC下胚轴中发光强度的定量。倍数变化是由时间点0的强度归一化的下胚轴的平均强度。

  3. 图像分析的细胞壁pH值
    1. 使用斐济分析质外pH值。我们使用Z-堆栈的SUM投影(图5A)。使用RFP通道设定质外体区域的阈值,以便仅选择细胞壁信号。使用“创建选择”命令创建选择,并在GFP和RFP通道中测量强度。在Excel程序中分析强度比。我们分析相对值中的质外pH值变化,GFP强度越低,外质体pH越低(Gjetting等人,2012)。
    2. 作为步骤C1的替代方案,可以使用AreaKymo MATLAB 脚本来显现和测量质外pH,图5B(补充文件1 )。 AreaKymo脚本基本上执行与步骤C1中所述相同的过程,但是在没有用户输入的情况下自动执行,允许一次快速处理几个胚轴。用户首先使用斐济程序('combine stacks'命令)将几个下胚轴SUM投影时间序列合并成一个'tif。'序列,并将它们转换成16位'tif'图像(MATLAB不能很好地处理32位SUM投影命令创建的图像)。然后,用户应该找到最适于选择质外体信号的RFP通道的阈值。在MATLAB中,运行AreaKymo脚本,选择组合的时间序列,并且用户指定RFP通道的阈值以及将表示单个时间范围的矩形的期望宽度。该脚本输出的外形pH值的可视化表示,也是一系列箱形图的形式。


      图5.对质外体pH值的分析。 :一种。将生长素施用于表达apo-pHusion传感器的下胚轴后的质外体pH。生长素用箭头表示,GFP用绿色表示,RFP用洋红色表示。 B. AreaKymo MATLAB ®脚本的输出。时间在上进,
      使用“FIRE”查找表显示两个荧光基团的比例

笔记

在处理下胚轴的所有步骤中(切割并转移到新的平板),对组织非常温和至关重要,而不是挤压它,而是用锋利的镊子舀它。需要保护组织不受干燥;必须尽可能保持密闭板以防止过度蒸发。

食谱

  1. 氯气杀菌
    100毫升家用漂白剂
    4.5ml 37%HCl
  2. 半强度MS琼脂培养基(AM +)
    半Murashige和Skoog基础盐
    1%蔗糖

    用KOH调节pH值至5.8 0.8%琼脂,植物细胞测试
    MiliQ水作为溶剂
  3. 耗尽媒介(DM)
    10 mM KCl
    1 mM MES
    用KOH调节pH值至6 1.5%phytagel
    MiliQ水作为溶剂
    注意:phytagel带来比普通琼脂更好的透明度,有助于提高扫描质量。

致谢

该协议摘自2016年的Fendrych et al。 ,该项目已获得欧盟Horizon 2020研究和创新计划的资助,该计划是根据MarieSkłodowska-Curie拨款协议第665385号和奥地利科学基金(FWF)[M 2128-B21]。作者声明没有利益冲突。

参考

  1. Fendrych,M.,Leung,J.和Friml,J。(2016)。 TIR1 / AFB-Aux / IAA植物生长素感知介导快速细胞壁酸化和生长 拟南芥 下胚轴。 5 。
  2. Fraas,S.,Niehoff,V。和Lüthen,H。(2014)。 一种用于根和下胚轴的高通量成像辅助测定仪 拟南芥 使用2D镂空算法。 Physiol Plant 151:112-118。
  3. Gjetting,K.S.,Ytting,C.K。,Schulz,A。和Fuglsang,A.T。(2012)。 使用pHusion(一种新的遗传编码的生物传感器)的植物内和植物细胞外pH值的实时成像。
    J Exp Bot 63(8):3207-3218。
  4. Moreno-Risueno,M.A.,Van Norman,J.M.,Moreno,A.,Zhang,J.,Ahnert,S.E。和Benfey,P.N。(2010)。 振荡基因表达决定了定期的能力 拟南芥 分支。科学 329(5997):1306-1311。
  5. 申克,D.,克里斯蒂安,米,琼斯,答,和吕滕,阁下(2010年)。 快速生长素诱导的细胞扩增和基因表达:一个四十年的老问题重新审视。植物生理学 152:1183-5。
  6. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P.和Cardona,A。(2012)。 斐济:生物图像分析的开源平台。 Nat方法 9(7):676-682。
  7. Spartz,A.K.,Ren,H.,Park,M.Y.,Grandt,K.N.,Lee,S.H.,Murphy,A.S.,Sussman,M.R.,Overvoorde,P.J.和Gray,W.M。 (2014)。 SAUR抑制PP2C-D磷酸酶激活质膜H href =“https://www.ncbi.nlm.nih.gov/pubmed/24858935”target =“_ blank”> + - 促进细胞扩张的ATP酶 拟南芥 植物细胞 26:2129-2142。
  8. Takahashi,K.,Hayashi,K.和Kinoshita,T。(2012)。 生长素在下胚轴伸长过程中通过磷酸化激活质膜H + -ATPase, 拟南芥 植物生理学 159:632-41。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright Li et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Li, L., Krens, S., Fendrych, M. and Friml, J. (2018). Real-time Analysis of Auxin Response, Cell Wall pH and Elongation in Arabidopsis thaliana Hypocotyls. Bio-protocol 8(1): e2685. DOI: 10.21769/BioProtoc.2685.
  2. Fendrych, M., Leung, J. and Friml, J. (2016). TIR1/AFB-Aux/IAA auxin perception mediates rapid cell wall acidification and growth of Arabidopsis hypocotyls. Elife 5.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。