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MIFE Technique-based Screening for Mesophyll K+ Retention for Crop Breeding for Salinity Tolerance
采用MIFE技术筛选持K+叶肉用于耐盐作物的育种   

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Abstract

Potassium is known as a rate-limiting factor for crop yield and plays an important role in plants response under abiotic stresses. Recently, cytosolic K+ retention ability in leaf mesophyll has emerged as an important component of plant salt tolerance mechanism (Wu et al., 2013; Wu et al., 2014; Wu et al., 2015). In this protocol, the procedure for screening leaf mesophyll for K+ retention by the MIFE (microelectrode ion flux estimation) technique is described in detail using wheat as an example. By measuring NaCl-induced K+ efflux in leaf mesophyll, a large number of plant accessions can be screened and categorised according to their salinity stress tolerance. The method provides a rapid and reliable tool that targets the activity of specific membrane transporters directly contributing to salinity tolerance trait and, because of this, has a competitive advantage over traditional whole-plant phenotyping. While the focus of this protocol is on wheat, the suggested method may be adopted for screening K+ retention in leaf mesophyll in any other crop species.

Keywords: K+ retention(钾潴留), Ion flux(离子通量), Salinity stress(盐胁迫), Mesophyll cells(叶肉细胞), Breeding(育种)

Materials and Reagents

  1. Two to three week old wheat seedlings
  2. NaCl (Sigma-Aldrich, catalog number: 746398 )
  3. KCl (Sigma-Aldrich, catalog number: 746436 )
  4. CaCl2 (Sigma-Aldrich, catalog number: C5670 )
  5. K+ LIX (liquid ion exchanger) (Sigma-Aldrich, catalog number: 60031 )
  6. Tributylchlorosilane (Sigma-Aldrich, catalog number: 282707 )
  7. ddH2O
  8. 95% ethanol (VWR International, catalog number CHESEA042-20L-P )
  9. Commercial bleach (contains 42 g/L NaClO)
  10. Agar (Oxoid, catalog number: LP0011 )
  11. Parafilm
  12. Basic salt medium (BSM) solution (mM) (see Recipes)
  13. Backfilling solution for K+ ion selective microelectrode (see Recipes)
  14. Filling solution for a reference electrode (see Recipes)
  15. K+ calibration solutions (μM) (see Recipes)
  16. Potting mix (see Recipes)

Equipment

  1. The MIFE (microelectrode ion flux estimation) system (designed, manufactured and distributed by the University of Tasmania)
    (http://www.phys.utas.edu.au/physics/biophys/mifecom/MIFEHome/Home.html)
  2. Glasshouse
  3. Vertical electrode puller (Narishige, model: PP-830 )
  4. Electrode filling station (contains a three-dimensional micromanipulator and a microscope)
  5. Laboratory fume cupboard (model: 1800)
  6. Inverted tissue culture microscope (Radical Instruments, model: RTC-6 )
  7. Microscope (Nikon, model: 100100 ) used in the electrode filling station
  8. Micromanipulator (Narishige, model: MMT-5 ) used in the electrode filling station
  9. Oven (Euromiad compact cooker, model: MC 110 T )
  10. Distiller (Labglass, model: 03DD )
  11. pH meter (Thermo Fisher Scientific, model: Orion 420 A +)
  12. Burner
  13. Magnetic stirrer (ISG® Hotplate and Magnetic stirrer, model: 153-005 ) and stirring bars
  14. Borosilicate glass capillaries (GC 150-10) (1.5 O.D. x 0.86 I.D. x 100 L mm) (Harvard Apparatus, catalog number: 30-0053 )
  15. Borosilicate glass capillaries (GC 100-10) (1.0 O.D. x 0.56 I.D. x 100 L mm) (Harvard Apparatus, catalog number: 30-0016 )
  16. Petri dishes (85 mm and 35 mm diameters)
  17. Perspex holder for immobilization of the leaf samples
  18. Metal electrode rack for electrode silanization
  19. Plastic electrode holder for storing silanized electrode blanks
  20. Standard surgical blades (Kiato stainless steel, model: BS 2982:1992, ISO 7740 )
  21. Syringe (Terumo, catalog number: SS-10L )
  22. Plastic needle (20 μl) (Eppendorf, catalog number: 5424 956.003 )
  23. Plastic-coated weights
  24. Silver wire (A-M Systems, catalog number: 787000 )
  25. 4.5 L PVC (polyvinyl chloride) pots

Procedure

  1. Plant material preparation
    1. Sow 12 to 14 seeds in a 4.5 L PVC pot (with a saucer) using the standard potting mix and grow them under glasshouse conditions (day/night temperature 23/18 °C; ~ 11 to 13 h photoperiod).
    2. After seedlings have emerged, thin plants to leave eight uniform plants per pot.
    3. Grow wheat plants until they are 15 to 20 days old (Figure 1).


      Figure 1. Two week- old wheat seedlings grown in a glasshouse

  2. Preparation of ion selective microelectrodes
    1. Electrode blank preparation
      1. Insert a non-filamentous borosilicate glass capillary (GC 150-10; 1.5 O.D. x 0.86 I.D. x 100 L mm) into a vertical electrode puller.
      2. Pull the glass capillary by heating the middle part into two blanks with external tip diameter of about 3-4 μm.
      3. Store the pulled blank electrodes in a stainless steel rack (Figure 2) in a vertical position with the tips positioned upwards.
      4. Put the steel rack with the blank electrodes into an oven located in a fume hood cabinet and heat them overnight at 225 °C.
      5. After 8-10 h of heating, cover the steel rack with a round shape steel lid and heat for a further 15 min.
      6. Inject a few drops of tributylchlorosilane under the lid (70 μl for a steel rack containing 36 blank electrodes, Figure 2) and heat for a further 10 min with the lid on. Depending on the number of blank electrodes in the steel rack, the volume of injected tributylchlorosilane should be adjusted.


        Figure 2. Pulled blank electrodes stored in the metal electrode rack

      7. Remove the lid from the steel rack and continue heating the blank electrodes in the oven for a further 30 min.
      8. Switch off the oven and let the electrode blanks cool down inside the oven.
      9. Transfer the silanized blank electrodes to electrodes holder (Figure 3) and cover with a lid. Prepared electrode blanks can be stored up to a month.


        Figure 3. Perspex-made electrode holder

    2. Filling up electrodes
      1. Prepare a K+ LIX containing tube by dipping a pulled borosilicate glass capillary (GC 100-10; 1.0 O.D. x 0.56 I.D. x 100 L mm) with a broken tip (~ 50 μm diameter) into a bottle containing K+ LIX.
      2. Immobilize the silanized electrode blank in a microelectrode filling station and break the electrode tip against a flat glass surface under a microscope (step 1 showed in Figure B4a) to achieve the electrode tip diameter of 2-3 μm.
      3. Immobilize prepared K+ LIX tube horizontally in the filling station against the prepared microelectrode blank (step 2 showed in Figure 4a). Align tips of the microelectrode and the LIX-containing tube under the microscope (Figure 4b).


        Figure 4. The blank electrode and a glass capillary containing K+ LIX are aligned at the filling station

      4. Back-fill the microelectrode with a K+ backfilling solution using a syringe with a plastic needle. Ensure the absence of any air bubble adhered to the glass capillary in the electrode tip. Add more back filling solution if the latter occurred.
      5. Front-fill the electrode by briefly putting the electrode tip in a contact with the open tip of the LIX-containing tube to achieve a column length of about 100-150 μm.
      6. Store prepared microelectrode in containing a basic salt medium (BSM) solution (Figure 5).


        Figure 5. Electrodes stored in BSM solution

  3. Preparation of a reference electrode
    1. Place pulled blank microelectrodes with broken tips (~ 50 μm tip diameter) into a wide neck glass container tips down.
    2. Prepare ~50 ml 2% (w/v) agar solution in 1 M KCl and melt it in a water bath.
    3. Pour the melted agar solution over prepared electrode blanks to immerse them completely in the agar solution. Ensure that internal parts of the glass capillaries are filled with the agar solution.
    4. Seal the top of the glass container once it has cooled down and store in a fridge for further use.
    5. For a reference electrode preparation, cut ~5 cm of a silver wire.
    6. Chlorinate the silver wire by immersing it into bottle containing commercial bleach for ~1-2 min leaving one end of the wire (~5 mm) exposed to air. Ensure that immersed part of the wire turned black.
    7. Insert the chlorinated silver wire into the prepared reference electrode blank (glass capillary filled with the 1 M KCl agar solution). Leave un-chlorinated part of the silver wire (5 mm) outside the glass capillary.
    8. Secure silver wire in the glass capillary with a strip of parafilm. Ensure that un-chlorinated part of the wire stays un-covered. Immerse the prepared reference electrode in a container with BSM and store them in a fridge until use.

  4. Microelectrode calibration
    Three calibration solutions with concentrations covering expected K+ concentration in the measured solution are used to calibrate K+ microelectrode. For example, for BSM containing 500 μM KCl, following KCl calibration solutions used are (μM): 250, 500, and 1,000.
    1. Place prepared K+ microelectrode in the MIFE electrode holder and ensure that the channel used is connected to an amplifier. Insert the electrode in BSM.
    2. Connect the reference electrode to a channel designated for the reference electrode and insert it in the same BSM.
    3. Run the MIFE software CHART from the appropriate directory (see http://www.phys.utas.edu.au/physics/biophys/mifecom/MIFEHome/Home.html for details).
    4. Press “Alt + S” to <s>tart creating a calibration file.
    5. Press F7 to specify channel used for K+ microelectrode. Press “Enter”.
    6. Insert K+ microelectrode into the first K+ calibration solution (250 μM). Press F7 to specify K+ concentration used. Record calibration outputs for 20-30 sec. Once the recorded line is strait, press F7 to accept it. In case the line is not acceptable, press “No” in F7 menu and repeat the calibration.
    7. Insert the K+ microelectrode into other calibration solutions (500 μM and 1,000 μM, one at a time) and repeat step D6.
    8. Press “Alt + H” to end the data acquisition. Then press “Y” for <Y>es to confirm your choice.
    9. On completion of K+ microelectrode calibration, its quality must be checked. Press “Alt + E” to enter the <E>lectrometer menu, then choose “A” to <A>average data, then press “C” for the <C>alibration average. Average calibration file <.AVC> will be created by the CHART software automatically, and also the information about a set of parameters, e.g. slop, intercept, and correlation coefficients, of K+ microelectrode will be displayed in the screen.
    10. Examine displayed parameters: only K+ microelectrode with a slope above 50 mV per decade and a correlation coefficient above 0.999 are accepted and can be used for further experiments. If the calibration result doesn’t meet the requirement, new electrode has to be prepared and the calibration process repeated.

  5. Preparing specimens
    1. Excise second youngest expanded leaf and place it in a beaker containing tap water.
    2. Using a sharp blade, cut the leaf segments angularly into small cross-sectional segments to expose mesophyll (5 to 8 mm each).
    3. Place leaf segments in an 85 mm diameter Petri dish containing BSM solution peeled side down and leave them floating on the surface in the darkness overnight to minimize possible confounding effects of tissue damage on ion fluxes (Shabala and Newman, 1999).
    4. After 10 to 12 h, mount the leaf segment in a Perspex holder and place it in a measuring chamber containing BSM solution.
    5. Press the holder to the bottom of the Petri dish with a plastic-coated weight.
    6. Leave the prepared sample to adapt for about 30 min.
    7. The leaf sample is ready for measurements now.

  6. Measuring NaCl-induced K+ efflux from the leaf mesophyll by the MIFE technique
    1. Place the measuring chamber on a microscope stage and insert the reference electrode into it.
    2. Position the calibrated K+ microelectrode 40 μm away from the exposed mesophyll (Wu et al., 2014; Wu et al., 2015) under high magnification (200x) of the inverted microscope using three dimensional micromanipulators of the MIFE technique.
    3. Start the MIFE program CHART as described above for calibration.
    4. Set up a travel range of 70 μm between two positions and moving cycle of 6 sec/ 6 sec to enable electrode movement in a 12 sec square-wave cycle by a computer-controlled hydraulic manipulator.
    5. Record K+ fluxes from leaf samples under control (BSM solution) conditions for 5 min.
    6. Add concentrated NaCl stock solution to achieve 100 mM NaCl in the BSM solution and mix well with a pipette.
    7. Record NaCl-induced K+ fluxes for 10 min.
    8. Press “Alt + H” to end the data acquisition followed by “Y” for <Y>es to confirm your choice.
    9. Create an average file by pressing “Alt + E” to enter the <E>lectrometer menu, then choose “A” for <A>average option and “M” for the <M>anipulator cycle average. A box with “Experimental parameter values” will open. Press <Z> and type radius of the root (in μm); press <U> and type distance between the root and the microelectrode (typically 40 μm), then press <OK>. A new box will appear in the screen with indication of a valid time. Accept all by pressing <Enter>. An <.AVM> file will be created.
    10. Quit the CHART program by pressing “Alt + Q”.

  7. Flux calculation
    1. Run MIFEFLUX software by typing “mflux” in the MIFE directory [see Shabala et al. (2012) for details].
    2. Type the name of the recorded AVC file (calibration of the electrodes). Follow the prompt, and then type the name of the AVM file for which flux calculation is required.
    3. Choose <C> for cylindrical diffusion geometry (Newman, 2001; Shabala et al., 2006) and press <Enter>. A flux file with an extension <.flx> will be generated. The same calibration file can be used for calculating more flux files. 
    4. Copy flux files to your working directory and use Excel for data analyses.

  8. Flux data analyses
    1. Open Excel and find a flux file of interest in , open it.
    2. Plot K+ flux values against time and assess flux changes. Negative values of the flux are associated with K+ efflux, the larger the value the higher K+ efflux through the plasma membrane. The lower is the value, the better is K+ retention ability (and, hence, salt tolerance).
    3. Pay specific attention to a peak and steady state values under different conditions (in the presence and the absence of NaCl in the BSM).

Notes

  1. Temperature-controlled room (~ 23 °C) is required.
  2. The K+ microelectrode must be changed daily.
  3. A capillary with the K+ LIX must be changed weekly.
  4. After silanizing, the flat side of the microelectrode must be flamed to avoid scratching the chlorinated silver wire.
  5. In general, the reference electrode must be changed weekly. Once the reference electrode is changed, the electrodes must be re-calibrated and the AVC file generated used for flux calculation of an AVM file generated using the same reference electrode.
  6. As tributylchlorosilane is toxic, electrode salinization should be conducted in a fume hood.
  7. An overview image of the whole measurement setup is available in our previously published papers (Wu et al., 2014; Wu et al., 2015).

Recipes

  1. Basic salt medium (BSM) solution (pH ~5.7)
    0.1 mM CaCl2
    0.5 mM KCl
  2. Backfilling solution for K+ ion selective microelectrode
    200 mM KCl
  3. Filling solution for a reference electrode
    1 M KCl
    2% Agar
  4. K+ calibration solutions (μM)
    250, 500, and 1000 μM KCl
  5. Potting mix
    80% composted pine bark
    10% sand and 10% coir peat, plus complete N:P:K (8:4:10), 1 kg/m3
    Dolomite, 8 kg/m3
    Gypsum, 1 kg/m3
    Iron sulphate, 1 kg/m3
    Isobutylenediurea, 1 kg/m3
    Trace element mix, 0.75 kg/m3
    Wetting agent, 0.75 Kg/m3
    Zeolite, 0.75 Kg/m3
    pH 6.0

Acknowledgments

This protocol was adapted from our previous publications (Wu et al., 2014; Wu et al., 2015; and Shabala et al., 2012). This work was supported by the Grain Research and Development Corporation grants to SS and MZ and by the Australian Research Council Discovery grant to SS.

References

  1. Newman, I. A. (2001). Ion transport in roots: measurement of fluxes using ion-selective microelectrodes to characterize transporter function. Plant Cell Environ 24(1): 1-14.
  2. Shabala, S., Cuin, T. A., Shabala, L. and Newman, I. (2012). Quantifying kinetics of net ion fluxes from plant tissues by non-invasive microelectrode measuring MIFE technique. Methods Mol Biol 913: 119-134.
  3. Shabala, S., Demidchik, V., Shabala, L., Cuin, T. A., Smith, S. J., Miller, A. J., Davies, J. M. and Newman, I. A. (2006). Extracellular Ca2+ ameliorates NaCl-induced K+ loss from Arabidopsis root and leaf cells by controlling plasma membrane K+ -permeable channels. Plant Physiol 141(4): 1653-1665.
  4. Shabala, S. and Newman, I. I. (1999). Light-induced changes in hydrogen, calcium, potassium, and chloride ion fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis. Plant Physiol 119(3): 1115-1124.
  5. Wu, H., Shabala, L., Barry, K., Zhou, M. and Shabala, S. (2013). Ability of leaf mesophyll to retain potassium correlates with salinity tolerance in wheat and barley. Physiol Plant 149: 515–527.
  6. Wu, H., Shabala, L., Zhou, M. and Shabala, S. (2014). Durum and bread wheat differ in their ability to retain potassium in leaf mesophyll: implications for salinity stress tolerance. Plant Cell Physiol 55(10): 1749-1762.
  7. Wu, H., Zhu, M., Shabala, L., Zhou, M. and Shabala, S. (2015). K+ retention in leaf mesophyll, an overlooked component of salinity tolerance mechanism: A case study for barley. J Integr Plant Biol 57(2): 171-185.

简介

钾被称为作物产量的限速因子,并且在植物在非生物胁迫下的应答中起重要作用。近来,叶肉叶中的细胞质K sup +保留能力已经成为植物盐耐受机制的重要组成部分(Wu等人,2013; Wu等人, ,2014; Wu et al。,2015)。在该方案中,使用小麦作为实例详细描述了通过MIFE(微电极离子通量估计)技术筛选叶子叶肉用于K sup +保留的程序。通过测量叶片叶肉中NaCl诱导的K + sup/+流出,可以根据其盐度胁迫耐受性筛选和分类大量植物种质。该方法提供了快速和可靠的工具,其靶向直接有助于耐盐性性状的特定膜转运蛋白的活性,并且因此,相对于传统的全植物表型具有竞争优势。虽然本议定书的焦点是小麦,但是可以采用所建议的方法来筛选任何其它作物品种的叶子叶肉中的K sup +保留。

关键字:钾潴留, 离子通量, 盐胁迫, 叶肉细胞, 育种

材料和试剂

  1. 两到三周龄的小麦幼苗
  2. NaCl(Sigma-Aldrich,目录号:746398)
  3. KCl(Sigma-Aldrich,目录号:746436)
  4. CaCl 2(Sigma-Aldrich,目录号:C5670)
  5. KIX +液体离子交换剂(Sigma-Aldrich,目录号:60031)
  6. 三丁基氯硅烷(Sigma-Aldrich,目录号:282707)
  7. ddH sub 2 O
  8. 95%乙醇(VWR International,目录号CHESEA042-20L-P)
  9. 商业漂白剂(含42g/L NaClO)
  10. 琼脂(Oxoid,目录号:LP0011)
  11. parafilm
  12. 碱性盐介质(BSM)溶液(mM)(参见配方)
  13. 用于K + 离子选择性微电极的回填溶液(参见配方)
  14. 参比电极的填充溶液(参见配方)
  15. K + 校准溶液(μM)(参见配方)
  16. 盆栽混合(见配方)

设备

  1. MIFE(微电极离子流量估计)系统(由塔斯马尼亚大学设计,制造和分发)
    http://www.phys.utas.edu。 au/physics/biophys/mifecom/MIFEHome/Home.html
  2. 温室
  3. 垂直电极拉出器(Narishige,型号:PP-830)
  4. 电极加油站(包含三维显微操作器和显微镜)
  5. 实验室通风橱(型号:1800)
  6. 倒置组织培养显微镜(Radical Instruments,型号:RTC-6)
  7. 显微镜(尼康,型号:100100)用于电极填充站
  8. 在电极填充站
    中使用的微型操作器(Narishige,型号:MMT-5)
  9. 烤箱(Euromiad compact compacter,型号:MC 110T)
  10. Distiller(Labglass,型号:03DD)
  11. pH计(Thermo Fisher Scientific,型号:Orion 420A +)
  12. 燃烧器
  13. 磁力搅拌器(ISG 热板和磁力搅拌器,型号:153-005)和搅拌棒
  14. 硼硅酸盐玻璃毛细管(GC 150-10)(1.5 O.D.x 0.86 I.D.x100Lmm)(Harvard Apparatus,目录号:30-0053)
  15. 硼硅酸盐玻璃毛细管(GC 100-10)(1.0 O.D.×0.56I.D.×100Lmm)(Harvard Apparatus,目录号:30-0016)
  16. 培养皿(直径85mm和35mm)
  17. 用于固定叶子样品的有机玻璃支架
  18. 用于电极硅烷化的金属电极架
  19. 用于存储硅烷化电极坯料的塑料电极夹
  20. 标准手术刀(Kiato不锈钢,型号:BS 2982:1992,ISO 7740)
  21. 注射器(Terumo,目录号:SS-10L)
  22. 塑料针(20μl)(Eppendorf,目录号:5424956.003)
  23. 塑料涂层重量
  24. 银线(A-M Systems,目录号:787000)
  25. 4.5 L PVC(聚氯乙烯)锅

程序

  1. 植物材料准备
    1. 使用标准品在4.5L PVC罐(具有碟)中播种12至14粒种子   灌封混合并在玻璃温室条件下生长(白天/黑夜 温度23/18℃; 〜11至13小时光周期)
    2. 幼苗出苗后,细植物每盆留下八个均匀的植株。
    3. 种植小麦植株,直到它们15到20天(图1)

      图1.在温室中生长的两周龄小麦幼苗

  2. 离子选择性微电极的制备
    1. 电极空白预处理
      1. 插入非丝状硼硅酸盐   玻璃毛细管(GC 150-10; 1.5 O.D.x 0.86 I.D.×100L mm) 垂直电极拉出器
      2. 通过将中间部分加热成两个外部尖端直径约3-4μm的毛坯来拉出玻璃毛细管
      3. 将拉出的空白电极存放在不锈钢架中(图 2)处于垂直位置,其中尖端向上定位。
      4. 将带有空白电极的钢架放入位于通风橱柜中的烤箱中,并在225℃下加热过夜。
      5. 加热8-10小时后,用圆形钢盖盖住钢架,再加热15分钟。
      6. 在盖子下注入几滴三丁基氯硅烷(70μl 对于包含36个空白电极的钢架,图2)和热量a   进一步打开盖子10分钟。 根据空白的数量 电极在钢架上,注入的体积 三丁基氯硅烷应调整

        图2.存放在金属电极架中的拉出的空白电极

      7. 从钢架上取下盖子,继续在烤箱中加热空白电极30分钟。
      8. 关闭烤箱,让电极坯料在烤箱内冷却。
      9. 将硅烷化的空白电极转移到电极夹 (图3)并用盖子盖住。 可以存储制备的电极坯料   最多一个月。


        图3.有机玻璃制电极夹

    2. 填充电极
      1. 通过浸渍拉制的硼硅酸盐制备含K LIX的管 玻璃毛细管(GC 100-10; 1.0OD×0.56I.D.×100Lmm),其中a 破碎的尖端(〜50μm直径)装入含有K on + LIX的瓶中。
      2. 将硅烷化的电极坯料固定在微电极填充物中 站和打破电极头对平面玻璃表面下   显微镜(步骤1在图B4a中显示)以实现电极尖端 直径为2-3μm。
      3. 水平制备K + LIX管 在填充站中对准制备的微电极坯体(步骤2   如图4a所示。 微电极的尖端和 LIX的显微镜下(图4b)

        图4.空白电极和含有K + LIX的玻璃毛细管在加注站对齐

      4. 用K + 回填溶液回填微电极,使用a 注射器用塑料针。 确保没有任何气泡 粘附在电极头中的玻璃毛细管上。 添加更多回 如果后者发生,则填充溶液
      5. 正面填充电极   通过短暂地使电极头与开口尖端接触 该含LIX的管实现约100-150μm的柱长度
      6. 将准备的微电极储存在含有碱性盐介质(BSM)溶液中(图5)

        图5.存储在BSM解决方案中的电极

  3. 参考电极的制备
    1. 将拉断的具有破碎尖端(约50μm尖端直径)的空白微电极放入宽颈玻璃容器尖端。
    2. 准备约50 ml 2%(w/v)琼脂的1M KCl溶液,并在水浴中融化。
    3. 将熔化的琼脂溶液倒在制备的电极坯料上 将它们完全浸没在琼脂溶液中。 确保内部零件   的玻璃毛细管用琼脂溶液填充。
    4. 一旦冷却后,将玻璃容器的顶部密封,存放在冰箱中备用
    5. 对于参考电极制备,切割〜5厘米的银线
    6. 通过将银线浸入装有瓶子的氯化银线 商业漂白剂〜1-2分钟,留下线的一端(〜5mm) 暴露于空气。 确保电线的浸入部分变黑。
    7. 将氯化银线插入准备的参考 电极空白(填充有1M KCl琼脂溶液的玻璃毛细管)。   将银线的未氯化部分(5 mm)留在玻璃外 毛细管
    8. 用带条将玻璃毛细管中的银线固定   的封口膜。 确保导线的未氯化部分停留 裸露。 将制备的参比电极浸入容器中   BSM并将其存储在冰箱中,直到使用。

  4. 微电极校准
    使用具有覆盖测量溶液中的预期K on +浓度的浓度的三种校准溶液来校准K sup +微电极。 例如,对于含有500μMKCl的BSM,所用的KCl校准溶液为(μM):250,500和1,000。
    1. 在MIFE电极夹中准备K + 微电极,并确保   所使用的通道连接到放大器。 插入 电极在BSM
    2. 将参考电极连接到为参考电极指定的通道,并将其插入同一BSM中
    3. 从相应的目录运行MIFE软件CHART(请参阅 http: //www.phys.utas.edu.au/physics/biophys/mifecom/MIFEHome/Home.html 了解详情)。
    4. 按"Alt + S"即可创建校准文件。
    5. 按F7以指定用于K + 微电极的通道。 按"Enter"。
    6. 将K + 微电极插入第一个K + 校准溶液   μM)。 按F7指定使用的K + 浓度。 记录校准 输出20-30秒。 一旦录制的线路是海峡,按F7到 接受。如果线路不可接受,请在F7菜单中按"否"  重复校准。
    7. 将K + 微电极插入其他校准溶液(500μM和1000μM,一次一个),并重复步骤D6。
    8. 按"Alt + H"结束数据采集。然后按"Y"表示< Y>以确认您的选择
    9. 在完成K + 微电极校准后,必须检查其质量。按"Alt + E"进入< E>测量仪菜单,然后选择"A"到< A>平均数据,然后按"C"进行< C&  平均。平均校准文件< .AVC>将由创建 CHART软件自动,以及有关一组的信息 将在屏幕中显示K + 微电极的参数,例如斜率,截距和相关系数。
    10. 检查 显示的参数:只有K + 微电极,斜率大于50 mV 每十年并且接受大于0.999的相关系数 可用于进一步的实验。 如果校准结果不是 满足要求,新电极必须做好准备 校准过程重复。

  5. 准备标本
    1. 消毒第二个最年轻的膨化叶,并将其放在含有自来水的烧杯中
    2. 使用锋利的刀片,将叶片段有角度地切成小 截面段以暴露叶肉(每个5至8mm)。
    3. 将叶节放置在包含BSM的85mm直径培养皿中 溶液剥离一面朝下,并使它们漂浮在表面上 黑暗过夜,以尽量减少组织的可能的混杂效应 对离子通量的损害(Shabala和Newman,1999)
    4. 10至12小时后,将叶扇段安装在有机玻璃支架中,并将其放置在含有BSM溶液的测量室中。
    5. 将支架按压到培养皿的底部,用塑料涂层的重量。
    6. 离开准备的样品适应大约30分钟。
    7. 叶样品现在准备测量。

  6. 通过MIFE技术测量NaCl诱导的K + 从叶肉叶的流出量
    1. 将测量室放在显微镜载物台上,将参比电极插入其中。
    2. 将校准的K + 微电极定位在距离40微米远的位置 暴露的叶肉(Wu等人,2014; Wu等人,2015)在高 使用三维的倒置显微镜的放大率(200x) MIFE技术的微操纵器
    3. 如上所述启动MIFE程序CHART以进行校准。
    4. 在两个位置之间设置移动范围为70μm 周期为6秒/6秒,以使电极在12秒内移动 方波周期由计算机控制的液压机械手
    5. 在控制(BSM溶液)条件下记录来自叶样品的K + 通量5分钟。
    6. 加入浓缩的NaCl储备溶液以在BSM溶液中达到100mM NaCl,并用移液管充分混合
    7. 记录NaCl诱导的K +通量10分钟。
    8. 按"Alt + H"结束数据采集,然后按"Y"确认您的选择。< Y>
    9. 通过按"Alt + E"进入< E>测量仪菜单,然后为 < A>平均选项选择"A",为< ; M>动物循环平均值。 将打开一个"实验参数值"框。 按   < Z> 和根的类型半径(以μm为单位); 按< U> 和类型之间的距离 根部和微电极(通常为40μm),然后按&OK; 屏幕中将出现一个新的框,其中显示有效时间。 按< Enter>接受全部。 < .AVM> 文件将被创建。
    10. 按"Alt + Q"退出CHART程序

  7. 流量计算
    1. 通过在MIFE目录中键入"mflux"运行MIFEFLUX软件[有关详细信息,请参阅Shabala 等(2012)]。
    2. 键入记录的AVC文件的名称(校准 电极)。 按照提示,然后键入AVM文件的名称 需要进行通量计算。
    3. 选择< C> 对于圆柱形扩散几何(Newman,2001; Shabala等人,2006),并按下< Enter>。   具有扩展< .flx>的通量文件 将生成。 一样 校准文件可用于计算更多通量文件。
    4. 将流量文件复制到工作目录,并使用Excel进行数据分析。


  8. 助焊剂数据分析
    1. 打开Excel并找到感兴趣的通量文件,打开它。
    2. 绘制K + 通量值与时间并评估通量变化。 负 通量的值与K + 流量相关,值越大 通过质膜的较高的K + sup/+流出。 较低的是 值,K + 保留能力(以及因此的耐盐性)越好
    3. 特别注意下面的峰值和稳态值 不同条件下(在存在和不存在NaCl的情况下) BSM)。

笔记

  1. 需要温控室(〜23°C)。
  2. K + 微电极必须每天更换。
  3. 具有K + LIX的毛细管必须每周更换一次
  4. 在硅烷化之后,微电极的平坦侧必须燃烧以避免刮擦氯化银线。
  5. 一般来说,参考电极必须每周更换一次。 一旦更改参考电极,必须重新校准电极,生成的AVC文件用于使用相同参考电极生成的AVM文件的通量计算。
  6. 由于三丁基氯硅烷有毒,电极盐化应在通风橱中进行
  7. 在我们以前发表的论文中可以获得整个测量设置的概述图像(Wu等人,2014; Wu等人,2015)。

食谱

  1. 碱性盐介质(BSM)溶液(pH〜5.7)
    0.1mM CaCl 2/v/v 0.5mM KCl
  2. K + 离子选择性微电极的回填溶液
    200 mM KCl
  3. 参比电极的填充溶液
    1 M KCl
    2%琼脂
  4. K + 校准溶液(μM)
    250,500和1000μMKCl
  5. 灌封混合料
    80%堆肥松树皮
    10%砂和10%椰皮,加上完全N:P:K(8:4:10),1kg/m 3 / 白云石,8kg/m 3
    石膏,1kg/m 3
    硫酸铁,1kg/m 3
    异丁烯二脲,1kg/m 3 微量元素混合物,0.75kg/m 3 润湿剂,0.75Kg/m 3
    沸石,0.75Kg/m 3 pH 6.0

致谢

该方案改编自我们以前的出版物(Wu等人,2014; Wu等人,2015;和Shabala等人)。 2012)。 这项工作得到粮食研究和发展公司给予SS和MZ以及澳大利亚研究委员会发现赠款给SS的支持。

参考文献

  1. Newman,I.A。(2001)。 根系中的离子迁移:使用离子选择性微电极测量转运蛋白功能的特征。 > Plant Cell Environ 24(1):1-14
  2. Shabala,S.,Cuin,T.A.,Shabala,L。和Newman,I。(2012)。 通过测量MIFE技术的非侵入性微电极对来自植物组织的净离子通量的动力学进行量化。 Methods Mol Biol 913:119-134
  3. Shabala,S.,Demidchik,V.,Shabala,L.,Cuin,T.A.,Smith,S.J.,Miller,A.J.,Davies,J.M.and Newman,I.A。(2006)。 细胞外Ca 2+ 2可以改善NaCl诱导的K + <通过控制质膜K + - 可渗透通道,从拟南芥根和叶细胞中丧失 ):1653-1665。
  4. Shabala,S。和Newman,I.I。(1999)。 氢,钙,钾和氯离子通量的光诱导变化和来自叶肉的浓度和豆叶的表皮组织。理解光致生物电生成的离子基础。植物生理学119(3):1115-1124。
  5. Wu,H.,Shabala,L.,Barry,K.,Zhou,M.and Shabala,S。(2013)。 叶肉叶保留钾的能力与小麦和大麦的耐盐性有关。 em> Physiol Plant 149:515-527。
  6. Wu,H.,Shabala,L.,Zhou,M。和Shabala,S。(2014)。 硬粒小麦和面包小麦在叶片叶肉中保留钾的能力不同:对盐分胁迫耐受性的影响。 植物细胞生理学 55(10):1749-1762
  7. Wu,H.,Zhu,M.,Shabala,L.,Zhou,M.and Shabala,S。(2015)。 K + 保留叶肉叶,这是一种被忽视的盐分耐受机制 :大麦的案例研究。 57(2):171-185。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wu, H., Shabala, L., Zhou, M. and Shabala, S. (2015). MIFE Technique-based Screening for Mesophyll K+ Retention for Crop Breeding for Salinity Tolerance. Bio-protocol 5(9): e1466. DOI: 10.21769/BioProtoc.1466.
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