Biomechanical Characterization of Onion Epidermal Cell Walls
洋葱表皮细胞壁的生物力学表征   

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Nature Plants
Apr 2017

Abstract

Here we describe two experimental protocols to measure the biomechanical properties of primary (growing) plant cell walls, with a focus on analyzing cell wall epidermal strips of onion scales. The first protocol measures cell wall creep (time-dependent irreversible extension) under constant force. Such creep is often mediated by the wall-loosening action of expansin or selective endoglucanases. The second protocol is based on two consecutive stretches of the wall and measures the wall’s elastic and plastic compliances, which depend on cell wall structure. These two assays provide complementary information that may be linked to cell wall structure and expansive growth of cells.

Keywords: Cell walls (细胞壁), Mechanics (力学), Elastic modulus (弹性模量), Plastic modulus (塑性模量), Creep (蠕变), Onion epidermis (洋葱表皮)

Background

The primary walls of growing plant cells are strong enough to resist the tensile forces generated by cell turgor pressure, yet can expand irreversibly during cell growth as a result of the selective loosening action of expansins or other catalysts necessary for irreversible cell wall enlargement (Cosgrove, 2016a and 2016b). Assessments of cell wall mechanical properties, such as elasticity and plasticity, are important for understanding cell wall structure and its modification during growth (cell enlargement) and after growth ceases. An important measure of wall mechanics is based on uniaxial tensile tests, as described below in our stress/strain assay, which measures elastic and plastic compliance (compliance is the reciprocal of modulus; modulus is a measure of stiffness). A second, complementary assay is based on the irreversible, time-dependent increase in length (creep) that occurs in primary cell walls when they are held at constant tension and continuously loosened by endogenous expansin or exogenous endoglucanase (Durachko and Cosgrove, 2009; Cosgrove, 2011; Cosgrove et al., 2017). In this protocol, we describe procedures for preparing epidermal cell wall strips from onion scales and testing them in these two assays. Onion epidermal cell walls provide a useful model to explore the connection between cell wall structure and biomechanics (Wilson et al., 2000; Suslov et al., 2009; Kim et al., 2015; Zhang et al., 2017; Zheng et al., 2017).

Materials and Reagents

  1. Single edge razor blades (e.g., VWR, catalog number: 55411-050 )
  2. Double edge razor blades (e.g., Safety Razor Company, catalog number: 74-0002 )
  3. Disposable Petri dishes (e.g., Corning, Falcon®, catalog number: 351007 )
  4. Double sided ½ inch wide ‘Scotch’ tape
  5. Masking tape or similar (e.g., VWR, catalog number: 89097-920 )
  6. 25 x 75 mm, 1.0 mm thick microscope slides (e.g., VWR, catalog number: 48300-025 )
  7. 22 x 22-1.5 microscope cover glass (e.g., Fisher Scientific, Fisherbrand, catalog number: 12-541-B )
  8. White onion (Allium cepa L.) bulbs; we use the Cometa cultivar
  9. Distilled-deionized water (ddH2O)
  10. Sodium acetate trihydrate (e.g., VWR, catalog number: BDH9278-500G )
  11. HEPES (e.g., VWR, catalog number: 97061-826 )
  12. Hydrochloric acid (e.g., Merck, catalog number: HX0603-75 )
  13. Acetic acid (e.g., Merck, catalog number: AX0073-6 )
  14. HEPES buffer (20 mM, pH 6.8) (see Recipes)
  15. Sodium acetate buffer (20 mM, pH 4.5) (see Recipes)

Equipment

  1. Microwave oven, hot plate, or Bunsen burner
  2. pH meter (e.g., Corning, model: Model 430 )
  3. Analytical balance (e.g., Mettler Toledo, model: XPE105 )
  4. Fine forceps (e.g., Dumont No. 5)
  5. Flat tip forceps (e.g., Rose Scientific, catalog number: FF-001 )
  6. Custom 3 mm slicing jig (see Notes section)
  7. Microcomputer
  8. Data acquisition hardware (e.g., Data Translation, model: DT9801 )
  9. Constant-force extensometer (Figure 1) (Durachko and Cosgrove, 2009; Cosgrove, 2011; Cosgrove et al., 2017)
  10. Stress/strain analyzer (Figure 2) (Cosgrove, 2011; Cosgrove et al., 2017)


    Figure 1. The constant-force extensometer. A vertically adjustable, open clamping chamber fixes the bottom of the wall sample and is capable of holding liquid with a capacity of roughly 150 microliters. A second clamp is applied to the upper portion of the wall sample and is connected to a balanced lever. Displacement of the lever and connecting rod is monitored using a linear variable displacement transducer (LVDT), e.g., Schaevitz 050HR. Weights are applied to the lever to modulate the tension on the sample. Custom software displays and records displacement and calculated extension rates. We use a bank of eight of these units connected in parallel to a microcomputer via a data acquisition module (Data Translation DT9801).


    Figure 2. The device used for stress/strain assays. A. The vertical stage is controlled by a PC serial port stepper driver (e.g., AutomationDirect STP-DRV-4850), stepper motor (e.g., Lin Engineering 4218L-01D-02), geared belt drive, and lead screw assembly. Stage position is monitored with an LVDT. Tension is monitored with an s-type load cell (e.g., Futek LSB200 100 g). Custom software controls the strain rate and tension. Two clamps hold the sample being measured. The upper clamp is fixed to the movable stage. The lower clamp is fixed to the load cell. B. Close up of peel affixed between clamps (peel stained blue to aid visualization for this image). C. Alternate view of device noting specific pieces.

Software

  1. Custom software written using Microsoft Visual Basic is used for acquiring data and some data analysis. Microsoft Visual Basic may be freely downloaded at http://www.microsoft.com. Alternatively, many commercial software packages are available both free and for purchase to allow data acquisition and analysis

Procedure

  1. Onion peel preparation
    Here we describe the protocol for preparing a cell wall strip from the onion. This is obtained from the abaxial epidermis of onion scales (from the outer or convex surface of the scale). The abaxial epidermis adheres tightly to the underlying cells and so when one makes a peel the outer (periclinal) epidermal wall separates from the rest of the cell, resulting in a strip made up of the outer wall only. This contrasts with the peeling behavior of the adaxial epidermis (from the inner or concave surface of the onion scale), which is only weakly attached to the underlying cells and consequently separates as a whole-cell layer with cells living and intact, e.g., Hepworth and Bruce, 2004; Vanstreels et al., 2005; Suslov et al., 2009; Beauzamy et al., 2015.
    1. Purchase fresh white onions roughly the size of a baseball (between 6 and 10 cm in diameter).
    2. Remove and discard the outermost dry, brown layers (Figure 3A).
    3. The remainder will be readily separable into layers called scales. We refer to these scales from outermost (oldest) to innermost by number (outer to inner, 1, 2..., n) (Figure 3B).
    4. Use a single edge razor blade to slice the onion vertically to obtain tapered scale sections of roughly 3 to 4 cm at their widest dimension (Figure 3B).
    5. Remove scales 1 through 4, leaving scale 5 on the onion from which we obtain our peels. It is also possible to make peels from the other scales.
    6. Use the 3 mm slicing jig to make shallow (~0.5 mm deep, 3 mm wide) incisions on the outer (abaxial) surface of scale #5 along the onion vertical axis. These cuts should be roughly 3 cm long and are centered over the middle of the onion vertical axis (Figure 3C).
    7. Use a single edge razor blade to make three similar incisions perpendicular to existing ones. These three cuts should be roughly 1 cm apart and centered about the onion vertical axis. This allows for four peels per set of incisions (Figure 3D, lines indicate incisions, one peel per rectangle).


      Figure 3. Preparing an onion for obtaining peel strips. A. Remove dry layers; B. Excise to scale #5; C. Make vertical incisions; D. Make horizontal incisions.

    8. Using the flat tip forceps, gently work under one end of the four incised rectangles around 0.5 mm deep into the subdermal tissue. Pinch this tissue and use it to gently peel off the epidermal layer approximately halfway along the long axis of one of your four incised rectangles. Then, working from the other end of the rectangle, peel the remainder to get an intact rectangle of epidermal tissue with chunks of subdermal tissue adhering to both ends. Float peels on HEPES buffer in a Petri dish (Figure 4).


      Figure 4. Peeling strips from the onion (A) and floating them on HEPES buffer (20 mM, pH 6.8) (B)

    9. Strips ~1 cm long and 3 mm wide are now ready for use in biomechanical assays. For the assays described in this paper, we clamp both ends into one of two devices, with an initial length of 3 or 5 mm between clamps.

  2. Constant-force extensometer (creep) assay
    This is the procedure for measuring cell wall creep, e.g., mediated by catalysts for cell wall loosening (Cosgrove, 2016a).
    1. Ensure all units of extensometer are clean and ready for a new experiment.
    2. We typically use 15 to 17 g of tension (0.15-0.17 N) for 3 mm wide abaxial onion epidermal peels from scale #5. Wall strips from younger and older scales have different properties, so you may need to adjust the extensometer tensioning system by trial and error (sufficient tension to obtain good creep rates, not so much that wall breakage becomes a problem).
    3. Prepare an appropriate number of peels and proceed with loading as many units as you desire for your particular experiment. We find 8 to 16 replicates to be typically adequate to account for sample variability.
    4. Heat inactivation
      Note: Steps B4a-B4g are only used when one wants to inactivate endogenous wall enzymes and they occur before loading the peels in the extensometer.
      1. Place the peels between microscope slides and secure with a rubber band (Figure 5).


        Figure 5. Three peels between slides secured with a rubber band

      2. Put peels in slides into a heat-safe vessel with 100 ml of distilled water so that they can lay flat and be completely submerged.
      3. Place into a microwave oven on its highest setting and start the oven.
      4. Watch carefully and once a rolling boil is established allow samples to heat for 12 sec.
      5. Remove immediately and quench by placing the slides with peels into another vessel containing room temperature water.
      6. A Bunsen burner or hot plate may be used but alter the procedure as follows: allow the water to come to a boil and then submerge the peels in slides into the already boiling water. After 12 sec quench as for the microwave protocol.
      7. Once the microscope slides are cool, place the peels into buffer as dictated by experimental requirements.
    5. Remove one peel from the Petri dish and place it onto a cover glass, which serves as a carrier for the peel (Figure 6A).
    6. Position the peel at the top center of the cover glass (Figure 6B).
    7. Open the upper clamp with one hand.
    8. Insert one end of the peel into the upper clamp of the extensometer and clamp it (Figure 6C).
    9. Slide the cover glass down away from the upper end of the peel so as to allow it to hang within the lower clamp. Position the sample so as to clamp a 5 mm length of tissue between the clamps (Figure 6D).
    10. Clamp the lower clamp onto the peel.
    11. Add buffer to the freshly hung sample as dictated by experimental requirements (Figure 6E).


      Figure 6. Procedures of a uniaxial wall extension (creep) assay. A. Transfer peel from solution to cover glass; B. Position peel at top center of cover glass; C. Insert peel into upper clamp and remove cover glass; D. Insert peel into lower clamp; E. Add buffer to peel.

    12. Adjust the lower clamp assembly so as to ‘zero’ the displacement transducer into the lower portion of its range.
    13. Proceed with remaining empty units.
    14. Begin acquiring length data.
    15. After recording stable baseline rates, solutions covering the samples are exchanged as dictated by experimental needs, e.g., different pH or addition of enzymes such as expansin (Cosgrove, 2015 and 2016a) or endoglucanases (Park and Cosgrove, 2012) (Figure 7).


      Figure 7. Uniaxial cell creep extension of native onion epidermal cell wall induced by acidic buffer. Native wall strip (that is, not inactivated by heat) clamped at 10 g tension exhibits acid-induced creep when the initial neutral buffer (20 mM HEPES, pH 6.8) is exchanged for 20 mM sodium acetate, pH 4.5, at the point indicated by the arrow.

  3. Stress/strain assay
    Here we describe a protocol for quantifying the cell wall mechanical properties based on stress/strain curves (Cosgrove et al., 2017). The wall is stretched until a defined force is attained, then returned to original length and stretched a second time to the same force. The second stretch is reversible and thus provides a way to measure wall elasticity. The first stretch is partly irreversible (plastic) and so may be combined with the second stretch to calculate plasticity.
    1. Adjust the extensometer software for a strain rate of 3 mm per minute, which is continued until a tension of 98 mN is reached. This tension may need to be smaller or larger, depending on the treatment and history of the cell wall strip. It should be set low enough to avoid tearing of the wall strip or slipping in the clamp, but large enough to produce plastic extension.
    2. We configure our device to use a starting distance of either 3 or 5 mm between clamps. This length of sample is convenient and provides sufficient material from which we can obtain a magnitude of measurable biomechanical response we require for these procedures.
    3. Prepare an appropriate number of peels as needed for your particular experiment. We typically find 12 to 15 replicates to be adequate to account for sample variability.
    4. Steps C4a-C4g are only used when one wants to inactivate endogenous wall enzymes.
      1. Place the peels between microscope slides and secure with a rubber band.
      2. Put peels in slides into a heat-safe vessel with 100 ml of distilled water so that they can lay flat and be completely submerged.
      3. Place into a microwave oven on its highest setting and start the oven.
      4. Watch carefully and once a rolling boil is established allow samples to be heated for 12 sec.
      5. Remove immediately and quench by placing the slides with peels into another vessel containing room temperature water.
      6. A Bunsen burner or hot plate may be used but alter the procedure as follows: allow the water to come to a boil and then submerge the peels in slides into the already boiling water. After 12 sec quench as for the microwave protocol.
      7. Once the microscope slides are cool, place the peels back into buffer as dictated by experimental requirements.
    5. Remove one peel from the Petri dish and place it onto a cover glass, which serves as a carrier for the peel (Figure 6A).
    6. Position the peel at the top center of the cover glass (Figure 6B).
    7. Open the upper clamp with one hand.
    8. Refer to Figure 6 panels C and D for Steps 14 through 16 below. The device clamps are similar.
    9. Insert one end of the peel into the upper clamp of the device and clamp it.
    10. Slide the cover glass down away from the upper end of the peel so as to allow it to hang within the lower clamp.
    11. Clamp the lower clamp onto the peel.
    12. Adjust the device to take any slack out of the sample.
    13. Stretch the sample twice consecutively until a stress of 98 mN is reached each time (see Figure 8 and Videos 1-3 for examples of what the stretching looks like).


      Figure 8. Representative stress-strain (force-extension) measurement. An onion epidermal cell wall strip was stretched in two cycles to a 98 mN load twice at a strain rate of 3 mm min-1.

      Here are three movies illustrating the two sequential extension cycles for a stress/strain assay of onion epidermal wall strips.

      Video 1. An example of a moderately stiff wall

      Video 2. An example of a less-stiff wall

      Video 3. An example with slippage at the top clamp (this is an error condition)

Data analysis

  1. Extensometer data is gathered as follows: For each displacement transducer a position sample is captured every 30 sec. Those thirty samples are then averaged and saved to a data file as a single average position data point. Each single extension rate data point is calculated from 3 consecutive position points and saved to the same data file.
  2. For stress/strain analysis the slope of each stress/strain line is calculated by fitting the final 10% of the data gathered up until the target stress is reached using a linear least squares fit. The slope of the line (% extension versus force) is known as a compliance (the reciprocal of stiffness or modulus).
  3. The slope of the second stretch represents the elastic compliance (% extension/change in tensile force). The slope of the first stretch represents the total (elastic + plastic) compliance. The plastic compliance may be obtained by subtracting the elastic compliance from the total compliance. 

Notes

  1. Incubation times for peels should always be minimized to attenuate any changes which might be caused by endogenous biological activities, unless that is the aim of the experiment. We also keep our samples on ice prior to use whenever it seems appropriate.
  2. Making the custom 3 mm slicing jig (Figure 9): This jig makes 3 mm wide peels. You will need three double edge razor blades, double stick ‘Scotch’ tape, masking tape (or similar) and 8 standard microscope slides (25 x 75 mm, 1.0 mm thick). On a clean, flat surface lay out the slides and razor blades. Put two pieces of double stick tape side-by-side on the upper surface of each slide to almost completely cover the slide surface. Place one blade near the end of a slide keeping a cutting edge parallel to the slide and protruding 5 mm from the edge of the slide. Place three slides on top of the first blade. Follow with another blade, 3 more slides, the final blade, and then the final slide. Be careful to assemble the parts in an orthogonal manner. You may wish to wrap the ends of the jig with additional tape.


    Figure 9. Custom 3 mm slicing jig

  3. When performing stress/strain analyses proceed at a pace so as not to allow tissue samples to dehydrate. Small amounts of buffer may be applied using a cotton-tipped swab or pipet if needed after clamping tissue and prior to collecting data.

Recipes

  1. HEPES buffer (20 mM, pH 6.8)
    Dissolve 4.77 g of (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in 0.95 L of ddH2O, adjust to pH 6.8 with 1 N HCl, and add ddH2O to make a final volume of 1 L
  2. Sodium acetate buffer (20 mM, pH 4.5)
    Dissolve 1.64 g of sodium acetate trihydrate in 0.95 L ddH2O and adjust to pH 4.5 with 1 M acetic acid, and add ddH2O to make a final volume of 1 L

Acknowledgments

We thank Ed Wagner, Dr. Sarah Kiemle and Xuan Wang for technical assistance. This work was supported by the Center for Lignocellulose Structure and Formation, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Basic Energy Sciences (grant No. DE-SC0001090). This protocol is adapted from Zhang et al. (2017). The authors declare no conflicts of interest or competing interests.

References

  1. Beauzamy, L., Derr, J. and Boudaoud, A. (2015). Quantifying hydrostatic pressure in plant cells by using indentation with an atomic force microscope. Biophys J 108(10): 2448-2456.
  2. Cosgrove, D. J. (2011). Measuring in vitro extensibility of growing plant cell walls. Methods Mol Biol 715: 291-303.
  3. Cosgrove, D. J. (2015). Plant expansins: diversity and interactions with plant cell walls. Curr Opin Plant Biol 25: 162-172.
  4. Cosgrove, D. J. (2016a). Catalysts of plant cell wall loosening. F1000Res 5.
  5. Cosgrove, D. J. (2016b). Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes. J Exp Bot 67(2): 463-476.
  6. Cosgrove, D. J., Hepler, N. K., Wagner, E. R. and Durachko, D. M. (2017). Measuring the biomechanical loosening action of bacterial expansins on paper and plant cell walls. Methods Mol Biol 1588: 157-165.
  7. Durachko, D. M. and Cosgrove, D. J. (2009). Measuring plant cell wall extension (creep) induced by acidic pH and by alpha-expansin. J Vis Exp(25): 1263.
  8. Hepworth, D. G. and Bruce, D. M. (2004). Relationships between primary plant cell wall architecture and mechanical properties for onion bulb scale epidermal cells. J Texture Stud 35: 586-602.
  9. Kim, K., Yi, H., Zamil, M. S., Haque, M. A. and Puri, V. M. (2015). Multiscale stress-strain characterization of onion outer epidermal tissue in wet and dry states. Am J Bot 102(1): 12-20.
  10. Park, Y. B. and Cosgrove, D. J. (2012). A revised architecture of primary cell walls based on biomechanical changes induced by substrate-specific endoglucanases. Plant Physiol 158(4): 1933-1943.
  11. Suslov, D., Verbelen, J. P. and Vissenberg, K. (2009). Onion epidermis as a new model to study the control of growth anisotropy in higher plants. J Exp Bot 60(14): 4175-4187.
  12. Vanstreels, E., Alamar, A. C., Verlinden, B. E., Enninghorst, A., Loodts, J. K. A., Tijskens, E., Ramon, H. and Nicolai, B. M. (2005). Micromechanical behaviour of onion epidermal tissue. Postharvest Biol Tec 37: 163-173.
  13. Wilson, R. H., Smith, A. C., Kacurakova, M., Saunders, P. K., Wellner, N. and Waldron, K. W. (2000). The mechanical properties and molecular dynamics of plant cell wall polysaccharides studied by Fourier-transform infrared spectroscopy. Plant Physiol 124(1): 397-405.
  14. Zhang, T., Vavylonis, D., Durachko, D. M. and Cosgrove, D. J. (2017). Nanoscale movements of cellulose microfibrils in primary cell walls. Nat Plants 3: 17056.
  15. Zheng, Y., Cosgrove, D. J. and Ning, G. (2017). High-resolution field emission scanning electron microscopy (FESEM) imaging of cellulose microfibril organization in plant primary cell walls. Microsc Microanal 23(5): 1048-1054.

简介

在这里,我们描述了两个实验协议来衡量的主要(成长)植物细胞壁的生物力学性能,重点是分析洋葱鳞片的细胞壁表皮带。 第一个协议测量细胞壁蠕变(时间依赖的不可逆扩展)在恒定的力量下。 这种蠕变通常由膨胀素或选择性内切葡聚糖酶的壁松弛作用介导。 第二种方案是基于两个连续的壁延伸并测量壁的弹性和塑性顺从性,这取决于细胞壁结构。 这两个测定提供了可能与细胞壁结构和细胞膨胀生长相关的补充信息。

【背景】生长的植物细胞的主壁足够强以抵抗由细胞膨压产生的张力,但由于扩展蛋白或其它不可逆细胞壁增大所必需的催化剂的选择性松动作用,细胞生长期间可能不可逆地膨胀(Cosgrove, 2016a和2016b)。细胞壁力学性质(如弹性和可塑性)的评估对于理解细胞壁结构及其在生长(细胞增大)和生长停止后的改变是重要的。墙体力学的一个重要测量方法是基于单轴拉伸试验,如我们在测量弹性和塑性柔量(柔量是模量的倒数;模量是刚度的度量)的应力/应变试验中所述。另一种互补测定法是基于不可逆的,随时间增长的长度(蠕变),其在原始细胞壁中发生,当它们保持恒定的张力并且被内源性膨胀素或外源内切葡聚糖酶持续松弛时(Durachko和Cosgrove,2009; Cosgrove ,2011; Cosgrove等人,2017)。在这个协议中,我们描述了从洋葱鳞片制备表皮细胞壁条的程序,并在这两个测定中测试它们。洋葱表皮细胞壁为研究细胞壁结构与生物力学之间的联系提供了有用的模型(Wilson等人,2000; Suslov等人,2009; Kim等人等人,2015; Zhang等人,2017; Zheng等人,2017)。

关键字:细胞壁, 力学, 弹性模量, 塑性模量, 蠕变, 洋葱表皮

材料和试剂

  1. 单刃剃须刀刀片( ,VWR,目录号:55411-050)
  2. 双刃剃刀刀片(例如,安全剃刀公司,目录号:74-0002)
  3. 一次性培养皿(如,Corning,Falcon ,产品目录号:351007)
  4. 双面半英寸“Scotch”胶带
  5. 美纹纸或类似物(例如,,VWR,目录号:89097-920)
  6. 25×75mm,1.0mm厚的显微镜载玻片(例如,VWR,目录号:48300-025)
  7. 22×22-1.5显微镜覆盖玻璃(例如,Fisher Scientific,Fisherbrand,目录号:12-541-B)
  8. 白洋葱(洋葱cepa L.)灯泡;我们使用Cometa品种
  9. 蒸馏去离子水(ddH2O)
  10. 醋酸钠三水合物(例如,VWR,目录号:BDH9278-500G)
  11. HEPES( ,VWR,目录号:97061-826)
  12. 盐酸(例如,Merck,目录号:HX0603-75)
  13. 醋酸(如emck,Merck,目录号:AX0073-6)
  14. HEPES缓冲液(20 mM,pH 4.5)(见食谱)
  15. 醋酸钠缓冲液(20毫米,pH值6.8)(见食谱)

设备

  1. 微波炉,电热板或本生燃烧器
  2. pH计(例如,Corning,型号:Model 430)
  3. 分析天平(例如,梅特勒 - 托利多,型号:XPE105)
  4. 细钳( ,Dumont 5号)
  5. 扁尖钳(,例如,Rose Scientific,目录号:FF-001)
  6. 自定义3毫米切片夹具(见注释部分)
  7. 微型计算机
  8. 数据采集硬件(例如,数据翻译,型号:DT9801)
  9. 恒力引伸计(图1)(Durachko和Cosgrove,2009; Cosgrove,2011; Cosgrove等,2017)
  10. 应力/应变分析仪(图2)(Cosgrove,2011; Cosgrove et al。,2017)


    图1.恒力引伸计垂直可调的敞开式夹紧室固定壁样品的底部,并能够容纳大约150微升容量的液体。第二个夹具被应用到壁样品的上部并连接到平衡杆。使用线性可变位移传感器(LVDT)(例如Schaevitz 050HR)监测杠杆和连杆的位移。重量被应用到杠杆来调节样品上的张力。自定义软件显示并记录位移和计算的延伸率。我们通过数据采集模块(Data Translation DT9801)将一组八个这些单元并联连接到一台微型计算机上。


    图2.用于应力/应变测定的装置A.垂直平台由PC串行端口步进驱动器(例如,AutomationDirect STP-DRV-4850 ),步进电机(例如Lin Engineering 4218L-01D-02),齿轮传动带和导螺杆组件。使用LVDT监视舞台位置。用s型称重传感器(例如Futek LSB200 100克)监测张力。定制软件控制应变率和张力。两个夹子夹住正在测量的样品。上夹具固定在可移动台上。下夹具固定在称重传感器上。 B.粘贴在夹子之间的果皮的特写(剥去蓝色以帮助可视化该图像)。 C.备用视图的设备注意特定的部分。

软件

  1. 使用Microsoft Visual Basic编写的自定义软件用于获取数据和一些数据分析。 Microsoft Visual Basic可以在 http://www.microsoft.com 上免费下载。另外,许多商业软件包可以免费和购买,以便进行数据采集和分析

程序

  1. 洋葱皮的准备
    这里我们描述从洋葱上制备细胞壁条的方案。这是从洋葱鳞片的外表面(从鳞片的外表面或凸面)获得的。下表皮紧紧地粘附在下面的细胞上,因此当一个脱落时,外(表皮)表皮壁与细胞的其余部分分离,导致仅由外壁组成的条带。这与近轴表皮(从洋葱鳞片的内表面或凹表面)的剥离行为形成对比,所述表皮仅与基础细胞弱附着,因此分离为具有活细胞和完整细胞的全细胞层,例如Hepworth和Bruce,2004; Vanstreels et al。,2005; Suslov 等。,2009; Beauzamy 等。,2015.
    1. 购买新鲜的白洋葱,大概是棒球的大小(直径在6到10厘米之间)。
    2. 删除并丢弃最外层的干燥棕色层(图3A)。
    3. 其余的将很容易分成称为尺度的层。我们指的是从最外层(最老的)到最内层的这些尺度(从外到内,1,2,...,n)(图3B)。
    4. 使用一个单一的边缘刀片垂直切洋葱,以获得最大尺寸大约3至4厘米的锥形部分(图3B)。
    5. 去掉秤1到4,在洋葱上留下刻度5,从中获得我们的果皮。也可以从其他尺度制作果皮。
    6. 使用3毫米的切片夹具沿洋葱垂直轴在刻度#5的外部(远轴)表面上制作浅的(〜0.5毫米深,3毫米宽)切口。这些削减应该是大约3厘米长,并在洋葱垂直轴的中间(图3C)。
    7. 使用一个单刃剃刀刀片,使三个相似的切口垂直于现有的。这三个切口应该大致相距1厘米,并以洋葱垂直轴为中心。这允许每组切口四个皮(图3D,线表示切口,每个矩形一个皮)。


      图3.准备一个洋葱获得剥离带A.去除干燥的层; B.消费量表#5; C.做垂直切口; D.做横向切口。

    8. 使用平头镊子,在深度约0.5mm的四个切开的矩形的一端下面轻轻地进入皮下组织。捏住这个组织,用它轻轻剥离表皮层,沿着四个切开的矩形之一的长轴的大约一半。然后,从矩形的另一端开始工作,将剩下的部分剥去,得到一个完整的表皮组织矩形,两侧粘附着皮下组织块。在培养皿中的HEPES缓冲液上漂浮(图4)。


      图4.从洋葱(A)剥离条带,并将其漂浮在HEPES缓冲液(20mM,pH 6.8)(B)上

    9. 〜1厘米长,3毫米宽的条纹现在可以用于生物力学分析。对于本文中描述的测定,我们将两端夹在两个装置中的一个中,夹具之间的初始长度为3或5mm。

  2. 恒力引伸计(蠕变)分析
    这是测量细胞壁蠕变的过程,例如由催化剂介导的细胞壁松动(Cosgrove,2016a)。
    1. 确保引伸计的所有单位都干净,准备好进行新的实验。
    2. 我们通常使用15到17克的张力(0.15-0.17 N),用于5号刻度的3毫米宽的洋葱表皮。年轻和年龄较大的标尺带有不同的属性,因此您可能需要通过反复试验来调整引伸计张紧系统(足够的张力以获得良好的蠕变速率,而不是墙壁破损成为问题)。
    3. 准备适当数量的果皮,然后按照您对特定实验的要求装入尽可能多的单位。我们发现8到16次重复通常足以解释样本的变化。
    4. 热灭活
      注意:步骤B4a-B4g仅在想要使内源性壁酶失活时才使用,并且在引伸计上装载剥离之前发生它们。
      1. 放置在显微镜幻灯片之间的果皮,并用橡皮筋固定(图5)。


        图5.用橡皮筋固定的幻灯片之间的三个剥离

      2. 将切片中的果皮放入装有100毫升蒸馏水的热保险容器中,使其可以平放并完全浸没。

      3. 放入微波炉的最高位置,启动烤箱。
      4. 仔细观察,一旦滚动沸腾建立允许样品加热12秒。

      5. 立即取出,并将带有剥离片的载玻片放入另一个含有室温水的容器中淬火。
      6. 可以使用本生灯或电热板,但是可以按照以下步骤改变步骤:让水沸腾,然后将滑板中的果皮浸入已经沸腾的水中。
        12秒钟后,如同微波协议
      7. 一旦显微镜载玻片凉快,根据实验要求的规定将果皮放入缓冲液。
    5. 从培养皿中取出一片果皮,放在盖玻片上,作为果皮的载体(图6A)。
    6. 将果皮放在盖玻璃的顶部中央(图6B)。

    7. 打开上方的夹子

    8. 将果皮的一端插入引伸计的上夹钳并夹紧(图6C)
    9. 将盖玻片向下滑动,使其远离果皮的上端,以便将其挂在下夹具内。定位样品,以夹紧夹子之间5mm长度的组织(图6D)。
    10. 将下夹钳夹在皮上。
    11. 按照实验要求(图6E)的规定,将缓冲液添加到新悬挂的样品中。


      图6.单轴延伸(蠕变)测定的程序A.转移溶液中的剥离物以覆盖玻璃; B.在玻璃盖顶部中心位置剥离; C.将剥离物插入上夹钳并取下盖玻片; D.将果皮插入下夹具; E.添加缓冲去皮。

    12. 调整下夹具组件,以便将位移传感器“归零”到其范围的下部。
    13. 继续使用剩余的空单元。
    14. 开始获取长度数据。
    15. 在记录稳定的基线速率之后,覆盖样品的溶液根据实验需要进行交换,例如不同的pH或添加酶如膨胀蛋白(Cosgrove,2015和2016a)或内切葡聚糖酶(Park和Cosgrove ,2012)(图7)。


      图7.由酸性缓冲液诱导的天然洋葱表皮细胞壁的单轴细胞蠕变延伸在10g张力下夹住的天然条带(即未被热灭活)表现出酸诱导的蠕变,中性缓冲液(20mM HEPES,pH6.8)在箭头所示的位置交换20mM乙酸钠,pH4.5。

  3. 应力/应变分析
    在这里,我们描述了基于应力/应变曲线量化细胞壁力学性质的方案(Cosgrove等人,2017)。墙被拉伸直到达到规定的力,然后恢复到原来的长度,第二次拉伸到相同的力量。第二次拉伸是可逆的,因此提供了一种测量壁弹性的方法。第一次拉伸部分是不可逆的(塑料),所以可以结合第二次拉伸计算可塑性。
    1. 调整引伸计软件的应变速率为每分钟3毫米,直到达到98毫牛的张力。这种张力可能需要更小或更大,这取决于细胞壁条的处理和历史。它应该设置得足够低,以避免撕裂墙壁条或在夹具中滑动,但是足够大以产生塑性延伸。
    2. 我们配置我们的设备使用夹子之间3或5毫米的起始距离。这个长度的样本是方便的,并提供足够的材料,从中我们可以获得这些程序所需的可测量的生物力学响应的量级。
    3. 根据您的特定实验需要准备适量的果皮。我们通常发现12到15次重复是足以解释样本变异的。
    4. 步骤C4a-C4g仅在想要灭活内源性壁酶时使用。
      1. 将显微镜载玻片之间的皮肤和橡皮筋固定。
      2. 将切片中的果皮放入装有100毫升蒸馏水的耐热容器中,使其可以平放并完全浸没。

      3. 放入微波炉的最高位置,启动烤箱。
      4. 仔细观察,一旦滚动沸腾建立允许样品加热12秒。

      5. 立即取出,并将带有剥离片的载玻片放入另一个含有室温水的容器中淬火。
      6. 可以使用本生灯或电热板,但是可以按照以下步骤改变步骤:让水沸腾,然后将滑板中的果皮浸入已经沸腾的水中。
        12秒钟后,如同微波协议
      7. 一旦显微镜载玻片凉快,根据实验要求,将皮放回缓冲液。
    5. 从培养皿中取出一片果皮,放在盖玻片上,作为果皮的载体(图6A)。
    6. 将果皮放在盖玻璃的顶部中央(图6B)。

    7. 打开上方的夹子
    8. 下面的步骤14至16参考图6的面板C和D.设备夹是相似的。

    9. 将果皮的一端插入设备的上部夹子并夹紧

    10. 。将玻璃盖从玻璃杯的上端向下滑动,以便将其挂在下夹具上。
    11. 将下夹钳夹在皮上。
    12. 调整设备从样品中取出任何松弛。
    13. 连续拉伸样品两次,直到每次达到98 mN的应力(参见图8和视频1-3,了解拉伸的例子)。


      图8.典型的应力 - 应变(力 - 拉伸)测量将洋葱表皮细胞壁条带以两个周期拉伸至98mN载荷两次,应变速率为3mm min- 1 。

      这里有三个电影说明了洋葱表皮壁带的压力/应变测定的两个连续的延伸周期。

      视频1

      视频2

      视频3

数据分析

  1. 引伸计数据收集如下:对于每个位移传感器,每30秒捕获一个位置样本。然后将这30个样本平均并作为单个平均位置数据点保存到数据文件中。每个扩展速率数据点都是从3个连续的位置点计算出来的,并保存到同一个数据文件中。
  2. 对于应力/应变分析,每个应力/应变线的斜率通过拟合最后10%的数据来计算,直到使用线性最小二乘拟合达到目标应力。线的斜率(伸长%对力)被称为顺应性(刚度或模量的倒数)。
  3. 第二次拉伸的斜率表示弹性顺应性(拉伸力的%伸长/变化)。第一次拉伸的斜率表示总(弹性+塑性)顺应性。

    塑性顺应性可以通过从总顺应性减去弹性顺应性来获得

笔记

  1. 果皮的孵化时间应该总是最小化,以减弱可能由内生生物活动引起的变化,除非这是实验的目的。
    在使用之前,我们也会将样品保存在冰上
  2. 制作定制的3毫米切片夹具(图9):该夹具制作3毫米宽的果皮。您将需要三个双刃刀片,双面“Scotch”胶带,遮蔽胶带(或类似物)和8个标准显微镜载玻片(25 x 75 mm,1.0 mm厚)。在一个干净,平坦的表面上放置幻灯片和剃刀刀片。将两块双面胶带并排放在每个滑板的上表面上,几乎完全覆盖滑板表面。将一个刀片放置在滑块末端附近,使切削刃平行于滑块,并从滑块边缘突出5 mm。在第一个刀片上放置三张幻灯片。跟着另一个刀片,3个更多的幻灯片,最后的刀片,然后是最后一张幻灯片。小心以正交方式组装零件。您可能希望用另外的胶带包裹夹具的两端。


    图9.自定义3毫米切片夹具

  3. 进行应力/应变分析时,应该保持一定的速度,以免组织样本脱水。
    少量缓冲液可以使用棉签或吸管,如果需要夹紧组织,然后收集数据。

食谱

  1. HEPES缓冲液(20 mM,pH 4.5)
    将4.77g(4-(2-羟乙基)-1-哌嗪乙磺酸)溶于0.95L ddH 2 O中,用1N HCl调pH至6.8,加入ddH 2 / sub> O来做1 L的最终音量
  2. 醋酸钠缓冲液(20 mM,pH 6.8)
    将1.64g三水合乙酸钠溶于0.95μLddH 2 O中,用1M乙酸调节至pH 4.5,加入ddH 2 O使终体积为1 L

致谢

我们感谢Ed Wagner,Sarah Kiemle博士和Xuan Wang的技术支持。这项工作得到了由美国能源部科学基础能源科学局资助的能源前沿研究中心木质纤维素结构和形成中心(批准号:DE-SC0001090)的支持。这个协议是从Zhang et al 改编的。 (2017年)。作者声明不存在利益冲突或利益冲突。

参考

  1. Beauzamy,L.,Derr,J。和Boudaoud,A。(2015)。 通过使用原子力显微镜的压痕来量化植物细胞中的静水压力 Biophys J 108(10):2448-2456。
  2. Cosgrove,D.J。(2011)。 测量生长的植物细胞壁的体外延伸性方法Mol Biol 715:291-303。
  3. Cosgrove,D.J。(2015)。 植物扩张蛋白:与植物细胞壁的多样性和相互作用 Curr Opin Plant生物学25:162-172。
  4. Cosgrove,D.J。(2016a)。 植物细胞壁松动的催化剂 F1000Res 5。
  5. Cosgrove,D. J.(2016b)。 植物细胞壁延伸性:连接植物细胞生长与细胞壁结构,力学和墙的作用 - 修饰酶。 J Exp Bot 67(2):463-476。
  6. Cosgrove,D.J.,Hepler,N.K。,Wagner,E.R。和Durachko,D.M。(2017)。 测量细菌扩展蛋白在纸张和植物细胞壁上的生物力学松动作用 Methods Mol Biol 1588:157-165。
  7. Durachko,D.M。和Cosgrove,D.J。(2009)。 测量由酸性pH和α-膨胀素引起的植物细胞壁延伸(蠕变) J Vis Exp (25):1263。
  8. Hepworth,D.G。和Bruce,D.M。(2004)。 植物细胞壁结构与洋葱灯泡机械特性的关系鳞状的表皮细胞。 <纹理螺柱 35:586-602。
  9. Kim,K.,Yi,H.,Zamil,M.S。,Haque,M.A。和Puri,V.M。(2015)。 湿润和干燥状态下洋葱外表皮组织的多尺度应力 - 应变特征 Am J Bot 102(1):12-20。
  10. Park,Y.B和Cosgrove,D.J。(2012)。 基于由底物特异性内切葡聚糖酶诱导的生物力学变化的原代细胞壁的修改的体系结构植物生理学 158(4):1933-1943。
  11. Suslov,D.,Verbelen,J.P.和Vissenberg,K。(2009)。 洋葱表皮作为研究高等植物生长各向异性控制的新模式。 J Exp Bot 60(14):4175-4187。
  12. Vanstreels,E.,Alamar,A.C.,Verlinden,B.E。,Enninghorst,A.,Loodts,J.K.A.,Tijskens,E.,Ramon,H.and Nicolai,B.M。(2005)。 洋葱表皮组织的微观机械行为 采后生物技术 37:163-173。
  13. Wilson,R.H.,Smith,A.C.,Kacurakova,M.,Saunders,P.K。,Wellner,N.and Waldron,K.W。(2000)。 傅里叶变换红外光谱研究植物细胞壁多糖的力学性质和分子动力学。植物生理学(Plant Physiol)124(1):397-405。
  14. Zhang,T.,Vavylonis,D.,Durachko,D.M。和Cosgrove,D.J。(2017)。 原生细胞壁中纤维素微原纤维的纳米级移动 Nat植物<
    3:17056
  15. Zheng,Y.,Cosgrove,D.J。和Ning,G。(2017)。 植物原代细胞壁中纤维素微原纤维组织的高分辨率场发射扫描电子显微镜(FESEM)成像。 Microsc Microanal 23(5):1048-1054。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Durachko, D. M., Park, Y., Zhang, T. and Cosgrove, D. J. (2017). Biomechanical Characterization of Onion Epidermal Cell Walls. Bio-protocol 7(24): e2662. DOI: 10.21769/BioProtoc.2662.
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William Nicolas
California Institute of Technology
Dear authors, I am currently trying to produce onion epidermal cell wall peels, exactly like in your Nature plants paper. This brought me to your detailed Bio-protocol paper. First of all, The pH at which the HEPES solution must be is not clear at all. In the materials in reagents section, it is said to be pH4.5, then in the recipe title, it is again 4.5 but in the description you say bring it to 6.8. Then figure 4, you say the peels are floating in HEPES buffer pH6.8. Moreover in the associated Nature plants paper, you say make a HEPES buffer solution pH7 + Tween-20 0.1%, which is not mentionned in this present protocol. I am especially asking this question because my first trials went quite well during the peeling stage, but when layed in the buffer, the peels would inevitably curl up into little tubes really hard to lay flat on a microscopy slide and the fact that this behaviour is not mentionned in your protocol leads me to think that you have not encountered such problem. I am thus suspecting my buffer, which does not contain the Tween-20 and is at pH6.8. In brief can you clear out the HEPES and sodium acetate buffer recipes please? Cheers! William Nicolas
1/24/2018 9:48:51 AM Reply
Daniel Cosgrove
Department of Biology and Center for LignoCellulose Structure and Formation, 208 Mueller Laboratory, Pennsylvania State University, USA, USA,

Thanks for your query and we are happy to clarify. In the Materials and Recipes sections, the pH of the HEPES and acetate buffers were incorrectly amended during the last stage of bio-protocol publication, and we missed this editorial change. HEPES buffer should be pH 6.8 and acetate buffer should be pH 4.5, throughout. Sorry for the confusion and we will ask bio-protocol to correct these typos. About curling: indeed the peeled strips have a strong tendency to curl. As described in step A.8 and shown in Figure 4B, it is important to leave chunks of subepidermal tissue at the two ends of the peel (like barbells). This helps a lot with the handling. Also, when you float the peels on a solution, ALWAYS place them with cuticle side up (facing the air). If you place the cuticle side down, they will curl. The power of surface tension of water! With a fine pair of forceps they can be uncurled under a dissecting scope, but it is challenging. The issue with curling should have been made clearer in the protocol. The curling behavior is not likely related to the solution composition. Also, we note that the tendency to curl varies greatly among onions, so try more than one onion. Good luck.

1/25/2018 8:05:56 AM


Ok thanks a lot, putting them with the cuticle facing up fixed the curling problem. Staining with DAPI also seems to indeed show the absence of the nuclei in the peeled part. The nuclei nonetheless remain in the thicker bordering parts, which is good. Thanks for the advice ! William Nicolas

1/25/2018 5:29:31 PM