Tensile Testing Assay for the Measurement of Tissue Stiffness in Arabidopsis Inflorescence Stem
拉伸强度测试用于检测拟南芥花序轴组织刚度   

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Nature Plants
Oct 2018

 

Abstract

Lignocellulosic biomass is a versatile renewable resource for fuels, buildings, crafts, and biomaterials. Strategies of molecularly designing lignocellulose for industrial application has been developed by the discoveries of novel genes after the screenings of various mutants and transformed lines of Arabidopsis whose cell walls could be modified in the inflorescence stem, a model woody tissue. The mechanical properties are used as a quantitative index for the chemorehological behavior of the genetically modified cell wall in the tissue. This parameter can be measured with tensile or bending tests of tissue explants, the vibration analysis of tissue behavior or using atomic force microscopy to probe the tissue surface. Here, we describe in detail the procedure to determine the stiffness of methanol-fixed, rehydrated and pronase-treated inflorescence explants with a tensile testing machine based on classical methods for the determination of cell wall extensibility.

Keywords: Arabidopsis (拟南芥), Cell wall (细胞壁), Compliance (服从), Inflorescence stem (花序轴), Mechanical properties (机械性能), Stiffness (刚度), Tensile tests (拉伸强度测试), Young’s modulus (杨氏模量)

Background

Plant biomechanics can be measured with different methods such as a tensile test (Burgert et al., 2003; Cosgrove, 2011), a bending test (Shah et al., 2017), vibration analysis (Niklas and Moon, 1988, Nakata et al., 2018) and atomic force microscopy (Radotić et al., 2012). These techniques are available to quantify the stiffness and strength properties of materials. Stiffness is defined by the force required to displace (stretch) the material over a unit length, while the strength is regarded as the amount of force required to ultimately rupture the material. The stiffness of a structure is affected by the material properties such as lignocellulosic composition and the bio-structural geometry such as a thickness of cell wall (Shah et al., 2017). The biomechanical tests designed to measure these properties are frequently used in timber grading for commercial sale as well as in tree breeding programs to select and breed superior trees with increased stiffness and strength. Such tests can be also used in Arabidopsis thaliana to identify candidate genes with effects on strength and stiffness in mutant screening as a wood model (Strabala and MacMillan, 2013, Figures 1A-1C). In fact, the tensile test was used to estimate the stiffness of inflorescence stems in Arabidopsis in our previous study (Sakamoto et al., 2018). The protocol in this article provides the procedure to measure the stiffness of mature regions of Arabidopsis inflorescence stem with a tensile testing machine (Figure 1D). It is based on the method to measure the cell wall extensibility (compliance) of tissue explants of dicot seedling stems and monocot coleoptiles (Olson et al., 1965; Cleland, 1967) performed intensively between the 1960s and the 1980s (Taiz, 1984; Masuda, 1990). This has been optimized to the determination of the stiffness of inflorescence stem in Arabidopsis and described in this article.


Figure 1. Overview of tensile test to measure the tissue stiffness of a model woody tissue in Arabidopsis. A. Cross sections of mature regions of Arabidopsis inflorescence stem as a model woody tissue. B and C. Stained cross section with Mäule reagent. Interfascicular fiber (If), Vascular bundles (vb), xylem vessels (xv), interfascicular xylem fibers (ixf). Bars indicate 200 μm. D. Flow chart of tensile testing assay for the measurement of tissue stiffness in Arabidopsis inflorescence stem.

Materials and Reagents

  1. Transparent tape
  2. PYREX® 55 ml Screw Cap Culture Tubes with PTFE Lined Phenolic Caps, 25 x 150 mm (Corning Co. Ltd., catalog number: 9826-25)
  3. Plastic transfer pipet (Transparent LDPE, 4 ml, Thin stem, Thermo Fisher Scientific Co. Ltd., catalog number: 13459118)
  4. Razor blade (Hi-Stainless Single Edge Blade, FEATHER Safety Razor Co. Ltd., catalog number: FHS-10)
  5. Plastic Petri dish (90 mm x 14.2 mm) (Thermo Fisher Scientific Co. Ltd., catalog number: 5184E)
  6. Kimtowel wipes (Kimberly-Clark Co. Ltd.)
  7. Kimwipe paper strips (Kimberly-Clark Co. Ltd., ca. 2 mm x ca.10 mm in length) (Figure 2)
  8. 2 ml microtube (Eppendorf Co. Ltd., catalog number: 0030120094)
  9. Aluminum foil
  10. Inflorescence stem of 8 week-old Arabidopsis thaliana
  11. Methanol (Kishida Chemical Co. Ltd. Japan, catalog number: 000-48666)
  12. KH2PO4 (Nakalai Co. Ltd. Japan, catalog number: 09582-75)
  13. K2HPO4 (Nakalai Co. Ltd. Japan, catalog number: 09583-65)
  14. Pronase E (PE) from Streptomyces griseus (Product name is “Actinase E”. Nacalai Co. Ltd. Japan, catalog number: 29003-51), store at 4 °C
  15. SDW: Sterilized ultrapure water (e.g., Milli-Q)
  16. Potassium phosphate buffer for PE solution (see Recipes)
  17. PE solution (see Recipes)


    Figure 2. Kimwipe paper strips used to fix sample with the clamps (ca. 2 mm x 10 mm in length, hand-made)

Equipment

  1. Tensile testing machine for max. 500 Newton (N) (T.S.E. Co. Ltd. Yokohama, Japan, Auto Com Series, catalog number: AC-500N-CM, Figure 3A, see Note 1) 
  2. Load cell for max.10 N (T.S.E. Co. Ltd., Catalog number: TC-10N-B) (Figure 3A)
  3. Upper and lower fiber clamps in lever action (Figures 3B and 3C, Clip jaw 5N, A & D Co. Ltd., Tokyo, Japan, catalog number: J-TZM-5N)


    Figure 3. Tensile testing machine and its clamps. A. Tensile testing machine for maximal 500 N force. B. Removable clamp with a hook and fixed clamp. Set the zero point of the load in this position of the removable clamp. C. Top-removable and bottom-fixed fiber clamps. The clamp can be opened by lever action and sample should be fixed with hand screw tightly.

  4. Oven (37 °C or 60 °C, EYELA Co. Ltd., Japan, Forced Air Flow Oven, WFO420)
  5. Water bath (TR-1α, ASONE Co. Ltd., Japan, catalog number: 1-5832-31)
  6. Chemical fume hoods
  7. Analytical balance (Mettler Toledo Co. Ltd., Balance XPR26, catalog number: 30355476)
  8. Universal Anti-Static Kit U-electrode (Mettler Toledo Co. Ltd., catalog number: 11107767)
  9. Sharp-Pointed Dissecting Scissor (Thermo Fisher Scientific. Co. Ltd., catalog number: 08-940)
  10. Digital camera
  11. Ruler (up to 150 mm)
  12. Tweezer (EBL Co. Ltd., model: #202)

Software

  1. Operation and analysis software for the tensile testing machine (T.S.E. Co. Ltd., Yokohama, Japan, UTPS-STD Single for windows 10, English version is available)
  2. Microsoft Excel (https://products.office.com/en-au/excel)

Procedure

  1. Plant material preparation (requires 2-3 days after the harvest of tissue) 
    1. Excise the basal parts (roughly 30 mm from bottom of stem) of inflorescence stems from 8 week-old Arabidopsis with scissors (Figures 4A-4C) and immediately and completely submerged the whole part of explant in 40 ml methanol in PYREX® 55 ml screw cap culture tubes. Plants are grown under 16-h light (60-80 μmol m-2 s-1)/8-h dark cycle in a constant 23 °C plant room with no humidity control.
      Note: More than fifteen explants of inflorescence stem are necessary for the evaluation of one plant line (see Note 2).


      Figure 4. Stem segment taken from basal part of the inflorescence stem of Arabidopsis. A. Eight-weeks old plant grown in soil. The basal end was cut at the position of red arrowhead with a pair of scissors. B. Harvested and fresh inflorescence stem. Excised stem segment which should be cut at the position of arrowhead. All branches should be removed. C. Digested fresh segment of inflorescence stem which should be submerged into methanol immediately. D. 30 mm-length inflorescence-stem segment after boiled in methanol, rehydration and Pronase E. Treatment. Longitudinal and vertical lengths of each sample should be determined later by Image J.

    2. Close the cap of culture tube tightly to avoid a methanol explosion from the tube during the heat treatment described in Step A3.
    3. Put the Pyrex glass tubes containing explants dipped in methanol into the water bath at 80 °C in the chemical fume hood. Incubate them for 15 min (see Note 3).
    4. After cooling the tubes at room temperature, discard the green-colored methanol extract and add 40 ml of fresh methanol to the glass tube. 
    5. Repeat Steps A3 and A4 until explants become white at least more than five times.
    6. Wash the methanol-fixed explants with SDW more than five times to remove methanol.
      Note: Make sure all explants sink to the bottom in SDW. If some explants are floating in the tube, stand for 5 min and change SDW again.
    7. Discard SDW completely in the tube with a plastic transfer pipet.
      Note: Don’t touch the tissue explants by a transfer pipet to avoid destruction of tissues.
    8. Add 40 ml of the Pronase E (PE) solution (see Recipes), which is pre-incubated for 2 h at 37 °C before use, to a glass tube.
      Note: Pre-incubation is essential to deactivate the glycosyl hydrolase contaminants in the PE products. Make sure all explants sink to the bottom of 50 ml glass tube in the PE solution.
    9. Incubate the glass tube containing explants in PE solution with tightly capped lids in the oven at 37 °C for 18 h (see Note 4).
    10. Discard PE solution from the tube. 
    11. Wash the PE-treated explants with SDW more than three times and remove the remaining solution from the 50 ml-glass tube as much as possible with a plastic transfer pipet.
      Note: Don’t touch the tissue explants with a transfer pipet to devoid any destruction of tissues.
    12. Trim the basal end of explants with a razor blade to make them exactly 30 mm in length (Figure 4D). 
    13. Determine the force and displacement curve of inflorescence explant (Procedure B) or store the 30 mm-length tissue explants in methanol at room temperature until use. Wash the explants with SDW at least three times before the experiment if the explants are stored in methanol (Step 10 of Procedure B)

  2. Determination of the force and displacement curve of inflorescence explants (ca. 5 min for the measurement of one sample).
    1. Turn on the tensile tester and the computer (Figure 3A)
    2. Set the “tensile test mode” in the operation software and the data sampling method to X-T mode with the maximal resolution of distance (0.001 mm) according to the manufacturer's manual.
    3. Set the initial distance between upper (top) and lower (bottom) fiber clamps to 10 mm after setting the upper clamp (without sample) to the original position (Figures 3B and 6F). 
    4. Calibrate the load cell (at both 0% and 50% of 10 N) without removing the upper removable clamp according to the manufacturer's manual (Figure 3B).
    5. Set the speed of upper movable clamp to 2 mm/min.
    6. Set the maximum load to 1 N (ca. 100 g) which is the load when the test is finished.
    7. Set the program to return upper movable clamp to the initial position after the test.
    8. Designate the data file format and enter the plant-line name of samples and the test number.
    9. Prepare 2 ml-micro tubes containing 2 ml fresh methanol in tube rack. Protect the tube label (plant line name and test number) from methanol with transparent tape.
    10. If samples have been stored in methanol prior to the test, wash and rehydrate the 30 mm length explants by at least five changes of SDW. Make sure all tissue explants sink to the bottom of the glass tube after the rehydration.
    11. Transfer tissue explants into the plastic Petri dish containing 40 ml SDW from the glass tube and keep them in the SDW until needed for the test.
    12. Take a tissue explant out from the SDW and immediately take a photograph of the tissue explant with a ruler and the label of the test number and sample name with a digital camera (Figure 4D). Determine the longitudinal and vertical lengths of explants later.
      Note: Avoid drying the sample from Steps B12 to B24.
    13. Put the tissue explant on Kimtowel wiper to remove excess water on the surface of the explant.
    14. Put the 2 mm x ca.10 mm strips of Kimwipe on both ends of the tissue explant (Figure 5A). Fold each paper strip at the middle to hold the both edges of the tissue explant (Figure 5B).
      Note: These paper strips are necessary to prevent tissue slippage in the fiber clamps during the test.


      Figure 5. Two small paper strips of Kimwipe are used to prevent the sample slippage in the clamps. A. After putting two paper strips under the sample, fold two paper fragments in the middle to the direction of red arrows. B. After that, fix the parts of the sample, which are indicated by blue boxes, with the upper and lower clamps.

    15. Fix one side of the paper strip-covered parts of the explant (Figure 5B) with the removable clamp by the lever action. Make sure the longitudinal axis of the tissue is vertically positioned at the center of the upper clamp (Figures 6A and 6B).
    16. Tighten the hand screw of the upper clamp to fix the tissue more. Do not tighten the screw completely to avoid destruction of the tissue (Figure 3C). Make sure the top-removable clamp fitting to the sample without any gap (upper arrowhead in Figure 6C) and any distortion (lower arrowhead and white dashed lines in Figure 6C). The gap and distortion could cause slightly wavy fluctuation of force extension curve during the tensile test. If gap and distortion are found, adjust the clamps and the sample by pushing the attachments of clamp with your thumbs (Figures 6D and 6E). This adjustment is also necessary to fix the sample with the bottom-fixed clamp in Steps B18 and B19.


      Figure 6. Tissue sample fastened between two clamps. A. Fix the sample with the upper removable clamp first (front view). See the position of one end of sample (Arrowhead). B. Make sure the angle between the sample and the edge of the upper clamp is ca. 90 degree (Side view). C-E. Make sure the top-removable and bottom-fixed clamps fitting to the sample without any gap (indicated by the upper arrowhead in C) and any distortion (indicated by the lower arrowhead and white dashed line in C). F and G. After hanging the upper clamp with the sample on the hook of the load cell, fix the sample with the lower clamp. Make sure both upper and lower ends of the sample protruding from clamps a little (Arrowheads in G).

    17. Hang the upper clamp with the explant on the hook of the loading cell.
    18. Fix another side of the paper strip-covered part of the explant with the lower clamp (Figure 6F). Make sure the longitudinal axis of the tissue is vertically positioned at the center of the lower clamp as for the upper clamp. Both ends of the sample should extrude a little from the clamps (Arrowheads in Figure 6G). 
    19. Tighten the hand screw of the lower clamp to fix the tissue securely (Figure 3C). Do not tighten the screw completely to avoid destruction of the tissue (see Note 5).
    20. Make sure the load between two clamps is almost 0 N (From -0.042 to 0.0183 N, Mean and standard deviation: -0.0059 N ± 0.0092 in our experiments) in order to avoid prestreching before the test. A strong prestrech could cause the plastic deformation of the sample before the estimation of stiffness (Cleland, 1967).
    21. If the load value before the test is not ignorable, loosen the hand screw of the lower clamp slightly. Repeat Steps B18 and B19 to get the load value close to 0. Alternatively, change the distance between two clamps to shorter than 10 mm with a manual controller (Figure 1A) to let the load value close to zero without touching the lower clamp (-0.01 mm to 0.05 mm in our experiments). In the latter case, record the changed distance between two clamps.
    22. Start loading the sample to 1 N by lifting the upper clamp.
    23. After reaching to 1 N, the loading should be stopped mechanically and the upper clamp should return to the initial position (10 mm distance) immediately. Prepare the next sample during this process (from Step B12).
    24. After the return of the upper and movable clamp to its original position, remove the tested sample from the upper and lower clamps by loosening the hand screws and levering action.
    25. Store each tested sample in methanol in a numbered 2 ml-micro tube.
    26. Repeat these steps (from Step B12 to Step B25) for all samples in one plant line.
    27. Store the series of all data for one plant line and export the relationship between displacement (mm) and load (N) for all samples in text format. The text data files should be analyzed with Microsoft Excel software or equivalent software (see Data analysis).
    28. Turn off the tensile testing machine and the computer.

  3. Determination of the sample dry weight
    1. Discard methanol from the 2 ml-microtubes containing a tissue explant (stored in Step B24) with a plastic transfer pipet avoiding destruction of the sample.
    2. Place the 2 ml-microtubes in the oven at 60 °C for 2 days without capping but with a small piece of aluminum foil sheet to prevent dust contamination. 
    3. Weigh the dried tested sample with an analytical balance equipped with an anti-static apparatus and the custom-made wind-proof cover (Note 6).

Data analysis

  1. Determination of inflorescence stiffness
    1. Open the text data file generated at the “Step 27” of Procedure B with Microsoft Excel or equivalent software. (An example of the text data file in our experiments is provided as a supplemental text files “Col0”.)
    2. Highlight all cells containing data. In our data, the first column (A) contains values of displacement (mm), and the second column (B) contains values of force (N) in the single tensile test and draw scatter plot using the software function.
    3. Observe the force and displacement curves (Figure 7). The curve should have two phases. At first, the displacement is accompanied by little increase of the load. This is, at least in part, due to some slack existing in the explant when it was fixed between the clamps and thus should be removed. Beyond a certain point, load increases in a linear manner as displacement proceeds (Cleland, 1967). Use the force and displacement values in the linear range to calculate stiffness.
    4. Draw the curves for all tests and compare the force ranges in a linear manner to extract the representative linear range. For example, we estimated that the representative linear force range was between 0.6 to 0.8 N in our experiment (Figure 7).


      Figure 7. Force and displacement curves for inflorescence explants in different plant lines of Arabidopsis. Col0: wild type, nst1nst3: double knockout mutant of NST1 and NST3 genes (Mitsuda et al., 2007), nst1nst3ERF35VP16: NST3 promoter-driven ERF035 gene with VP16 activation domain in the double knockout mutant (Sakamoto et al., 2018).

    5. Highlight the cells of displacement column and force column in the fixed force range (for example, from 0.6 to 0.8 N) and create a scatter plot.
    6. Add the linear fit (a straight line fit) using the software function. In Microsoft Excel, click once anywhere inside the graph area and select the “Layout” tab from “Chart Tools”. Click “Trendline” icon and select the “Linear Trendline” option.
    7. To show the equation, click “Trendline” and select “More Trendline Options…” Then check the “Display Equation on chart” and “Display R-squared value on chart” boxes. (Figure 8).
    8. The slope of the equation indicates stiffness (N/mm, Note 7). Examine the fitness of a linear plot with R-squared value (R2: 0.9969 to 1 in our experiment).


      Figure 8. Linear plots and equations of force (F) and displacement (D) curves prepared by Microsoft Excel. Round-shaped actual plots indicate the values between 0.6 and 0.8 N, which were used for the linear fit. “n” indicates the number of used plots. The slope in the equation surrounded by the red box gives the stiffness of each sample. R square-values are also indicated.

  2. Determination of mass per u nit length of tissue explants and the estimation of the effects of introduced or mutated genes on the inflorescence-stem stiffness
    1. Determine the longitudinal and the vertical length of tissue explants from the photograph taken at Step 12 of Procedure B (Figure 4D).
    2. Divide the dry weight (obtained at the Step 3 of Procedure C) by the longitudinal length of each tissue explant to obtain mass per unit length.
    3. Plot the mass per unit length and the stiffness of the investigated plant lines on a graph to see if there is any trend (i.e., effects of genes on the material properties such as cell wall compositions and/or the bio-structural geometry such as wall thickness, Figure 9).


      Figure 9. Scatter plot between dry weight/length and force/extension (stiffness) of different genotype plants. Wild-type (n = 17 biologically independent samples), nst1nst3 (n = 27 biologically independent samples) and nst1nst3 NST3pro::ERF035-VP16 (n = 76 biologically independent samples) (Sakamoto et al., 2018).

  3. Representative data
    One set of raw text data of the force and displacement curve of wild-type Arabidopsis inflorescence-stem explant is provided as a supplemental text file. Three test results of our experiments (Figures 7 and 8) are also provided as a supplemental Excel file (Force and displacement.xls). For the plot between mass per length and stiffness, see Figure 9.

Notes

  1. The tensile testing machine, AC-500N-CM, was exported from Japan to destinations such as Singapore and Thailand before. English manual is available for the operation of this machine with a computer. Other tensile testing machines are also available in the local manufacturer in your country such as Instron (https://www.instron.us/en-us). The detailed performance of the tensile testing machine in our laboratory is shown in Table 1.

    Table 1. Performance of the tensile testing machine AC-500N-CM


  2. We used seventeen plants in the wild type, twenty-seven plants in nst1nst3 mutant line, fifteen to twenty-two plants for each of four independent lines of transgenic NST3 promoter-driven ERF035-VP16 (120 plants in total) in the previous publication (Sakamoto et al., 2018).
  3. The heat treatment with methanol kills the inflorescence stem and deactivates endogenous cell wall hydrolases. However, expansin, a wall loosening protein remains active in this treatment (Cosgrove, 2011). There is a method to remove protoplasts from plant tissues without using methanol if it is required to see in-vitro effects of exogenously applied expansin or glycosyl hydrolase on the mechanical properties (Cosgrove, 2011). Non-metal materials for the fiber clamps in the tensile testing machine were recommended in some experiments (Cosgrove, 2011). If unexpected results of the mechanical properties are obtained, fixation method of tissues and the appropriate material for the clamps may need to be considered.
  4. Pronase E (Actinase E) is a nonspecific protease (Nomoto et al., 1960) and generally used for the removal of proteins from the polysaccharide samples (Schmidt et al., 2014; Higashi et al., 2015). This treatment removes the remaining cytoplasmic proteins and cell wall proteins including expansins from the explants. According to Cleland (1967), pronase treatment removed 85-90% of total protein from the stem explants, which was measured by either Kjeldahl nitrogen or proline, but it was less effective to solubilize the structural wall protein such as extensin; only 45% of the hydroxyproline was removed. 
  5. If the tightening by the hand screw is not appropriate (too strong or too weak), a slippage or breakdown of the sample could be observed during the loading of the sample (Figure 10).


    Figure 10. Example of a sample slippage or breakdown. Arrow indicates the displacement where the slippage or the breakdown of the sample has occurred.

  6. Dry weight of inflorescence explants (ca. 30 mm in longitudinal length) ranged from 1.02 to 4.91 mg in our experiments. Therefore, we need an analytical balance equipped with an anti-static apparatus and a custom-made wind-proof cover to achieve the stable measurement of the dry weight for each sample (Figure 11).


    Figure 11. Analytical balance with custom-made wind-proof cover and antistatic apparatus

  7. The obtained equation is also available to calculate the cell wall compliances of tissues by using the mass per unit length as an arbitrary unit of the cross-sectional area (Cleland, 1967). The cell wall compliance is a reciprocal of Young’s modulus. If each sample is loaded twice to obtain first and second force extension curves, compliances of elastic extensibility and plastic extensibility can be determined from these two curves (Masuda, 1969; Cleland, 1984; Park and Cosgrove, 2012; Phyo et al., 2017).

Recipes

  1. Potassium phosphate buffer for PE
    1. Dissolve 14.3 g KH2PO4 and 25.5 g K2HPO4 into SDW and adjust the entire volume to 500 ml to obtain 0.5 M potassium phosphate buffer (pH 7.0)
    2. Autoclave the buffer stock at 121 °C for 15 min
  2. PE solution
    1. PE powder bottle must be at room temperature before use to avoid its moisture absorption
    2. Dissolve 0.02 g of PE powder in 85 ml of SDW
    3. Add 5 ml of ethanol and 10 ml 0.5 M potassium phosphate buffer stock
    4. The prepared PE solution should be incubated for 2 h at 37 °C to deactivate possibly contaminating glycosyl hydrolases
    5. After incubation, use PE solution for the protein removal from tissue explants

Acknowledgments

We appreciate T. Kuroki, S. Watanabe, Y. Ono (TS-engineering Co. Ltd.), A. Nabeshima (Soft-brain Co. Ltd.) and A. Tanaka (BLW Co. Ltd.) for their technical support of the tensile testing machine. We also thank A. Hosaka, Y. Sugimoto, Dr. H. Takasaki and M. Yamada for technical support of plant material preparation. This protocol was made by optimizing the procedure for tensile tests performed by Dr. Yoshio Masuda, professor emeritus of Osaka City University (1928.01.01-2017.03.22) and his colleagues. K.Y. appreciates Dr. Chikashi Shimoda and Dr. Masayuki Katsumi for kindly providing the information of Dr. Y. Masuda. This work was supported by the JST ALCA program (grant no. JPMJAL1107) and JSPS KAKENHI Grant Number JP18H02259 to N.M.

Competing interests

The authors declare no competing interests.

References

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  20. Taiz, L. (1984). Plant cell expansion: regulation of cell wall mechanical properties. Ann Rev Plant Physiol 35: 585-657.

简介

木质纤维素生物质是用于燃料,建筑物,工艺品和生物材料的通用可再生资源。 用于工业应用的木质纤维素的分子设计策略已经通过筛选各种突变体和拟南芥的转化系列后的新基因的发现而得到发展,其细胞壁可以在花序茎中修饰,模型木本组织。 机械性质用作组织中遗传修饰细胞壁的化学病学行为的定量指标。 该参数可以通过组织外植体的拉伸或弯曲测试,组织行为的振动分析或使用原子力显微镜来探测组织表面来测量。 在这里,我们详细描述了使用基于经典方法测定细胞壁延伸性的拉伸试验机确定甲醇固定,再水合和链霉蛋白酶处理的花序外植体的刚度的方法。
【背景】植物生物力学可以用不同的方法测量,如拉伸试验(Burgert et al。,2003; Cosgrove,2011),弯曲试验(Shah et al。,2017)振动分析(Niklas and Moon,1988,Nakata et al。,2018)和原子力显微镜(Radotić et al。,2012)。这些技术可用于量化材料的刚度和强度特性。刚度由在单位长度上移位(拉伸)材料所需的力来定义,而强度被认为是最终使材料破裂所需的力的大小。结构的刚度受材料性质的影响,例如木质纤维素组合物和生物结构几何形状,例如细胞壁的厚度(Shah 等,,2017)。用于测量这些特性的生物力学测试经常用于商业销售的木材分级以及树木育种计划中以选择和培育具有增加的刚度和强度的优质树木。此类测试也可用于拟南芥中,以鉴定作为木材模型的突变体筛选中对强度和刚度有影响的候选基因(Strabala和MacMillan,2013,图1A-1C)。事实上,在我们之前的研究中,拉伸试验用于估算拟南芥中花序茎的刚度(Sakamoto et al。,2018)。本文中的方案提供了用拉伸试验机测量拟南芥花序茎的成熟区域的刚度的程序(图1D)。它是基于测量双子叶植物茎和单子叶植物胚芽组织外植体的细胞壁延伸性(顺应性)的方法(Olson et al。,1965; Cleland,1967)在20世纪60年代和20世纪80年代(Taiz,1984; Masuda,1990)。这已被优化用于确定拟南芥中花序茎的刚度,并在本文中进行了描述。


图1.测量拟南芥模型木本组织组织硬度的拉伸试验概述 A.拟南芥成熟区域的横断面花序茎作为模型木质组织。 B和C.用Mäule试剂染色横截面。束间纤维(If),维管束(vb),木质部导管(xv),束间木质部纤维(ixf)。条表示200μm。 D.用于测量拟南芥花序茎中组织硬度的拉伸测试分析的流程图。

关键字:拟南芥, 细胞壁, 服从, 花序轴, 机械性能, 刚度, 拉伸强度测试, 杨氏模量

材料和试剂

  1. 透明胶带
  2. PYREX ® 55 ml螺旋盖培养管,带PTFE衬里酚醛胶囊,25 x 150 mm(Corning Co. Ltd.,目录号:9826-25)
  3. 塑料转移移液管(透明LDPE,4 ml,薄柄,Thermo Fisher Scientific Co. Ltd.,目录号:13459118)
  4. 剃刀刀片(Hi-Stainless Single Edge Blade,FEATHER Safety Razor Co. Ltd.,目录号:FHS-10)
  5. 塑料培养皿(90 mm x 14.2 mm)(Thermo Fisher Scientific Co. Ltd.,目录号:5184E)
  6. Kimtowel擦拭巾(Kimberly-Clark Co. Ltd.)
  7. Kimwipe纸条(Kimberly-Clark有限公司, ca。 2 mm x ca。长10 mm)(图2)
  8. 2毫升微管(Eppendorf Co. Ltd.,目录号:0030120094)
  9. 铝箔
  10. 8周龄拟南芥(Arabidopsis thaliana)的花序茎
  11. 甲醇(日本Kishida Chemical Co. Ltd.,目录号:000-48666)
  12. KH 2 PO 4 (Nakalai Co. Ltd. Japan,目录号:09582-75)
  13. K 2 HPO 4 (Nakalai Co. Ltd. Japan,目录号:09583-65)
  14. 来自 Streptomyces griseus <(em)的链霉蛋白酶E(PE)(产品名称为“Actinase E”.Nacalai Co. Ltd. Japan,目录号:29003-51),储存于4°C
  15. SDW:灭菌超纯水(例如,Milli-Q)
  16. 用于PE溶液的磷酸钾缓冲液(参见食谱)
  17. PE解决方案(见食谱)


    图2.使用夹具固定样品的Kimwipe纸条( ca。 2 mm x 10 mm,手工制作)

设备

  1. 最大拉力试验机500牛顿(N)(T.S.E.Co. Ltd. Yokohama,日本,Auto Com系列,目录号:AC-500N-CM,图3A,见注1)&nbsp;
  2. 称重传感器最大10 N(T.S.E. Co. Ltd.,目录号:TC-10N-B)(图3A)
  3. 上部和下部纤维夹具在杠杆作用下(图3B和3C,夹爪5N,A&amp; D Co.Ltd。,东京,日本,目录号:J-TZM-5N)


    图3.拉伸试验机及其夹具。 A.拉伸试验机最大500 N力。 B.带钩和固定夹的可拆卸夹具。在可移动夹具的此位置设置负载的零点。 C.顶部可拆卸和底部固定的光纤夹具。夹具可以通过杠杆作用打开,样品应用手拧紧。

  4. 烤箱(37°C或60°C,EYELA Co. Ltd.,日本,强制气流烘箱,WFO420)
  5. 水浴(TR-1α,ASONE Co. Ltd.,日本,目录号:1-5832-31)
  6. 化学通风柜
  7. 分析天平(Mettler Toledo Co. Ltd.,Balance XPR26,目录号:30355476)
  8. 通用防静电套件U型电极(Mettler Toledo Co. Ltd.,目录号:11107767)
  9. Sharp-Pointed Dissecting Scissor(赛默飞世尔科技有限公司,产品目录号:08-940)
  10. 数码相机
  11. 标尺(最大150 mm)
  12. Tweezer(EBL Co. Ltd.,型号:#202)

软件

  1. 拉力试验机的操作和分析软件(T.S.E. Co. Ltd.,Yokohama,Japan,UTPS-STD Single for windows 10,英文版可用)
  2. Microsoft Excel( https://products.office.com/en-au/excel

程序

  1. 植物材料制备(收获组织后需要2-3天)&nbsp;
    1. 用剪刀从8周龄的拟南芥切下花序茎的基部(距茎的底部大约30mm)(图4A-4C),立即将整个部分外植体浸没在40ml中。 PYREX ®中的甲醇55 ml螺旋盖培养管。植物在16小时光照下(60-80μmolm -2 s -1 )/ 8小时黑暗循环在恒定的23°C植物室中生长,没有湿度控制。
      注意:一个植物系的评估需要超过15个花序茎外植体(见注2)。


      图4.茎段取自拟南芥的花序茎的基部。 A.在土壤中生长的8周龄植物。用一把剪刀在红色箭头的位置切割基端。 B.收获的和新鲜的花序茎。切除的茎段应在箭头位置切割。应删除所有分支。 C.消化的新鲜花序茎段应立即浸入甲醇中。 D.在甲醇中煮沸后,30毫米长的花序茎段,再水化和链霉蛋白酶E.处理。每个样品的纵向和垂直长度应在稍后由Image J.确定。

    2. 在步骤A3中描述的热处理过程中,紧紧关闭培养管盖以避免管中的甲醇爆炸。
    3. 将含有外植体的Pyrex玻璃管浸入80℃的化学通风橱中的水浴中浸入甲醇中。孵育15分钟(见注3)。
    4. 在室温下冷却管后,弃去绿色甲醇提取物并在玻璃管中加入40ml新鲜甲醇。&nbsp;
    5. 重复步骤A3和A4,直到外植体变白至少五次以上。
    6. 用SDW洗涤甲醇固定的外植体超过5次以除去甲醇。
      注意:确保所有外植体都沉入SDW的底部。如果一些外植体漂浮在管中,则静置5分钟并再次更换SDW。
    7. 使用塑料移液管将SDW完全丢弃在管中。
      注意:请勿用移液管接触组织外植体,以免损坏组织。
    8. 加入40毫升Pronase E(PE)溶液(见食谱),在使用前于37℃预孵育2小时,放入玻璃管中。
      注意:预孵育对于去除PE产品中的糖基水解酶污染物至关重要。确保所有外植体都沉到PE溶液中50 ml玻璃管的底部。
    9. 将含有外植体的玻璃管在带有盖紧盖子的PE溶液中在37°C的烘箱中孵育18小时(见注4)。
    10. 从管中丢弃PE溶液。&nbsp;
    11. 用SDW洗涤PE处理的外植体三次以上,并用塑料移液管尽可能多地从50ml玻璃管中取出剩余的溶液。
      注意:请勿使用移液管接触组织外植体,以免组织破坏。
    12. 用剃刀刀片修剪外植体的基端,使其长度正好为30毫米(图4D)。&nbsp;
    13. 确定花序外植体的力和位移曲线(程序B)或将30mm长的组织外植体在室温下储存在甲醇中直至使用。如果外植体储存在甲醇中,则在实验前用SDW洗涤外植体至少三次(程序B的步骤10)

  2. 确定花序外植体的力和位移曲线(大约 5分钟,用于测量一个样品)。
    1. 打开拉伸试验机和计算机(图3A)
    2. 根据制造商的手册,将操作软件中的“拉伸测试模式”和数据采样方法设置为X-T模式,最大分辨率为(0.001 mm)。
    3. 将上夹具(无样品)设置到原始位置后,将上(上)和下(下)光纤夹具之间的初始距离设置为10 mm(图3B和6F)。&nbsp;
    4. 根据制造商的手册(图3B)校准称重传感器(10 N的0%和50%),而不拆下上部可拆卸夹具。
    5. 将上部可动夹具的速度设定为2 mm / min。
    6. 将最大负载设置为1 N( ca。 100 g ),这是测试完成时的负载。
    7. 设置程序以在测试后将上部可移动夹具返回到初始位置。
    8. 指定数据文件格式并输入样品的工厂线名称和测试编号。
    9. 在管架中制备含有2ml新鲜甲醇的2ml微量管。用透明胶带保护管标签(工厂管线名称和测试编号)免受甲醇的影响。
    10. 如果样品在测试前已经储存在甲醇中,则将30 mm长度的外植体洗涤并再水化至少5次SDW变化。在再水化后,确保所有组织外植体都沉入玻璃管的底部。
    11. 将组织外植体转移到来自玻璃管的含有40ml SDW的塑料培养皿中,并将它们保持在SDW中直至需要进行测试。
    12. 从SDW取出组织外植体,立即用尺子拍摄组织外植体的照片,用数码相机拍摄测试编号和样品名称的标签(图4D)。稍后确定外植体的纵向和纵向长度。
      注意:避免从步骤B12到B24干燥样品。
    13. 将组织外植体放在Kimtowel擦拭器上以除去外植体表面上的多余水分。
    14. 将2 mm x ca。 10 mm的Kimwipe条带放在组织外植体的两端(图5A)。折叠中间的每个纸条以保持组织外植体的两个边缘(图5B)。
      注意:在测试过程中,这些纸条是防止纤维夹组织滑动所必需的。


      图5. Kimwipe的两个小纸条用于防止样品在夹具中滑动。 A.在样品下方放置两个纸条后,将中间的两个纸屑折叠到红色方向箭头。 B.之后,用上部和下部夹子固定样品的部分,用蓝色框表示。

    15. 通过杠杆作用用可移除的夹具固定外植体(图5B)的纸带覆盖部分的一侧。确保组织的纵轴垂直位于上夹具的中心(图6A和6B)。
    16. 拧紧上夹具的手拧螺丝,以更好地固定组织。不要完全拧紧螺钉以避免损坏组织(图3C)。确保顶部可拆卸夹具与样品无任何间隙(图6C中的上箭头)和任何变形(图6C中的下箭头和白色虚线)。在拉伸试验期间,间隙和变形可能引起力延伸曲线的轻微波动。如果发现间隙和变形,请用拇指推动夹具的附件来调整夹具和样品(图6D和6E)。在步骤B18和B19中,使用底部固定夹具固定样品也需要进行此调整。


      图6.将组织样品固定在两个夹具之间。 A.首先用上部可拆卸夹具固定样品(前视图)。查看样品一端的位置(箭头)。 B.确保样品与上夹具边缘之间的角度 ca。 90度(侧视图)。 C-即确保顶部可拆卸和底部固定的夹具适合样品,没有任何间隙(由C中的上箭头指示)和任何变形(由C中的下箭头和白色虚线表示)。 F和G.将上夹具上的样品悬挂在称重传感器的钩子上后,用下夹具固定样品。确保样品的上端和下端从夹子上突出一点(G中的箭头)。

    17. 将上夹具与外植体悬挂在装载单元的钩子上。
    18. 用下夹具固定外植体的纸带覆盖部分的另一侧(图6F)。确保组织的纵轴垂直位于下夹具的中心,与上夹具一样。样品的两端应从夹具中挤出一点(图6G中的箭头)。&nbsp;
    19. 拧紧下夹具的手拧螺丝,牢牢固定组织(图3C)。不要完全拧紧螺丝,以免损坏组织(见注5)。
    20. 确保两个夹具之间的载荷几乎为0 N(从-0.042到0.0183 N,平均值和标准偏差:在我们的实验中为-0.0059 N±0.0092),以避免在测试前进行预拉伸。在估算刚度之前,强烈的预拉伸可能导致样品的塑性变形(Cleland,1967)。
    21. 如果测试前的负载值不可忽略,请稍微松开下夹具的手拧螺丝。重复步骤B18和B19,使负载值接近0.或者,使用手动控制器将两个夹具之间的距离改为短于10 mm(图1A),使负载值接近零而不接触下夹具( - 在我们的实验中0.01mm至0.05mm)。在后一种情况下,记录两个夹具之间改变的距离。
    22. 通过抬起上夹具开始将样品加载到1N。
    23. 达到1 N后,应机械停止加载,上夹具应立即返回初始位置(10 mm距离)。在此过程中准备下一个样品(从步骤B12开始)。
    24. 在将上夹具和可动夹具返回其原始位置后,通过松开手拧螺丝和撬动动作,从上夹具和下夹具中取出测试样品。
    25. 将每个测试样品在甲醇中储存在编号为2ml的微管中。
    26. 对一个工厂生产线中的所有样品重复这些步骤(从步骤B12到步骤B25)。
    27. 存储一个工厂生产线的所有数据系列,并以文本格式导出所有样品的位移(mm)和负载(N)之间的关系。应使用Microsoft Excel软件或等效软件分析文本数据文件(请参阅数据分析)。
    28. 关闭拉力试验机和电脑。

  3. 测定样品干重
    1. 用塑料转移移液管从含有组织外植体(存储在步骤B24中)的2ml微管中丢弃甲醇,避免破坏样品。
    2. 将2毫升微量管置于60°C的烘箱中2天,不加盖,但使用一小块铝箔片以防止灰尘污染。&nbsp;
    3. 用配有防静电装置的分析天平和定制的防风罩(注6)称量干燥的测试样品。

数据分析

  1. 花序刚度的测定
    1. 使用Microsoft Excel或同等软件打开在程序B的“步骤27”生成的文本数据文件。 (我们实验中的文本数据文件示例以补充文本文件”Col0“。)
    2. 突出显示包含数据的所有单元格在我们的数据中,第一列(A)包含位移值(mm),第二列(B)包含使用软件函数的单个拉伸测试和绘制散点图中的力(N)值。
    3. 观察力和位移曲线(图7)。曲线应该有两个阶段。首先,位移伴随着负载的少量增加。这至少部分是由于当外植体固定在夹具之间时存在一些松弛,因此应该将其移除。超过某一点,随着位移的进行,负荷以线性方式增加(Cleland,1967)。使用线性范围中的力和位移值来计算刚度。
    4. 绘制所有测试的曲线并以线性方式比较力范围以提取代表性线性范围。例如,我们估计在我们的实验中代表性的线性力范围在0.6到0.8N之间(图7)。


      图7. 拟南芥的不同植物系中花序外植体的力和位移曲线。 Col0:野生型, nst1nst3 :<的双敲除突变体 NST1 和 NST3 基因(Mitsuda et al。,2007), nst1nst3ERF35VP16 : NST3 启动子驱动的 ERF035 基因,在双敲除突变体中具有VP16激活结构域(Sakamoto et al。,2018)。

    5. 在固定力范围内(例如,从0.6到0.8 N)突出显示位移列和力柱的单元格,并创建散点图。
    6. 使用软件功能添加线性拟合(直线拟合)。在Microsoft Excel中,单击图形区域内的任意位置,然后从“图表工具”中选择“布局”选项卡。单击“趋势线”图标,然后选择“线性趋势线”选项。
    7. 要显示公式,请单击“趋势线”并选择“更多趋势线选项...”然后选中“在图表上显示公式”和“在图表上显示R平方值”框。 (图8)。
    8. 等式的斜率表示刚度(N / mm,注7)。在我们的实验中检查具有R平方值的线性图的适合度(R 2 :0.9969比1)。


      图8.由Microsoft Excel准备的线性图和力(F)和位移(D)曲线的方程。圆形实际图表示0.6到0.8 N之间的值,用于线性适合。 “n”表示使用的图的数量。由红色框包围的等式中的斜率给出了每个样本的刚度。还表示了R平方值。

  2. 组织外植体的每单位长度质量的确定和引入或突变基因对花序茎刚度的影响的估计
    1. 从程序B的步骤12(图4D)拍摄的照片确定组织外植体的纵向和纵向长度。
    2. 将干重(在方法C的步骤3中获得)除以每个组织外植体的纵向长度,以获得每单位长度的质量。
    3. 在图表上绘制每单位长度的质量和所研究的植物系的刚度,以查看是否存在任何趋势(即,基因对材料特性的影响,例如细胞壁组成和/或生物结构几何,如壁厚,图9)。


      图9.不同基因型植物的干重/长度和力/伸长(硬度)之间的散点图。野生型(n = 17个生物学独立样本), nst1nst3 ( n = 27个生物学独立样本)和 nst1nst3 NST3pro :: ERF035-VP16 (n = 76个生物学独立样本)(Sakamoto et al。,2018)。

  3. 代表性数据
    野生型拟南芥花序茎外植体的力和位移曲线的一组原始文本数据以补充文本文件。我们的实验的三个测试结果(图7和图8)也作为补充Excel文件(Force和displacement.xls)。有关每长度质量和刚度之间的关系图,请参见图9.

笔记

  1. 拉伸试验机AC-500N-CM之前从日本出口到新加坡和泰国等目的地。英语手册可用于使用计算机操作本机。其他拉伸试验机也可在您所在国家/地区的当地制造商处获得,例如Instron( https://www.instron页面没有自动跳转/ EN-US )。我们实验室中拉伸试验机的详细性能如表1所示。

    表1.拉伸试验机AC-500N-CM的性能


  2. 我们在野生型中使用了17种植物,在 nst1nst3 突变体系中使用了27种植物,对于转基因 NST3 启动子驱动的4种独立系中的每一种使用了15至22种植物 ERF035-VP16 (以前的出版物中共120株植物)(Sakamoto et al。,2018)。
  3. 用甲醇热处理杀死花序茎并使内源细胞壁水解酶失活。然而,扩张蛋白,一种壁松动蛋白在这种治疗中仍然活跃(Cosgrove,2011)。如果需要观察外源施加的扩展蛋白或糖基水解酶对机械性质的体外效应,有一种方法可以在不使用甲醇的情况下从植物组织中去除原生质体(Cosgrove,2011)。在一些实验中推荐用于拉伸试验机中的纤维夹具的非金属材料(Cosgrove,2011)。如果获得机械性能的意外结果,则可能需要考虑组织的固定方法和夹具的适当材料。
  4. Pronase E(Actinase E)是一种非特异性蛋白酶(Nomoto et al。,1960),通常用于从多糖样品中去除蛋白质(Schmidt et al。, 2014; Higashi et al。,2015)。该处理从外植体中去除剩余的细胞质蛋白和细胞壁蛋白,包括扩展蛋白。根据Cleland(1967)的观点,链霉蛋白酶处理从茎外植体中去除了85-90%的总蛋白,这是通过凯氏氮或脯氨酸测量的,但它对结肠壁蛋白如伸香蛋白的溶解效果较差;仅去除了45%的羟脯氨酸。&nbsp;
  5. 如果手动螺钉拧紧不合适(太强或太弱),在装载样品时可能会发现样品滑动或破裂(图10)。


    图10.样品滑动或破裂的示例。箭头表示样品滑移或破裂的位移。

  6. 在我们的实验中,花序外植体的干重( ca. 30mm,纵向长度)范围为1.02至4.91mg。因此,我们需要一个配有防静电设备和定制防风罩的分析天平,以实现每个样品干重的稳定测量(图11)。


    图11.使用定制防风罩和防静电设备的分析天平

  7. 通过使用每单位长度的质量作为横截面积的任意单位,所获得的等式也可用于计算组织的细胞壁顺应性(Cleland,1967)。细胞壁顺应性是杨氏模量的倒数。如果每个样品加载两次以获得第一和第二力延伸曲线,则可以从这两条曲线确定弹性延展性和塑性延展性的一致性(Masuda,1969; Cleland,1984; Park和Cosgrove,2012; Phyo 等。,2017)。

食谱

  1. 用于PE的磷酸钾缓冲剂
    1. 将14.3 g KH 2 PO 4 和25.5 g K 2 HPO 4 溶解到SDW中,并将整个体积调整为500毫升,获得0.5 M磷酸钾缓冲液(pH 7.0)
    2. 将缓冲液在121℃下高压灭菌15分钟
  2. PE解决方案
    1. PE粉瓶在使用前必须在室温下,以避免其吸湿
    2. 将0.02g PE粉末溶于85ml SDW中
    3. 加入5毫升乙醇和10毫升0.5M磷酸钾缓冲液
    4. 制备的PE溶液应在37℃下孵育2小时以使可能污染的糖基水解酶失活
    5. 孵育后,使用PE溶液从组织外植体中去除蛋白质

致谢

我们感谢T. Kuroki,S。Watanabe,Y。Ono(TS-engineering Co. Ltd.),A。Nabeshima(Soft-brain Co. Ltd.)和A. Tanaka(BLW Co. Ltd.)的技术支持拉力试验机。我们还要感谢A. Hosaka,Y。Sugimoto,H. Takasaki博士和M. Yamada对植物材料制备的技术支持。该协议是通过优化大阪市立大学名誉教授Yoshio Masuda博士(1928.01.01-2017.03.22)及其同事进行的拉伸试验程序而制定的。 K.Y.感谢Chikashi Shimoda博士和Masayuki Katsumi博士友好地提供Y. Masuda博士的信息。这项工作得到了JST ALCA计划(授权号JPMJAL1107)和JSPS KAKENHI拨款号JP18H02259对N.M.的支持。

利益争夺

作者宣称没有竞争利益。

参考

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引用:Yoshida, K., Sakamoto, S. and Mitsuda, N. (2019). Tensile Testing Assay for the Measurement of Tissue Stiffness in Arabidopsis Inflorescence Stem. Bio-protocol 9(15): e3327. DOI: 10.21769/BioProtoc.3327.
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