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Jul 2016
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Pneumatic Method to Measure Plant Xylem Embolism
气动法检测植物木质部栓塞   

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Abstract

Embolism, the formation of air bubbles in the plant water transport system, has a major impact on plant water relations. Embolism formation in the water transport system of plants disrupts plant water transport capacity, impairing plant functioning and triggering plant mortality. Measuring embolism with traditional hydraulic methods is both time-consuming and requires large amounts of plant material. While the stem hydraulic methods measure loss of xylem hydraulic conductance due to embolism formation, the pneumatic method directly quantifies the amount of emboli inside the xylem as changes in xylem air content. The pneumatic method is an easy and fast (8+ embolism curves per day) method to measure plant embolism requiring minimal plant material. Here, we provide detailed descriptions and recent technical improvements on the pneumatic method.

Keywords: Embolism resistance (抗栓性), Plant drought stress (植物干旱胁迫), Hydraulic failure (水力学失败), Plant pneumatics (植物气体力学)

Background

Plant xylem embolizes due to the entry of air into the xylem vessels under drought conditions. Resistance to embolism formation is one of the most important plant traits strongly determining species distribution, mortality and evolution (Choat et al., 2012; Rowland et al., 2015; Larter et al., 2017) and has been recently suggested as a key trait to model plant function and predict plant responses to global changes (Sperry and Love, 2015; Brodribb, 2017). Most methods used to estimate embolism resistance measures the hydraulic conductivity of embolized branch segments and relate it to the hydraulic conductance of the branch segment without embolism (Sperry et al., 1988; Melcher et al., 2012). These methods are usually time-consuming and prone to several artifacts (Wheeler et al., 2013; Trifilò et al., 2014; Beikircher and Mayr, 2016).

The pneumatic method has been recently proposed as an alternative method to estimate embolism resistance from a different point of view, not from the water flow perspective but from the direct consequence of embolism-air presence in the xylem (Pereira et al., 2016). As plant embolizes, air spaces inside the xylem increase. Embolism thus changes the pneumatic properties of branch segments. In this method, a vacuum is applied to a cut branch and the air flowing outside the branch is measured as an estimate of xylem air content. A strong relationship exists between air flow outside the branch segment and the amount of emboli in the branch xylem (Pereira et al., 2016; Zhang et al., 2018). The vacuum method presents a simple, low cost, fast and practical method to measure plant embolism. Additionally, the pneumatic method does not require rehydrating (flushing water) through samples in which the effects of drought are being studied.

Materials and Reagents

  1. Pneumatic apparatus
    1. Adapter Luers (Cole-Parmer, catalog numbers: EW-30800-06 and EW-30800-24)
    2. 1 L Kitasato flask (Prolab, catalog number: PL287)
    3. Silicone tubing (3 mm ID and 5.2 mm OD and 4.9 mm ID and 9.7 mm OD, larger or smaller sizes depending on sample diameter, with preferences for thick walled tubes, as they seal better after clamping)
    4. Rigid tubing (Cole-Parmer, catalog number: EW-30600-62)
    5. Vacuum source, either a syringe or vacuum pump (Prolab, catalog numbers: 032357 and VAC29-110, respectively)
    6. Three-way stopcock (Cole-Parmer, catalog number: EW-30600-07)
    7. Vacuum reservoir (a container or tube with rigid walls to store vacuum; 1-10 ml volume is usually enough)
    8. Vacuum meter, 30 to 110 kPa recommended (Honeywell, catalog numbers: 142PC05D or 26PCCFA6D or MPX5100AP; NXP Semiconductors; Netherlands. Farnell, catalog numbers: 1386589, 731766 and 1457156, respectively)

  2. Sample preparation and handling
    1. Plastic glue
    2. Plastic paraffin film (Prolab, catalog number: PM996)
    3. Plastic clamps (Cole-Parmer, catalog number: RZ-06832-02) and/or zip ties

Equipment

  1. Voltage meter
    1. Pliers
    2. Sharp razors
    3. Voltmeter or voltage logger (1 mV precision for 142PC05D or MPX5100AP, 0.01 mV precision for 26PCCFA6D)
    4. Alternative: Pneumatic Shield for Arduino UNO microcontroller board (Plant Technology and Environmental Monitoring–PLANTEM), to use with 26PCCFA6D (see details in the Figure 4)

  2. Xylem water potential measurement
    1. Pressure chamber (PMS Instruments, model: PMS 1000, or other)
    2. Notebook and USB digital microscope (Jiusion, or other)
      Note: We discourage the use of magnifying glasses because of safety issues.

Procedure

Pneumatic measurements require construction of the pneumatic apparatus and measurement of air discharge and xylem water potential. Each step is described below and in Video 1.

Video 1. Demonstration of pneumatic apparatus assemblage and an air discharge measurement routine using a voltmeter

  1. Pneumatic apparatus (Figure 1)
    The pneumatic apparatus consists basically of a container filled with vacuum where the branch will be connected, a meter to read vacuum pressure and a vacuum source to fill the reservoir.


    Figure 1. Pneumatic apparatus. A. Vacuum apparatus. a) common syringe used as vacuum source; b) rigid tubing also used as the vacuum reservoir; c) vacuum meter (142PC05D) coupled to a 12 V power source and a voltage regulator; d) voltmeter probe connected to a sensor output. B. Branch connection details. e) plastic clamp to tighten the elastic tubing to the sample; f) plastic film (and glue if necessary) to avoid leakage; g) elastic tubing that deforms when pressed by the plastic clamp. C. Alternative setup for the vacuum source. A vacuum pump is used to store vacuum in the vacuum storage (h; a 1 L Kitasato flask in this example). The vacuum storage is used to apply vacuum to the vacuum reservoir through a third three-way stopcock (i). S1 and S2 are the three-way stopcock 1 and 2, respectively.

    1. Connect the vacuum reservoir to the first three-way stopcock (S1). Connect a silicone tubing (enough to fit the branch stem tight) to one exit of S1 and leave the other exit of S1 connected to the atmosphere (Figures 1A and 1B).
    2. Connect the vacuum reservoir to the second three-way stopcock (S2). Connect the vacuum meter to one exit of S2 and the vacuum source to the other exit of S2 (Figure 1A).
      1. Choose a vacuum reservoir volume (Vr; L) that ensures enough precision in the vacuum meter. Vr must be either measured or calculated from the tubing and stopcock datasheets (each rigid tubing [Cole-Parmer, USA] has 1.304 ml). A few milliliters of vacuum reservoir is usually enough (the tubing of the apparatus alone may be enough as a vacuum reservoir).
      2. The vacuum source can be either a syringe or a vacuum pump. Large quantities of vacuum can be stored in a container (Kitasato flasks, for example) and used to replenish the apparatus vacuum reservoir for easier use (Figure 1C).
      3. The vacuum meter may be read: with a voltmeter; with a voltage logger coupled to a computer; or with the Pneumatic Shield and Arduino UNO microcontroller.
      4. All tubing, except the one connecting the branch, must be rigid to avoid volume change under vacuum. The tube connecting the branch to S1 should be elastic so the branch can be tightly connected. We recommend using Luer lock connections for easy use and to prevent leakage.

  2. Air discharge measurements (Figure 2)
    Air discharged from the branch (AD) is the amount of air leaving the branch to the vacuum reservoir. Air pressure changes in the vacuum reservoir are measured and converted to volume using the ideal gas law. AD increases with the amount of emboli of the branch segment being measured until the branch is fully embolized. Multiple AD points must be measured with the same sample in different xylem water potentials so the data can then be standardized and an embolism resistance curve can be estimated in the next section.


    Figure 2. Air discharge curves measurement. Steps to construct and air discharge curve. 1) Fill the vacuum reservoir with air. 2) Measure initial pressure. 3) Apply pressure to sample, wait 2.5 minutes and measure final pressure. 4) Measure sample water potential. 5) Let sample to desiccate. Repeat step one after further desiccation.

    1. Select a fully hydrated and non-embolized branch segment with some leaves.
    2. Cut the branch from the plant with a sharp razor.
    3. Connect the branch to the elastic tubing in S1 using a plastic clamp/plastic film to ensure no or insignificant leakage. 
    4. Open the branch to the atmosphere in S1 and close the branch to the vacuum reservoir in S2 (Figure 2.1).
    5. Reduce the vacuum reservoir pressure to 35-40 kPa (atmospheric pressure minus 55-60 kPa) using the vacuum source and close the vacuum reservoir to the vacuum source in S2 (Figure 2.1).
    6. Read the initial pressure (Pi; kPa) at the vacuum reservoir (Figure 2.2).
    7. Close the branch segment to the atmosphere in S1 and connect it to the vacuum reservoir in S2 (Figure 2.3).
    8. After 2.5 min, read the final pressure at the vacuum reservoir (Pf; Figure 2.3).
    9. Calculate the number of moles of air Δn (mole) discharged from the branch using the ideal gas law:

      (1) Δn = PiVr/RT – PfVr/RT;

      where R is the gas constant (8.314 kPa L mol-1 k-1); T is apparatus/room temperature (K) during measurement, and Vr is the reservoir volume (L).

    10. (Optional) Transform moles of air discharged to the volume of air discharged at atmospheric pressure (AD; μl) using the ideal gas law:

      (2) AD = (ΔnRT/Patm) x 106

      where Patm is atmospheric pressure (kPa) and 106 is to change from L to μl.

    11. Measure the xylem water potential (Ψx) of the branch sample and seal the leaf cut with glue (Figure 2.4). See “Note 11” regarding sample handling and leaf and xylem water potential equilibrium issues.
    12. Leave the branch to dry in the bench (Figure 2.5).
    13. Bag the sample for leaf water potential to equilibrate with Ψx.
    14. Start again from Step B5 to obtain another data point. The last data point for a branch sample should be one where it is fully embolized as all data will be standardized by this last point.
    Notes:
    1. The branch must be connected to the atmosphere whenever a measurement is not happening to ensure the initial air pressure inside the branch is atmospheric. After an AD measure, leave the branch connected to the atmosphere for at least 2.5 min before another measure to ensure no vacuum remains inside the branch.
    2. The branch may shrink as it dehydrates, and the connection to S1 may get loose. Tighten the connection to ensure no leakage.

Data analysis

Data processing requires the use of pneumatic measurements to estimate the percentage loss of conductance (PLC). PLC is proportional to the percentage of maximum air discharged (PAD) of a branch sample. An example spreadsheet and an R script using base packages (R Core Team, 2011 [version 2.1.2]) for data processing and analysis are presented in the annex (“data.example.xls” and “data.analysis.r”). To obtain PAD, AD must be standardized for each branch sample (see Figures 3A and 3B).


Figure 3. Air discharge and percentage air discharge curves. A. Example of air discharge measurements from branch samples in different xylem water potentials (Ψx). B. Air discharge measures from (A) were standardized by maximum air discharge to obtain percentage maximum air discharged (PAD) as a function of Ψx. Red line is the regression for the PAD data. Blue line is the percentage loss of conductivity (PLC; points not shown) curve obtained with the hydraulic method. Different symbols represent data from different samples in the pneumatic method. The branch lost its leaves at around minus 3.5-4.5 MPa.

Data.example.xlsx. Example spreadsheet for writing data and calculating AD and PAD.
Data.analysis.docx. R script with routines for processing and analyzing data collected with the pneumatic method.


  1. Standardize each AD measurement (ADi; where i represents each individual measurement) by the maximum AD (ADmax) of the branch segment being measured:

    (3) PAD = 100 x (ADi/ADmax);

    where PAD is the percentage of maximum air discharged from the sample and is related to PLC.
  2. For some species, even when fully hydrated and with no embolism present, AD may be significant, possibly due to natural air spaces in the xylem. In this case, the initial AD (ADini) must be subtracted from each AD measurement so PAD is related to PLC:

    (4) PAD = 100 x (ADi – ADini)/(ADmax – ADini)

  3. The construction of the embolism curves follow the standard methods used to calculate PLC curves (fitting sigmoidal curves, exponential curves, etc.) but using the relationship between PAD and Ψx to estimate the PLC curves. A quality curve must present a plateau at high water potentials where PAD changes very little, a transition zone, and a second plateau where PAD stops changing (Figure 3B).

Notes

  1. We recommend using the Pneumatic Shield for Arduino UNO microcontroller developed by Plant Technology and Environmental Monitoring (PLANTEM, Brazil, Figure 4) as it allows for two samples to be measured at the same time and removes the need for manually taking notes on vacuum meter voltage, effectively doubling measurement velocity.


    Figure 4. Pneumatic apparatus shield. A. Model of Pneumatic Shield for Arduino UNO developed by PLANTEM. B. Pneumatic Shield prototype is automatically logging two vacuum meters (26PCCFA6D). In this example, the left branch is being measured (indicated by blue LED) while the data is displayed in the LCD display and logged to an SD card. Measurement starts when a button associated with each vacuum meter is pressed, and data is logged each second.

  2. Stopcocks also have air spaces inside them. If for one measurement you close the stopcock one direction and the other using a different direction the total amount of air may be slightly different.
  3. Leakage: If leakage is significant, it can be calculated and subtracted from each measurement.
  4. Sensor voltage supply: a simple voltage regulator can be used to supply power the vacuum meter. We recommend further controlling sensor input voltage using an LM78XX voltage regulator (usually LM7810 or 7812) as noise in input voltage will change voltmeter output. This is particularly important when using the lower output voltage 26PCCFA6D sensor. An LM78XX circuit and an assembled sensor (142PC05D) are shown in Figure 5 below:


    Figure 5. Pressure sensor and a simple voltage regulator. A. An LM7810 voltage regulator setup. B and C. Circuit board used in C). The circuit in B) was hand-drawn using a permanent marker on a virgin printed circuit board and corroded with ferric perchlorate. Holes were made for the components, and then they were welded on the board. 7810 is the LM7810 voltage regulator. C1 and C2 are electrolytic capacitors used to filter noise and reduce transient changes in input voltage.

  5. The vacuum meter has very low power consumption. It can be powered from a 12 V car battery for days in the field or remote areas. In this case, a voltage regulator is fundamental.
  6. AD measurements can be expressed in mols of air or equivalent volume at atmospheric pressure. We recommend the expression in volume as it is in the same unit as anatomical measures and equipment descriptions.
  7. We use PAD instead of AD for estimating embolism because variability between samples of the same species can be substantial. If there is enough control of the samples so variability is small or the entire curve is made of one branch sample, AD can be used directly to estimate embolism.
  8. We use a discharge time of 2.5 min because we found it is enough to ensure precision. Shorter or longer times can be used. AD is not linear in time, so care should be taken when comparing different times with the same species/samples. If you want to measure all the volume of air inside a sample, you can use a long discharge time or extrapolate the discharge curve to infinity, find out the pressure at equilibrium and use it to calculate the total air space inside the sample.
  9. The branch connection (Figure 1B) can be removed from S1 without removing the sample from the elastic tubing for easier handling of multiple samples. For example, if you measure 10 samples on the same day, use 10 individual connections (Luer lock, elastic tubing, plastic clamp and sample setup) and simply connect the sample that you are going to measure each time to S1.
  10. Many plant species produce resins or latex in the bark/xylem. Secretion of resin/latex clogs the vessels and changes air discharge. We recommend verifying if this occurs in each species studied. If it occurs, cutting the branch with a sharp razor, waiting 1 h while the sample is bagged for the cessation of resin/latex secretion, and cutting the stem again prior to connecting to the apparatus usually solves the problem.
  11. To ensure xylem and leaf water potential to equilibrate, so leaf water potential can be used as an indicator of xylem water potential, before measuring leaf water potential the sample must stay the minimum amount of time possible outside of the plastic bag. The best way to do this is to measure the air discharge while the sample is bagged and afterward remove the bag to measure the water potential.
  12. The sample connection (Figure 1B) can hold a significant amount of air that can inflate AD measurements. We found this can be corrected by discounting the first second of measurement. Discounting the first second of measurement leads to a small underestimation of the total air inside the plant, but this has no effect on estimating PAD values, as they are relative. An alternative to measuring AD without discounting the first second is to discount the sample connection volume from the total air discharge. The total amount of discharged air (Δn), in mols, can then be calculated as:

    Δn = (nr + nsc) – nf

    where nr and nsc are the initial amount of air inside the reservoir and the sample connector, respectively, which sums to the total amount of air in the apparatus before measurement. nf is the final amount of air in the apparatus. As nsc is at atmospheric pressure prior to measurement, the above equation [equivalent of equation (1) in this situation] becomes:

    Δn = (PiVr/RT + PatmVsc/RT) – PfVr/RT

    where Vsc is the volume of the sample connection. All other procedures are equal.

  13. Units, terms and definitions used in the protocol:
    AD – air discharged from the branch segment (μl)
    ADmax – maximum air discharged from the branch segment (μl). It is the AD of the sample when it is fully cavitated
    ADini – air discharged when fully rehydrate and without cavitation (μl)
    Pi – initial pressure of the vacuum reservoir (kPa)
    Pf – final pressure of the vacuum reservoir (kPa)
    Patm – atmospheric pressure (101.3 kPa at sea level approximately)
    PAD – percentage of maximum air discharged from the branch
    PLC – percentage loss of hydraulic conductance of the xylem
    S1 – three-way stopcock connecting the branch segment to the vacuum reservoir and the atmosphere
    S2 – three-way stopcock connecting the vacuum reservoir to the vacuum source and vacuum meter
    R – gas constant (8.314 kPa L mol-1 k-1)
    T – apparatus/room temperature (K)
    Vr – vacuum reservoir volume (L)
    Δn – change in the number of mols in the vacuum reservoir (ni minus nf; mol)
    Ψx – xylem water potential (MPA)

Acknowledgments

This research was funded by FAPESP/Microsoft research (grant 11/52072-0) awarded to R.O. We thank the UNICAMP post-graduate programs in Ecology and Plant Biology and the Higher Education Co-ordination Agency (CAPES) scholarship award to P.R.L.B. We thank Newton International Fellowship (grant NF170370) who funded P.R.L.B. in the final writing of this manuscript and São Paulo Research Foundation for the fellowship granted to L.P. (FAPESP, Grant No. 2017/14075-3). We thank David Bartholomew for reviewing and proofreading the manuscript.

Competing interests

The authors declare they have no conflict of interest.

References

  1. Beikircher, B. and Mayr, S. (2016). Avoidance of harvesting and sampling artefacts in hydraulic analyses: a protocol tested on Malus domestica. Tree Physiol 36(6): 797-803. 
  2. Brodribb, T. J. (2017). Progressing from 'functional' to mechanistic traits. New Phytologist 215: 97-112.
  3. Choat, B., Jansen, S., Brodribb, T. J., Cochard, H., Delzon, S., Bhaskar, R., Bucci, S. J., Feild, T. S., Gleason, S. M., Hacke, U. G., Jacobsen, A. L., Lens, F., Maherali, H., Martinez-Vilalta, J., Mayr, S., Mencuccini, M., Mitchell, P. J., Nardini, A., Pittermann, J., Pratt, R. B., Sperry, J. S., Westoby, M., Wright, I. J. and Zanne, A. E. (2012). Global convergence in the vulnerability of forests to drought. Nature 491(7426): 752-755.
  4. Larter, M., Pfautsch, S., Domec, J. C., Trueba, S., Nagalingum, N. and Delzon, S. (2017). Aridity drove the evolution of extreme embolism resistance and the radiation of conifer genus Callitris. New Phytol 215(1): 97-112.
  5. Melcher, P. J., Michele Holbrook, N., Burns, M. J., Zwieniecki, M. A., Cobb, A. R., Brodribb, T. J., Choat, B. and Sack, L. (2012). Measurements of stem xylem hydraulic conductivity in the laboratory and field: Measurements of stem xylem hydraulic conductivity. Methods Ecol Evol 3: 685-694.
  6. Pereira, L., Bittencourt, P. R., Oliveira, R. S., Junior, M. B., Barros, F. V., Ribeiro, R. V. and Mazzafera, P. (2016). Plant pneumatics: stem air flow is related to embolism - new perspectives on methods in plant hydraulics. New Phytol 211(1): 357-370.
  7. R Core Team (2011). R: A language and environment for statistical computing. Vienna, Austria: the R Foundation for Statistical Computing.
  8. Rowland, L., da Costa, A. C., Galbraith, D. R., Oliveira, R. S., Binks, O. J., Oliveira, A. A., Pullen, A. M., Doughty, C. E., Metcalfe, D. B., Vasconcelos, S. S., Ferreira, L. V., Malhi, Y., Grace, J., Mencuccini, M. and Meir, P. (2015). Death from drought in tropical forests is triggered by hydraulics not carbon starvation. Nature 528(7580): 119-122.
  9. Sperry, J. S., Donnelly, J. R. and Tyree, M. T. (1988). A method for measuring hydraulic conductivity and embolism in xylem. Plant Cell Environ 11: 35-40.
  10. Sperry, J. S and Love, D. M. (2015). What plant hydraulics can tell us about responses to climate-change droughts. New Phytol 207: 14-27.
  11. Trifilò, P., Raimondo, F., Lo Gullo, M. A., Barbera, P. M., Salleo, S. and Nardini, A. (2014). Relax and refill: xylem rehydration prior to hydraulic measurements favours embolism repair in stems and generates artificially low PLC values. Plant Cell Environ 37(11): 2491-2499.
  12. Wheeler, J. K., Huggett, B. A., Tofte, A. N., Rockwell, F. E. and Holbrook, N. M. (2013). Cutting xylem under tension or supersaturated with gas can generate PLC and the appearance of rapid recovery from embolism. Plant Cell Environ 36(11): 1938-1949.
  13. Zhang, Y., Lamarque, L. J., Torres-Ruiz, J. M., Schuldt, B., Karimi, Z., Li, S., Qin, D. W., Bittencourt, P., Burlett, R., Cao, K. F., Delzon, S., Oliveira, R., Pereira, L. and Jansen, S. (2018). Testing the plant pneumatic method to estimate xylem embolism resistance in stems of temperate trees. Tree Physiol 38(7): 1016-1025.

简介

栓塞,在植物水运系统中形成气泡,对植物水分关系产生重大影响。 植物水分运输系统中的栓塞形成破坏了植物水分运输能力,损害了植物功能并引发植物死亡。 用传统的液压方法测量栓塞既耗时又需要大量的植物材料。 虽然茎部液压方法测量由于栓塞形成而导致的木质部水力传导损失,但气动方法直接量化木质部内栓子的量随木质部空气含量的变化而变化。 气动方法是一种简单快速(每天8+栓塞曲线)方法,用于测量需要最少植物材料的植物栓塞。 在这里,我们提供有关气动方法的详细描述和最近的技术改进。

【背景】由于在干旱条件下空气进入木质部导管,植物木质部栓塞。抗栓塞形成是决定物种分布,死亡率和进化的最重要的植物性状之一(Choat et al。>,2012; Rowland et al。>,2015; Larter et al。>,2017)最近被认为是模拟植物功能和预测植物对全球变化的反应的关键特征(Sperry和Love,2015; Brodribb,2017)。大多数用于评估栓塞阻力的方法测量栓塞分支节段的水力传导率,并将其与无栓塞的分支节段的水力传导相关联(Sperry et al。>,1988; Melcher et al。 >,2012)。这些方法通常很耗时并且容易产生多种伪影(Wheeler et al。>,2013;Trifilò et al。>,2014; Beikircher and Mayr,2016)。

气动方法最近被提出作为从不同的角度估计栓塞抗性的替代方法,而不是从水流的角度来看,而是从栓塞的直接后果 - 空气存在于木质部(Pereira 等人< / em>,2016)。随着植物的栓塞,木质部内的空气空间增加。因此栓塞改变了分支段的气动特性。在该方法中,对切割分支施加真空,并且测量在分支外流动的空气作为木质部空气含量的估计。分支区段外的气流与分支木质部中的栓子数量之间存在很强的关系(Pereira et al。>,2016; Zhang et al。>,2018)。真空法提供了一种简单,低成本,快速且实用的方法来测量植物栓塞。另外,气动方法不需要通过正在研究干旱影响的样品进行再水化(冲洗水)。

关键字:抗栓性, 植物干旱胁迫, 水力学失败, 植物气体力学

材料和试剂

  1. 气动设备
    1. 适配器Luers(Cole-Parmer,目录号:EW-30800-06和EW-30800-24)
    2. 1 L Kitasato烧瓶(Prolab,目录号:PL287)
    3. 硅胶管(3 mm内径和5.2 mm外径,4.9 mm内径和9.7 mm外径,更大或更小尺寸取决于样品直径,优先选择厚壁管,因为它们在夹紧后密封性更好)
    4. 刚性管(Cole-Parmer,目录号:EW-30600-62)
    5. 真空源,注射器或真空泵(Prolab,目录号分别为032357和VAC29-110)
    6. 三通旋塞(Cole-Parmer,目录号:EW-30600-07)
    7. 真空容器(具有刚性壁的容器或管以存储真空; 1-10ml体积通常就足够了)
    8. 建议使用30至110 kPa的真空计(霍尼韦尔,目录号:142PC05D或26PCCFA6D或MPX5100AP;恩智浦半导体;荷兰.Farnell,目录号:1386589,731766和1457156)

  2. 样品制备和处理
    1. 塑胶
    2. 塑料石蜡膜(Prolab,目录号:PM996)
    3. 塑料夹(Cole-Parmer,目录号:RZ-06832-02)和/或拉链

设备

  1. 电压表
    1. 锋利的剃须刀
    2. 电压表或电压记录仪(142PC05D或MPX5100AP精度为1 mV,26PCCFA6D精度为0.01 mV)
    3. 替代方案:用于Arduino UNO微控制器板(Plant Technology and Environmental Monitoring-PLANTEM)的气动屏蔽,与26PCCFA6D一起使用(详见图4)

  2. 木质部水势测量
    1. 压力室(PMS仪器,型号:PMS 1000,或其他)
    2. 笔记本和USB数码显微镜(Jiusion,或其他)
      注意:由于安全问题,我们不鼓励使用放大镜。>

程序

气动测量需要构造气动装置并测量空气排放和木质部水势。每个步骤在下面和视频1中描述。


视频1.使用电压表演示气动设备组合和空气放电测量程序

  1. 气动仪器(图1)
    气动装置基本上由一个装有真空的容器连接,一个用于读取真空压力的仪表和一个用于填充储液器的真空源。


    图1.气动设备。 :一种。真空设备。 a)用作真空源的普通注射器; b)刚性管也用作真空容器; c)真空计(142PC05D)耦合到12V电源和电压调节器; d)电压表探头连接到传感器输出。 B.分支连接细节。 e)用塑料夹紧住弹性管到样品; f)塑料薄膜(必要时涂胶)以避免泄漏; g)弹性管在被塑料夹压紧时变形。 C.真空源的替代设置。真空泵用于在真空储存器中储存真空(h;在该实施例中为1L Kitasato烧瓶)。真空储存器用于通过第三个三通旋塞(i)向真空容器施加真空。 S1和S2分别是三通旋塞阀1和。

    1. 将真空容器连接到第一个三通旋塞(S1)。将硅胶管(足以使分支杆紧固)连接到S1的一个出口,并将S1的另一个出口连接到大气(图1A和1B)。
    2. 将真空容器连接到第二个三通旋塞(S2)。将真空计连接到S2的一个出口,将真空源连接到S2的另一个出口(图1A)。
      1. 选择真空容器容积(V r ; L),以确保真空计的足够精确度。必须从管道和旋塞阀数据表(每个刚性管[Cole-Parmer,USA]具有1.304 ml)测量或计算V r 。几毫升的真空容器通常就足够了(单独的设备的管道可能足以作为真空容器)。
      2. 真空源可以是注射器或真空泵。可以将大量真空储存在容器(例如Kitasato烧瓶)中,并用于补充设备真空容器以便于使用(图1C)。
      3. 可以读取真空计:用电压表;电压记录器连接到计算机;或者使用Pneumatic Shield和Arduino UNO微控制器。
      4. 除连接分支的管道外,所有管道必须是刚性的,以避免真空下的体积变化。将分支连接到S1的管应该是弹性的,因此分支可以紧密连接。我们建议使用鲁尔锁连接,以方便使用并防止泄漏。

  2. 空气排放测量(图2)
    从分支(AD)排出的空气是离开分支到真空容器的空气量。测量真空容器中的空气压力变化并使用理想气体定律将其转换成体积。 AD随着测量的分支节段的栓子量而增加,直到分支完全栓塞。必须使用不同木质部水势中的相同样品测量多个AD点,以便数据可以标准化,并且可以在下一部分估计栓塞阻力曲线。


    图2.空气流量曲线测量。构建和排气曲线的步骤。 1)用真空填充真空容器。 2)测量初始压力。 3)对样品施加压力,等待2.5分钟并测量最终压力。 4)测量样本水势。 5)让样品干燥。进一步干燥后重复第一步。

    1. 选择一个完全水合和非栓塞的分支段与一些叶子。
    2. 用锋利的剃刀从植物上切下树枝。
    3. 使用塑料夹/塑料薄膜将分支连接到S1中的弹性管,以确保无泄漏或无明显泄漏。&nbsp;
    4. 在S1中将分支打开到大气,并将分支关闭到S2中的真空容器(图2.1)。
    5. 使用真空源将真空容器压力降低至35-40 kPa(大气压减去55-60 kPa),并将真空容器关闭到S2中的真空源(图2.1)。
    6. 读取真空容器的初始压力(P i ; kPa)(图2.2)。
    7. 在S1中将分支段关闭到大气,并将其连接到S2中的真空容器(图2.3)。
    8. 2.5分钟后,读取真空容器的最终压力(P f ;图2.3)。
    9. 使用理想气体定律计算从分支排出的空气摩尔数Δn(摩尔):

      (1)Δn= PiVr / RT - PfVr / RT;

      其中R是气体常数(8.314 kPa L mol -1 k -1 ); T是测量期间的设备/室温(K),V r 是储层体积(L)。

    10. (可选)使用理想气体定律将排出的空气摩尔数转换为在大气压(AD;μl)下排出的空气量:

      (2)AD =(ΔnRT/ P atm )x 10 6

      其中P atm 是大气压(kPa),10 6 是从L变为μl。

    11. 测量分支样品的木质部水势(Ψ x )并用胶水密封叶片(图2.4)。关于样品处理和叶片和木质部水势平衡问题,请参见“注11”。
    12. 让支管在工作台上干燥(图2.5)。
    13. 将样品的叶水势包装起来,用Ψ x 平衡。
    14. 从步骤B5再次开始以获得另一个数据点。分支样本的最后一个数据点应该是完全栓塞的数据点,因为所有数据都将由最后一个点标准化。
    注意:>
    1. 每当没有进行测量时,必须将分支连接到大气,以确保分支内的初始气压是大气压。在AD测量之后,将分支连接到大气至少2.5分钟,然后再进行另一项测量,以确保分支内没有真空。>
    2. 分支在脱水时可能会收缩,与S1的连接可能会松动。拧紧连接以确保无泄漏。>

数据分析

数据处理需要使用气动测量来估计电导率(PLC)的百分比损失。 PLC与分支样品的最大空气排放百分比(PAD)成比例。使用基础软件包(R Core Team,2011 [版本2.1.2])进行数据处理和分析的示例电子表格和R脚本在附录中提供(“ data.example.xls ”和“ data.analysis.r ”)。要获得PAD,必须对每个分支样本标准化AD(见图3A和3B)。


图3.空气排放和百分比空气排放曲线。 A.不同木质部水势(Ψ x )中分支样品的空气排放测量示例。 B.来自(A)的空气排放措施通过最大空气排放标准化,以获得作为Ψ x 的函数的最大空气排放百分比(PAD)。红线是PAD数据的回归。蓝线是用液压方法获得的电导率(PLC;点未显示)曲线的百分比损失。不同的符号表示气动方法中来自不同样品的数据。树枝在零下3.5-4.5 MPa左右失去了叶子。

的 Data.example.xlsx。 用于写入数据和计算AD和PAD的示例电子表格。
Data.analysis.docx。 R脚本,包含用于处理和分析使用气动方法收集的数据的例程。


图4.气动设备护罩。 A.由PLANTEM开发的Arduino UNO气动护罩模型。 B. Pneumatic Shield原型自动记录两个真空计(26PCCFA6D)。在此示例中,正在测量左分支(由蓝色LED指示),同时数据显示在LCD显示屏中并记录到SD卡。当按下与每个真空计相关联的按钮时,测量开始,并且每秒记录数据。

  • 旋塞阀内部也有空隙。如果对于一次测量,您将一个方向关闭活塞而另一个方向使用不同的方向,则总空气量可能略有不同。
  • 泄漏:如果泄漏很严重,可以计算并从每次测量中减去泄漏。
  • 传感器电压供应:可以使用简单的电压调节器为真空计供电。我们建议使用LM78XX电压调节器(通常为LM7810或7812)进一步控制传感器输入电压,因为输入电压中的噪声会改变电压表输出。使用较低输出电压26PCCFA6D传感器时,这一点尤为重要。 LM78XX电路和组装好的传感器(142PC05D)如下图5所示:


    图5.压力传感器和简单的电压调节器。 A. LM7810电压调节器设置。 B和C. C)中使用的电路板。 B)中的电路是在原始印刷电路板上使用永久性标记手工绘制的,并用高氯酸铁腐蚀。为部件制造孔,然后将它们焊接在板上。 7810是LM7810稳压器。 C1和C2是电解电容,用于滤除噪声并减少输入电压的瞬态变化。

  • 真空计具有非常低的功耗。它可以在现场或偏远地区使用12 V汽车电池供电数天。在这种情况下,电压调节器是基础。
  • AD测量值可以用空气的摩尔数或大气压下的等效体积表示。我们建议将体积表达为与解剖学测量和设备描述相同的单位。
  • 我们使用PAD代替AD来估计栓塞,因为相同物种的样品之间的可变性可能很大。如果对样本有足够的控制,因此变异性很小或整个曲线由一个分支样本组成,AD可以直接用于估计栓塞。
  • 我们使用2.5分钟的放电时间,因为我们发现它足以确保精度。可以使用更短或更长的时间。 AD在时间上不是线性的,因此在比较不同时间与相同物种/样品时应该小心。如果要测量样品中的所有空气体积,可以使用较长的放电时间或将放电曲线外推至无穷大,找出平衡时的压力并使用它来计算样品内的总空气空间。
  • 可以从S1移除分支连接(图1B),而无需从弹性管中取出样品,以便于处理多个样品。例如,如果您在同一天测量10个样品,请使用10个单独的连接(鲁尔锁,弹性管,塑料夹和样品设置),并将每次要测量的样品连接到S1。
    1. 通过被测分支段的最大AD(ADmax)标准化每个AD测量(AD i ;其中i代表每个单独的测量):

      (3)PAD = 100 x(AD i / AD max );

      其中PAD是从样品中排出的最大空气百分比,与PLC有关。
    2. 对于某些物种,即使在完全水合并且没有栓塞存在的情况下,AD也可能是重要的,可能是由于木质部中的自然空气空间。在这种情况下,必须从每个AD测量值中减去初始AD(AD ini ),以便PAD与PLC相关:

      (4)PAD = 100 x(AD i - AD ini )/(AD max - AD ini )

    3. 栓塞曲线的构建遵循用于计算PLC曲线的标准方法(拟合S形曲线,指数曲线,等>),但使用PAD和Ψ x 之间的关系估计PLC曲线。质量曲线必须在PAD变化很小的高水势,过渡区和PAD停止变化的第二个平台处呈现平台(图3B)。


    笔记

    1. 我们建议使用由Plant Technology and Environmental Monitoring(PLANTEM,巴西,图4)开发的Arduino UNO微控制器的Pneumatic Shield,因为它允许同时测量两个样品,无需手动记录真空计电压,有效地使测量速度加倍。


      4660/5000 许多植物物种在树皮/木质部中产生树脂或乳胶。树脂/乳胶的分泌会堵塞容器并改变空气排放。我们建议验证每种研究中是否发生这种情况。如果发生这种情况,用锋利的剃刀切割分支,等待1小时,同时将样品装袋以停止树脂/乳胶分泌,并在连接到装置之前再次切割茎干通常可以解决问题。
    2. 为了确保木质部和叶片水势平衡,叶水势可以用作木质部水势的指标,在测量叶片水势之前,样品必须在塑料袋外保持尽可能少的时间。最好的方法是在装袋样品时测量空气排放,然后取出袋子测量水势。
    3. 样品连接(图1B)可以容纳大量的空气,可以使AD测量值膨胀。我们发现这可以通过折扣测量的第一秒来纠正。折扣测量的第一秒会导致对工厂内部总空气的低估,但这对估计PAD值没有影响,因为它们是相对的。在不折扣第一秒的情况下测量AD的替代方案是从总空气排放中减去样品连接体积。排出的空气总量(Δn),以摩尔计,然后可以计算为:

      Δn=(n r + n sc ) - n f

      其中n r 和n sc 分别是储液器和样品连接器内的初始空气量,其与测量前设备中的空气总量相加。 n f 是设备中的最终空气量。由于n sc 在测量前处于大气压力下,上述等式[在这种情况下等式(1)]变为:

      Δn=(P i V r / RT + P atm V sc / RT) - P f V r / RT
      其中V sc 是样本连接的体积。所有其他程序都是平等的。

    4. 协议中使用的单位,术语和定义:
      AD - 从分支段(μl)排出的空气
      AD max - 从分支段(μl)排出的最大空气量。当它完全空化时,它是样品的AD AD ini - 完全再水化且无气蚀(μl)时排出的空气
      P i - 真空容器的初始压力(kPa)
      P f - 真空容器的最终压力(kPa)
      P atm - 大气压(海平面约101.3 kPa)
      PAD - 从分支排出的最大空气百分比
      PLC-木质部水力传导百分比损失
      S1 - 将分支段连接到真空容器和大气的三通旋塞 S2 - 三通旋塞将真空容器连接到真空源和真空计
      R - 气体常数(8.314 kPa L mol -1 k -1 )
      T - 仪器/室温(K)
      V r - 真空容积(L)
      Δn-真空容器中摩尔数的变化(ni减去nf; mol)
      Ψ x - 木质部水势(MPA)

    致谢

    该研究由FAPESP /微软研究(授予11 / 52072-0)资助,授予R.O.我们感谢UNICAMP生态学和植物生物学研究生课程以及高等教育协调机构(CAPES)奖学金给P.R.L.B.我们感谢资助P.R.L.B.的牛顿国际奖学金(资助NF170370)。在本手稿和圣保罗研究基金会的最终撰写中,授予L.P.(FAPESP,Grant No. 2017 / 14075-3)奖学金。我们感谢David Bartholomew审阅和校对手稿。

    利益争夺

    作者声明他们没有利益冲突。

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    引用:Bittencourt, P. R. L., Pereira, L. and Oliveira, R. S. (2018). Pneumatic Method to Measure Plant Xylem Embolism. Bio-protocol 8(20): e3059. DOI: 10.21769/BioProtoc.3059.
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