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Photometric Assays for Chloroplast Movement Responses to Blue Light
光度法测定叶绿体对蓝光的运动反应   

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Journal of Experimental Botany
Sep 2016

Abstract

Assessment of chloroplast movements by measuring changes in leaf transmittance is discussed, with special reference to the conditions necessary for reliable estimation of blue light–activated chloroplast responses.

Keywords: Arabidopsis thaliana (拟南芥), Blue light (蓝光), Chloroplast movements (叶绿体运动), Leaf transmittance (叶片透光率), Photometric method (光度法), Phototropins (向光素)

Background

Following the discovery of phototropins, chloroplast movements activated with blue light absorbed by these photoreceptors began to arouse much interest. Quantitative assessment of chloroplast redistribution in a multilayer leaf relies mainly on measuring transmittance changes of the tissue that are a consequence of this redistribution. Various devices have been used for that purpose, including a recently adapted commercial microplate reader (Wada and Kong, 2011; Johansson and Zeidler, 2016), particularly suited for screening large number of samples. However, samples must be properly pretreated and characterized to make their comparison reliable. This aspect has not been referred to in many papers making use of the transmittance technique. Hence, we found it important to discuss these issues in the current protocol.

Materials and Reagents

  1. Thin microscope slide with double wells (Ted Pella, catalog number: 260242 )
  2. Cling film
  3. Tissue paper
  4. Optionally, for measurements of water plants or infiltrated leaf pieces
    1. Parafilm M (Sigma-Aldrich, catalog number: P7793 )
    2. Silicon grease (Baysilone-Paste, GE Bayer Silicones)
    3. 2 ml syringe

Equipment

  1. Custom-made photometer, based on Walczak and Gabryś (1980). This device is a prototype that contains the following commercially available parts (Figure 1):
    1. Luxeon Royal Blue LXHL-FR5C LED (460 nm) (Luxeon Star, catalog number: LXHL-FR5C )–the source of the blue actinic beam (Figure 1A)
      Note: This product has been discontinued.
    2. L-793SRD-B LED (660 nm) (Kingbright, catalog number: L-793SRD-B )–the source of the red measuring light (Figure 1B)
    3. BPW20RF Planar Silicon PN photodiode (Vishay, catalog number: BPW20RF )–the detector (Figure 1E)
    4. NI USB-6001 DAQ board (National Instruments, model: USB-6001 )–for signal digitization (Figure 1F)


      Figure 1. A double-beam photometer used for measurements of transmittance changes resulting from blue light–activated chloroplast movements. Key components: A. blue LED housing; B. red LED housing; C. measuring chamber; D. 660 nm interference filter; E. receiver photodiode; F. electronic controller.

Software

  1. Software appropriate for analysis of numerical data, e.g., R, Octave, MATLAB, or Mathematica, equipped with a digital filter appropriate for numerical differentiation (e.g., Savitzky-Golay filter, available in the R software as ‘sgolayfilt’, part of the ‘signal’ library)

Procedure

  1. Principle of the experiment
    The photometric method is based on the measurement of changes in leaf transmittance that result from chloroplast relocations in the cells. In terrestrial angiosperms, chloroplast movements are activated only by blue light and depend on its fluence rate and direction. In weak blue light, chloroplasts redistribute to cell walls perpendicular to the direction of incident light. This response, called accumulation, leads to a decrease in the leaf transmittance. Strong blue light induces chloroplast avoidance. Chloroplasts redistribute to cell walls parallel to the light direction, which results in an increase of the leaf transmittance. Photoreceptors responsible for chloroplast movements are called phototropins. Two phototropins are encoded in the genome of Arabidopsis thaliana: phototropin1 and phototropin2. While the accumulation response is mediated by both phototropins, the avoidance response is primarily controlled by phototropin2 (Jarillo et al., 2001; Kagawa et al., 2001; Sakai et al., 2001). Red light does not induce chloroplast movements in terrestrial angiosperms, thus it is used as measuring light. The measuring red light intensity (0.3 µmol m-2 sec-1) is ca. 40 times less than the light compensation point of photosynthesis (12.8 µmol m-2 sec-1 for red light, Takemiya et al., 2005). Thus, the activity of photosynthesis induced by the measuring light is negligible.

  2. Description of the equipment
    1. Transmittance measurements are carried out using a custom-made photometer (Figure 1). A detached leaf is mounted on a holder (Figure 2) fitting the measuring chamber (Figure 1C). Two concentric light beams are delivered perpendicular to the dorsal surface of the leaf. These are the actinic blue light of 460 nm from a Luxeon Royal Blue LXHL-FR5C LED (Philips Lumileds Lighting Comp.) (Figure 1A) and the measuring red light of 660 nm, 0.3 µmol m-2 sec-1, from an L-793SRD-B LED (Kingbright) (Figure 1B). Fluence rate of the actinic beam is controlled electronically using voltage-controlled current-source electronics (Figure 1F). An aperture in the sample holder limits the diameter of the transmitted light beam to about ½ of the incident one to ensure uniform illumination of the measured tissue fragment. The aperture diameter of 3.5 mm is used for Arabidopsis thaliana (Figure 2).
      Note: Measurements on leaves of other plant species, such as aquatic angiosperms (Banaś and Gabryś, 2007) and mosses (e.g., Lemna trisulca and Funaria hygrometrica, respectively), may require smaller apertures.


      Figure 2. An Arabidopsis leaf on the sample holder

  3. An opal glass–topped light guide is used to deliver the transmitted light to the signal detector (Figure 1E). This is a planar Silicon PN photodiode, model BPW20RF (Vishay Semiconductor) designed for high precision linear applications. The photodiode is integrated with a 660 nm interference filter and a double lens system (Figure 1D). The measuring beam is square-wave modulated with the frequency of 2 kHz to eliminate contributions from the photodiode dark current and external light sources. This also allows to supplement the actinic blue light with other selected wavelengths (Gabryś, 1985) or to use white light to activate chloroplast movements. The device selectively records changes in the modulated beam due to the application of the lock-in detection technique. The actinic beam intensity can be set to a desired level at any time after the start of the experiment. The device can also generate short pulses of actinic light. Pulse generation is hardware-controlled to ensure repeatability of the pulse duration. The software used to run the photometer is written in LabVIEW (National Instruments)–a software environment for deployment of measurement and control systems.

  4. Calibration of the equipment
    1. Before measurements, the instrument must be calibrated. The receiver photodiode signal is measured with the measuring light path blocked by an opaque lid. This value corresponds to 0% transmittance.
    2. Next, the signal is measured with the sample holder inside the measuring chamber, but without the leaf. This value corresponds to 100% transmittance. The receiver photodiode signal depends linearly on the fluence rate of the transmitted light.

  5. Plant growth conditions
    Note: Leaves used for chloroplast movement measurements should be optically thin. Each cell layer absorbs light, so that a fluence rate gradient is formed across the leaf blade. The distal cell layer receives less light. Thus, its chloroplasts respond to weaker light than those in the layer facing the light source. Optically denser layers produce a steeper gradient. To minimize light attenuation through the leaf blade, the transmittance of leaves used for experiments is at least 8%. It is possible to measure thicker leaves after infiltration with water or buffer using a syringe, which increases leaf transmittance. It is also a convenient way of applying inhibitors (Aggarwal et al., 2013) or other chemicals (Anielska-Mazur et al., 2009).
    1. For Arabidopsis thaliana, moderate transmittance levels of leaves are assured by growing at 80-100 µmol m-2 sec-1 of white light in a short day (10 h light/14 h dark).
    2. Leaves from 4-5-week-old plants are used for photometric measurements.

  6. Sample preparation
    1. Plants are dark-adapted in a darkroom for at least 7 h to allow chloroplasts to obtain the stationary dark position. Dark adaptation is necessary to ensure that chloroplasts start from the same positions in the cell, which is crucial for quantitative comparison of chloroplast responses in various leaves. Otherwise transmittance changes correspond to different phases of redistribution and cannot be compared. Routinely, leaves are transferred to darkness at the end of the photoperiod light cycle.
    2. All experimental manipulations are performed under dim green light (< 0.05 µmol m-2 sec-1).
    3. Following dark adaptation, a single leaf is detached from the plant and placed on a thin microscope slide mounted on the holder (Figure 2).
    4. The leaf is flattened and positioned in the middle of the slide directly above the measuring aperture. It is important to avoid putting the major vein into the measuring area.
    5. The leaf is covered with cling film stretched over a metal ring and its petiole is wrapped in a piece of water-soaked tissue paper to avoid drying during the experiment.
      Note: Big leaves, e.g., Nicotiana tabaccum, may be cut into small fragments and immersed in a drop of water for the measurement. In that case, the leaf fragment is placed in a chamber formed by a Parafilm ring and the cling film (Anielska-Mazur et al., 2009). To prevent evaporation, the chamber is sealed with a high viscosity silicon grease.
    6. The holder with the leaf is placed inside the measuring chamber and the initial transmittance level is recorded immediately.
      Note: Ideally, the dark transmittance values of all leaves used in the same study should be similar. As the photometric method is an indirect method of chloroplast movement assessment, the reliability of the results may be adversely affected by differences in leaf anatomy. This must be taken into account while comparing chloroplast responses in mutant and wild type leaves (Eckstein et al., 2016). Variation in leaf anatomy is often reflected in different dark transmittance. The effect of dark transmittance on the movement parameters calculated from the photometric curves (e.g., amplitudes and velocities, see below) should always be determined. Thus, it is advisable to plot the parameters against the dark transmittance. This effect can also be taken into account at the stage of statistical analysis of results (see the Data analysis section).

  7. Measurements under continuous light–multiple illumination steps of increasing intensity
    Note: Chloroplast relocations in response to increasing light intensities can be measured in one leaf. The outcome is a fluence rate–response curve. For Arabidopsis, typical light intensities used start from 0.1 µmol m-2 sec-1 and increase stepwise up to 120 µmol m-2 sec-1 (Jarillo et al., 2001) (Figure 3A).


    Figure 3. Typical curves obtained using the photometric method. A. A fluence rate–response curve. A dark-adapted leaf of A. thaliana with initial transmittance of 12% was exposed to continuous blue light of fluence rates 0.4-120 µmol m-2 sec-1. Each irradiation step lasted 50 min. B. A typical recording trace, explaining the way in which amplitudes and velocities are calculated. Arrows indicate start of illumination with weak (↓1.6 µmol m-2 sec-1) or strong (↑120 µmol m-2 sec-1) blue light. ΔTac, ΔTav–amplitudes, and (dT/dt)ac, (dT/dt)av–velocities of transmittance changes corresponding to the accumulation and avoidance response, respectively.

    1. The transmittance of a dark-adapted leaf is recorded for several minutes in physiological darkness to make sure that its level is stable.
    2. Blue light of the desired fluence rate is turned on and the changes in transmittance are recorded for at least 45 min.
      Note: This is a compromise time, usually too short to obtain the stationary phase of the response. However, the transmittance drift in the final stage of the response is very small and can be neglected. In the case of slow responses, e.g., at low temperature, the time of light exposure/measurement should be increased (Łabuz et al., 2015).
    3. After the response reaches a plateau, the blue light intensity can be changed.
      Note: To measure only the maximal accumulation and avoidance, saturating light for each response can be used. A typical experiment consists of recording the dark transmittance level for 5 min, followed by weak blue light (1.6 µmol m-2 sec-1) for at least 45 min and strong blue light (120 µmol m-2 sec-1) for at least 45 min (Figure 3B).

  8. Measurements under continuous light–one intensity at a time
    Another approach is to measure chloroplast responses only to one light intensity at a time. In this case, we always illuminate a dark-adapted leaf and obtain a family of curves, each corresponding to one intensity (Jarillo et al., 2001). This is a standard approach if we want to compare the kinetics of chloroplast responses in various samples under various fluence-rates, as it ensures that chloroplasts start from the same position in the cell. For response amplitudes, both approaches are equivalent provided that amplitudes do not depend significantly on previous illumination but only on the current fluence-rate. This must be verified experimentally for the studied species. In the case of A. thaliana wild type, amplitudes depend only on the current illumination. However, mutants may behave differently. For example, the phot2 mutant shows practically no response to strong blue light when chloroplasts have already reached the accumulation position, while a biphasic response is observed when strong light is applied directly after dark adaptation (Jarillo et al., 2001).

  9. Measurements of responses to light pulses
    Note: Transmittance changes after light pulses are lower in magnitude than responses to continuous light, therefore they require a good signal-to-noise ratio of the recording system.
    1. The dark transmittance level is recorded for 5 min.
    2. A very short pulse of blue light is applied and chloroplast relocations are recorded in darkness for the following 45 min. For experiments with A. thaliana, we typically use a series of strong blue light (120 µmol m-2 sec-1, saturating the avoidance response) pulses for 0.1, 0.2, 1, 2, 10 and 20 sec (Sztatelman et al., 2016).
      Note: The pulse duration and light intensity can be adjusted in broad ranges. For pulses resulting only in transient accumulation, the chloroplast response follows the Bunsen-Roscoe reciprocity law. Thus, it is determined by the total amount of radiant energy delivered (product of fluence rate and the exposure time) (Gabryś et al., 1981).
    3. If the goal of the experiment is to compare effects of different pulse durations on chloroplast movements in the same plant line, it is convenient to record the whole series on the same leaf. After the transmittance curve is recorded, the leaf is transferred back to darkness for recovery and the response in a second leaf is measured. This dark recovery may be shorter than the initial adaptation period because the responses to pulses and the respective transmittance changes are small and usually level off within 1.5 h. Next, the first leaf is taken back and challenged with a light pulse of increased duration.

Data analysis

  1. Calculation of movement parameters
    1. To quantify the chloroplast movements, amplitudes (ΔT) and maximal velocities (dT/dt) of transmittance change during each illumination phase are calculated. These parameters can be estimated on a curve print-out using a ruler. Alternatively, they can be determined using software appropriate for analysis of numerical data, e.g., R, MATLAB or Mathematica. Amplitudes are calculated relative to the dark transmittance level (Figure 3B). If the noise is substantial, the accuracy of the calculated values of amplitudes can be improved by smoothing the curves with a low-pass digital filter. Savitzky Golay filter (available e.g., in the free software R) is appropriate for curves with points spaced at equal distances, as is usually the case. If the points are irregularly spaced, the curve can be smoothed using local regression (also available in R). Maximal velocity can be determined by drawing a straight line, tangent to the transmittance curve at the point where the response is the fastest. The slope of the line is equal to the magnitude of the velocity. Numerical estimation of the velocity is possible with an appropriate digital filter. Savitzky-Golay filter can also be used for this purpose. It works by fitting a polynomial to points within a sliding window. The point in its center is replaced with a value predicted by the polynomial fit (for smoothing) or with the derivative of the fit (for velocity calculation). The degree of the polynomial should be low. We use quadratic polynomials. The width of the window is also specified by the user. Wider window allows for more efficient noise removal. However, the use of a too wide window may result in underestimation of the maximal velocity of movements. Thus, the width should be adjusted to the noise level and sampling rate of the equipment. For velocity calculation, we use the window width that corresponds to a 2 min interval.
      Note: To better characterize the kinetics of chloroplast movements, it is advisable to plot rate (dT/dt) against time as in Łabuz et al., 2015 (Figures 1C and 1E-1F, therein). This facilitates estimation of the maximum velocity and the time after which the maximum velocity is achieved. This also helps to assess whether the new light intensity was applied after reaching the stationary phase of the preceding response.
    2. The chloroplast movements triggered by pulses can be quantified similarly to responses to continuous light. However, the response to a single pulse often consists of two phases–a fast, transient avoidance phase followed by a slower accumulation response (Sztatelman et al., 2006). Thus, two amplitudes and two velocities should be calculated for a single pulse.
      Note: Additional parameters may be calculated from photometric curves. The time between the onset of the pulse and the maximum of transient avoidance or accumulation is useful in characterizing the dynamics of chloroplast responses to light pulses (Sztatelman et al., 2006).

  2. Randomization and experimental design
    1. Reproducibility of the chloroplast movement assay requires both randomization and proper control over plant growth conditions. The conditions in the growth chamber (availability of water, light intensity, etc.) may vary from place to place. To reduce the risk of systematic errors, plants from different lines should be placed in the chamber in a randomized manner. To reduce variance due to differences in growth, pots may be reshuffled several times during plant growth; this is especially important in growth chambers with side illumination. If the experiment requires use of several growth chambers, plants from all lines (or, in general, all experimental groups) should be grown simultaneously in every chamber. The results obtained from different batches of plants may vary. To ensure the reproducibility of results, the measurements should be repeated at least three times, using batches of plants that were sown and grown independently.
    2. In experiments that involve leaf pretreatment (e.g., infiltration with an inhibitor), whole plants may be either allocated randomly to each treatment level (e.g., inhibitor concentration), or leaves from the same plant, similar in size and shape, may be allocated to each level. This choice determines the type of the statistical method that can be used for data analysis. These two methods of allocation to treatment levels must not be mixed in a single experiment. Otherwise it would be impossible to analyze the results statistically.
    3. The number of plants necessary for an experiment depends on the magnitude of the studied effects, the variability of plants and the number of experimental groups. A group is a combination of levels of different factors, e.g., in an experiment on a mutant and the wild type plants, treated with an inhibitor (two concentrations plus control), which was repeated three times on different batches of plants, there are three factors (the plant line, the inhibitor concentration, the plant batch) and 18 groups (2 x 3 x 3). In experiments on A. thaliana, we use 10 or more plants per group when we look for effects of moderate magnitude. Large effects require fewer replicates.

  3. Statistical tests
    1. The choice of statistical tests depends on the type of variables that may affect the movement parameters. Most often, the variables controlled by the experimenter are fixed categorical variables (factors), which means that their values are levels specified in advance. Examples are plant lines (two levels: wild type vs. mutant plants) or stress treatment (stressed vs. control plants). If the whole experiment was repeated on multiple batches of plants, the batch can be treated as a random variable. Variables may also be continuous, e.g., dark transmittance. The movement parameters can be treated as linearly dependent on the variables. If all variables included in the model are categorical factors, regular Analysis of Variance (ANOVA) is used to test whether the effects of the factors are significant. The differences in means between all pairs of groups can be tested with Tukey test. A selected level, treated as the control can be compared to every other level, with Dunnett’s test. When similar leaves, usually selected from the same plant, are allocated to different treatment levels, a new blocking variable (plant) must be introduced into the ANOVA model. In the special case of just one treatment level and the control, the paired-sample t-test is appropriate. 
    2. The effects of dark transmittance on chloroplast responses may also be included in the model. Usually, the relationship between movement parameters and dark transmittance can be fitted well with a line. In such a case, the effect of dark transmittance can be reduced at the stage of data analysis, by treating dark transmittance as an additional, continuous variable (a covariate), alongside other variables of experimental interest. If the slopes of the best linear fits are similar for all plant lines or treatments, the standard analysis of covariance (ANCOVA), available in the free software R, can be employed.

Acknowledgments

This work was supported from European Union funds within the framework of FP7, Marie Curie ITN CALIPSO, grant No. [GA 2013-ITN-607-607] and from financial resources for science in the years 2013-2017, allocated to the realization of a co-financed international project.
Both the equipment and the protocol have been modified from previous work by Walczak and Gabrys (1980). New type of photometer for measurement of transmission changes corresponding to chloroplast movements in leaves. Photosynthetica 14, 65-72.

References

  1. Aggarwal, C., Łabuz, J. and Gabrys, H. (2013). Phosphoinositides play differential roles in regulating phototropin1- and phototropin2-mediated chloroplast movements in Arabidopsis. PLoS One 8(2): e55393.
  2. Anielska-Mazur, A., Bernaś, T. and Gabryś, H. (2009). In vivo reorganization of the actin cytoskeleton in leaves of Nicotiana tabacum L. transformed with plastin-GFP. Correlation with light-activated chloroplast responses. BMC Plant Biol 9: 64.
  3. Banaś A. K and Gabryś, H. (2007). Influence of sugars on blue light-induced chloroplast movements. Plant Signal Behav 4(2): 221-230.
  4. Eckstein, A., Krzeszowiec, W., Waligórski, P. and Gabryś, H. (2016). Auxin and chloroplast movements. Physiol Plant 156(3): 351-366.
  5. Gabryś, H. (1985). Chloroplast movement in Mougeotia induced by blue light pulses. Planta 166:134-140.
  6. Gabryś, H., Walczak, T. and Zurzycki J. (1981). Chloroplast translocations induced by light pulses: Effects of single light pulses. Planta 152: 553-556.
  7. Jarillo, J. A., Gabrys, H., Capel, J., Alonso, J. M., Ecker, J. R. and Cashmore, A. R. (2001). Phototropin-related NPL1 controls chloroplast relocation induced by blue light. Nature 410(6831): 952-954.
  8. Johansson, H. and Zeidler, M. (2016). Automatic chloroplast movement analysis. Methods Mol Biol 1398: 29-35.
  9. Kagawa, T., Sakai, T., Suetsugu, N., Oikawa, K., Ishiguro, S., Kato, T., Tabata, S., Okada, K. and Wada, M. (2001). Arabidopsis NPL1: a phototropin homolog controlling the chloroplast high-light avoidance response. Science 291: 2138-2141.
  10. Łabuz, J., Hermanowicz, P. and Gabryś, H. (2015). The impact of temperature on blue light induced chloroplast movements in Arabidopsis thaliana. Plant Sci 239: 238-249.
  11. Sakai, T., Kagawa, T., Kasahara, M., Swartz, T. E., Christie, J. M., Briggs, W. R., Wada, M. and Okada, K. (2001). Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation. PNAS 98: 6969-6974.
  12. Sztatelman, O., Łabuz, J., Hermanowicz, P., Banaś, A. K., Bażant, A., Zgłobicki, P., Aggarwal, C., Nadzieja, M., Krzeszowiec, W., Strzałka, W. and Gabryś, H. (2016). Fine tuning chloroplast movements through physical interactions between phototropins. J Exp Bot 67: 4963-4978.
  13. Takemiya, A., Inoue, S., Doi, M., Kinoshita, T. and Shimazaki, K. (2005). Phototropins promote plant growth in response to blue light in low light environments. Plant Cell 17: 1120-1127.
  14. Wada, M. and Kong, S. G. (2011). Analysis of chloroplast movement and relocation in Arabidopsis. Methods Mol Biol 774: 87-102.
  15. Walczak, T. and Gabrys, H. (1980). New type of photometer for measurements of transmission changes corresponding to chloroplast movements in leaves. Photosynthetica 14: 65-72.

简介

通过测量叶片透光率的变化来评估叶绿体运动,特别参考了可靠估计蓝光活化叶绿体反应所需的条件。

背景 在光皮质素的发现之后,由这些光感受器吸收的蓝光激活的叶绿体运动开始引起很大的兴趣。多层叶片中叶绿体再分布的定量评估主要依赖于测量组织的透射率变化,这是该再分布的结果。已经使用了各种装置,包括最近改编的商业酶标仪(Wada和Kong,2011; Johansson和Zeidler,2016),特别适用于筛选大量样品。然而,样品必须进行适当的预处理和表征,以使其比较可靠。在使用透射技术的许多论文中没有提到这个方面。因此,我们认为在当前协议中讨论这些问题很重要。

关键字:拟南芥, 蓝光, 叶绿体运动, 叶片透光率, 光度法, 向光素

材料和试剂

  1. 带双孔的薄型显微镜载玻片(Ted Pella,目录号:260242)
  2. 贴膜
  3. 组织纸
  4. 任选地,用于测量水植物或渗透的叶片
    1. Parafilm M(Sigma-Aldrich,目录号:P7793)
    2. 硅油(Baysilone-Paste,GE拜耳有机硅)
    3. 2 ml注射器

设备

  1. 定制光度计,基于Walczak和Gabryś(1980)。该设备是包含以下市售零件的原型(图1):
    1. Luxeon Royal Blue LXHL-FR5C LED(460nm)(Luxeon Star,目录号:LXHL-FR5C) - 蓝色光化射束的来源(图1A)
      注意:本产品已停产。
    2. L-793SRD-B LED(660nm)(Kingbright,目录号:L-793SRD-B) - 红色测量光源(图1B)
    3. BPW20RF平面硅PN光电二极管(Vishay,目录号:BPW20RF) - 检测器(图1E)
    4. NI USB-6001 DAQ板(National Instruments,型号:USB-6001) - 用于信号数字化(图1F)


      图1.用于测量由蓝色光活化叶绿体运动导致的透射率变化的双光束光度计。关键部件:A.蓝色LED外壳; B.红色LED外壳; C.测量室; D. 660 nm干涉滤光片;接收光电二极管; F.电子控制器。

软件

  1. 适用于数值数据分析的软件,例如,具有适合数值微分的数字滤波器(例如,Savitzky-Golay)的R,Octave,MATLAB或Mathematica过滤器,在R软件中可用作“sgolayfilt”,“signal”库的一部分)

程序

  1. 实验原理
    测光方法是基于细胞中叶绿体重新定位导致的叶片透光率变化的测量。在陆地被子植物中,叶绿体运动仅由蓝光激活,取决于其通量率和方向。在弱蓝光下,叶绿体重新分布到垂直于入射光方向的细胞壁上。这种响应称为积累,导致叶片透光率的降低。强蓝光诱导叶绿体避免。叶绿体重新分布到与光方向平行的细胞壁上,这导致叶片透射率的增加。负责叶绿体运动的光感受器称为光皮质素。在拟南芥的基因组中编码两种光皮质素:光皮质素1和光诱导素2。虽然积累反应是由光皮质素介导的,但避免反应主要由光皮素2控制(Jarillo等人,2001; Kagawa等人,2001; Sakai et al。,2001)。红光不会诱导陆生被子植物中的叶绿体运动,因此被用作测量光。测量红光强度(0.3μmol/ m 2) sec -1。 Takemiya等人,2005年光合作用的光补偿点(12.8μmolm -2 sec -1 的40倍) )。因此,由测量光引起的光合作用活动可以忽略不计。

  2. 设备说明
    1. 透射率测量使用定制的光度计进行(图1)。分离的叶片安装在安装测量室的支架(图2)上(图1C)。两个同心的光束垂直于叶子的背面递送。这些是来自Luxeon Royal Blue LXHL-FR5C LED(Philips Lumiled Lighting Comp。)(图1A)的460nm的光化蓝光,660nm,0.3μmol/ m 2的测量红光, sec -1 ,从L-793SRD-B LED(Kingbright)(图1B)。使用电压控制的电流源电子器件(图1F)可以电子控制光化光束的光通量。样品架中的孔径将透射光束的直径限制为入射光束的约1/2,以确保测量的组织片段的均匀照明。 3.5 mm的孔径直径用于拟南芥(图2)。
      注意:其他植物物种的叶片,如水生被子植物(Banaś和Gabryś,2007)和苔藓(例如,三叶草和Funaria hygrometrica)的叶片的测量可能需要较小的孔径。 >

      图2.样品架上的拟南芥叶

  3. 使用蛋白石玻璃顶光导,将透射光传递到信号检测器(图1E)。这是一种平面硅PN光电二极管,型号为BPW20RF(Vishay Semiconductor),专为高精度线性应用而设计。光电二极管与660 nm干涉滤光片和双透镜系统集成(图1D)。测量光束以2 kHz的频率进行方波调制,以消除光电二极管暗电流和外部光源的贡献。这也允许用其他选择的波长补充光化蓝光(Gabryś,1985)或使用白光激活叶绿体运动。由于应用锁定检测技术,该装置选择性地记录调制束的变化。激光束强度可以在实验开始后的任何时间被设定为期望的水平。该装置还可以产生短的光化光脉冲。脉冲发生是硬件控制的,以确保脉冲持续时间的重复性。用于运行光度计的软件是用LabVIEW(National Instruments)编写的,用于部署测量和控制系统的软件环境。

  4. 校准设备
    1. 测量前,必须校准仪器。接收光电二极管信号是用不透明的盖子阻挡的测量光路测量的。该值对应于0%透光率。
    2. 接下来,用测量室内的样品架测量信号,但没有叶。该值对应于100%的透光率。接收器光电二极管信号线性地依赖于透射光的注量率
  5. 植物生长条件
    注意:用于叶绿体运动测量的叶子应该是光学薄的。每个单元层吸收光,从而在叶片上形成通量密度梯度。远端细胞层受光较少。因此,其叶绿体对面对光源的层响应较弱的光。光学密度较高的层产生更陡峭的梯度。为了使通过叶片的光衰减最小化,用于实验的叶片的透射率至少为8%。使用注射器在用水或缓冲液渗透后可以测量更厚的叶子,这增加了叶片的透光率。它也是应用抑制剂(Aggarwal等,2013)或其他化学物质(Anielska-Mazur等人,2009)的一种方便的方法。
    1. 对于拟南芥,通过在80-100μmol/平方米的白光中生长,确保了叶片的适度透光率水平短时间(10小时光/ 14小时黑暗)。
    2. 来自4-5周龄植物的叶子用于光度测量。

  6. 样品制备
    1. 植物在暗室中暗适应至少7小时以允许叶绿体获得静止的黑暗位置。黑暗适应是必要的,以确保叶绿体从细胞中的相同位置开始,这对于各种叶中叶绿体反应的定量比较是至关重要的。否则透光率变化对应于再分布的不同阶段,不能比较。常规地,在光周期光循环结束时,叶子被转移到黑暗中。
    2. 所有的实验操作都是在浅绿色的光(<0.05μmol/ m 2以上)下进行的。
    3. 在黑暗适应之后,将一片叶子从植物上分离并放置在安装在支架上的薄的显微镜载玻片上(图2)。
    4. 叶片变平,并定位在测量孔正上方的滑块中间。重要的是避免将主要静脉放入测量区域。
    5. 叶子被覆在金属环上的保鲜膜覆盖,其叶柄被包裹在一块水浸的薄纸中,以避免在实验期间干燥。
      注意:大叶子,例如烟草,可以切成小碎片,并浸入一滴水中进行测量。在这种情况下,将叶片放置在由帕拉菲尔环和粘膜形成的室中(Anielska-Mazur等人,2009)。为了防止蒸发,室内用高粘度硅油脂密封。
    6. 具有叶片的支架放置在测量室内,并且立即记录初始透射率水平。
      注意:理想情况下,同一研究中使用的所有叶子的透光率值应该相似。由于光度法是叶绿体运动评估的间接方法,结果的可靠性可能受到叶解剖学差异的不利影响。在比较突变体和野生型叶中的叶绿体反应时必须考虑这一点(Eckstein等,2016)。叶解剖学的变化通常反映在不同的透光率。应始终确定暗透射对由光度曲线计算的运动参数(例如振幅和速度,见下文)的影响。因此,建议根据暗透射率绘制参数。在结果统计分析阶段也可以考虑到这种影响(见数据分析部分)。

  7. 在连续的光 - 多个照明步骤下测量增加强度
    注意:可以在一片叶子中测量响应增加的光强度的叶绿体重新定位。结果是注量率 - 响应曲线。对于拟南芥,所使用的典型光强度从0.1μmol/平方米开始,并逐渐升高至120μmol/平方米以上, sup> -1 (Jarillo等人,2001)(图3A)。


    图3.使用光度法获得的典型曲线。 A.注量率 - 响应曲线。一个黑暗适应的叶子。初始透射率为12%的连续蓝光以0.4-120μmol/ m 2的上限连续的蓝光曝光。每次照射步骤持续50分钟。 B.一个典型的记录轨迹,解释了幅度和速度的计算方式。箭头表示弱的开始(↓1.6μmolm -2 sec -1 )或强(↑120μmolm -2 sup> -1 )蓝光。 (dT / dt)>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>透光率的变化分别与累积和回避响应相对应
    1. 黑暗适应叶片的透光率在生理黑暗中记录数分钟,以确保其水平稳定。
    2. 打开所需注射率的蓝光,记录透射率的变化至少45分钟。
      注意:这是一个妥协时间,通常太短,无法获得响应的稳定阶段。然而,响应的最后阶段的透射率漂移非常小,可以忽略不计。在缓慢响应的情况下,例如在低温下,应该增加曝光/测量的时间(Łabuz等人,2015)。
    3. 响应达到高原后,可以改变蓝光强度。
      注意:为了仅测量最大累积和避免,可以使用每个响应的饱和光。一个典型的实验包括:将透明度较低的5分钟记录下来,然后将弱蓝光(1.6μmol/ m 2)至少记录45分钟,蓝光(120μmol/ m 2)至少45分钟(图3B)。

  8. 一次连续测光 - 一次强度 -
    另一种方法是一次只测量一个光强度的叶绿体反应。在这种情况下,我们总是照亮一个暗适应的叶子,并获得一系列曲线,每个对应一个强度(Jarillo et al。,2001)。如果我们想要以各种注射率比较各种样品中叶绿体反应的动力学,这是一个标准方法,因为它确保叶绿体从细胞中的相同位置开始。对于响应振幅,两种方法都是等效的,只要振幅不依赖于以前的照明,而仅仅依赖于当前的通向率。这必须通过实验验证研究的物种。在 A的情况下。野生型,幅度仅取决于当前的照度。然而,突变体的行为可能不同。例如,当叶绿体已经达到积累位置时, phot2突变体对强蓝光几乎没有反应,而在暗适应后直接施加强光时观察到双相反应(Jarillo et al。,2001)。

  9. 对光脉冲响应的测量
    注意:光脉冲的幅度比对连续光的响应更低,因此它们需要良好的记录系统的信噪比。
    1. 记录暗透光率5分钟。
    2. 应用非常短的蓝光脉冲,并在黑暗中记录叶绿体重定位,持续45分钟。对于具有 A的实验。我们通常使用一系列强蓝光(120μmol/ m 2,sup-sup),使避光响应饱和为0.1,0.2, 1,2,10和20秒(Sztatelman等人,2016)。
      注意:可以在宽范围内调整脉冲持续时间和光强度。对于只产生瞬时积累的脉冲,叶绿体反应遵循本生 - 罗斯托互逆定律。因此,由辐射能量的总量(注量率和曝光时间的乘积)决定(Gabryś等,1981)。
    3. 如果实验的目标是比较不同脉冲持续时间对同一植株系中叶绿体运动的影响,则将整个系列记录在同一叶上是方便的。记录透射率曲线后,将叶转移回黑暗进行恢复,并测量第二叶中的响应。这种黑暗恢复可能比初始适应期短,因为对脉冲的响应和各自的透射率变化很小,并且通常在1.5小时内达到平衡。接下来,第一片叶片被带回并用持续时间增加的光脉冲进行挑战。

数据分析

  1. 计算运动参数
    1. 为了量化叶绿体运动,计算每个照射阶段的透射率变化幅度(ΔT)和最大速度(dT / dt)。可以使用标尺在曲线打印输出上估算这些参数。或者,可以使用适于分析数值数据的软件来确定它们,例如,R,MATLAB或数学。相对于暗透射率水平计算幅度(图3B)。如果噪声很大,则可以通过用低通数字滤波器平滑曲线来提高计算值的幅度精度。 Savitzky Golay滤波器(可用于自由软件R)适用于通常情况下以相等距离间隔的点的曲线。如果点不规则间隔,则可以使用局部回归(也可用于R)平滑曲线。最大速度可以通过在响应最快的点绘制与透射率曲线相切的直线来确定。线的斜率等于速度的大小。使用合适的数字滤波器可以对速度进行数值估计。 Savitzky-Golay过滤器也可用于此目的。它通过将多项式拟合到滑动窗口内的点来起作用。其中心点被用多项式拟合(用于平滑)或拟合导数(用于速度计算)预测的值代替。多项式的程度应该很低。我们使用二次多项式。窗口的宽度也由用户指定。更宽的窗口允许更有效的噪声消除。然而,使用太宽的窗口可能导致低估运动的最大速度。因此,宽度应调整到设备的噪声水平和采样率。对于速度计算,我们使用对应于2分钟间隔的窗口宽度。
      注意:为了更好地表征叶绿体运动的动力学,建议在Łabuz等人,2015年(图1C和1E-1F)中绘制速率(dT / dt)与时间的关系。这有助于估计最大速度和实现最大速度的时间。这也有助于评估在达到上述响应的稳定阶段之后是否应用新的光强度。
    2. 由脉冲触发的叶绿体运动可以类似于对连续光的响应进行量化。然而,对单个脉冲的响应通常由两个阶段组成 - 快速,短暂的回避阶段,随后是较慢的累积响应(Sztatelman等人,2006)。因此,应该为单个脉冲计算两个幅度和两个速度。
      注意:可以通过光度曲线计算附加参数。脉冲开始与瞬时避免或累积的最大值之间的时间在表征叶绿体对光脉冲反应的动力学方面是有用的(Sztatelman et al。,2006)。

  2. 随机化和实验设计
    1. 叶绿体运动测定的重现性需要随机化和对植物生长条件的适当控制。生长室中的条件(水的可用性,光强度,等等)可能因地而异。为了减少系统性错误的风险,不同生产线的植物应以随机的方式放置在室内。为了减少由于生长差异造成的差异,在植物生长过程中,盆可能被重新洗牌数次;这在具有侧面照明的生长室中尤其重要。如果实验需要使用几个生长室,则应在每个室中同时生长来自所有系(或一般所有实验组)的植物。从不同批次的植物获得的结果可能有所不同。为了确保结果的重复性,测量应重复至少三次,使用批量播种和独立种植的植物。
    2. 在涉及叶片预处理(例如,用抑制剂浸润)的实验中,整个植物可以随机分配到每个处理水平(例如,抑制剂浓度),或来自同一植物的叶片的大小和形状相似,可以分配给每个级别。此选择决定了可用于数据分析的统计方法的类型。这两种处理水平的分配方法不能在单个实验中混合使用。否则不可能统计分析结果。
    3. 实验所需的植物数量取决于研究的影响的大小,植物的变异性和实验组的数量。一组是在用抑制剂(两个浓度加上对照)处理的突变体和野生型植物的实验中的不同因素水平的组合,其重复三次不同批次的植物,有三个因素(植物系,抑制剂浓度,植物批次)和18个组(2×3×3)。在实验中。当我们寻找中等程度的影响时,我们每组使用10个或更多的植物。大效果需要较少的重复。

  3. 统计测试
    1. 统计测试的选择取决于可能影响运动参数的变量的类型。大多数情况下,由实验者控制的变量是固定的分类变量(因子),这意味着它们的值是预先规定的水平。实例是植物系(两个等级:野生型与突变体植物)或胁迫处理(胁迫与对照植物)。如果在多批植物上重复了整个实验,则该批次可以作为随机变量处理。变量也可以是连续的,例如,暗透射率。运动参数可以视为线性依赖于变量。如果模型中包含的所有变量都是分类因子,则使用常数方差分析(ANOVA)来检验因子的影响是否显着。所有组之间的平均值差异可以用Tukey检验进行测试。选择的级别,被视为对照可以与Dunnett的其他级别进行比较。当通常选自相同植物的类似叶分配给不同的处理水平时,必须将新的阻断变量(植物)引入方差分析模型。在一个治疗水平和对照的特殊情况下,配对样本测试是适当的。
    2. 暗透射对叶绿体反应的影响也可能包括在模型中。通常,运动参数和暗透光率之间的关系可以很好地适应一条线。在这种情况下,通过将暗透射作为附加的连续变量(协变量)与实验感兴趣的其它变量一起处理,在数据分析阶段可以减少暗透射的影响。如果所有工厂生产线或处理中最佳线性拟合的斜率相似,则可以使用在自由软件R中可用的协方差(ANCOVA)的标准分析。

致谢

这项工作得到欧盟基金在FP7框架内的支持,Marie Curie ITN CALIPSO,授权号[GA 2013-ITN-607-607]和2013-2017年科学财政资源,分配给实现一个共同资助的国际项目 这些设备和协议都是由Walczak和Gabrys(1980)以前的工作进行了修改的。新型光度计用于测量叶片叶绿体运动的变化。光合作用14,65-72。

参考

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引用:Gabryś, H., Banaś, A. K., Hermanowicz, P., Krzeszowiec, W., Leśniewski, S., Łabuz, J. and Sztatelman, O. (2017). Photometric Assays for Chloroplast Movement Responses to Blue Light. Bio-protocol 7(11): e2310. DOI: 10.21769/BioProtoc.2310.
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