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Measurement of Stomatal Conductance in Rice
水稻气孔导度测量   

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Abstract

Stomatal conductance, the reciprocal of stomatal resistance, represents the gas exchange ability of stomata. Generally, the stomatal conductance is higher when stomata open wider, and vice versa. In this protocol, we describe how to measure stomatal conductance in rice using Li-6400 (Licor, USA).

Keywords: Stomatal conductance(气孔导度), Li-6400XT(Li-6400XT), Rice(水稻), Red light(红光), Blue light(蓝光)

Background

Stomata consist of a pair of guard cells and open in response to blue light as a signal. Using epidermal fragments from the kidney-shaped guard cells (found mainly in dicots), the blue light-induced stomatal opening can be observed readily under a microscope. However, direct measurement of the stomatal aperture from the dumbbell-shaped guard cells in monocots, such as rice, maize, wheat, and oats, is very difficult, due to their uneven leaf surface. Thus, a gas-exchange system is useful for the measurement of blue light-induced stomatal opening in the leaves of monocots.

Materials and Reagents

  1. Rice (Oryza sativa) cultivar Nipponbare
    Note: Rice (O. sativa) cultivar Nipponbare plants were grown at 28 °C under a photoperiod of 14 h light/10 h dark or in a greenhouse at room temperature (25-32 °C). Mature leaves, from 4-week-old plants, were used in gas-exchange measurements.

Equipment

  1. Portable gas-exchange system, Li-6400XT (Licor, USA), with standard chamber
    Licor gas-exchange systems, especially the Li-6400 and Li-6400XT, are used widely in photosynthesis and stomatal conductance measurements. The standard chamber, as a basic accessory for the Li-6400XT, is assembled with a high-precision temperature sensor and an internal light sensor (see Figure 1). Because stomatal conductance is a function of leaf temperature (von Caemmerer and Farquhar, 1981), precise control of the leaf temperature is important. Moreover, to induce specific stomatal blue light response, an internal light sensor to measure extra red light and blue light (for the light source see below) is needed. Therefore, the standard chamber, with a high precision temperature sensor and an internal light sensor, is suitable for this experiment.
  2. Light source
    Light is provided via a fiber-optic illuminator with a halogen projector lamp (15 V/150 W; Philips, Netherlands) as the light source. A power supply (MORITEX, catalog number: MHAB-150W-100V ) was used to power the lamp. Red light (660 nm) and blue light (470 nm) are obtained with filters (#2-61 and #5-60, Corning, USA)

Procedure

  1. Plants are kept overnight in the dark for before gas-exchange measurements. The overnight dark-treatment lasts at least 14 h to ensure complete stomatal closure.
    Note: In our case, we keep the plant in the dark in the same experiment room as the gas-exchange system.
  2. Warm up and calibrate the Li-6400XT, following the official instruction manual https://www.licor.com/env/support/resources?p=6400XT.
  3. Set the control for the fixed flow model, at 500 μmol sec-1.
    Important: For the standard chamber, a flow rate lower than 500 μmol sec-1 will cause insufficient gas circulation, especially when the leaf area is smaller than the chamber area (6 cm2).
  4. Adjust the desiccant to keep the reference relative humidity (RH_R) at about 65%.
    Note: In our case, a relative humidity lower than 60% will cause unstable stomatal conductance (low humidity stress).
  5. Set the reference CO2 at ambient CO2 concentration (400 μmol mol-1) using the CO2 mixer.
  6. Set leaf temperature at room temperature.
    Note: In our case, we set the leaf temperature at 24 °C.
    Important: Do not set the ‘block temperature’ (chamber temperature) to constant instead of the ‘leaf temperature’, because even when the ‘block temperature’ is stable the leaf temperature will still fluctuate. According to the equation for stomatal conductance (see the official instruction manual for the Li-6400XT), the leaf temperature is one of the key factors for calculating stomatal conductance. A stable leaf temperature makes the stomatal conductance value representative of stomatal transpiration.
  7. After these parameters become stable, match the system as described in the official instruction manual, then clamp the rice leaf in the center of the chamber (Figure 1B). After all the parameters have stabilized, match the system again.
    Note: Make sure the lower side of the leaf touches the temperature sensor (see Figure 1).


    Figure 1. How to clamp the rice leaf in the chamber and images of the illumination system. A. A picture of the standard chamber. Arrows indicate the position of the temperature sensor and the inside light sensor. B. A picture of clamping the rice leaf in the center of the chamber. Make sure the leaf surface touches the temperature sensor. C. and D. Images of the red and red + blue light illumination system.

  8. Open a log file, name it, and add some necessary notes, such as the name of the researcher performing the measurement. Then, open Auto Program (see Figure 2), set the logging frequency (Log every_ _ secs), duration (Run for _ _ _ minutes), and match frequency (Match every _ _ _ minutes).
    Note: In our case, we set those parameters as shown in Figure 2 (right panel).


    Figure 2. Parameters of Auto Program. A. A picture of the content in the Auto Program. The highlight option ‘AutoLog’ is the program used in this measurement. B. A picture of the parameter settings on entering the ‘AutoLog’ program.

Note: Data in steps 9 and 10 are recorded by Auto Program.

  1. First measure stomatal conductance under dark condition for 30 min, and then illuminate the leaf with 700 μmol m-2 sec-1 red light from the top of the chamber, until the stomatal conductance and photosynthetic rate reach steady states (stable for at least 10 min).
    Note: In our case, it always takes more than 120 min to reach the steady state.
  2. Under the red light background, add a weak blue light (3 μmol m-2 sec-1) for 30 min and then turn the blue light off, keeping the leaf illuminated under red light for another 90 min or more. For images of red light and red + blue light illumination see Figures 1C and 1D.
  3. Close the Auto Program and the data log file.
  4. Open the chamber and measure the width of the leaf. Because the length of the chamber is 3 cm, the area of the leaf sample is equal to the leaf width x 3 cm.
  5. Transfer the data from equipment to computer.
  6. Open the data file (.xls) with Excel (Microsoft, USA). Change the default leaf area of 6 cm2 to the actual leaf area determined following step 12 (the data file is shown in Figure 3). The data sheet will recalculate the stomatal conductance and other parameters automatically with the new leaf area.


    Figure 3. Example of data in Excel: changing the data for leaf area in the Excel file. The highlighted column ‘Area’ shows the default leaf area (6 cm2) that should be changed to the actual leaf area following step 12. 

Data analysis

The stomatal conductance is calculated automatically by the software of the instrument. Select the ‘Obs’ column (refers to the number of objects logged in this experiment) and ‘Cond’ column (refers to the stomatal conductance), using the Chart tool to make a scatter (x, y) chart (Figure 4). Because the objects were logged every minute, the chart reflects the time course of changes in stomatal conductance.


Figure 4. Data analysis of stomatal conductance. Select the highlighted columns ‘Obs’ and ‘Cond’ to make a scatter (x, y) chart. The red arrow and blue arrows show the points when the light sources were switched on and off.

Notes

  1. The portable gas-exchange system, Li-6400XT, is not a simple instrument. Researchers who want to use this system for their experiments should have basic training from Licor Company (or sales agent) at first. Basic manipulations including warm up, calibration, and match the system etc. are included in the basic training. Therefore, in this protocol we omit the details of these parts in the procedure.
  2. Because stomata are sensitive to light, all gas-exchange experiments should be conducted in a dim-light (less than 3 μmol photon m-2 sec-1) room or a dark room.
  3. This protocol is also applicable to other plant species, monocot and dicot plants, such as corn, Arabidopsis thaliana etc. For plants with irregularly shaped leaves (e.g., Arabidopsis), we recommend using a scanner or professional instrument such as the LI-3100C Area Meter (Licor, USA) to calculate the leaf area accurately.

Acknowledgments

This protocol was adapted from Toda et al. (2016). This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (15H05956 to T.K.), and by the Japan Science and Technology Agency [the Advanced Low Carbon Technology Research and Development Program].

References

  1. Toda, Y., Wang, Y., Takahashi, A., Kawai, Y., Tada, Y., Yamaji, N., Feng Ma, J., Ashikari, M. and Kinoshita, T. (2016). Oryza sativa H+-ATPase (OSA) is involved in the regulation of dumbbell-shaped guard cells of rice. Plant Cell Physiol 57(6): 1220-1230.
  2. von Caemmerer, S. V. and Farquhar, G. D. (1981). Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153(4): 376-387.

简介

气孔导度是气孔阻力的倒数,代表气孔的气体交换能力。 一般来说,当气孔开阔时气孔导度较高,反之亦然。 在本协议中,我们描述了如何使用Li-6400测量水稻的气孔导度(Licor,USA)。

Stomata由一对保护单元组成,响应于蓝光作为信号而打开。 使用来自肾形保卫细胞(主要在双子叶植物中发现)的表皮碎片,可以在显微镜下容易观察到蓝光诱导的气孔开放。 然而,由于叶片表面不均匀,因此直接测量单子叶植物(如稻米,玉米,小麦和燕麦)中哑铃状保卫细胞的气孔开度是非常困难的。 因此,气体交换系统可用于测量单子叶植物叶片中蓝光诱导的气孔开放。

关键字:气孔导度, Li-6400XT, 水稻, 红光, 蓝光

材料和试剂

  1. 水稻(Oryza sativa)品种日本晴
    注意:水稻(O. sativa)栽培品种Nipponbare植物在室温(25-32℃)下在28℃,光照14小时/10小时的光周期下生长。来自4周龄植物的成熟叶子用于气体交换测量。

设备

  1. 便携式气体交换系统,Li-6400XT(Licor,USA),带标准室
    Licor气体交换系统,特别是Li-6400和Li-6400XT,广泛用于光合作用和气孔导度测量。标准室作为Li-6400XT的基本配件,采用高精度温度传感器和内部光传感器组装(见图1)。因为气孔导度是叶片温度的函数(von Caemmerer and Farquhar,1981),对叶片温度的精确控制是重要的。此外,为了诱导特定的气孔蓝光响应,需要内部光传感器来测量额外的红光和蓝光(对于光源见下文)。因此,具有高精度温度传感器和内部光传感器的标准室适用于本实验
  2. 光源
    光源通过带有卤素投影灯(15 V/150 W;荷兰飞利浦)的光纤照明器提供,作为光源。电源(MORITEX,目录号:MHAB-150W-100V)用于为灯供电。用滤光片(#2-61和#5-60,Corning,USA)获得红光(660nm)和蓝光(470nm)。

程序

  1. 在气体交换测量之前,将植物在黑暗中保持过夜。过夜黑暗治疗持续至少14小时,以确保气孔完全闭合。
    注意:在我们的例子中,我们将工厂保持在与气体交换系统相同的实验室内的黑暗中。
  2. 按照正式使用说明书 https://www.licor.com/env/support/resources?p=6400XT
  3. 设置固定流量模型的控制,为500μmolsec -1
    重要提示:对于标准室,流量低于500μmol/sec的气流将导致气体循环不足,特别是当叶面积小于室面积(6 cm 2 )。
  4. 调整干燥剂以保持参考相对湿度(RH_R)约为65%。
    注意:在我们的情况下,低于60%的相对湿度将导致气孔导度不稳定(低湿度胁迫)。
  5. 使用CO 2混合器将环境CO 2浓度(400μmolmol -1,以上)设定参考CO 2 。
  6. 将叶温度设置在室温。
    注意:在我们的例子中,我们将叶子温度设置在24°C。
    重要提示:不要将"块温度"(室温度)设置为常数而不是"叶温度",因为即使"块温度"稳定,叶片温度仍会波动。根据气孔导度方程式(参见Li-6400XT官方使用手册),叶片温度是计算气孔导度的关键因素之一。稳定的叶片温度使得气孔导度值代表气孔蒸腾。
  7. 在这些参数变得稳定后,按照官方说明书中的说明与系统相符合,然后将米片夹在室中心(图1B)。所有的参数都已经稳定后,再次匹配系统。
    注意:确保叶子的下侧接触温度传感器(见图1)。


    图1.如何夹住室内的稻叶和照明系统的图像。 A.标准室的图片。箭头表示温度传感器和内部光传感器的位置。 B.将米叶夹在室中心的图片。确保叶面接触温度传感器。 C.和D.红色和红色+蓝光照明系统的图像。

  8. 打开一个日志文件,将其命名,并添加一些必要的注释,例如执行测量的研究人员的名称。然后,打开自动程序(见图2),设置测井频率(Log every_ _ secs),持续时间(_ _ _分钟运行)和匹配频率(每_ _ _分钟匹配)。
    注意:在我们的例子中,我们设置这些参数,如图2所示(右图)。


    图2.自动程序的参数。 A.自动程序中内容的图片。高亮度选项"AutoLog"是此测量中使用的程序。 B.输入"AutoLog"程序时参数设置的图片。

注意:步骤9和10中的数据由Auto Program记录。

  1. 首先在黑暗条件下测量30分钟的气孔导度,然后从室顶部以700μmol/m 2以上的红光照亮叶子,直到气孔导度和光合速率达到稳定状态(稳定至少10分钟)。
    注意:在我们的例子中,总是需要超过120分钟才能达到稳定状态。
  2. 在红光背景下,加入弱蓝光(3μmolm〜sup sec -1 )30分钟,然后关闭蓝灯,保持叶亮在红灯下还有90分钟以上。对于红光和红+蓝光照明的图像,请参见图1C和1D
  3. 关闭自动程序和数据日志文件。
  4. 打开房间并测量叶子的宽度。因为室的长度是3厘米,叶片的面积等于叶宽x 3厘米
  5. 将数据从设备传输到计算机。
  6. 用Excel(Microsoft,USA)打开数据文件(.xls)。将6厘米 2 的默认叶面积更改为步骤12确定的实际叶面积(数据文件如图3所示)。数据表将自动重新计算新叶面积的气孔导度等参数。


    图3. Excel中的数据示例:更改Excel文件中叶面积的数据。突出显示的列"区域"显示默认叶面积(6厘米 2 )应该在步骤12之后更改为实际的叶面积。

数据分析

气孔导度由仪器软件自动计算。选择"Obs"列(指本实验中记录的对象数)和"Cond"列(指气孔导度),使用"图表"工具制作散点图(x,y)图(图4)。由于物体每分钟记录一次,图表反映了气孔导度变化的时间过程。


图4.气孔导度的数据分析。选择突出显示的列"Obs"和"Cond"作出分散(x,y)图。红色箭头和蓝色箭头显示光源打开和关闭时的点数。

笔记

  1. 便携式气体交换系统Li-6400XT不是简单的仪器。想要使用该系统进行实验的研究人员应该首先从Licor公司(或销售代理)进行基本培训。基本操作包括预热,校准和匹配系统等。被纳入基础训练。因此,在本协议中,我们省略了程序中这些部分的细节。
  2. 由于气孔对光敏感,因此所有气体交换实验应在暗光(小于3μmol的光子m -2 sec -1 )室或黑暗的房间。
  3. 该方案也适用于其他植物物种,单子叶植物和双子叶植物,如玉米,拟南芥等。对于具有不规则形状叶子的植物(例如,,拟南芥),我们建议使用扫描仪或专业仪器,如LI-3100C面积计(Licor,USA)来计算叶面积准确。

致谢

该协议改编自Toda等人。 (2016)。这项工作部分由日本教育,文化,体育,科技部科学研究资助单位(15H05956至TK)和日本科技厅[先进低碳技术研究开发计划]。

参考文献

  1. Toda,Y.,Wang,Y.,Takahashi,A.,Kawai,Y.,Tada,Y.,Yamaji,N.,Feng Ma,J.,Ashikari,M.and Kinoshita,T。(2016) ; Oryza sativa H + -ATPase(OSA)参与水稻哑铃状保卫细胞的调节。植物细胞生理学57(6):1220-1230。 >
  2. von Caemmerer,SV和Farquhar,GD(1981)。一些关系在光合作用的生物化学和叶片的气体交换之间。植物 153(4):376-387。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, Y. and Kinoshita, T. (2017). Measurement of Stomatal Conductance in Rice. Bio-protocol 7(8): e2226. DOI: 10.21769/BioProtoc.2226.
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