Stomatal Bioassay in Arabidopsis Leaves

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Plant Physiology
Mar 2013



Stomata embedded in the epidermis of terrestrial plants are important for CO2 absorption and water transpiration, and are possible points of entry for pathogens. Thus, the regulation of stomatal apertures is extremely important for the survival of plants. Furthermore, stomata can respond via accurate change of stomatal apertures to a series of extracellular stimuli such as phytohormones, pathogens, ozone, drought, humidity, darkness, CO2, visible light and UV-B radiation, so stomatal bioassay is widely used to dissect signal transduction mechanisms of plant cells in responses to multiple stimuli. This protocol describes how to measure stomatal apertures in leaves of model plant Arabidopsis thaliana under multiple treatments.

Materials and Reagents

  1. Leaves of Arabidopsis (Arabidopsis thaliana)
  2. Ethanesulfonic acid (Mes)-KOH
  3. MES/KCl buffer, pH 6.15 (see Recipes)


  1. Light microscope
  2. Eyepiece micrometer and stage micrometer
  3. Glass slide and cover glass
  4. 6-cm diameter Petri plates
  5. Eyelbrow brush (Yellow wolf hair, length of hair: 0.8 cm, width of hair: 0.8 cm)
  6. Tweezers


  1. Sampling
    Arabidopsis seedlings were gown in plant growth chambers under a 16-h light/8-h dark cycle, a photon flux density of 0.1 mmol/m2/s, and a day/night temperature cycle of 18 °C/22 °C for 4-6 weeks. The youngest, fully expanded and flat leaves were harvested for immediate use.
    1. To avoid any potential rhythmic effects on stomatal aperture, sampling was always started at the same time of day and avoided at midday.
    2. For all treatments, the leaves at same developmental stage were always sampled.
    3. For each treatment, at least three leaves originated from different plants and at same developmental stage were harvested.
  2. Opening the stomata
    For stomatal closing experiments, to ensure stomata at fully opened stage before starting of treatments, the fresh sampled flat leaves were first floated with their abaxial surfaces facing up on MES/KCl buffer (15 ml) in 6-cm diameter Petri plates for 2-3 h at 22 °C under light condition (0.1 mmol/m2/s) to open the stomata, and then for subsequent treatments.
  3. Treating samples
    Once the stomata were fully open (checked by microscope as the following 5 procedure), the leaves were then floated on MES/KCl buffer alone or containing various compounds or inhibitors for required time at 22 °C under the same white light condition mentioned above or under the desired conditions. Control treatments involved addition of buffer or appropriate solvents used with inhibitors.
    1. For each treatment, at least three leaves originated from different plants were treated.
    2. As the epidermal strips is easier peeled from the abaxial surface than the paraxial surface of Arabidopsis leaves, we only peeled epidermal strips from abaxial surface of leaves for subsequent measurement of stomatal aperture. Thus, for treatments of UV-B radiation as well as other lights, to ensure the abaxial surface of leaves receiving same dose of UV-B radiation as well as other lights, the leaves were floated with their abaxial surfaces facing up and perpendicular to the light on MES/KCl buffer in all treatments including opening stomata.
  4. Peeling epidermal strips and making slides
    After the above treatments, the leaf was taken out from MES/KCl buffer and a piece of filter paper was used to absorb the MES/KCl buffer on the surface of leaf. The leaf were flatly placed on a glass slide with its abaxial surfaces facing up, a tweezers was used to clamp a part of abaxial epidermis and mesophyll cells near the tip of leaf and the epidermal strips were quickly peeled along with the direction of the main leaf veins. Then, the peeled epidermal strips were immediately immersed in the corresponding treated buffer and pushed on the bottom of Petri plates by a forceps, the remained mesophyll cells were gently removed from epidermal strips by an eyebrow brush (Figure 1), and the tip of epidermal strip clamped by tweezers with more mesophyll cells was cut off, then slides were made with the corresponding treating buffer.

    Figure 1. Eyebrow brush

  5. Measurement of stomatal apertures with a light microscope
    Install eyepiece micrometer and stage micrometer in your used light microscope, calibrate micrometer scale, and fifty to ninety randomly selected stomatal apertures were scored under the calibrated microscope in each replicate and treatments were repeated at least three times. The data are presented as means ± SE derived from one-way ANOVA.

    Figure 2. Eyepiece micrometer and stage micrometer


  1. MES/KCl buffer, pH 6.15 (500 ml)
    1.86375 g 50 mM KCl
    5.549 mg 0.1 mM CaCl2
    1.066 g 10 mM Mes-KOH (pH 6.15)
    Stored at room temperature and used for one week


This work was supported by the National Science Foundation of China (grant no. 31170370) and the Fundamental Research Funds for the Central Universities (grant no. GK200901013). This protocol was adapted from previously published paper He et al. (2013).


  1. He, J. M., Ma, X. G., Zhang, Y., Sun, T. F., Xu, F. F., Chen, Y. P., Liu, X. and Yue, M. (2013). Role and interrelationship of Galpha protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol 161(3): 1570-1583.


埋藏在陆生植物表皮中的气孔对于CO 2吸收和水蒸腾是重要的,并且是病原体的可能进入点。 因此,气孔孔径的调节对于植物的存活是极其重要的。 此外,气孔可以通过气孔孔径的准确变化对一系列细胞外刺激如植物激素,病原体,臭氧,干旱,湿度,黑暗,CO 2,可见光和UV-B辐射作出反应,因此 气孔生物测定广泛用于解剖植物细胞响应多种刺激的信号转导机制。 该协议描述了如何在多种处理下测定模拟植物拟南芥(Arabidopsis thaliana)叶片中的气孔孔径。


  1. 拟南芥(拟南芥)的叶子
  2. 乙磺酸(Mes)-KOH
  3. MES/KCl缓冲液,pH6.15(参见配方)


  1. 光学显微镜
  2. 目镜千分尺和平台千分尺
  3. 玻璃滑盖和盖玻璃
  4. 直径6cm的培养皿
  5. 眼眉刷(黄狼毛,头发长度:0.8cm,头发宽度:0.8cm)
  6. 镊子


  1. 抽样
    将拟南芥幼苗在16小时光照/8小时黑暗循环下在植物生长室中长成,光子通量密度为0.1mmol / m 2 /s,昼/夜温度周期为18°C/22°C 4-6周。 收获最年轻,完全展开和扁平的叶子以供立即使用 注意:
    1. 为了避免对气孔孔径的任何潜在节律效应,取样总是在一天的同一时间开始,并在中午避免。
    2. 对于所有处理,始终对处于相同发育阶段的叶子进行取样。
    3. 对于每次处理,至少收获三片来自不同植物并处于相同发育阶段的叶子。
  2. 打开气孔
    对于气孔关闭实验,为了在开始处理之前确保气孔处于完全打开的阶段,首先将新鲜取样的平叶漂浮,其背面朝上在6cm直径培养皿中的MES/KCl缓冲液(15ml)在光照条件(0.1mmol/m 2/s)下在22℃温育3小时以打开气孔,然后用于随后的处理。
  3. 处理样品
    一旦气孔完全打开(如下面的程序通过显微镜检查),然后将叶子在上述相同的白光条件下在22℃漂浮在单独的MES/KCl缓冲液或含有各种化合物或抑制剂所需的时间,或在所需条件下。控制处理涉及添加缓冲液或与抑制剂一起使用的适当溶剂 注意:
    1. 对于每次处理,处理来自不同植物的至少三片叶。
    2. 因为表皮条比拟南芥叶的近轴表面更容易从后轴表面剥离,所以我们仅从叶的背轴表面剥离表皮条,用于随后测量气孔孔径。因此,对于UV-B辐射以及其它光的处理,为了确保接受相同剂量的UV-B辐射的叶子的背面表面以及其它光,叶子浮动,它们的背面朝上并垂直于所有处理中的光照在MES/KCl缓冲液上,包括打开气孔。
  4. 剥皮表皮条和制作载玻片


  5. 用光学显微镜测量气孔孔径



  1. MES/KCl缓冲液,pH6.15(500ml) 1.86375g 50mM KCl
    5.549mg 0.1mM CaCl 2 v/v 1.066g 10mM Mes-KOH(pH6.15)


这项工作得到中国国家科学基金会(拨款号31170370)和中央大学基础研究基金(拨款号GK200901013)的支持。 该协议改编自以前发表的论文He (2013)。


  1. He,J. M.,Ma,X. G.,Zhang,Y.,Sun,T. F.,Xu,F. F.,Chen,Y. P.,Liu,X.和Yue, Galpha蛋白,过氧化氢和一氧化氮在紫外线B诱导的气孔关闭中的作用和相互关系 植物生理 161(3):1570-1583。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Li, X., Ma, X. and He, J. (2013). Stomatal Bioassay in Arabidopsis Leaves. Bio-protocol 3(19): e921. DOI: 10.21769/BioProtoc.921.
  2. He, J. M., Ma, X. G., Zhang, Y., Sun, T. F., Xu, F. F., Chen, Y. P., Liu, X. and Yue, M. (2013). Role and interrelationship of Galpha protein, hydrogen peroxide, and nitric oxide in ultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol 161(3): 1570-1583.



Kai Jiang
The Univ. of Tokyo
Dear Sir or madam,

Many thanks for sharing of your protocols.
I still have some questions about how to peel the abaxial epidermis smoothly.
Could you share much more details of peeling the epidermis please? Such as that should I make a cutting edge to facilitate to separate the epidermis and mesophyll cells? Or is there any techniques to make it easier? I have tried many times but I always destroyed the leaves and could not get good samples for further observation. I will appreciate for your help.

Best regards.

Sincerely Kai Jiang

9/5/2014 6:09:14 AM Reply