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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.

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Stomatal Bioassay in Arabidopsis Leaves
模式植物拟南芥中气孔开张度的测定

植物科学 > 植物生理学 > 组织分析
作者: Xuan Li
Xuan LiAffiliation: School of Life Sciences, Shaanxi Normal University, Xi’an , China
Bio-protocol author page: a896
Xian-Ge Ma
Xian-Ge MaAffiliation: School of Life Sciences, Shaanxi Normal University, Xi’an, China
Bio-protocol author page: a870
 and Jun-Min He
Jun-Min HeAffiliation: School of Life Sciences, Shaanxi Normal University, Xi’an, China
For correspondence: hejm@snnu.edu.cn
Bio-protocol author page: a871
Vol 3, Iss 19, 10/5/2013, 5090 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.921

[Abstract] 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.

[Abstract]

Materials and Reagents

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

Equipment

  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

Procedure

  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.
    Notes:
    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.
    Notes:
    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

Recipes

  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

Acknowledgments

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).

References

  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.

材料和试剂

  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)

设备

  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

程序

  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
    在上述处理之后,从MES/KCl缓冲液中取出叶子,并使用一片滤纸吸收叶子表面上的MES/KCl缓冲液。将叶片平放在载玻片上,其背面朝上,用镊子夹住叶尖附近的背轴表皮和叶肉细胞的一部分,并且将表皮条带随主叶的方向快速剥离静脉。然后,将剥离的表皮条立即浸入相应的处理缓冲液中,并通过镊子推到培养皿的底部,通过眉毛刷轻轻地从表皮条上除去剩余的叶肉细胞(图1),并且将表皮尖切下带有更多叶肉细胞的镊子夹住的条,然后用相应的处理缓冲液制成载玻片。


    图1.眉笔

  5. 用光学显微镜测量气孔孔径
    在您使用的光学显微镜中安装目镜测微计和台式千分尺,校准微米刻度,并且在每个重复中在校准的显微镜下对五十至九十个随机选择的气孔孔径进行评分,并且重复治疗至少三次。数据表示为从单因素方差分析得到的平均值±SE

    图2.目镜千分尺和台式千分尺

食谱

  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)
    在室温下储存并使用一周

致谢

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)。

参考文献

  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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How to cite this protocol: 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; Full Text
  2. He, J. M., Ma, X. G., Zhang, Y., Sun, T.F., Xu, F. F., Chen, Y. P., Liu, X. and Yue, M. (2013). Roleand interrelationship of Galpha protein, hydrogen peroxide, and nitric oxide inultraviolet B-induced stomatal closure in Arabidopsis leaves. Plant Physiol 161(3): 1570-1583.




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    登陆 | 注册
    9/5/2014 6:09:14 AM  

    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

    Reply

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