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Stomata pores are mainly localized in the lower epidermis of plant leaves and transpirational water loss occurs through these pores. ABA inhibits light-promoted stomatal opening and promotes stomatal closure. This protocol describes how to measure stomatal apertures following ABA treatment.

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[Bio101] Arabidopsis Stomatal Opening and Closing Experiment

Plant Science > Plant physiology > Tissue analysis
Author: Zhixin Zhao
Zhixin ZhaoAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: zxzhao@stanford.edu
Bio-protocol author page: a33
5/20/2011, 8304 views, 1 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.74

[Abstract] Stomata pores are mainly localized in the lower epidermis of plant leaves and transpirational water loss occurs through these pores. ABA inhibits light-promoted stomatal opening and promotes stomatal closure. This protocol describes how to measure stomatal apertures following ABA treatment.

Materials and Reagents

  1. ABA
  2. KCl
  3. CaCl2
  4. Iminodiacetic acid
  5. Mes-KOH
  6. Solution for stomatal opening (see Recipes)
  7. Solution for stomatal closure (see Recipes)

Equipment

  1. Light microscope
  2. Aluminium foil
  3. Culture dish

Procedure

  1. Stomata opening
    1. Sampling
      Sample three leaves for each treatment before turning the light on in the morning and put them under a small container enveloped with aluminium foil.
      Put them into a culture dish and place a cover glass on them to make them immersed in the opening medium, envelop with aluminium foil, and then put the culture plate under dark for at least 2.5 h to ensure that most of the stomata are fully closed.
    2. ABA treatment
      Transfer the culture plate to the light, in the mean time add 50 μM ABA (20 °C, 450 μmol/ m2/s), treat for at least 2 h to ensure that most of the stomata of the control are fully open.
    3. Peel experiment:
      Peel abaxial epidermis, make slides, and then observe and measure under microscope. (*40 objective, unit/4.5 in the field). One scale in the sight field corresponding to one circle of the screw attached to eyepiece.


  2. Stomata closing
    1. Sampling
      Sample two leaves for each treatment, put them into culture dish and place a cover glass on them to immerse them in the opening medium, and then put the culture plate under the light for 2.5 h to ensure that most of the stomata are fully open.
    2. ABA treatment
      Add 50 μM ABA and treat under the light for at least 3 h.
    3. Peel experiment
      Peel abaxial epidermis, make slides, and then observe and measure under microscope.

Recipes

  1. Solution for stomatal opening (200 ml)
    Reagent
    Stock or FW
    Weight or volume
    10 mM KCl
    3 M
    0.67 ml
    7.5 mM Iminodiacetic acid
    133.1
    0.2 g
    10 mM Mes-KOH (pH 6.15)

    0.3904 g

  2. Solution for stomatal closure (200 ml)
    Reagent
    Stock or FW
    Weight or volume
    20 mM KCl
    75.5
    0.2982 g
    1 mM CaCl2
    0.5 M
    0.4 ml
    5 mM Mes-KOH (pH 6.15)

    0.1952 g

References

  1. Allen, G. J., Kuchitsu, K., Chu, S. P., Murata, Y. and Schroeder, J. I. (1999). Arabidopsis abi1-1 and abi2-1 phosphatase mutations reduce abscisic acid-induced cytoplasmic calcium rises in guard cells. Plant Cell 11(9): 1785-1798.
  2. Leymarie, J., Lasceve, G., Vavasseur, A. (1998). Interaction of stomatal responses to ABA and CO2 in Arabidopsis thaliana. Aust J Plant Physiol 25: 785-791.


How to cite this protocol: Zhao, Z. (2011). Arabidopsis Stomatal Opening and Closing Experiment. Bio-protocol Bio101: e74. DOI: 10.21769/BioProtoc.74; Full Text



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6/4/2013 4:03:01 PM  

Vladimir Muro
Instituto Potosini de Investigación Científica y Tecnológica; IPICyT

Hi!

I have doubt about the peel experiments

How i could do it?

Whit a little razor?

Thanks

Vladimir

6/4/2013 4:09:58 PM  

Vladimir Muro
Instituto Potosini de Investigación Científica y Tecnológica; IPICyT

I forgot mention two more questions

What is the recomended plant age for the experiment? and, rossette leaves or cauline leaves?

thanks again

Vladimir

9/10/2014 10:15:24 PM  

Kai Jiang
The Univ. of Tokyo

Hello!
Have you solved the problem in peeling the epidermis now?
I'm also confused with this technique. Furthermore I would like to observe the movement of stomata in a real time manner.
Do you have any recommendation please?

Thanks a lot

Kevin

Reply

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