Arabidopsis Stomatal Opening and Closing Experiment   

Abstract

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
   
   4. 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.
      
   5. ABA treatment
      Add 50 μM ABA and treat under the light for at least 3 h.
      
   6. 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.