Abstract
The opening of stomata in plants in response to blue light is driven by the plasma membrane H+-ATPase in guard cells. To evaluate the activation of the H+-ATPase in vivo, we can use H+-pumping by guard cells in response to blue light and fusicoccin. To do this, it is required to prepare a large amount of guard cell protoplasts and measure H+-pumping in the protoplasts. It is also necessary to determine the protein amount of H+-ATPase. In this protocol, we describe the procedures required for these preparations and measurements.
Keywords: Arabidopsis thaliana, Blue light, Fusicoccin, H+-pumping, Plasma membrane H+-ATPase
Background
The opening of stomata in response to blue light is driven by membrane hyperpolarization mediated through H+-pumping across the plasma membrane in guard cells (Assmann et al., 1985; Shimazaki et al., 1986), and is brought about by the plasma membrane H+-ATPase (Kinoshita and Shimazaki, 1999). The H+-ATPase generates an electrochemical gradient across the membrane, and provides the energy required for numerous secondary transports in plant cells. However, it is not easy to measure the activity of H+-ATPase in vivo. Taking advantage of the blue light-sensitive properties of guard cells, our method makes it possible to measure H+-pumping as an in vivo H+-ATPase activity using Arabidopsis guard cell protoplasts (Ueno et al., 2005). Together with H+-ATPase quantification by Western blot (Yamauchi et al., 2016), this method allows comparing H+-ATPase activity under different conditions or mutant backgrounds.
Materials and Reagents
Equipment
*Note: This product has been discontinued.
Software
Procedure
Data analysis
Calculation of the magnitude and the maximum rate of H+-pumping (see Figure 13).
Notes
The H+-pumping activities vary with the viability of guard cell protoplasts. An important criterion to obtain viable guard cell protoplasts is enzyme quality (Cellulase RS and R-10). Activity and toxicity, which often cause reduction in protoplast recovery, vary with enzyme lot numbers. We usually check the enzyme activity on a small scale through preparation of the protoplasts and H+-pumping measurement. If the lot produces viable protoplasts, we purchase it in larger amounts.
Recipes
Note: Enzyme solutions for 1st and 2nd digestion should be prepared fresh.
Acknowledgments
This protocol is adapted from Shimazaki et al. (1986), Ueno et al. (2005), and Yamauchi et al. (2016). This work was supported by JSPS KAKENHI Grant Number 26251032 (to K.S.). There is no conflict of interest.
References
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