Abstract
Trehalose is a nonreducing disaccharide. It is a common sugar in bacteria, fungi and yeast, where it functions as a carbon source and stress protectant. In contrast, plants, although encoding large trehalose biosynthesis gene families, contain only small amounts of trehalose. The intermediate compound of trehalose, trehalose-6-phosphate (T6P), is a signaling molecule in plants, regulating metabolism, growth, and development. Most plants contain only a single trehalase, the enzyme that specifically hydrolyzes trehalose into two glucose molecules. High trehalase activity has been suggested to be part of the defense mechanism in plants hosting mycorrhizal fungi, rhizobia, and the plant pathogen Plasmodiophora brassica. Recently, it was shown in Arabidopsis thaliana that high trehalase activity is associated with an increase in drought stress tolerance and that trehalase fulfills an important role in stomatal regulation. Here we describe a protocol for measuring trehalase activity in Arabidopsis tissues, optimized for 96-well plates. Dialyzed protein extracts will be incubated with trehalose, followed by the quantitation of the released glucose using glucose oxidase-peroxidase.
Keywords: Arabidopsis thaliana, Trehalose, Trehalase
Materials and Reagents
Equipment
Procedure
For samples
For blanks
For samples and blanks
For proteins (Lowry procedure, Van Houtte et al., 2013)
Data analysis
Here we show an example how to calculate the trehalase activity from a protein extract of Arabidopsis Col-0 seedlings.
Recipes
Acknowledgments
This protocol was developed in the framework of the following paper: Van Houtte et al. (2013). It was developed based on two previous publications: Brodmann et al. (2002) and Pernambuco et al. (1996). Hilde Van Houtte was supported by the KU Leuven industrial research fund (IOF/KP/08/001). This work was supported by a grant from the FWO (G.0859.10).
References
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I think this can indeed be done by a proportional scale-up. If you want to use less coloring reagents or less protein extract, that might also be possible, as long as you do the same for your calibration curve and calculate everything again. Good luck!
A membrane with a pore size between 12- 24 kD MWCO should also retain the Arabidopsis trehalase (64 kD). Note: In some cases, the dialysis step can be omitted, for instance when using plant material with low amounts of carbohydrates and ions.