Abstract
Plant proteins can be targeted to intracellular (i.e., cytosol, vacuole, organelles etc.) or extracellular (i.e., cell walls, apoplast) compartments. Dual targeting is a key mechanism with important implications for plant metabolism, growth, development and defense etc. Harsh Hakea (Hakea prostrata R.Br.) is a perennial species and member of the Proteaceae family that thrives on extremely phosphate impoverished soils of southwestern Australia. Harsh Hakea is not a common model organism, but has been widely developed for physiological and molecular/biochemical studies of the endogenous adaptations of an ‘extremophile’ plant species to abiotic stress, including low phosphorus tolerance. Tissues of Harsh Hakea contain large amounts of compounds (e.g., phenolics) that interfere with the extraction of soluble proteins. We previously optimised extraction of intracellular proteins from Harsh Hakea proteoid roots to improve soluble protein yield by at least 10-fold (Shane et al., 2013). Here, we describe the protocol for extraction and separation of intracellular from ‘loosely bound’ cell-wall proteins in Harsh Hakea.
Materials and Reagents
Equipment
Procedure
This protocol applies to extraction of intracellular and cell-wall proteins from Harsh Hakea leaves, but can also be applied to roots (e.g., Shane et al., 2014; Shane et al., 2013). For protocols optimized for extracting intracellular and cell wall proteins from leaves, stems etc. of the model plant Arabidopsis thaliana see Shane et al. (2014); Shane et al. (2013) and Robinson et al. (2012).
Recipes
All buffers are made up fresh the day before and stored at 4 °C until used, but no sterilization was done.
Acknowledgments
This work was supported by the Australian Research Council (grant no. DP1092856 to M. W. S.), as well as grants from the Natural Sciences and Engineering Research Council of Canada and Queen’s Research Chairs program (to W. C. P.).
References
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