A New and Simple Technique for the Isolation of Plasma Membrane Lipids from Root-Tips(Plant Nutrition)

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It has been suggested that plasma membrane (PM) lipids play a major role in aluminum (Al) tolerance; however, no direct investigations have been carried out using PM lipids from root-tips. Here we report a new technique for PM isolation as an alternative to the laborious two-polymer phase partitioning method that is commonly applied, as follows: 1) separation of protoplasts from 1-cm root-tip portions by enzymatic digestion, 2) attachment of the purified protoplasts to glass plates coated with polylysine, 3) preparation of PM ghosts by successive burst of the attached protoplasts using three separate buffer solutions (25 mM PIPES, 5 mM EDTA, and 2 mM MgCl_2, at pH 7.0) with slow stirring for 60s. The PMs were confirmed to be devoid of organelle membranes by fluorescence microscopy, thin layer chromatography (TLC) and western blot analysis. The PM lipids obtained were found to be useful for studies on their differential permeability and lipid composition between lines of triticale or cultivars of maize under Al stress.
社団法人日本土壌肥料学会の論文