Flow Cytometric Fluorescence Anisotropy of Lipophilic Probes in Epidermal and Mesophyll Protoplasts from Water-Stressed Lupinus albus L

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Abstract
The blue emission anisotropy, r, of two lipophilic probes, diphenylhexatriene (DPH) and its trimethyl-ammonium derivative (TMA-DPH), has been measured in foliar Lupinus albus L. protoplasts for the first time by flow cytometry. Distinctive values have been obtained for protoplasts of epidermal and mesophyll origin, identified by their intensities of chlorophyll fluorescence. Fluorescence microscopy confirmed that TMA-DPH remained in the plasma membrane while DPH penetrated into intracellular lipid domains. Typical emission anisotropy values at 22°C for mesophyll and epidermal protoplasts, respectively, were 0.225 and 0.312 with TMA-DPH, and 0.083 and 0.104 with DPH. This indicates that epidermal cells—and notably their plasma membranes (TMA-DPH)—have higher lipid microviscosity and/or more ordered lipid structure. Two lupin genotypes characterized as resistant or susceptible to drought were analyzed with or without 9 days of water stress shown to increase ion leakage from foliar discs. Water stress greatly increased the apparent fluidity, and more so in the susceptible genotype; the effect was more pronounced in the chlorophyll-containing mesophyll cells than in the epidermal cells.