Meckel, Tobias (2004)
Endocytosis against the high turgor of guard cells.
Technische Universität Darmstadt
Dissertation, Erstveröffentlichung

Kurzbeschreibung (Abstract)

Stomata are found in the epidermis of photosynthetic active plant organs. They are formed by pairs of guard cells which create a pore to facilitate CO2 and water exchange with the environment. In order to control this gas exchange, guard cells actively change their volume and, consequently, surface area to alter the aperture of the stomatal pore. These changes are achieved by an uptake or release of K+ through K+-selective channels followed by the respective osmotic water fluxes. The quantification of such osmotically driven changes on 3D reconstructions revealed that guard cells of open and closed stomata of Vicia faba L., which show a 50 % change in aperture, differ in volume and surface area by 25 % and 15 %, respectively. Since biological membranes only have a limited elasticity of about 2 %, such excursions in surface area during stomatal movement require an addition or retrieval of membrane to or from the plasma membrane (PM). However, the relevance of endocytosis in plants has frequently been questioned on the basis of energetic considerations, because high turgor poses a barrier on the budding of the PM into vesicles. To investigate this process in highly turgescent guard cells, the dynamics of the PM were examined by monitoring with confocal microscopy the fate of membrane-affine styryl dyes (FM4-64, FM2-10, FM1-43) and the fluid-affine dye Alexa 488 hydrazide under natural and constant osmotic conditions. As a third marker, a relevant transporter for stomatal function was observed by following the retrieval of a fluorescent chimera of the K+-channel KAT1 (KAT1::GFP). Over some minutes of incubation in FM-dyes, endocytic vesicles in the cortical cytoplasm beneath the PMwere fluorescently labelled. The identification was based on the observation that the size distribution of these structures is very similar to that of endocytic vesicles obtained from patch-clamp capacitance recordings. All markers, whether membrane- (FM-dyes, KAT1::GFP) or fluid-phase-affine (Alexa 488 hydrazide), are taken up into structures of diffraction-limited size and similar localisation in the cortical cytoplasm. The calculated size of Alexa 488 hydrazide labelled structures was 60 - 80 nm. This is well in accordance with sizes reported for clathrin-coated vesicles in plants. Moreover, a subset of single vesicles was labelled with the externally supplied membrane marker FM4-64 and the intracellular produced KAT1::GFP. Consequently these vesicles carry K+-channels, which had already been delivered to the PM and are now retrieved via endocytosis. To summarize, the data provide strong evidence that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K+-channel KAT1 from the PM via endocytic vesicles.

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