TU Darmstadt / ULB / TUbiblio

Endocytosis against the high turgor of guard cells

Meckel, Tobias (2004)
Endocytosis against the high turgor of guard cells.
Technische Universität Darmstadt
Ph.D. Thesis, Primary publication

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.

Item Type: Ph.D. Thesis
Erschienen: 2004
Creators: Meckel, Tobias
Type of entry: Primary publication
Title: Endocytosis against the high turgor of guard cells
Language: English
Referees: Holstein, Prof. Dr. Thomas W. ; Dencher, Prof. Dr. Norbert A. ; Kubitscheck, Prof. Dr. Ulrich
Advisors: Thiel, Prof. Dr. Gerhard
Date: 13 December 2004
Place of Publication: Darmstadt
Publisher: Technische Universität
Refereed: 29 October 2004
URL / URN: urn:nbn:de:tuda-tuprints-5076
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.

Alternative Abstract:
Alternative abstract Language

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.

English
Uncontrolled Keywords: endocytosis, vesicle, confocal laser scanning microscopy, guard cell, green fluorescent protein, potassium channel, live cell imaging, plants, Vicia faba L.
Classification DDC: 500 Science and mathematics > 570 Life sciences, biology
Divisions: 10 Department of Biology
?? fb10_botanik ??
10 Department of Biology > Plant Membrane Biophyscis (20.12.23 renamed in Biology of Algae and Protozoa)
10 Department of Biology > Membrane Dynamics
Date Deposited: 17 Oct 2008 09:21
Last Modified: 05 Mar 2013 09:26
PPN:
Referees: Holstein, Prof. Dr. Thomas W. ; Dencher, Prof. Dr. Norbert A. ; Kubitscheck, Prof. Dr. Ulrich
Refereed / Verteidigung / mdl. Prüfung: 29 October 2004
Export:
Suche nach Titel in: TUfind oder in Google
Send an inquiry Send an inquiry

Options (only for editors)
Show editorial Details Show editorial Details