Endocytosis against high turgor: intact guard cells of Vicia faba constitutively endocytose fluorescently labelled plasma membrane and GFP‐tagged K⁺‐channel KAT1

Abstract

SummaryThe relevance of endocytosis in plants against high turgor pressure has frequently been questioned on the basis of energetic considerations. Here, we examine the dynamics of the plasma membrane (PM) in turgid guard cells of Vicia faba by monitoring with confocal microscopy the fate of¯uorescent styryl dyes (FM1-43, FM2-10 and FM4-64). As a second marker, we also observe the retrieval of a¯uorescent chimaera of the K -inward rectifying channel from Arabidopsis thaliana and the green¯uorescent protein (KAT1::GFP). Analysis of cytoplasmic structures, which became labelled by the different styryl dyes, revealed that only FM4-64, the most hydrophobic dye, was a reliable marker of endocytosis, whereas the two other styryl dyes resulted also in an unspeci®c labelling of different cytoplasmic structures including mitochondria. Over some minutes of incubation in continuous presence of these dyes, endocytic vesicles in the cortical cytoplasm beneath the PM were¯uorescently labelled. The identi®cation is 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. Also, these structures are frequently co-labelled with KAT1::GFP. Taken together, the data show that turgid guard cells undergo vigorous constitutive endocytosis and retrieve membrane including the K -channel KAT1 from the PM via endocytic vesicles.

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…This may be attributable to physical constraints placed on the endocytic machinery in these two very different types of cells. For instance, it has been proposed that actin plays an obligatory role in walled yeast endocytosis because high membrane tension resulting from turgor pressure elicits more work to invaginate the membrane as compared with mammalian cells (Meckel et al 2004;Aghamohammadzadeh and Ayscough 2009;Boulant et al 2011). Indeed, there is evidence that membrane tension may modulate the requirement for actin in mammalian CME and yeast protoplasts wherein regimes of high membrane tension, generated by hyperosmotic challenge or physical stretching, invoke a requirement for actin (Aghamohammadzadeh and Ayscough 2009;Boulant et al 2011).…”

Participation of Endomembrane Cation/H+ Exchanger AtCHX20 in Osmoregulation of Guard Cells

“…These changes indicate incorporation of the exocytic vesicle with the PM. Guard cells undergo constitutive endocytosis and exocytosis even in turgid cells as shown by the uptake of impermeant fluorescent FM4-64 dyes into vesicles beneath the PM (Meckel et al, 2004). Although direct biophysical measurements are not yet available to measure vacuolar dynamics, electron microscopy (e.g.…”

Endocytosis and degradation of BOR1, a boron transporter ofArabidopsis thaliana, regulated by boron availability

“…In addition, early studies using electron-dense markers, such as cationized ferritin, and recent studies using lipophilic styryl FM dyes and maker proteins such as Rab GTPase and SNAREs allowed us to visualize the endocytic route from the plasma membrane to the vacuole in plant cells (29,30). FM4-64 confers red fluorescence upon insertion into the plasma membrane and then labels all other membranes in the endocytic pathway down to the vacuole in yeast (32) as well as in plant cells (33,34). In A. thaliana cells, FM4-64 stained distinct populations of endosomes in which three members of Rab5-related GTPases were localized (21,33).…”