Ulrike Homann

Osmotically evoked shrinking of guard-cell protoplasts causes vesicular retrieval of plasma membrane into the cytoplasm

Abstract. The dye FM1-43 was used alone or in combination with measurements of the membrane capacitance (Cm) to monitor membrane changes in protoplasts from Viciafaba L. guard cells. Confocal images of protoplasts incubated with FM1-43 (10 μM) at constant ambient osmotic pressure (πo) revealed in confocal images a slow internalisation of FM1-43-labelled membrane into the cytoplasm. As a result of this process the relative fluorescence intensity of the cell interior (fFM,i) increased with reference to the total fluorescence (fFM,t) by 7.4 × 10−4 min−1. This steady internalisation of dye suggests the occurrence of constitutive endocytosis under constant osmotic pressure. Steady internalisation of FM1-43 labelled membrane caused a prominent staining of a ring-like structure located beneath the plasma membrane. Abrupt elevation of πo by 200 mosmol kg−1 caused, over the first minutes of incubation, a rapid internalisation of FM1-43 fluorescence into the cytoplasm concomitant with a decrease in cell perimeter. Within the first 5 min the cell perimeter decreased by 7.9%. Over the same time fFM,i/fFM,t increased by 0.13, reflecting internalisation of fluorescent label into the cytoplasm. Combined measurements of Cm and total fluorescence of a protoplast (fFM,p) showed that an increase in πo evoked a decrease in Cm but no change in fFM,p. This means that surface contraction of the protoplast is due to retrieval of excess membrane from the plasma membrane and internalisation into the cytoplasm. Further inspection of confocal images revealed that protoplast shrinking was only occasionally associated with internalisation of giant vesicles (median diameter 2.7 μm) with FM1-43-labelled membrane. But, in all cases, osmotic contraction was correlated with a diffuse distribution of FM1-43 label throughout the cytoplasm. From this, we conclude that endocytosis of small vesicles into the cytoplasm is the obligatory process by which cells accommodate an osmotically driven decrease in membrane surface area.

Diacidic Motif Is Required for Efficient Transport of the K+ Channel KAT1 to the Plasma Membrane

For a number of mammalian ion channels, trafficking to the plasma membrane was found to be controlled by intrinsic sequence motifs. Among these sequences are diacidic motifs that function as endoplasmic reticulum (ER) export signals. So far it is unclear if similar motifs also exist in plant ion channels. In this study we analyzed the function of four diacidic DXE/DXD motifs of the plant K+ channel KAT1. Mutation of the first diacidic DXE motif resulted in a strong reduction of the KAT1 conductance in both guard cell protoplasts and HEK293 cells (human embryonic kidney cells). Confocal fluorescence microscopy of guard cells expressing the mutated KAT1 fused to green fluorescent protein revealed localization of the mutated channel only in intracellular structures around the nucleus. These structures could be identified as part of the ER via coexpression of KAT1 fused to yellow fluorescent protein with an ER-retained protein (HDEL) fused to cyan fluorescent protein. Block of vesicle formation from the ER by overexpression of the small GTP-binding protein Sar1 fixed in its GDP-bound form led to retention of wild-type KAT1 in similar parts of the ER. Mutation of the three other diacidic motifs had no effect. Together, the results demonstrate that one diacidic motif of KAT1 is essential for ER export of the functional channel in both guard cell protoplasts and HEK293 cells. This suggests that trafficking of plant plasma membrane ion channels is controlled via a conserved mechanism.

Unitary exocytotic and endocytotic events in guard-cell protoplasts during osmotically driven volume changes.

Osmotically driven swelling and shrinking of guard‐cell protoplasts (GCPs) requires adjustment of surface area which is achieved by addition and removal of plasma membrane material. To investigate the mechanism for adaptation of surface area we have used patch‐clamp capacitance measurements. The recorded membrane capacitance (C m) trace of swelling and shrinking GCPs occasionally revealed discrete upward and downward deflecting capacitance steps, respectively, with a median value of about 2 fF. The observed capacitance steps resulted from the fusion and fission of single vesicles with a diameter of around 300 nm. We conclude that exo‐ and endocytosis of these vesicles accommodate for osmotically driven surface area changes in GCPs.

Interaction of the K(+)-channel KAT1 with the coat protein complex II coat component Sec24 depends on a di-acidic endoplasmic reticulum export motif.

The correct functioning of ion channels depends not only on the control of their activity but also on the regulation of the number of channels in the membrane. For example, it has been proposed that the density of the plant K(+)-channel KAT1 may be adjusted by controlling its export from its site of synthesis, the endoplasmic reticulum (ER). Efficient transport of the channel to the plasma membrane was found to depend on a di-acidic ER export signal in the C-terminus of the protein. Studies in yeast and mammals indicate that di-acidic ER export motifs are essential for enrichment of proteins into ER-derived coat protein complex II (COPII) vesicles and are recognized by Sec24 a component of the COPII coat. To investigate whether similar mechanisms also exist in plants we have analysed the interaction of KAT1 with Sec24 in vivo using fluorescence resonance energy transfer (FRET) measurements in Vicia faba guard cells. These measurements revealed a FRET signal between KAT1 and Sec24 fused to the cyan fluorescent protein and the yellow fluorescent protein, respectively, indicating an interaction between KAT1 and Sec24. The FRET signal only occurred in the perinuclear region of the ER and was dependent on the di-acidic ER export motif of KAT1. Together, the results point to a highly conserved mechanism for ER export of KAT1 whereby the channel is recruited into COPII vesicles via binding of the di-acidic motif to Sec24.

Ion channel activity during the action potential in Chara: new insights with new techniques

The dynamics of macroscopic currents underlying the electrically triggered action potential (AP) in the giant alga Chara corallina were directly recorded with an action potential clamp method. In this technique an AP is recorded and repetitively replayed as the command voltage to the same cell under voltage control. Upon adding the channel blockers niflumic acid and/or Ba(2+) to the bath, the excitation current, i.e. the current crossing the membrane during an AP, can be dissected into a transient, fast-appearing Cl(-) inward current and a transient delayed K(+) outward current. The delayed onset of the K(+) outward current demands the postulation of an additional outward current in order to balance the excess Cl(-) inward current at the onset of the AP. The capacitive current that alters the charge on the membrane during excitation is several orders of magnitude too small to be relevant for charge balance. Measurements of single channel activity in the plasma membrane of C. corallina by the patch clamp method shows two types of Cl(-) channel (15 and 38 pS with 100 mM Cl(-) in the pipette) and one type of K(+) channel (about 40 pS with 100 mM K(+) in the pipette) which become transiently active during an AP. Typically, variable numbers of CI(-) channels activate in a random fashion for short periods of time when favoured by positive voltages in combination with high concentrations of extracellular Ca(2+) (Ca(2+)(o)) or during an AP of the whole cell. The peak values of these Cl(-) channel currents measured in a patch are such that they can account quantitatively for the peak of the whole cell Cl(-) excitation current studied under comparable ionic conditions. Furthermore, the short dura- tion of channel activity, as well as the fast rising and somewhat slower trailing kinetics is similar in duration and dynamics to AP-associated changes in membrane permeability of the whole Chara cell to Cl(-) (P(Cl(-))). Taken together, the data stress that the characteristic, transient activation of random numbers of Cl(-) channels seen in membrane patches is the elementary unit of the Cl(-) excitation current. However, due to the random nature of this transient activity, gating of Cl(-) channels can not be explained on the basis of previous models for excitation: gating can neither be due to intrinsic voltage sensitivity of the Cl(-) channels, nor to a voltage-dependent influx of Ca(2+) and subsequent activation of Ca(2+)-sensitive Cl(-) channels. To account for the short life-time and for the randomness of Cl(-) channel activity, the putative gating factors Ca(2+) and voltage must be uncoupled in time. This could be explained by a random release of Ca(2+) from stores, the latter being filled in a voltage-sensitive manner via non-specific cation channels from the outside. A 4 pS non-selective cation channel in the plasma membrane may serve this purpose. The 40 pS K(+) channel, which becomes transiently active in C. corallina during a cell AP, is an outward rectifier. At negative resting voltages the channel has a low open probability (< <1%). At voltages reached during an AP the open probability rises significantly reaching half-maximal open probability at -25 mV. The elevated activity of the 40 pS channel associated with membrane excitation relaxes at the end of an AP with a time constant of about 2.5 s. A comparable time constant of 2 s can be obtained for the decay of the transiently elevated permeability of the membrane to K(+) (P(K(+))), stressing that the kinetic properties of the 40 pS K(+) channel are responsible for the course of whole cell P(K(+)) changes. Voltage sensitivity of the K(+) channels suggests that they are activated during an AP by the drop in membrane voltage in order to aid repolarization. However, the rise and decay of P(K(+)) during an AP also shares similarity with the time-course of transient changes in cytoplasmic concentration of free Ca(2+), [Ca(2+)](cyt), the latter being measured in parallel experiments with the Ca(2+)-sensitive fluorescent dye, Fura-2, in excited C. corallina cells. This similarity could suggest that gating of the 40 pS K(+) channel is also sensitive to [Ca(2+)](cyt) and that the latter sensitivity is rate-limiting for activity during an AP.

Na+/H+ antiporters are differentially regulated in response to NaCl stress in leaves and roots of Mesembryanthemum crystallinum.

Salinity tolerance in plants involves controlled Na(+) transport at the site of Na(+) accumulation and intracellular Na(+) compartmentation. The focus of this study was the identification and analysis of the expression of Na(+)/H(+) antiporters in response to NaCl stress in one particular plant, the facultative halophyte Mesembryanthemum crystallinum Na(+)/H(+) antiporters of M. crystallinum were cloned by RACE-PCR from total mRNA of leaf mesophyll cells. Functional complementation of Saccharomyces cerevisiae and Escherichia coli mutants was performed. The kinetics of changes in the expression of antiporters were quantified by real-time PCR in leaves and roots. Five Na(+)/H(+) antiporters (McSOS1, McNhaD, McNHX1, McNHX2 and McNHX3) were cloned, representing the entire set of these transporters in M. crystallinum. The functionality of McSOS1, McHX1 and McNhaD was demonstrated in complementation experiments. Quantitative analysis revealed a temporal correlation between salt accumulation and expression levels of genes in leaves, but not in roots, which was most pronounced for McNhaD. Results suggest a physiological role of McSOS1, McNhaD and McNHX1 in Na(+) compartmentation during plant adaptation to high salinity. The study also provides evidence for salt-induced expression and function of the Na(+)/H(+) antiporter McNhaD in chloroplasts and demonstrates that the chloroplast is one of the compartments involved in the response of cells to salt stress.

Microscopic elements of electrical excitation in Chara: Transient activity of Cl− channels in the plasma membrane

The plasma membrane of Chara corallina was made accessible for patch pipettes by cutting a small window through the cell wall of plasmolyzed internodal cells. With pipettes containing Cl− as Ca2+ or Ba2+ (50 or 100 mm), but not as Mg2+ or K+ salt, it was possible to record in the cell-attached mode for long periods with little channel activity, randomly interspersed with intervals of transient activation of two Cl− channel types (cord conductance at +50 mV: 52 and 16 pS, respectively). During these periods of transient channel activity, variable numbers (up to some 10) of the two Cl− channel types activated and again inactivated over several 100 msec in a coordinated fashion. Transient Cl channel activity was favored by voltages positive of the free running membrane voltage (> −45 mV); but positive voltage alone was neither a sufficient nor a necessary condition for activtion of these channels. Neither type of Cl− channel was markedly voltage dependent. A third, nonselective 4 pS channel is a candidate for Ca2+ translocation. The activity of this channel does not correlate in time with the transient activity of the Cl− channels. The entire set of results is consistent with the following microscopic mechanism of action potentials in Chara, concerning the role of Ca2+ and Cl− for triggering and time course: Ca2+ uptake does not activate Cl− channels directly but first supplies a membrane-associated population of Ca2+ storage sites. Depolarization enhances discharge of Ca2+ from these elements (none or few under the patch pipette) resulting in a local and transient increase of free Ca2+ concentration ([Ca2+]cyt) at the inner side of the membrane before being scavenged by the cytoplasmic Ca2+ buffer system. In turn, the transient rise in [Ca2+]cyt causes the transient activity of those Cl− channels, which are more likely to open at an elevated Ca2+ concentration.

Clathrin‐independent endocytosis contributes to uptake of glucose into BY‐2 protoplasts

In eukaryotic cells, several pathways exist for the internalization of plasma membrane proteins and extracellular cargo molecules. These endocytic pathways can be divided into clathrin-dependent and clathrin-independent pathways. While clathrin-dependent pathways are known to be involved in a variety of cellular processes in plants, clathrin-independent pathways have so far only been identified in animal and yeast cells. Here we show that internalization of fluorescent glucose into BY-2 cells leads to accumulation of the sugar in compartments of the endocytic pathway. This endocytic uptake of glucose was not blocked by ikarugamycin, an inhibitor of clathrin-dependent endocytosis, suggesting a role for clathrin-independent endocytosis in glucose uptake. Investigations of fusion and fission of single vesicles by membrane capacitance measurements revealed stimulation of endocytic activity by extracellular glucose. Glucose-stimulated fission of vesicles was not affected by addition of ikarugamycin or blocking of clathrin coat formation by transient over-expression of HUB1 (the C-terminal part of the clathrin heavy chain). These data demonstrate that clathrin-independent endocytosis does occur in plant cells. This pathway may represent a common mechanism for the uptake of external nutrients.

The number of K+ channels in the plasma membrane of guard cell protoplasts changes in parallel with the surface area

The activity of the two dominant K+ channels in the plasma membrane of Vicia faba guard cell protoplasts was examined during pressure-driven swelling. For this purpose, the K+ currents and the membrane capacitance (Cm) of guard cell protoplasts were recorded in parallel. A rise in Cm, reflecting an increase of the membrane surface area, was coupled to a proportional rise in conductance of both the K+ inward and K+ outward rectifier. The activation kinetics of the K+ channels were not affected during this process. The quantitative and temporal coupling of Cm and K+ conductance can hence be interpreted as the result of the addition of active inward and outward rectifier K+ channels to the plasma membrane during an increase in surface area.

Guard cells undergo constitutive and pressure-driven membrane turnover.

Summary.During stomatal movement, guard cells undergo large and reversible changes in cell volume and consequently surface area. These alterations in surface area require addition and removal of plasma membrane material. How this is achieved is largely unknown. Here we summarize recent studies of membrane turnover in guard cells using electrophysiology and fluorescent imaging techniques. The results implicate that membrane turnover in guard cells and most likely in plant cells in general is sensitive to changes in membrane tension. We suggest that this provides a mechanism for the adaptation of surface area of guard cells to osmotically driven changes in cell volume. In addition, guard cells also exhibit constitutive membrane turnover. Constitutive and pressure-driven membrane turnover were found to be associated with addition and removal of K+ channels. This implies that some of the exo- and endocytic vesicles carry K+ channels. Together the results demonstrate that exo- and endocytosis is an essential process in guard cell functioning.