Here, we discuss why guard cells in intact plants respond to environmental signals in a different way than guard cells in epidermal strips, or protoplasts thereof.
In intact leaves stomatal opening is counteracted by epidermal cells that press against the guard cells. Changes in the turgor of epidermal cells therefore can alter the stomatal aperture.
In addition, stomatal opening may be modulated by the solute composition of the guard cell wall. Changes in apoplastic K+, Cl− and Ca2+ occur after light–dark transitions, but not in such a way that it would support stomatal opening.
Organic anions may play a role, since they enhance the open probability of anion channels in the plasma membrane.
Furthermore, studies with auxin-resistant and abscisic acid-insensitive mutants show that light-induced stomatal opening is modulated by these hormones.
Using the newly developed method in which guard cells in the intact plant are impaled with double-barreled electrodes, the role of these apoplastic factors now can be studied on single guard cells that are still in their natural environment.
Drought induces stomatal closure, a response that is associated with the activation of plasma membrane anion channels in guard cells, by the phytohormone abscisic acid (ABA). In several species, this response is associated with changes in the cytoplasmic free Ca2+ concentration. In Vicia faba, however, guard cell anion channels activate in a Ca2+-independent manner.
Because of potential differences between species, Nicotiana tabacum guard cells were studied in intact plants, with simultaneous recordings of the plasma membrane conductance and the cytoplasmic free Ca2+ concentration. ABA triggered transient rises in cytoplasmic Ca2+ in the majority of the guard cells (14 out of 19).
In seven out of 14 guard cells, the change in cytoplasmic free Ca2+ closely matched the activation of anion channels, while the Ca2+ rise was delayed in seven other cells. In the remaining five cells, ABA stimulated anion channels without a change in the cytoplasmic Ca2+ level.
Even though ABA could activate anion channels in N. tabacumguard cells independent of a rise in the cytoplasmic Ca2+ concentration, patch clamp experiments showed that anion channels in these cells are stimulated by elevated Ca2+ in an ATP-dependent manner.
Guard cells thus seem to have evolved both Ca2+-independent and -dependent ABA signaling pathways. Guard cells of N. tabacum apparently utilize both pathways, while ABA signaling in V. faba seems to be restricted to the Ca2+-independent pathway.
Cytoplasmic calcium elevations, transients, and oscillations are thought to encode information that triggers a variety of physiological responses in plant cells. Yet Ca(2+) signals induced by a single stimulus vary, depending on the physiological state of the cell and experimental conditions.
We compared Ca(2+) homeostasis and stimulus-induced Ca(2+) signals in guard cells of intact plants, epidermal strips, and isolated protoplasts.
Single-cell ratiometric imaging with the Ca(2+)-sensitive dye Fura 2 was applied in combination with electrophysiological recordings. Guard cell protoplasts were loaded with Fura 2 via a patch pipette, revealing a cytoplasmic free Ca(2+) concentration of around 80 nM at -47 mV. Upon hyperpolarization of the plasma membrane to -107 mV, the Ca(2+) concentration increased to levels exceeding 400 nM.
Intact guard cells were able to maintain much lower cytoplasmic free Ca(2+) concentrations at hyperpolarized potentials, the average concentration at -100 mV was 183 and 90 nM in epidermal strips and intact plants, respectively. Further hyperpolarization of the plasma membrane to -160 mV induced a sustained rise of the guard cell cytoplasmic Ca(2+) concentration, which slowly returned to the prestimulus level in intact plants but not in epidermal strips.
Our results show that cytoplasmic Ca(2+) concentrations are stringently controlled in guard cells of intact plants but become increasingly more sensitive to changes in the plasma membrane potential in epidermal strips and isolated protoplasts.
Stomatal closure is known to be associated with early defence responses of plant cells triggered by microbe-associated molecular patterns (MAMPs). However, the molecular mechanisms underlying these guard cell responses have not yet been elucidated.
We therefore studied pathogen-induced changes in ion channel activity in Hordeum vulgareguard cells. Barley mildew (Blumeria graminis) hyphae growing on leaves inhibited light-induced stomatal opening, starting at 9 h after inoculation, when appressoria had developed.
Alternatively, stomatal closure was induced by nano-infusion of chitosan via open stomata into the sub-stomatal cavity.
Experiments using intracellular double-barreled micro-electrodes revealed that mildew stimulated S-type (slow) anion channels in guard cells. These channels enable the efflux of anions from guard cells and also promote K+ extrusion by altering the plasma membrane potential.
Stimulation of S-type anion channels was also provoked by nano-infusion of chitosan.
These data suggest that MAMPs of mildew hyphae penetrating the cuticle provoke activation of S-type anion channels in guard cells.
In response, guard cells extrude K+ salts, resulting in stomatal closure. Plasma membrane anion channels probably represent general targets of MAMP signaling in plants, as these elicitors depolarize the plasma membrane of various cell types.
During stress, plant cells activate anion channels and trigger the release of anions across the plasma membrane. Recently, two new gene families have been identified that encode major groups of anion channels. The SLAC/SLAH channels are characterized by slow voltage-dependent activation (S-type), whereas ALMT genes encode rapid-activating channels (R-type). Both S- and R-type channels are stimulated in guard cells by the stress hormone ABA, which leads to stomatal closure.
Besides their role in ABA-dependent stomatal movement, anion channels are also activated by biotic stress factors such as microbe-associated molecular patterns (MAMPs). Given that anion channels occur throughout the plant kingdom, they are likely to serve a general function as master switches of stress responses.
The stomatal complex of Zea mays is composed of two pore-forming guard cells and two adjacent subsidiary cells. For stomatal movement, potassium ions and anions are thought to shuttle between these two cell types. As potential cation transport pathways, K+-selective channels have already been identified and characterized in subsidiary cells and guard cells. However, so far the nature and regulation of anion channels in these cell types have remained unclear.
In order to bridge this gap, we performed patch–clamp experiments with subsidiary cell and guard cell protoplasts. Voltage-independent anion channels were identified in both cell types which, surprisingly, exhibited different, cell-type specific dependencies on cytosolic Ca2+ and pH. After impaling subsidiary cells of intact maize plants with microelectrodes and loading with BCECF [(2′,7′-bis-(2-carboxyethyl)-5(and6)carboxyflurescein] as a fluorescent pH indicator, the regulation of ion channels by the cytosolic pH and the membrane voltage was further examined.
Stomatal closure was found to be accompanied by an initial hyperpolarization and cytosolic acidification of subsidiary cells, while opposite responses were observed during stomatal opening. Our findings suggest that specific changes in membrane potential and cytosolic pH are likely to play a role in determining the direction and capacity of ion transport in subsidiary cells.