Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir12[CC-BY]

Clustering of stomata suppresses guard cell ion transport independent of ion stores in neighboring epidermal cells. Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K+ channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K+ ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K+ and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K+ accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells.

Stomata are pores found in the epidermis of most aerial parts of plants and are formed between a specialized pair of cells, the guard cells. Stomata facilitate the uptake of CO 2 at the expense of water vapor release via transpiration (Hetherington and Woodward, 2003). Hence, stomata play a crucial role in the physiology of plants. They permit gaseous exchange between the environment and the inside of the leaf for photosynthesis and, in turn, they influence the water use efficiency and growth of the plant. Mathematical models have suggested that historical changes in the freshwater resources can be attributed to stomatal transpiration, and it has been argued that the manipulation of stomata will be an important factor in ensuring water availability over the next 20 to 30 years (UNESCO World Water Development Report, 2015). Efforts to develop crops with higher water use efficiency through conventional breeding strategies have led to some successes, including the Drysdale wheat (Triticum aestivum) variety (Condon et al., 2002). It is likely that further advances will be possible as we gain insights into the physiology of stomata in situ.
The regulation of gas exchange is achieved by controlling the stomatal pore. Stomata respond dynamically to environmental changes, including light quality and intensity, ambient CO 2 concentration, and humidity (Aphalo and Jarvis, 1991;Hetherington and Woodward, 2003;Shimazaki et al., 2007). Stomatal movements result from changes in guard cell volume and turgor and are driven by solute and water fluxes across the plasma membrane and tonoplast of guard cells. In particular, fluxes of the predominant inorganic cation K + are mediated by the voltage-dependent inward-rectifying K + channels (I KIN ), in Arabidopsis (Arabidopsis thaliana) by the channel proteins KAT1 and KAT2, during stomatal opening Clint and Blatt, 1989;Kwak et al., 2001;Dreyer and Blatt, 2009) and by outward-rectifying K + channels (I KOUT ), notably GORK in Arabidopsis, during stomatal closure Clint and Blatt, 1989;Blatt, 1991;Hosy et al., 2003;Wang et al., 2012).
It has generally been argued that the osmotic solutes required for stomatal movements are provided by the surrounding leaf tissues, which act as both a source and sink for ions (Raschke and Fellows, 1971;MacRobbie and Lettau, 1980;Outlaw, 1983;Wilmer and Fricker, 1996;Franks and Farquhar, 2007). For example, previous studies showed that the accumulation of K + in Commelina communis guard cells was accompanied by its loss from the surrounding epidermal cells during stomatal opening, and the reverse was observed during the closing process (MacRobbie and Lettau, 1980). The presence of epidermal neighboring cells to provide an exchange of osmotic solute also is argued to eliminate the mechanical back pressure from guard cells. Indeed, Franks and Farquhar (2007) have noted the distinct arrangements of stomatal complexes between species and their association with adjacent epidermal cells allowing the ion exchange required for the opening process.
The majority of plant species follow a one-cell spacing rule during epidermal development that leads to the separation of stomata by at least one epidermal cell (Geisler et al., 2000;Peterson et al., 2010;Pillitteri and Dong, 2013). However, there are several genera that diverge from this rule. For instance, stomatal clustering in Begonia has been considered to be an adaptation for growth in ecological niches with low water availability (Hoover, 1986;Tang et al., 2002). Even so, no quantitative data are available confirming an advantage of species with stomatal clusters to grow in dry environments. To date, only one study with Arabidopsis transgenic lines has reported on the impact of stomatal clustering in plant physiology, suggesting a negative correlation between gaseous exchange and the degree of clustering (Dow et al., 2014). Those authors speculated that the much-reduced availability of adjacent epidermal cells could explain the altered stomatal behavior in plants with stomatal clusters.
We have revisited the physiological impact of stomatal clustering, making use of the Arabidopsis mutant too many mouths (tmm1; Yang and Sack, 1995;Geisler et al., 2000) that exhibits stomatal clusters. We find that stomatal clustering of the Arabidopsis tmm1 mutant affects stomatal behavior. We also provide evidence that this impairment is linked to changes in ion transport at the guard cell plasma membrane and is independent of the presence of neighboring epidermal cells that mediate ionic exchange with the guard cells. These results emphasize the importance of spacing between stomata to ensure proper stomatal behavior and indicate that its impact goes beyond sole mechanical, spatial, or source-sink relations.

Stomatal Clustering Affects Maximum Stomatal Conductance
Stomatal patterning was analyzed in epidermal peels from Arabidopsis wild-type Columbia-0 (Col-0), the tmm1 mutant, and the complementation line PTMM1 (Fig. 1). The mean stomatal density ranged between 290 to 740 stomata per mm 2 for Arabidopsis. The tmm1 plants showed significantly higher stomatal density compared with wild-type and PTMM1 plants. The lines showed an inverse correlation between stomatal density and size, with smaller stomata being more numerous (Fig. 1A), as has been reported previously (Hunt and Gray, 2009;Doheny-Adams et al., 2012). The differences in the number and size of stomata also were apparent in the stomatal index, which is the ratio of the Figure 1. Stomatal characteristics of Arabidopsis lines. The images at top display representative micrographs from the abaxial side of Arabidopsis wild-type (Col-0), tmm1, and PTMM1 plants. Bar = 20 mm. A to C, Stomatal patterning was determined from epidermal peels of wildtype (white symbols), tmm1 (black symbols), and PTMM1 (gray symbols) plants. Graphs represent correlation between stomatal density and stomatal size (r 2 = 0.99; A), mean stomatal index (B), and mean maximum stomatal aperture (C). Data are means 6 SE of n . 60 stomata from three independent experiments. Letters indicate statistical differences among Arabidopsis lines (P , 0.05) as determined by the Student-Newman-Keuls test. D, Steady-state diffusional stomatal conductance was measured from Arabidopsis leaves treated with saturating light of 400 mmol m 22 s 21 . Data are means 6 SE of n = 3 plants. No significant differences among Arabidopsis lines (P , 0.05) were detected. E and F, G Wmax showed a positive correlation with stomatal density (E; r 2 = 0.98) and a negative correlation with stomatal size (F; r 2 = 0.97). Data are means 6 SE of n . 60 stomata from three independent experiments. number of stomata over the number of nonstomatal cells. The tmm1 mutant showed a roughly 4-fold greater stomatal index than wild-type and PTMM1 plants (Fig. 1B).
We examined stomatal opening in stomatal clusters by treating epidermal peels with depolarizing buffer (60 mM KCl-MES, pH 6.1; Papanatsiou et al., 2015) under high light intensity for 2 h. Maximum opening of stomatal pores was measured by incorporating the geometry of individual guard cells as well as the whole stoma to calculate the area within the pore, as described previously (Doheny-Adams et al., 2012;Meckel et al., 2007). The wild-type and PTMM1 plants yielded stomatal apertures of 22.6 6 1.5 and 24.6 6 1.7 mm 2 , respectively (Fig. 1C). By contrast, the stomatal aperture of the tmm1 mutant was 24% smaller than that of the wild type, yielding values of 16.9 6 1.7 mm 2 .
We also carried out infrared gas analysis at the leaf level to estimate the diffusional stomatal conductance using saturating light as the opening stimulus. In selecting the level of photosynthetic active radiation (PAR), we performed light curves that show the response of the assimilation of CO 2 over different quantum flux densities, ranging from 0 to 800 mmol m 22 s 21 (Supplemental Fig.  S1). The mean diffusional stomatal conductance at saturating light (400 mmol m 22 s 21 ) was indistinguishable between plants with single stomata and stomatal clusters (Fig. 1D). We also calculated the theoretical maximum conductance for water vapor (G Wmax ) of each line based on the measurements of maximum stomatal aperture as well as the spatial specifications of stomata. G Wmax was positively correlated with the stomatal density ( Fig. 1E), whereas it was negatively correlated with stomatal size (Fig. 1F). Both observations were consistent with previous reports ). We noted that the G Wmax of tmm1 was greater than that of the plants with single stomata but that measured conductances were indistinguishable among the lines, indicating that the mutant was unable to approach its theoretical maximum as closely as the wild-type and PTMM1 plants.

Spacing between Stomata Is Essential for Stomatal Closure
Evidence for the effects of stomatal clustering on stomatal function is scarce. Only Dow et al. (2014) examined the effects of stomatal clustering and reported the reduced gas exchange and photosynthetic capacity when plants were subjected to different CO 2 concentrations, ranging from 200 to 1,000 mmol CO 2 mol -1 . We carried out complementary measurements, examining the effects of light and dark transitions on CO 2 assimilation and transpiration associated with the dynamics of stomatal movements (Supplemental Fig. S2).
The kinetics of gas-exchange responses upon exposure to light/dark treatments are summarized in Table I and Figure 2. Stomatal clustering in Arabidopsis plants did not influence the kinetics of changes in gas exchange under any light or dark treatment (Table I), but it did affect the steady-state rates of gas exchange (Fig. 2). CO 2 assimilation of tmm1 appeared lower when plants were subjected to saturating light (400 mmol m 22 s 21 ). The tmm1 plants exhibited a 20% lower maximum rate of CO 2 assimilation, corresponding to a value of 7.8 6 0.23 mmol CO 2 m 22 s 21 compared with 9.3 6 0.5 mmol CO 2 m 22 s 21 in wild-type plants and 9.0 6 0.1 mmol CO 2 m 22 s 21 in PTMM1 plants. No differences between the Arabidopsis lines were observed in the maximum rate of CO 2 assimilation in the photosynthetically limited low-light conditions (70 mmol m 22 s 21 ), suggesting that the altered CO 2 assimilation response was dependent on stomatal aperture, in line with conclusions from Dow et al. (2014).
The steady-state rates of transpiration under the two light treatments from the tmm1 mutant were not substantially different from those of Arabidopsis plants with single stomata, despite the higher stomatal numbers. However, the tmm1 plants exhibited higher steadystate rates of transpiration in the dark than the plants with single stomata. When the tmm1 plants were transferred to dark from the low-light regime, the transpiration rate in the dark was 1.21 6 0.02 mmol m 22 s 21 compared with 0.73 6 0.06 mmol m 22 s 21 in the wild type. Similarly, when the plants were transferred from saturating light intensity, the transpiration rate in the dark of tmm1 was approximately 20% higher than that of plants with single stomata.
To directly assess the impact of stomatal clustering on stomatal closing, we measured stomatal apertures after treatments with the well-established closing stimuli Ca 2+ and abscisic acid (ABA; Grabov and Blatt, 1998;Eisenach et al., 2012;Papanatsiou et al., 2015). Stomata of wild-type and PTMM1 plants closed to similar extents, whereas stomata of the tmm1 mutant Table I. Half-times of gas-exchange responses in plants with single stomata and stomatal clusters Gas-exchange data from Arabidopsis plants under low and saturating light intensity were fitted to nonlinear regression models to extrapolate half-times for CO 2 assimilation, transpiration under light, and transpiration under darkness. Data are presented in minutes. PAR was measured in mmol m 22 s 21 . Data are means 6 SE of n = 3 plants per genotype. No significant differences among Arabidopsis lines (P , 0.05) were detected. were impaired in this process (Fig. 3A). The tmm1 mutant showed a significantly smaller dynamic range of apertures in comparison with wild-type plants following a closing stimulus. We also carried out timecourse analysis of stomatal closure in which epidermal peels from Arabidopsis lines were treated with 20 mM ABA and stomata were imaged on a cell-by-cell basis at intervals for 60 min (Fig. 3B). The closing process was attenuated significantly in the tmm1 mutants, which exhibited 2.5-fold smaller decrease in stomatal aperture than the Arabidopsis lines with single stomata, indicating defects in the movements of stomata in clusters.

Stomatal Clustering Influences Ion Transport in Guard Cells
Stomatal movements depend on the changes in the guard cell turgor pressure and volume that ensue from K + fluxes through I KIN and I KOUT at the plasma membrane (Blatt, 1992(Blatt, , 2000a(Blatt, , 2000bBlatt and Armstrong, 1993). We were interested, therefore, in examining whether ion transport at the guard cell plasma membrane was affected by stomatal clustering. We recorded currents through K + channels under voltage clamp from Arabidopsis wild-type, tmm1, and PTMM1 guard cells from isolated epidermal peels continuously bathed in solution with a fixed K + concentration. . The tmm1 mutant is defective in stomatal closing. Epidermal peels from wild-type, tmm1, and PTMM1 plants were placed in opening buffer and irradiated to ensure maximum stomatal opening before they were treated with buffer containing 6 mM CaCl 2 (A) or 20 mM ABA (B) for 60 min to induce stomatal closure. Relative stomatal closing was measured on a cell-by-cell basis. Data are means 6 SE of n . 80 stomata from three independent experiments. Letters indicate statistical differences between Arabidopsis lines (P , 0.001) as determined by the Student-Newman-Keuls test. Figure 2. The tmm1 mutant suppresses gas-exchange responses induced by dark treatment. Steady state gas-exchange triggered by light or darkness is shown. Three-week-old dark-adapted plants were treated with PAR of 70 (black bars) and 400 (gray bars) mmol m 22 s 21 and then exposed to darkness. Graphs present maximum rates of CO 2 assimilation (A max ; A), transpiration under light (E max ; B), and transpiration under darkness (E min ; C). Data are means 6 SE of n = 3 plants per species. Letters indicate significant differences among Arabidopsis lines at each light condition (P , 0.05) as determined by the Student-Newman-Keuls test. Figure 4 presents the current traces and average steady-state currents as a function of voltage (currentvoltage curves) from guard cells of the Arabidopsis lines. In each case, the currents yielded gating characteristics similar to previous reports (Garcia-Mata et al., 2003;Eisenach et al., 2012;Papanatsiou et al., 2015), albeit with some notable differences in the tmm1 plants. Voltages positive of 240 mV were marked by strong outward-directed currents, with the current amplitude increasing with increasing voltages, while shifting the voltages to more negative values than 2100 mV generated inward-directed currents of increasing amplitude.
To extract the characteristics of changes in the K + transport in guard cells found in clusters, we fitted current-voltage curves for each of the currents jointly to a Boltzmann function (see Eq. 4 below) and determined the maximum conductance and gating characteristics of the K + channels (Table II). Jointly fitted curves minimize the number of free parameters and allow comparisons between fitting with common parameter values (Honsbein et al., 2009;Wang et al., 2012;Papanatsiou et al., 2015). For I KOUT and I KIN , this analysis yielded statistically and visually satisfactory fittings with the gating charge held in common to give values of 1.9 6 0.3 and 2.1 6 0.3, respectively. The maximum conductance for I KOUT from tmm1 guard cells was reduced by approximately 60% compared with wild-type and PTMM1 cells, whereas no statistically significant alteration was observed for the I KIN among the lines. The amplitude differences aside, we found that the voltage dependence of the I KIN from the tmm1 mutant was displaced by approximately +30 mV to more positive voltages compared with wild-type Arabidopsis guard cells, indicating that the stomatal clustering also affected the voltage dependence of the gating of the K + channels. We carried out quantitative PCR to compare the transcript levels of the plasma membrane H + -ATPase and three major plasma membrane K + channels, the outward-rectifier GORK and the two inward-rectifiers KAT1 and KAT2. This analysis uncovered no substantial difference in transcript levels, suggesting that the alterations in ion transport were not due to altered population sizes or their distributions (Supplemental Fig. S3).
Finally, we made use of the voltage clamp to determine the K + content of the guard cells from wild-type and clustered tmm1 stomata. Tail current analysis provides a direct measure of current reversal and, for any given ensemble of K + channels, defines the effective equilibrium voltage that can be used to determine the cytosolic K + content. Figure 5A presents current traces and average tail currents for the I KOUT as a function of voltage from guard cells. The point at which no current relaxation was observed defines the reversal voltage. As shown in Figure 5B, the reversal potential was displaced toward more positive values by about 50 mV in the tmm1 mutant compared with the wild type. From the Nernst equation, the K + content of the tmm1 guard cells was calculated as 54 mM, which is significantly lower than the 199 mM calculated for the wild-type guard cells. Collectively, the results indicate that stomatal clustering affects the K + fluxes and the accumulation of K + ions in guard cells even under the unrestricted K + availability afforded by superfusion with millimolar concentrations in electrophysiological experiments.

DISCUSSION
Substantial evidence shows that stomatal density affects gas exchange, which, in turn, translates to assimilation rates and water use efficiency in plants (Schlüter et al., 2003;Lawson, 2009;Yoo et al., 2010;Fanourakis et al., 2011;Drake et al., 2013;Tanaka et al., 2013). Only recently has attention turned to questions of the spacing of stomata on the leaf surface and its implications for environmental physiology and adaptation. The stomata of most plants occur singly, one over each substomatal cavity within the leaf mesophyll layer, and are separated by at least one pavement cell within the leaf epidermis. However, in a few plant species, stomata are found to cluster naturally in groups of two or more over a substomatal cavity and may be separated by a narrow wedge only of a pavement cell. Mutants, too, are known in which stomata develop in clusters, often without a separating pavement cell. To date, a single study has shown that Arabidopsis mutants with a high degree of clustering show reductions in gas exchange and are impaired in their capacity for carbon assimilation (Dow et al., 2014). However, until now, no information has been forthcoming on the mechanisms underlying these differences.
We examined the physiology of Arabidopsis and its tmm1 mutant, which develops clusters without intervening pavement cells (Yang and Sack, 1995;Geisler et al., 2000). These studies compared the gas-exchange characteristics as well as the dynamics of stomatal movements. Additionally, we used voltage clamp analysis to assess the capacities for K + flux and to compare the K + contents in the Arabidopsis lines. Like Dow et al. (2014), we found that clustering in the Arabidopsis tmm1 mutant compromises carbon assimilation and stomatal closure. Our studies go further to show that the effects in the mutant are largely a consequence of a reduced dynamic range for stomatal movement and that they are associated with decreases in both the capacity for K + flux and the K + content of the guard cells. Most interestingly, the differences in stomatal behavior cannot be understood on the basis of channel transcript levels (Supplemental Fig. S3) or solely on the availability of K + for exchange with the apoplast and surrounding cells, since the electrophysiological experiments were carried out with isolated epidermal peels in unconstrained K + concentration. In addition, the isolation of epidermal peels results in the complete destruction of epidermal cells. Thus, we conclude that spacing between stomata may be important for stomatal function but that the effects on gas-exchange kinetics, suppressed K + channel activities, and K + content in guard cells of the Arabidopsis tmm1 mutant are clearly independent of any source-sink relations with the surrounding epidermal cell layer.

Gas Exchange and Stomatal Patterning
It is well documented that stomatal density across dicotyledonous species is balanced against stomatal size and that reducing stomatal size is an important strategy to facilitate stomatal dynamics without increasing the evaporative area of the leaf (Drake et al., 2013). Reducing the size of the guard cells surrounding the stomatal pore has the effect of increasing the ratio of membrane surface area to guard cell volume. Provided that the density of membrane transporters per unit of surface area is nearly constant, a decrease in guard cell size can be expected to accelerate the solute flux per unit of volume proportionally, thereby allowing for faster responses to environmental transients. This strategy is well documented in studies of Arabidopsis (Schlüter et al., 2003;Tanaka et al., 2013) and other plant species (Franks and Farquhar, 2007;Drake et al., 2013;Lawson and Blatt, 2014) and is consistent with mathematical models that take into account the geometry of guard   Wang et al., 2012;Lawson and Blatt, 2014). In general, such analysis assumes the physical and spatial independence of each stoma and its positioning over a substomatal cavity.
Our data pointed to a mismatch between measured stomatal conductance and the theoretical maximum stomatal conductance of the tmm1 mutant (Fig. 1). This finding indicates that the presence of epidermal cells between stomata is crucial to allow stomatal opening and underlines the importance of spacing between stomata. Dow et al. (2014) proposed that the decreased CO 2 assimilation rates in plants with clustered stomata might depend on structural misplacement of stomata over mesophyll tissue. Although we cannot exclude this interpretation, we found that stomata from isolated epidermal peels of tmm1 failed to open when exposed to opening stimuli (Fig. 1C), suggesting that stomatal impairment in the tmm1 mutant is not dependent on the substomatal cavity or the underlying photosynthetic tissue. More importantly, we identified that the transpiration rate of tmm1 plants in the dark was higher (Fig. 2C) and that their response to closing stimuli was attenuated (Fig. 3), indicating a dependence on stomatal aperture rather than on leaf structure. Why assimilation was affected despite the lack of effect on transpiration in the light is puzzling. One possible explanation is that the tmm1 mutant additionally targets tissue development or function independent of the stomata, which, in turn, might influence water flux through the plant.
The tmm1 Mutant Affects K + Channel Activity and K + Content Of our findings, the most surprising were of alterations in K + channel activities and of decreases in the K + contents of clustered guard cells in the Arabidopsis tmm1 mutant (Figs. 4 and 5). Stomatal movements depend on changes in turgor, which is driven largely by the transport of osmotically active solutes and water across the plasma membrane of the guard cells (Blatt, 2000a(Blatt, , 2000bFranks and Farquhar, 2007). Potassium makes up by far the largest fraction of the inorganic osmotica, and its transport often leads to changes of whole-cell K + concentrations from 150 to 400 mM between the closed and open states of the guard cell, respectively (Blatt, 2000a(Blatt, , 2000bMacRobbie, 1998;Hills et al., 2012;Chen et al., 2012). Guard cells are packed together in the tmm1 mutant, however, implying a need for substantially greater solute accumulation to drive stomatal opening. The surrounding epidermal or subsidiary cells are thought to serve as a source for K + and other ions during stomatal opening and as a sink for the same ions when stomata close (Raschke and Fellows, 1971;MacRobbie and Lettau, 1980;Outlaw, 1983;Franks and Farquhar, 2007). MacRobbie and Lettau (1980) used K + -sensitive microelectrodes to demonstrate that the K + content in the epidermal cells of C. communis was reduced from between 180 and 300 mM to 80 mM when the nearby stomata opened, consistent with solute uptake by the guard cells from adjacent subsidiary cells (Penny and Bowling, 1974). However, when isolated in epidermal peels and superfused with solution containing millimolar KCl, any source-sink relations between guard cells and the surrounding epidermis are short-circuited by the nearly infinite volume of the buffer solution. As a consequence, we can rule out any effects associated with epidermal cell placement or their presence as a source of osmoticum and can eliminate any dependency of guard cells for ionic exchange on these neighboring cells. Yet, we observed that the ensemble conductances of both K + channels (Fig. 4) and the low K + concentrations indicated by tail current Figure 5. Stomatal clustering alters the cytosolic K + content of guard cells. A, Currents through I KOUT recorded under voltage clamp from wild-type (white circles) and tmm1 (black circles) guard cells. Voltages were clamped from a holding voltage of +50 mV in 5-s steps between 2220 and +40 mV. Data are means 6 SE of n = 5 guard cells bathed in 5 mM Ca 2+ -MES buffer, pH 6.1, with 10 mM KCl. B, Reversal potential of I KOUT as depicted from the tail current analysis. Decrease in voltage steps led to a negative reversal potential around 2110 6 16 mV for wild-type plants, whereas stomatal clustering of the tmm1 mutant shifted the reversal potential to a more positive value of 258 mV 6 9 mV. From the Nernst equation, the K + concentrations of wild-type and tmm1 guard cells were calculated as 199 and 54 mM, respectively. The asterisk indicates a statistical difference as determined by Student's t test (P , 0.05). analysis (Fig. 5) were reduced in the tmm1 mutant. Nor can the effects on the K + current be understood as the result of the reduced K + content or gene expression (Pilot et al., 2003a;Sutter et al., 2007;Eisenach et al., 2012Eisenach et al., , 2014. Analysis of the I KIN , especially, showed a substantial shift in the voltage dependence for channel gating, which is independent of cytosolic K + concentration (Wilmer and Fricker, 1996;Hills et al., 2012), and no appreciable difference in K + channel expression was observed compared with the wild type. The obvious conclusion from these findings is that the tmm1 mutant affects the channels via posttranslational regulation of their activities. Such control may well arise from the pleiotropic changes in protein kinase and phosphatase signal cascades or more directly through alterations in metabolism that lead to changes in cytosol-free [Ca 2+ ] and pH that modulate these channels, as has been demonstrated for the slac1 Cl 2 channel mutant of Arabidopsis (Wang et al., 2012). Regardless of the final explanation, it is clear that the effects of stomatal clustering are not a simple consequence of the physical spacing of the stomata within the epidermis or their placement in relation to the underlying mesophyll cells.

Gas Exchange
Gas exchange was carried out using the LI-COR 6400 XT Infrared Gas Analyzer (LICOR Biosciences) and whole-plant Arabidopsis chamber (LI-COR 6400-17). Pots of Arabidopsis were sealed with cling film to prevent any water vapor and CO 2 diffusion from the soil. All measurements were carried out at 22°C, 60% relative humidity, and 390 mg mL 21 CO 2 . Gas-exchange responses were measured using an integrated light source (LI-COR 6400-18) after plant materials were adapted to dark for 1.5 h. At least three plants per genotype were measured on different days at the same time of the diurnal cycle. Arabidopsis plants were normalized to the whole rosette area using ImageJ version 1.43u (http://rsb.info.nih.gov/ij; Rasband and Bright, 1995).

Stomatal Assays
Stomatal pattern and stomatal aperture measurements were carried out using epidermal peels isolated as described before (Wang et al., 2012). Images were recorded by digital photomicrography using a Zeiss Axiovert 200 microscope with LD Achroplan 403/NA0.75 and Planapo 203/0.80 objectives and an AxioCam HRc digital camera (Zeiss).
Stomatal apertures were recorded from epidermal peels following preincubations in depolarizing buffer (5 mM MES-NaOH, pH 6.15, and 60 mM KCl) for 2 h under 100 mmol m 22 s 21 light to fully open stomata (Papanatsiou et al., 2015). After imaging, epidermal peels were incubated for 5 min in depolarizing buffer supplemented with 20 mM fluorescein diacetate and examined for fluorescence to confirm vitality. Only stomata that showed fluorescence under confocal microscopy were analyzed further.
Maximum stomatal aperture (a max ) was estimated as where W a is the aperture width and L a is the aperture length. Stomatal size (S s ) was obtained as where W s is the stoma width and L s is the stoma length. The theoretical G Wmax was calculated as described by  as where d is the diffusivity of water vapor in air (m 2 s 21 ), v is the molar volume of air at 1 atm and 22°C (m 3 mol 21 ), S D is stomatal density (m 22 ), and l is the pore depth, estimated as the width of fully inflated guard cell. Stomatal closure was initiated after guard cells were fully open by superfusion with 10 mM MES-KCl, pH 6.1, supplemented with 6 mM CaCl 2 or 20 mM ABA. Measurements were carried out on a cell-by-cell basis, and results are reported as means 6 SE of n . 80 stomata.

Guard Cell Electrophysiology
Currents were recorded under two-electrode voltage clamp using Henry's EP software (Y-Science; http://www.psrg.org.uk). Microelectrodes were constructed to give tip resistances of 300 to 500 MV for Arabidopsis impalements, and they were filled with 200 mM K + acetate, pH 7.5, to minimize interference arising from anion leakage from the microelectrode (Blatt and Slayman, 1983;Blatt, 1987;Wang and Blatt, 2011). Electrolyte-filling solutions also were equilibrated against the resin-bound Ca 2+ buffer BAPTA (Ca 2+ sponge; Invitrogen) to avoid Ca 2+ loading of the cytosol from the microelectrode. Arabidopsis guard cells from isolated epidermal peels were pretreated with depolarizing buffer and light of 150 mmol m 22 s 21 to subsequently record K + channel currents in bathing solution of 5 mM Ca 2+ -MES, pH 6.1, supplemented with 10 mM KCl. Current and voltage under clamp were recorded using a set of mP electrometer amplifiers (WyeScience) with an input impedance of greater than 5 3 10 11 V (Blatt, 1987). Recordings were typically obtained by clamping in cycles with a holding voltage of 2 s at 2100 mV and 6-s steps either to voltages from 2120 to 2240 mV for currents from I KIN or voltages from 280 to +40 mV for currents from I KOUT . Recordings were corrected for background currents determined as the instantaneous current recorded on stepping from the holding voltage, much as described previously (Blatt, 1987;Wang et al., 2012;Papanatsiou et al., 2015).
Current-voltage curves were fitted by joint, nonlinear least squares and the Marquardt-Levenberg algorithm using a Boltzmann function of the form where g max is the maximum conductance, V is the membrane voltage, E K is the equilibrium voltage for K + , V 1/2 is the voltage at which half-maximum activation of channels occurs, z is the ionic valence, d is the gating charge, and F, R, and T have their usual meanings. Fitting current-voltage curves with the Boltzmann function results in the determination of d, which describes the apparent sensitivity of current (I) to a change in membrane voltage, and V 1/2 , which defines the midpoint voltage characteristic of this voltage sensitivity. Tail current analysis was used to estimate the cytosolic concentration of K + . For this purpose, the membrane was clamped in cycles with a holding voltage of 5 s at +50 mV followed by steps to voltages between +30 and 2120 mV. The relaxation amplitudes were plotted as a function of clamp voltage to identify the reversal voltage, at which no current relaxation was seen. This voltage was used as the equilibrium voltage for the K + channel, and the cytosolic K + concentration was then calculated from the Nernst equation and the known external K + concentration as where E x is the voltage at which the current crosses the voltage axis.

Data and Statistical Analyses
Data analysis and curve fitting were carried out using SigmaPlot 12 (Systat Software). Statistical significances were determined using one-way ANOVA and Student's t test at P , 0.05.

Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number BT005892.

Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. Effect of photosynthetic radiation on CO 2 assimilation.
Supplemental Figure S2. Time series of gas-exchange responses from Arabidopsis plants.
Supplemental Figure S3. Transcript levels of major plasma membrane transport genes in Arabidopsis lines.