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OPEN Vacuolar control of stomatal

opening revealed by 3D imaging
of the guard cells
Filippo Maria Mirasole 1,2, Sara Paola Nastasi 1,3, Paloma Cubero‑Font 1
&
Alexis De Angeli 1*

Land plants regulate their photosynthesis and water transpiration by exchanging gases ­(CO2 and
­ 2Ovapour) with the atmosphere. These exchanges take place through microscopic valves, called
H
stomata, on the leaf surface. The opening of the stomata is regulated by two guard cells that actively
and reversibly modify their turgor pressure to modulate the opening of the stomatal pores. Stomatal
function depends on the regulation of the ion transport capacities of cell membranes as well as on
the modification of the subcellular organisation of guard cells. Here we report how the vacuolar and
cytosolic compartments of guard cells quantitatively participate in stomatal opening. We used a
genetically encoded biosensor to visualise changes in ionic concentration during stomatal opening.
The 3D reconstruction of living guard cells shows that the vacuole is the responsible for the change in
guard cell volume required for stomatal opening.

Land plants regulate gas exchanges ­(CO2, ­O2, and H ­ 2Ov) through microscopic pores on the leaf surface, the
stomata. These pores, delimited by two guard cells, control the diffusion of gases between the leaf and the atmos-
phere. In all land plants, the two guard cells can actively modify their turgor pressure and shape and thus change
the stomatal pore aperture. The guard cell turgor changes depend on the concerted activation of solute (ions and
sugar) transporters residing in the plasma membrane, the tonoplast, and other endomembrane s­ ystems1–4. Several
ion transport systems in the plasma membrane ­(K+ and ­Cl− channels, proton pumps, and sugar transporters)
and in the vacuolar membrane ­(Cl− and malate channels, and ­H+ coupled ­K+, ­Cl−, ­NO3− antiporters) are now
­identified5. These ion transporters and channels are regulated by signaling cascades decoding different stimuli
(e.g. light, ABA, C­ O2, humidly, pathogens) and participate in the coordination of the ion transport network of
guard ­cells2,6.
Ion transport modifies the osmotic potential of guard cells by inducing massive water fluxes to open and
close stomata. The highly efficient intracellular solute transport machinery allows reversible changes of the turgor
pressure of the guard cells from 1 to 4.5 ­MPa7,8. Despite these large changes in turgor pressure, the guard cell
volume is only modified by 20%9 in Vicia faba. The combination of turgor pressure and anisotropic properties
of the cell wall induces the elastic deformation of the guard cells that regulates the stomatal a­ perture10. Model-
ling studies have demonstrated the functional links existing between the turgor pressure, the cell wall and the
stomatal ­aperture10–12.
During stomatal opening/closure guard cells undergo major and rapid rearrangements of their intracellu-
lar ­organization13–16. The cytoskeleton experiences these rearrangements with both the actin and microtubule
networks being differently organized in open or closed stomata. Indeed, the cortical microtubules are radially
oriented in open stomata but not in closed ­ones17,18. However, in guard cells the more dramatic intracellular
modification concerns the vacuole m ­ orphology13,15,16. Indeed, the vacuole is deeply reorganized, with both its
volume and structure being rapidly modified. In closed stomata, the vacuole is fragmented in several vesicles
that can be ­interconnected13,19. In contrast, in open stomata the vacuole is a single compartment occupying a
large proportion of the whole cell ­volume15,16. The vacuole stores the major part of the solutes accumulated by
the guard cells and is therefore central for the control of the turgor pressure. Indeed, the solute concentration
in guard cells reaches the molar range, a concentration too high to be tolerated by the c­ ytosol8,20. The vacuolar
expansion and shrinking relies on its capacity to accumulate i­ons15. In parallel to the vacuole, the nuclear and
cytosolic compartments also reorganize within the limited space of the guard cell to accommodate the vacuolar

1
IPSiM, CNRS, INRAE, Institut Agro, Université Montpellier, Montpellier, France. 2Present address:
Department of Plant and Microbial Biology, University of Zurich, Zollikerstrasse 107, CH‑8008 Zürich,
Switzerland. 3Present address: Department of Biosciences, University of Milan, Via G. Celoria 26, 20133 Milano,
Italy. *email: [email protected]

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rearrangements during stomatal movements. However, surprisingly the nuclear and cytosol dynamics in the
guard cells are almost unknown. Further, in guard cells the functional connection between the subcellular
organization and the ion transport machinery of the cellular membranes is still poorly understood.
Here we investigated in Arabidopsis thaliana guard cells, from a quantitative point of view, the link between
ion transport reactions and the rearrangement of subcellular compartments. We therefore developed a strategy
based on a genetically encoded biosensor and 3D imaging to follow, in living guard cells, ­pHcyt and ­[Cl−]cyt dur-
ing stomatal opening in parallel with the volumetric changes of the subcellular compartments. We quantified,
in individual guard cells, the volume and the morphological modifications of the vacuolar and cytosolic com-
partments together with the ­pHcyt and ­[Cl−]cyt dynamics. Surprisingly, our data show that the cytosolic volume
is constant and that the vacuole is responsible of the guard cell volume changes. Consequently, our data show
that the morphological and volume modifications of the vacuole are the processes driving stomatal opening.

Results
Cytosolic pH and ­[Cl−] dynamics in fusicoccin‑induced stomatal opening.. We induced stomatal
opening under a confocal laser scanning microscope with fusicoccin. This fungal toxin activates the plasma
membrane proton pumps, stimulating ion fluxes directed into the guard c­ ells21 (Fig. 1a). We designed stomatal

Figure 1.  pHcyt and ­[Cl−]cyt dynamics during stomatal opening. (a) Schema of fusicoccin-induced stomatal
opening. fusicoccin directly activates the plasma membrane ­H+-ATPases inducing extrusion of ­H+ to the
apoplast and a hyperpolarization of the plasma membrane. This induces the influx of solutes (i.e. K ­ + and
−
­Cl ) inside the guard cell, decreasing its water potential and driving the influx of water. (b) Representative
transmitted light (top) and false color images of ­RpH (bottom) representing ­pHcyt in a stomata expressing
ClopHensor at different time points before (control) and after fusicoccin-induced opening. The black space
within the guard cells in the ­RpH images represents the vacuole. During the whole experiments stomata were
exposed to 0.1% DMSO. Scale bars 5 µm. (c) Time resolved stomatal aperture kinetics over 3 h, and after
application of fusicoccin (grey area, n = 11 stomata). Data were fitted (solid blue line) with plateau followed by
an exponential function. Opening rate was calculated as the first derivative of the exponential fit (black dotted
line). Arrows indicate the time points corresponding to the acquisition for 3D reconstruction (see Fig. 3). (d)
Time resolved p ­ Hcyt changes represented as ΔRpH/RpH,i, (n = 11 s stomata) during stomatal opening (grey area).
After fusicoccin application ­pHcyt rapidly increased before decreasing to values close to the initial ones. (e)
Time-resolved changes of [­ Cl−]cyt represented by ΔRanion/Ranion,I (n = 11 stomata). After fusicoccin (grey area)
ΔRanion/Ranion,I was stable for the first 60 min. After it raised, indicating an increase of [­ Cl−]cyt. In (c–e) data are
presented as mean ± S.D.

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opening experiments using Arabidopsis thaliana plants expressing the biosensor ClopHensor to measure the
changes of ­pHcyt and ­[Cl−]cyt22 (Fig. 1). ClopHensor allows simultaneous visualization and quantification of ­pHcyt
and ­[Cl−]cyt in living guard cells (Fig. 1). To start the stomatal opening experiments with guard cells in a defined
physiological state, Arabidopsis leaf epidermal peels were prepared at the end of the dark period (i.e. 1 h before
light onset), when stomata were still closed (Fig. 1b). Firstly, stomata were imaged for 30 min in a control buffer
with 30 mM KCl (see “Material and methods”). Then, 10 µM fusicoccin was applied and the stomatal opening
process was followed for a total of 195 min (Fig. 1b–e). The stomata opened to a pore width of 2.2 ± 0.6 µm (n = 9
stomata) after 110 min in fusicoccin (Fig. 1c and Fig S1). The stomatal aperture kinetics were fitted with a plateau
followed by an exponential function and we estimated an opening half-time of about 43 min that is in the same
range of those observed in whole leaves after exposure to ­light23 (Fig. 1c). Notably the stomatal opening rate was
maximal just after fusicoccin application and afterwards decreased over time (Fig. 1c).
In the same stomata the fluorescence of ClopHensor was imaged after excitation at 488 nm, 458 nm and
561 nm. These data were used to calculate the two ratios: ­RpH (F488/F458) and ­Ranion (F458/F561), which report
­pHcyt and ­[Cl−]cyt, ­respectively22 (Fig. 1b). ClopHensor is sensitive to ­Cl− and N ­ O3−, with different ­affinities22,24.
In the present set of experiments, we used a buffer based on 30 mM KCl and, therefore, R ­ anion variations can be
interpreted as ­[Cl−]cyt changes. After application of fusicoccin, ­pHcyt rapidly increased, reaching a maximum
within 5–10 min (Fig. 1d). Faster image acquisition confirmed that ­pHcyt maximum was within the 5 min after
fusicoccin application (Fig. S2). This behaviour of p ­ Hcyt was previously o ­ bserved22 and shows that, when the
stomatal pore is still closed, fusicoccin rapidly activates the plasma membrane H ­ +-ATPases inducing H ­ + extru-
sion and a rapid ­pHcyt increase. In a second step, ­pHcyt decreased and reached its initial values (Fig. 1c, d). This
phase coincides with the maximum pore aperture. In contrast, [­ Cl−]cyt was mostly constant in the first 60 min
after fusicoccin application (Fig. 1e). Only after this period, a raise of [­ Cl−]cyt was observed (Fig. 1e). Interestingly,
the ­[Cl−]cyt increased only when the stomatal pore aperture was above the half of its maximum, i.e. when the
turgor pressure of the guard cell was m ­ aximal10. Anions like C­ l− significantly contribute to the osmotic potential
8 −
of guard c­ ells , explaining why C­ l concentration and pore aperture maximum coincided and, consequently, the
turgor pressure too.

3D reconstruction of the vacuole with a “negative staining” approach. The ratiometric images of
the stomata obtained at different time points suggest that the relative importance of the nucleus-cytsolic and of
the vacuolar compartments varies during stomatal opening (Fig. 1b). In these 2D images the cytosol appears to
undergo a size reduction while the vacuole increases. To better study these changes, we quantified the volume of
the vacuolar compartment of guard cells during stomatal opening (Fig. 2). Indeed, in guard cells, the vacuole is
the subcellular compartment undergoing the major modifications during stomatal opening/closure13,15,16. Nota-
bly, the morphological modifications of vacuoles have been investigated in many plant cell t­ ypes19,25. However,
in guard cells no quantitative volumetric changes of both the vacuole and of the nuclei-cytosolic compartment
have been reported so far. To tackle this issue, we used a 3D reconstruction approach mixing “direct and negative
staining” of the subcellular compartments by ClopHensor. Indeed, ClopHensor expression in both the cytosol
and the nucleus directly labels these compartments, and it additionally provides a “negative staining” of the
vacuole (Fig. 2). Thus, the vacuole can be then identified as the intracellular region where no fluorescent signal of
the ­E2GFP moiety of ClopHensor is visible, i.e. “negative staining” (Fig. 2). This strategy enables the quantifica-
tion of the volumes of the two major compartments in a single guard cell during stomatal opening. Thus, to test
this possibility we quantified the vacuolar volumes obtained from guard cells expressing ClopHensor and from
guard cells expressing the tonoplast marker YFP-VAMP71125 (Fig. 2). Epidermal samples were prepared at the
end of the dark period, as previously described. The samples were either kept in the dark, or exposed to fusicoc-
cin 10 µM or to light for 3 h (Fig. 2a,b). We acquired z-Stacks of stomata in the different treatments exciting at
488 nm the E ­ 2GFP moiety of ­ClopHensor22,26, or the YFP in VAMP711-YFP (Fig. S3). The z-Stacks were then
manually segmented using 3D Slicer software (https://​www.​slicer.​org)27 to reconstruct the vacuole in each guard
cell (see “Material and methods”).
In closed stomata the vacuole was fragmented in guard cells expressing both ClopHensor (n = 24 guard cells)
and VAMP711-YFP (n = 12 guard cells) (Fig. 2a,b). The vacuolar compartment was divided in multiple vesicles
of different sizes (Fig. 2a, b). Interestingly, in all cases we observed the presence of two major vacuolar vesicles
systematically localized at the two poles of each guard cell (Fig. 2a,b). The nuclear region was in the central area of
the guard cell separating the two polar vacuolar vesicles (Fig. 2a,b). In the guard cells expressing VAMP711-YFP,
we observed fluorescence in the area surrounding the nucleus suggesting the presence of tonoplastic structures
(Fig. 2b). In open stomata the vacuole became a single compartment occupying a large part of the guard cell
(Fig. 2a,b). Notably, in stomata expressing ClopHensor, the nuclear region moved to the side of the cell (Figs. 2a,
4). The distance separating the two guard cells showed that the stomata were open at the end of both the light and
fusicoccin treatments (Fig. 2c). After the 3D reconstruction of the vacuolar compartment, the vacuolar volume
from ClopHensor “negative staining” and VAMP711-YFP was calculated (Fig. 2; Table S1). The calculated vacu-
olar volumes were not statistically different (Fig. 2d; 1-way Anova p < 0.01) between the two marker lines in both
closed stomata (­ VvacClopHensor = 130.9 ± 46.1 µm3 and V ­ vacVAMP711 = 143.5 ± 64.8 µm3) and fusicoccin-open stomata
ClopHensor 3 VAMP711
­(Vvac = 313.0 ± 67.9 µm and V ­ vac = 397.7 ± 92.7 µm3). Moreover, the mean values of the vacuolar
volume of light-opened stomata ­(VvacClopHensor = 303.5 ± 111.6 µm3) were similar to those induced by fusicoccin
(Fig. 2d; Table S1). Interestingly, the vacuolar volume was about 2.5 times higher in open stomata compared to
the closed ones. The similar values of the vacuolar volumes observed in guard cells expressing ClopHensor and
VAMP711-YFP show that our “negative staining” used method is suitable to quantify the volume of the vacuole.

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Figure 2.  3D reconstruction of vacuole in guard cells. (a,b) Representative transmitted light images (top)
and confocal images with superimposed reconstructed vacuolar 3D models (bottom) of guard cells. (a) Guard
cells expressing ClopHensor in the cytosol and the nucleus. (b) Guard cells expressing the tonoplast marker
VAMP711-YFP. In (a,b) images were obtained after incubation in darkness for 180 min in control buffer (left
column, closed) and in presence of 10 µM fusicoccin (right column, open). Scale bars 5 µm. (c) Distance
between guard-cells (yellow line in a,b) of each stomata measured from the middle fluorescence z-stack
image with or without fusicoccin. Samples were left 180 min under white light (n = 6 stomata, open green
triangles), fusicoccin (n = 6 stomata, filled green triangles) and in darkness (n = 6 stomata, filled blue triangles).
(b) Vacuolar volumes calculated from the 3D models of guard-cells expressing VAMP711-YFP (circles) and
ClopHensor (triangles). Blue symbols, samples kept in dark for 180 min (n = 12–24 guard cells); green symbols,
samples kept in the dark with10 µM fusicoccin for 180 min (n = 12 guard cells); open triangle samples exposed
for 180 min to light (n = 12 guard cells). Statistical significance was determined with a one-way ANOVA with
non-parametric Dunnett’s post-hoc test, letters indicate significant differences (p < 0.01). (c,d) Horizontal line in
the dot plots indicates the mean.

3D reconstruction of the whole guard cell subcellular volumes. In a next step, we quantified the
volumetric changes of different compartments (nucleus + cytosol, vacuole and chloroplasts) of a living guard
cells during stomatal opening in the same cell (Table S1). Indeed, we built 3D models of the whole guard cell
divided in vacuole, nucleus-cytosol and chloroplasts (see “Material and methods”).
We imaged the same stomata closed (i.e. at the end of the dark period) and open after 3 h in the presence of
fusicoccin (Fig S4). We acquired stomatal z-Stacks and 3D models of the whole guard cell and of the subcellular
compartments were built (Fig. S3; Table S1). The chloroplasts fluorescence could be detected simultaneously with
the ­E2GFP using two separated emission windows (see “Material and methods”). We therefore build 3D models
of the chloroplasts and found that their volume was stable during opening being 27.4 ± 5.5 µm3 and 25.3 ± 3.7
µm3 in closed and open stomata, respectively (Fig. S4a). In contrast, the whole guard cell volume expanded from
­VGCclosed = 379.5 ± 50.6 µm3 (n = 6 guard cells) in closed stomata to V
­ GCopen = 533.4 ± 64 µm3 in open stomata (n = 6
guard cells; Fig. S4a, b). This expansion corresponds to an increase of + 154 µm3, i.e. 1.4 times its initial volume.
In the same guard cells, the vacuolar volume increased from ­VVacclosed = 92.9 ± 11.6 µm3 to a ­VVacopen = 249.5 ± 24.8
µm3, corresponding to a 2.7 times increase of the initial volume (Fig. S4a, b). Interestingly, the volume of the
vacuole increase by + 156 µm3, which coincides with the volume increase of the whole-guard cell (Fig. S4). These
data uncover an intriguing characteristics of stomatal opening: the whole guard cell volume increase relies on

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Figure 3.  Vacuolar volume changes drive stomatal opening. (a) Representative transmitted light images
(top) and 3D models (bottom) of single stomata before (left), 30 min (middle) and 180 min (right) after the
application of 10 µM fusicoccin. The 3D models show the same stomata from the mesophyll facing side (left
panel) and from the lateral side (right panel) at three time points. Blue, vacuole; green, cytosol + nucleus; red,
chloroplasts. Scale bars 5 µm. (b) Frequencies of the different vacuolar conformations in guard cells. (c) Volume
changes of the subcellular compartments during stomatal opening. Left, whole guard cell; center, vacuole; right,
cytosol + nucleus (mean and S.D., n = 10 guard cells). Statistical significance was determined with a two tailed
t-test, the obtained p values are reported. (d) Percentage of the guard cell volume occupied by the vacuole during
opening induced by fusicoccin (grey area). Data were fitted (solid line) by a plateau followed by exponential
function like in Fig. 1c (see “Material and methods”). (a–c) Stomatal opening was induced by exposing the
guard cells to 10 µM fusicoccin at t = 30 min (vertical dashed line) for 180 min. Error bars are S.D.

the vacuolar expansion. Indeed, in contrast with the suggestion of the 2D confocal images (Fig. 1b) the volume
occupied by nucleo-cytosolic compartment remains constant during stomatal opening.
We further analysed the kinetics of the subcellular changes by measuring the guard cell volumes in closed,
fully open stomata, and in stomata in an intermediate state (Fig. 3; Table S1). ClopHensor fluorescence was
simultaneously imaged to measure p ­ Hcyt, ­[Cl−]cyt, and for obtaining 3D reconstructions (Figs. 1, 3). We acquired
z-Stacks of the stomata at only three time points to limit the effects of phototoxicity. We reconstructed 3D models
30 min after fusicoccin application, when the stomatal aperture was approximatively halfway from its maximum
(Fig. 1c). At this time point ­pHcyt was at its maximum (i.e. the activity of the ­H+-pumps dominate), and the ­[Cl−]cyt
was similar to the initial level (Fig. 1d, e). Before fusicoccin application, the vacuoles were fragmented in 86%
of the guard cells, with two major vesicles at the poles of the guard cells (Fig. 3a). After 30 min in fusicoccin,
the stomatal pore opening could be observed (Fig. 3a middle). The two polar vacuolar vesicles were connected
at the level of the nuclear region in at least one guard cell in 80% of the stomata (Fig. 3a). Finally, 200 min after
fusicoccin application, the opening was maximal and in 86% of the guard cells the vacuoles were fused in a single
compartment. The vacuolar volume increased from ­VVacclosed = 125.7 ± 37.8 µm3 to ­VVac30min = 202.0 ± 42.4 µm3 after
30 min, and reached V ­ Vac200min = 304.9 ± 45.6 µm3 at 200 min. In this set of experiments, the vacuole increased
2.4 times (Fig. 3b), while the whole guard cell volume increased 1.3 times between closed stomata and open
at 200 min (Fig. 3b). Particularly, the volume increase of the whole guard cell and of the vacuole were similar,

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Figure 4.  The guard cell nuclei move during stomatal opening. (a) Representative images of guard cell
expressing ClopHensor during stomatal opening before (left column) and 180 min (right column) after applying
10 µM fusicoccin. Transmitted light (top) and fluorescence images with superimposed 3D nuclear model
(bottom, purple). Scale bars 5 µm. (b) Nuclear volumes calculated from the 3D models, before fusicoccin
application (squares; n = 10 nuclei from 5 stomata), and after 180 min in fusicoccin (triangles; n = 10 nuclei).
Statistical significance was tested with a tailed t-test. (c) 3D model of a guard cells showing the position of the
nucleus in the guard cell before (red) and after (yellow) opening. (d) Quantification of the position change of the
nucleus (arrows) referred to the geometric center of the nucleus calculated from the 3D model of the nucleus
before stomatal opening. x, z projection (left) and y, z projection (right) view of nuclei movements (n = 8 nuclei).

being + 150 µm3 and + 180 µm3 respectively. The vacuole occupied 27 ± 6% of the cell volume when stomata were
closed, and 50 ± 7% when stomata were fully open (Fig. 3c). Interestingly, the nucleus-cytosolic volume (see
“Material and methods”) was only marginally modified during stomatal opening with ­Vcytclosed = 297.5 ± 56.3 µm3
before fusicoccin, V ­ cyt30min = 294.4 ± 38.2 µm3 and ­Vcyt200min = 270.9 ± 80.2 µm3 after fusicoccin (Fig. 3d).
We noticed that the nuclear region was in a different position within the guard-cells in closed or fully open
stomata (Figs. 3 and 4). In guard cells expressing ClopHensor, the nuclei could be identified as the area with the
higher fluorescence intensity (Fig. 4a). In closed stomata the nuclear region was positioned in the central part
of the cells, while in open stomata it moved to a peripheral area (Fig. 4a). Thus, during opening the nuclei move
meanwhile the vacuole increases its volume and becomes a single compartment. We also built 3D models of the
guard cells nuclei and quantified the displacement of the barycenter of the nuclei within the guard cell (Fig. 3c,d).
Starting from a central position, the nuclei moved mainly in the z axis and underwent a slight decrease of the
volume from 21.8 ± 2.0 to 14.9 ± 1.3µm3 (Fig. 4c,d).

Discussion
The driving force regulating the stomatal aperture is the osmotic potential of the guard ­cells1,2. The osmotic
potential changes stimulate water fluxes across the plasma and vacuolar membranes of guard cells, modifying
the turgor pressure. In guard cells, the combination of the turgor pressure and the anisotropic properties of the
cell-wall modifies the shape of the cells to open and close the stomatal ­pore7,10–12. The subcellular organization
rearranges to accommodate the massive fluxes of ions and water that are required for stomatal opening. However,
the morphological coordination of the different intracellular compartments was not clear so far, although it is

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essential for stomatal aperture regulation. Indeed, how the different subcellular compartments quantitatively
participate to the change of volume of the guard cell is still to be understood.
Here, we provide a quantitative analysis of the volume dynamics of the major guard cell compartments (i.e.,
nucleus-cytosol, vacuole and chloroplasts) during stomatal opening. By using an original approach, we simul-
taneously measured, in individual living guard cell, the subcellular compartments volumes at different stages
of stomatal opening. Further, we coupled volumetric measurements with the visualization of the activity of key
ion transport systems (i.e. H ­ l− transport; Fig. 1). We used A. thaliana stomata expressing ClopHensor, a
­ + and C
biosensor allowing the simultaneously quantification of the changes of p ­ Hcyt and ­[Cl− or ­NO3−]cyt22,24. Addition-
ally, we reconstructed 3D models of the whole-guard cell, the vacuole, the cytosol, and of the nucleus in the
same guard cell by using the fluorescence emitted by ClopHensor (Figs. 2, 3 and 4). To induce stomatal opening
we used ­fusiccocin28, a toxin directly activating the plasma membrane proton pump (Fig. 1a). This triggers ­H+
extrusion from the guard cell and hyperpolarizes the plasma membrane, mimicking stomatal opening induced
by blue l­ ight29. Importantly, we observed overlapping vacuolar volume changes in fusicoccin- and light-induced
stomatal opening (Fig. 2). The application of fusicoccin stimulated a rapid increase of the p ­ Hcyt (Phase I), fol-
lowed by a slow return to its initial values (Phase II; Fig. 1d). The p ­ Hcyt alkalinization of Phase I depends on the
activation of the plasma membrane ­H+ pumps by fusicoccin, and it coincides with the maximal pore opening
rate (Fig. 1c,d). The subsequent decrease of ­pHcyt is likely caused by the activation of secondary active transport-
ers coupling solute transport to H ­ +, such as ­AtCLCa22,30, and mediating a net flux of ­H+ into the cytosol. Thus
the ­pHcyt is controlled by several actors, including H ­ +-coupled ion transport systems residing in the different
cellular membranes of guard cells.
The stomatal aperture showed an increase of + 0.66 µm and of + 1.1 µm in Phase I and II, respectively (Fig. 1c).
Since the pore aperture directly depends on the turgor pressure, our results indicate a higher turgor pressure
increase in Phase I­ I10,20. Interestingly, the vacuolar volume followed the same trend (Fig. 3). In Phase I (i.e.
the first 30 min after fusicoccin application) the vacuolar volume increased by of 1.6 times with a rate of 2.54
µm3·min−1 (Fig. 3c). In Phase II (i.e. the last 120 min) the vacuole grew also 1.6 times, but showing a three times
slower rate of 0.85 µm3·min−1 (Fig. 3). Since the pressure and volume are inversely related, the reduced volume
growth rate in Phase II is in line with a larger turgor pressure increase in the guard cells. Accordingly, in Phase
II we also observed a significant increase of the ­[Cl−]cyt (Fig. 1e), indicating a higher osmotic load of the cell and
thus a higher osmotic pressure.
Our results shed light on the subcellular morphological changes of the guard cells during opening. Impor-
tantly, we found that the volume of the nucleus-cytosolic compartment was constant during the whole stomatal
opening process (Fig. 3). This observation is paralleled by the fact that the change in total guard cell volume
quantitatively coincides with the increase in vacuolar volume. (Table S1). Taken together these data show that
the vacuole provides the driving force of stomatal opening. It is interesting to note that roots cell expansion dur-
ing growth similarly depends on the vacuolar ­size25,31. Recently, it was found that stomata differentiation relies
on vacuolar fusion g­ rowth19. These data, together with ours, highlight the role of the vacuolar compartment in
regulating plant cell volume. We investigated a cellular process, stomatal opening, involving fully developed
cells. In both developing cells and mature guard cells, the vacuolar size and whole-cell size are tightly linked
and coordinated. Nonetheless, the regulation of the stomatal aperture presents important specificities such as
reversible and large turgor pressure changes in a relatively short time. These changes depend on massive uptake
of solutes generating turgor pressure variations in the MPa r­ ange7. In these processes the whole cell is involved
and the cytoskeleton plays an important role in remodelling the vacuole in both cell development and stomatal
­aperture14,18,32.
To conclude, we followed in vivo and in single cells the ion concentration dynamics of the nucleus-cytosolic
compartment in parallel with the volume changes of different guard cells’ compartments. We have correlated the
three-dimensional organization with the ion transport activity across the two major membranes of the guard
cell: the plasma and the vacuolar membranes. We found that during stomatal opening the nucleus-cytosolic
compartment remains constant while the vacuole accounts for the required whole guard cell volume increase,
highlighting the importance of the vacuole in stomatal opening. More in general, our data open the question
of how the relative volumes occupied by the cytosol and the vacuole within plant cells are regulated. Our data
provide a step further in understanding the cellular process regulating leaf gas exchanges for both photosynthetic
carbon fixation and water loss by land plants.

Material and methods

Plant material and growth conditions. The Arabidopsis thaliana transgenic lines pUBI10::ClopHensor
and Col-0 pUBI10::VAMP711-YFP, in the Col-0 background, were kindly provided by Dr J. Kleine-Vehn , Uni-
versity of Freiburg, Germany. Seeds were vernalized 3 days in water at 4 °C in the dark before being sown in pots
in a growth chamber (22 °C/20 °C, 8 h/16 h day/night photoperiod, 65% humidity, 150 μmol photons·s−1). GMO
plants were cultivated and collected following the relevant institutional, national, and international guidelines
and legislation.

Sample preparation. Experiments were conducted on leaves from 4–5 week-old plants. The leaves were
selected in order to have the same size and development. Each set of experiments were performed on stomata
from at least 2 independent plants. Epidermal peel samples were prepared 1 h before light turned on in the
growth chamber to have closed stomata. Epidermal strips were prepared under green light (LED Osram star
classic A green) as follow: firstly, the leaf abaxial side was glued on a microscope cover slip by using a medical
glue (Adapt™ 7730, Hollister, EEUU). Then the mesophyll and the adaxial side of the leaf were gently removed.
To keep stomata alive, a buffer (30 mM KCl, 5 mM MES, 0.1 mM C ­ aCl2, 0.1% DMSO, pH 5.7)33 was applied to

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the samples. The coverslip containing the sample was placed on a homemade perfusion chamber designed for
imaging. After epidermal strips preparation, samples were kept 20 min in darkness. The buffer was regularly
(every 10–15 min) refreshed either with fresh buffer (control) or with buffer solution supplemented with fusico-
ccin (30 mM KCl, 5 mM MES, 0.1 mM ­CaCl2, 10 μM fusicoccin, 0.1% DMSO, pH 5.7).
For experiments in Fig. 2 samples were incubated 3 h in the dark before image acquisition. In light-induced
experiments, samples were incubated under light (LED-240 μM·photons−1·m−2; ­Ledmo® LED strip Light) for 3 h.
Throughout the experiments in Figs. 3 and 4, after the 20 min of incubation in the dark, samples were imaged
under the microscope. Buffer solutions were applied with a manual pump system.

Image acquisition. Stomata were imaged in vivo with an inverse confocal laser scanning microscope
(CLSM) Leica SP8 (Leica, Germany) with a 63× oil objective (HCX Plan Apochromat CS 1.4 NA). Fluorescence
was detected using a GaAsP Hybrid photon detector in photon counting mode. For 3D reconstruction z-Stacks
were composed by 8 bit images with an image size of 512 × 512 pixels acquired with a 1 airy units pinhole. The
optical section was 0.849 μm, the z-step size was 0.29 μm and the voxel size was 0.12 × 0.12 × 0.285 μm. Thus,
datasets of 30–50 frames/stomata were obtained. For 3D reconstruction, the fluorescence emission of the ­E2GFP
moiety of ClopHensor was detected between 500 and 550 nm after excitation at 488 nm by using an Argon laser.
Chloroplasts were detected by exciting the chlorophyll at 488 nm, and the emitted fluorescence was collected in
the 650–675 nm range. Transmitted light images were obtained with a PMT (Photomultiplier tube) detector. For
cytosolic pH and ­Cl− quantifications, the same CLSM setup was used with a sequential excitation mode (Excita-
tion at 561, 488, and 458 nm). Upon excitation at 458 nm, 488 nm (Argon laser) and 561 nm (Diode Pump Solid
State laser, DPSS), the emission was detected at 500–550 nm and 600–625 nm, respectively. Images were 12 bit
with an image size of 256 × 256, pinhole 3 Airy units. The CLSM was driven by Las X software (Leica).

3D reconstruction of guard cell

Image preparation. Image stacks were processed by using Fiji (https://​imagej.​net/​Fiji). The pixel with the
lowest value was identified with the Pixel Inspection tool, and it was subtracted to the entire stack to reduce the
background. To remove noise and enhance boundaries, a median filter equal to 1 was applied to the stack. This
process was common for reconstructing all subcellular compartments, except the vacuole. For the vacuole, we
firstly enhanced the contours, then the brightness and contrast were automatically adjusted using the middle
image each stoma as reference (Fig. S3). All processed files were saved in ’.nrrd format.

3D segmentation. Segmentation for 3D reconstruction was performed semi-automatically by using the

3D Slicer software (https://​www.​slicer.​org)27. Firstly, we assigned a single colour to each ROI (Region Of Inter-
est) corresponding to a subcellular compartment. We semi-automatically selected the voxels forming the ROI
of each compartment depending on their fluorescence intensity in the x, y images. For the cytosol + nucleus
compartment, we selected fluorescent voxels (i.e. direct labelling method). For the vacuole, we selected non-
fluorescent voxels within the cell (i.e. “negative staining”). The chloroplasts were identified based on their auto-
fluorescence on separated x, y images. Finally, for each x, y image of the z-Stack the ROI of each compartment
was refined taking into account the z, x and z, y projections (Fig. 2b). To build the 3D model of each compart-
ment, the z-stack with the selected ROIs was processed by the Model Maker module of 3D Slicer (Fig. 2b). Files
were saved in ’.stl format.

Geometrical measurements. The volume of the subcellular compartments was calculated by using the
Segment Statistic tool from 3D slicer. The stomatal pore width was manually measured with Fiji from the middle
image of the stomata. The geometric centre of 3D nuclear models was obtained importing the ’.stl files of models
in the software MeshLab (https://​www.​meshl​ab.​net/). Nuclear displacement was visualized by using the 3D cal-
culator tool from the software GeoGebra (https://​www.​geoge​bra.​org/).

Software. All software used (Fiji, 3D Slicer, MeshLab and GeoGebra) are free and open source.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reason-
able request.

Received: 8 March 2023; Accepted: 27 April 2023

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Acknowledgements
We acknowledge the imaging facility MRI, member of the France-BioImaging national infrastructure supported
by the French National Research Agency (ANR-10-INBS-04, «Investments for the future»). We acknowledge C.
Alcon (IPSiM, Montpellier) for her assistance, J. Jaslan (IPSiM, Montpellier), R. Doireau (IPSiM, Montpellier),
J-M Frachisse (I2BC, Gif-sur-Yvette), A. Costa (University of Milano, Italy) for critical discussions and K. Bertaux
(IPSiM, Montpellier) for technical assistance. A.D.A. discloses support for the research of this work from CNRS
[ATIP-AVENIR 2018] and ANR [NetFlux project].

Author contributions
F.M.M., S.P.N., P.C.-F. performed experiments and analysed the data. F.M.M. and A.D.A. designed the experi-
ments. A.D.A. wrote the manuscript with the help of F.M.M. and P.C.-F. A.D.A. designed the project.

Competing interests
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at https://​doi.​org/​
10.​1038/​s41598-​023-​34273-x.
Correspondence and requests for materials should be addressed to A.A.
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