This raises the question of what function it might play. The finite element modeling approach allows prediction of strain/stress patterns within the guard cells as they undergo movement. These data indicate that, in the baseline model, large gradients of strain/stress are generated across the inner radial wall of the guard cell during stomatal opening ( Figures 2 E and 2F). The geometry (and associated differential wall thickening) in the VWT model leads to a non-intuitive dissipation of these strain/stress gradients so that the maximum stress occurs away from the inner radial wall and the stress gradient is diminished ( Figures 2 G and 2H). Due to the vital role that stomata play in the control of plant gas and water relations, they must repeatedly adjust their aperture to the ambient environment []; thus, the guard cells must undergo extensive and repeated strain. We suggest that the differential thickening of the inner radial wall in mature stomata generated as a consequence of guard cell geometry acts primarily to alleviate the potential for mechanical failure, helping to maintain cell wall integrity as it undergoes repeated stress/strain cycles. This structural modification has an associated outcome of slightly altering the aperture response to turgor pressure.

An increased thickening of the inner radial wall of guard cells was observed in early botanical studies, leading to the widely accepted view that this leads to a stiffening of the wall, which is required for the curling displayed by guard cells as they expand due to increased turgor pressure []. Although this interpretation has been challenged [], lack of measurement of guard cell mechanics has limited the scope for discussion. AFM provides a means of assessing mechanical properties that has become increasingly used in the analysis of biological material, including plants []. Although care must be taken in the interpretation of such data (since the values obtained are influenced by a range of factors, including the geometry and mechanical properties of the tips used, and factors intrinsic to the complex composition and geometry of the tissue), AFM provides a robust method for estimating relative stiffness across cellular dimension []. We report stiffness as an apparent modulus, E, not inferring a specific modulus of the material being indented. Our results support the interpretation that the observed thickening of the inner radial wall leads to a gradient of stiffening across the guard cell []; however, this gradient is only observed in relatively mature cells. Younger guard cells do not display any consistent gradient of radial stiffening, yet our measurements of pore aperture indicate that these stomata are able to respond to appropriate triggers by opening the stomatal pore at least as widely as the calculated theoretical maximum ( Figure S1 B). Coupled with our modeling indicating that increased stiffening of the inner radial wall has a minimal outcome on stomatal movement, we propose that radial stiffening of guard cells is not required for stomatal opening.

To extend our understanding of the physics of stomatal opening/closing, we exploited a recently developed finite element model ( STAR Methods ). In the baseline model, the guard cells have a circular cross-section and uniform wall thickness and, thus, uniform mechanical properties ( Figure 2 A). Under these conditions, as the epidermal and internal pressure (turgor) of the guard cells are increased from zero, the system moves slightly away from the starting geometry, but even as the guard cell turgor pressure rises above the epidermal pressure (limited here to 0.5 MPa), there is initially no increase in pore aperture ( Figure 2 B). When the guard cell turgor pressure reaches about 1.3 MPa, the stomatal aperture starts to increase, approaching a maximum as pressure increases above 5 MPa. When the model is adjusted so that the cells have a geometry more in keeping with that described in the literature [], leading to differential wall thickness along the inner radial wall (variable wall thickness, VWT model) ( Figure 2 A), there is a slight shift in the aperture-response curve, favoring larger aperture at a lower pressure and smaller aperture at higher pressure, but the changes are relatively small ( Figure 2 B). When we explored the sensitivity of the VWT model to altered wall thickness, there was a very limited response to this parameter. Thus, increasing or decreasing inner wall thickness in the VWT model by 10% had essentially no outcome on the aperture/pressure response curve ( Figure 2 C).

(J) Effective stress pattern in the guard cell modeled in (I). Steeper stress gradients now form toward the guard cell poles compared to (F). In (E), (G), and (I) the strain is dimensionless and is capped at 1 for consistency across the figures. Only the regions immediately neighboring the point at which the pore adjoins the polar wall exceed this value. Strain is a dimensionless tensor describing the deformation of the material, which in simple cases is defined as length change per length. Stress is a tensor which characterizes the internal forces within a material as force per area. In (F), (H), and (J) the unit of stress is MPa, where 1 Pa = 1 Nm −2 .

(I) As in (E) but with the stomatal poles fixed (as in D). The pattern is modified from (E) so that high strain gradients form in localized regions toward the guard cell poles.

(H) Effective stress pattern in the guard cell modeled in (G). The stress pattern observed in (F) is dissipated so that less extreme gradients are formed, with maximal stress occurring in a medial region.

(G) As in (E) but with VWT parameters used in the model. A decreased strain gradient occurs across the cell.

(F) Effective stress pattern in the guard cell modeled in (E). A radial stress pattern is generated with high stress at points along the inner radial wall.

(E) Effective Lagrange strain for the inside of a guard cell modeled using the baseline parameters. The colored scale indicates the range of strain calculated in different regions of the cell, with a gradient of strain occurring across the cell radius with the inner radial wall having a high strain.

(D) Modification of the baseline model (purple) so that the poles of the guard cells are fixed to prevent stomatal complex elongation leads to a modified output curve (blue) in which pore opening occurs at a lower turgor pressure and the final aperture attained is larger than the baseline model.

(C) Exploration of the VWT model by increasing or decreasing the inner (ventral) wall thickness by 10% indicates essentially no outcome on the aperture/pressure response curve (lines superimposed).

(B) Modeled relationship of stomatal aperture to guard cell turgor pressure. In the baseline model (purple), aperture increases as pressure increases above about 1.3 MPa, reaching a maximum value as pressure exceeds 5 MPa. Both epidermal and guard cell turgor are increased initially (gray area) after which only guard cell turgor increases. Modification of the model to include a VWT (shown in A) leads to a slight alteration in curve shape (green) so that opening occurs at a slightly lower turgor pressure and the maximal aperture attained is slightly lower.

(A) Cross-sections through guard cells modeled using the baseline parameters (circular cross-section and uniform wall thickness) or the variable wall thickness (VWT) model in which a rounded triangular geometry leads to differential inner wall thickness. The cell wall is modeled as an anisotropic material, parameterized by cellulose micro-fibrils embedded in an isotropic matrix. The micro-fibrils are oriented circumferentially in all models.

Finite Element Modeling Indicates Only a Minor Role for Radial Stiffening in Stomatal Function but Demonstrates that Fixing Stomatal Poles Has a Major Influence on Aperture Response to Change of Turgor Pressure

Figure 2 Finite Element Modeling Indicates Only a Minor Role for Radial Stiffening in Stomatal Function but Demonstrates that Fixing Stomatal Poles Has a Major Influence on Aperture Response to Change of Turgor Pressure

To investigate whether the observed differences in radial Ebetween young and mature guard cells reflected any difference in function, we performed bioassays on epidermal strips, using depleted COto trigger stomatal opening and elevated COto close stomata []. These results indicated that both young and mature stomata are able to open and close in response to an external trigger ( Figure 1 B). The absolute values for pore aperture were clearly lower for young stomata compared with mature stomata. Comparison of the measured maximal pore aperture attained under low COconditions with the theoretical maximal aperture predicted from pore geometry indicated that the younger stomata were just as capable as mature stomata of opening their pores; thus, the lower absolute values for pore aperture most likely simply reflected stomatal size differences between young and mature stomata ( Figure S1 B).

Atomic force microscopy (AFM) was performed on leaves of Arabidopsis using a 5-nm-diameter pyramidal indenter on a cantilever of nominal 45 N/m stiffness mounted on a JPK Nano Wizard 3 instrument. Probing the surface generated force maps in which it was possible to identify stomata at various stages of development ( Figure 1 A) [], ranging from guard mother cells (GMCs) undergoing the final symmetrical division to form two guard cells ( Figure 1 C), young stomata (characterized by an approximately equal length:width ratio) ( Figure 1 F), and more mature stomata (complex length greater than width; Figure 1 I). Visual observation of the stiffness patterns indicated by apparent modulus values (E) suggested that although the more mature guard cells had the expected gradient of stiffness in which the inner radial region of each guard cell was stiffer than the outer radial part of the cell ( Figure 1 I), this pattern was not obvious in the younger stomata ( Figure 1 F). Quantitative analysis of Eacross the maximum diameter of stomata supported these observations. Thus, the Eof mature stomata showed clear peaks in the inner radial regions of the guard cells relative to the outer radial regions ( Figure 1 J). A similar analysis of younger stomata did not reveal any such gradient ( Figure 1 G). By determining the difference in max Eat the inner and outer radial regions across the width of the guard cells, values for Egradient were calculated ( Figure S1 A). For the more mature guard cells, the median Egradient was 4 MPa/μm (n = 14), whereas for younger guard cells, the median gradient was essentially 0 MPa/μm (n = 18). Statistical analysis using a Mann-Whitney test indicated that the mature guard cells displayed a significantly higher stiffness gradient (p < 0.001). We were able to analyze only two GMCs, and these showed a single peak of Ein the center of the forming stomatal complex in the position of the dividing wall ( Figure 1 D). The value of the Efor the dividing wall of GMCs was not higher than the outer cell wall of the GMCs, suggesting that there is no radial gradient of stiffness in the guard cells at formation.

(K) Distribution of E a around the circumference of the stomatal complex shown in (I). Two main peaks of E a are observed at the poles of the stomatal complex. The minor third peak corresponds to the junction with the epidermal cell on the right-hand guard cell. Representative images and analyses of young (F–H) and mature (I–K) guard cells are shown. Force maps were obtained from a total of 14 young and 18 mature guard cells. Scale bars in (A), (C), (F), and (I), 10 μm.

(J) Distribution of E a across the diameter of the stomatal complex shown in (I). Four peaks are detected, corresponding to the pairs of walls defining the guard cells. The maximum E a value for the inner radial walls is higher than the peak E a for the outer radial walls.

(H) Distribution of E a around the circumference of the stomatal complex shown in (F). Two main peaks of E a are observed at the poles of the stomatal complex. The shoulder on the second peak corresponds to the junction with the epidermal cell on the right-hand guard cell.

(G) Distribution of E a across the diameter of the stomatal complex shown in (F). Four peaks are detected, corresponding to the pairs of walls defining the guard cells. The maximum peak value is similar for all four walls.

(E) Distribution of E a around the circumference of the GMC shown in (C), with the start point at the equator (as shown in the schematic). A series of peaks of E a are observed.

(D) Distribution of E a across the diameter (as shown in schematic) of the GMC shown in (C). Three peaks of E a of similar value are detected, corresponding to the three walls of the GMC.

(B) Bioassays of young and mature stomata indicate that they both respond to low CO 2 by increasing pore area and to high CO 2 by decreasing pore area. Single asterisk indicates significant difference p < 0.01, n > 23; double asterisk indicates significant difference p < 0.001, n > 23 (ANOVA was performed on “young” or “mature” datasets, followed by a Tukey test). Error bars indicate SEM.

(A) Force map of a leaf epidermis showing the spatial pattern of E a . Stomata (indicated by asterisks) at different stages of differentiation are distributed across the epidermis and show different patterns of E a , indicated by relative signal value (yellow, high; red/black, low).

Polar Stiffening Modulates Stomatal Function

a ( 2 concentrations to open or close the stomatal pore indicated no trend for change in complex length at different pore widths ( 18 Rui Y.

Anderson C.T. Functional analysis of cellulose and xyloglucan in the walls of stomatal guard cells of Arabidopsis. Figure 3 Measured Change in Stomatal Dimensions during Opening and Closing Supports a Fixed Position of the Stomatal Poles Show full caption (A) Modeled change in stomatal complex length with increase in guard cell pressure predicts a gradual increase in length at pressures above 1 MPa, both for the baseline (purple) and the VWT model (green), whereas the fixed pole model imposes a constant complex length (blue). (B) Measured complex length in mature stomata triggered to close by elevated CO 2 (red), open by depleted CO 2 (green), or incubated under ambient CO 2 levels (blue). Complex length does not overtly change relative to pore width. Regression analysis was used to calculate the line indicated but is supported with only a low confidence value (p = 0.354, n = 360), suggesting a very limited relationship of complex length and pore width. (C) Measured pore length in mature stomata triggered to close by elevated CO 2 (red), open by depleted CO 2 (green), or incubated under ambient CO 2 levels (blue). Pore length increases with pore width. Regression analysis was used to calculate the line indicated, which is supported with p < 0.0001 (n = 360), suggesting a close relationship of pore length and pore width. Note that the size parameters used for the model are based on those from the literature for Vicia faba, thus the absolute magnitudes of stomatal complex length are greater in (A) than in (B). An unexpected observation from our analysis was the apparent stiffening of the polar regions of both young ( Figure 1 H) and mature ( Figure 1 K) stomata. As far as we are aware, this has not previously been observed. An analysis of tomato and maize leaves revealed comparable patterns of stiffening, suggesting that this phenomenon might be widespread ( Figures S2 A–S2D), and higher-resolution imaging did not reveal any overt surface features that might lead to such localized regions of high E Figures S2 E and S2F). To investigate the function of such polar stiffening, we further explored the model described in Figure 2 . As shown in Figure 3 A, both the baseline and VWT models predict that as turgor pressure increases (and, as a consequence, pore width increases), stomatal complex length should increase. However, analysis of samples incubated under differing COconcentrations to open or close the stomatal pore indicated no trend for change in complex length at different pore widths ( Figure 3 B), as also observed by other authors []. This is in contrast to measured pore length, which showed a strong positive correlation with pore width under the same treatments (p < 0.0001, n = 360) ( Figure 3 C). The experimental data suggested to us that the measured local stiffening observed in Figures 1 F and 1I might reflect a pinning down of the poles so that complex length does not change during opening/closure of the stomata. We therefore modified the model to impose a restriction on stomatal complex length change during pore opening/closure (blue line in Figure 3 A), better capturing experimental reality. This had a dramatic outcome on aperture change in response to increase in turgor pressure, with opening occurring at a lower pressure, a greater increase in aperture per unit pressure being achieved, and a larger final aperture being attained (“fixed poles” curve in Figure 2 D).

488) probe that enables localization of de-esterified homogalacturonic polymers in plant cell walls [ 19 Mravec J.

Kračun S.K.

Rydahl M.G.

Westereng B.

Miart F.

Clausen M.H.

Fangel J.U.

Daugaard M.

Van Cutsem P.

De Fine Licht H.H.

et al. Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes. 488 binding ( 20 Amsbury S.

Hunt L.

Elhaddad N.

Baillie A.

Lundgren M.

Verhertbruggen Y.

Scheller H.V.

Knox J.P.

Fleming A.J.

Gray J.E. Stomatal function requires pectin de-methyl-esterification of the guard cell wall. 21 Verhertbruggen Y.

Marcus S.E.

Haeger A.

Ordaz-Ortiz J.J.

Knox J.P. An extended set of monoclonal antibodies to pectic homogalacturonan. 488 and LM19 detect de-esterified pectin, it is likely that the signal observed depends on the degree of de-esterification and the local matrix conformation, which may restrict probe access [ 19 Mravec J.

Kračun S.K.

Rydahl M.G.

Westereng B.

Miart F.

Clausen M.H.

Fangel J.U.

Daugaard M.

Van Cutsem P.

De Fine Licht H.H.

et al. Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes. 22 Hervé C.

Rogowski A.

Gilbert H.J.

Paul Knox J. Enzymatic treatments reveal differential capacities for xylan recognition and degradation in primary and secondary plant cell walls. 23 Altartouri B.

Geitmann A. Understanding plant cell morphogenesis requires real-time monitoring of cell wall polymers. 24 Zhang T.

Zheng Y.

Cosgrove D.J. Spatial organization of cellulose microfibrils and matrix polysaccharides in primary plant cell walls as imaged by multichannel atomic force microscopy. Figure 4 Polar Cell Wall Structure Plays a Role in Stiffening and Stomatal Function Show full caption (A) Labeling of stomata with the COS488 probe reveals a high level of signal (green) at the stomatal poles (left). Treatment of tissue with polygalacturonase (4 hr) leads to loss of COS488 binding (right). (B) Bioassays after pre-treatment with buffer (control), cellulose, or polygalacturonase (PGase) indicate that stomata retain the ability to open in response to low CO 2 after all treatments, but the stomatal aperture attained after PGase treatment is significantly smaller, both at ambient and low CO 2 , relative to the control. ANOVA was performed across all samples with post hoc Tukey. Columns indicated with the same letter cannot be distinguished from each other at the 0.05 confidence limit (n = 40). Error bars indicate SEM. (C) Force map of epidermis from a control sample showing the spatial pattern of E a after 4 hr incubation of tissue in buffer. Relative signal value is indicated by high (yellow) to low (red/black). a across the diameter (as shown in schematic in (D) Distribution of Eacross the diameter (as shown in schematic in Figure 1 ) of the stomata indicated by asterisk in (C). Four peaks are detected, corresponding to the pairs of walls defining the guard cells. The maximum peak value for the inner radial walls (peaks 2 and 3) is higher than the peak value for the outer radial walls (peaks 1 and 4). a around the circumference (as shown in schematic in a are observed at the poles of the stomatal complex. (E) Distribution of Earound the circumference (as shown in schematic in Figure 1 ) of the stomatal complex shown in (C). Two main peaks of Eare observed at the poles of the stomatal complex. (F) Force map of epidermis showing the spatial pattern of E a after 4 hr incubation of tissue in cellulase. Relative signal value is indicated by high (yellow) to low (red/black). (G) Distribution of E a across the diameter of the stomata indicated by asterisk in (F). Four peaks are detected, corresponding to the pairs of walls defining the guard cells. The maximum E a for the inner radial walls is higher than the peak value for the outer radial walls. (H) Distribution of E a around the circumference of the stomatal complex shown in (F). Two main peaks of E a are observed at the poles of the stomatal complex. (I) Force map of epidermis showing the spatial pattern of E a after 4 hr incubation of tissue in polygalacturonase. Relative signal value is indicated by high (yellow) to low (red/black). (J) Distribution of E a across the diameter of the stomata indicated by asterisk in (I). Two broad, asymmetric peaks are detected, with the highest values at the inner radial walls of the two guard cells. The peaks corresponding to the outer radial wall (peaks 1 and 4) are only barely detectable. a around the circumference of the stomatal complex shown in (I). Two main peaks of E a are observed at the poles of the stomatal complex. The E a value of these peaks is lower than those observed in (E) and (H). Representative images and analysis are shown for control (C–E), cellulase (F–H), and polygalacturonase-treated tissue (I–K). The analyses were repeated at least three times with similar results (data shown in (K) Distribution of Earound the circumference of the stomatal complex shown in (I). Two main peaks of Eare observed at the poles of the stomatal complex. The Evalue of these peaks is lower than those observed in (E) and (H). Representative images and analysis are shown for control (C–E), cellulase (F–H), and polygalacturonase-treated tissue (I–K). The analyses were repeated at least three times with similar results (data shown in Figure S4 ). Scale bars in (A), (C), (F), and (I), 10 μm. See also Figures S3 and S4 To investigate the molecular structure of the stomatal poles that might underpin the observed stiffening, we took an in situ labeling approach to characterize the spatial pattern of cell wall epitopes. Recent work has identified a chitosan oligosaccharide (COS) probe that enables localization of de-esterified homogalacturonic polymers in plant cell walls []. Incubation of this probe with intact leaf epidermal tissue revealed binding to the epidermal pavement cells and especially strong signal at the stomatal poles, with apparent exclusion from the outer radial walls of the guard cells ( Figure 4 A; Figure S3 A). Treatment of tissue with polygalacturonase led to loss of COSbinding ( Figure 4 B; Figure S3 B), corroborating that the probe was detecting a pectin motif in this region. Our previous work using antibodies raised against pectins revealed that guard cell walls are distinguished by the exclusion of epitopes corresponding to methylated pectin and the accumulation of epitopes corresponding to de-esterified pectin []. Interestingly, following treatment with polygalacturonase, the uniform signal observed around guard cells with antibodies JIM7 and LM19 (which detect general levels of pectin and de-esterified pectin, respectively []) was replaced by a pattern of weak signal around the stomatal poles ( Figures S3 C–S3F). Although both COSand LM19 detect de-esterified pectin, it is likely that the signal observed depends on the degree of de-esterification and the local matrix conformation, which may restrict probe access [], complicating interpretation of the patterns in signal observed. As a consequence of such technical challenges, our detailed understanding of plant cell wall molecular architecture is still somewhat limited []. However, taken together, the data in Figure 4 A and Figure S3 are consistent with the hypothesis that stomatal poles in Arabidopsis have a distinct cell wall pectin structure, which might define the localized regions of stiffness detected in our AFM analysis. Modeling of the fixed pole model indicated that it would lead to an altered pattern of strain/stress during stomatal opening, with a focusing of gradients toward the polar regions of the guard cells ( Figures 2 I and 2J). Whether guard cell wall composition/structure is modified in these regions to cope with these predicted strain/stress patterns awaits further analysis.

25 Jones L.

Milne J.L.

Ashford D.

McQueen-Mason S.J. Cell wall arabinan is essential for guard cell function. a across the diameter and around the circumference of mature stomata revealed normal patterns, with a radial gradient in the guard cells and two peaks of E a in the polar regions ( a substantiated these observations. Thus, the E a peaks corresponding to the outer radial walls of the guard cells tended to be diminished ( a tended to be narrower and much smaller in absolute value ( To investigate whether the localized difference in pectin structure was related to the observed polar stiffening, and thus the role of polar stiffening in stomatal function, we treated leaf explants with cell-wall-modifying enzymes []. Treatment of tissue with buffer alone did not overtly change the pattern of stiffness observed in mature stomata ( Figure 4 C). Quantitative analysis of Eacross the diameter and around the circumference of mature stomata revealed normal patterns, with a radial gradient in the guard cells and two peaks of Ein the polar regions ( Figures 4 D and 4E). Similarly, treatment with exogenous cellulase for 4 hr did not alter the stiffness patterns in a major fashion from those observed in control tissue ( Figures 4 F–4H). However, treatment with polygalacturonase led to major changes in stiffness pattern. With respect to the stomata, there was an accentuation in the apparent relative gradient of radial stiffening of the guard cells, and polar stiffening was less marked ( Figure 4 I). Quantitation of the radial and circumferential patterns of Esubstantiated these observations. Thus, the Epeaks corresponding to the outer radial walls of the guard cells tended to be diminished ( Figure 4 J; Figure S4 A), and the polar peaks of Etended to be narrower and much smaller in absolute value ( Figure 4 K; Figure S4 B).

2 levels ( We performed opening/closing assays to test the outcome of enzyme treatment on stomatal function. After all treatments, guard cells retained the ability to increase pore aperture following exposure to depleted (low) COlevels ( Figure 4 B); however, the basal aperture under ambient conditions was significantly lower in the polygalacturonase-treated stomata than in those treated with cellulase or buffer alone (ANOVA with post hoc Tukey, p < 0.001, n = 40, experiment repeated three times). The maximal aperture achieved by both polygalacturonase- and cellulase-treated stomata was smaller than that achieved in control tissue (ANOVA with post hoc Tukey, p < 0.05, n = 40).

26 Franks P.J.

Farquhar G.D. The mechanical diversity of stomata and its significance in gas-exchange control. 27 Franks P.J.

Cowan I.R.

Farquhar G.D. A study of stomatal mechanics using the cell pressure probe. 28 Jones L.

Milne J.L.

Ashford D.

McCann M.C.

McQueen-Mason S.J. A conserved functional role of pectic polymers in stomatal guard cells from a range of plant species. A decrease in stomatal pore aperture relative to control after enzyme treatment could occur via a number of mechanisms. For example, treatment with polygalacturonase led to an altered gradient of stiffness across guard cells ( Figures 4 I and 4J), but our finite element modeling suggested that alteration in radial stiffness has only a very moderate effect on stomatal opening ( Figure 2 C). It was also apparent that treatment with polygalacturonase led to a decreased relative stiffness in all epidermal cell walls (compare Figure 4 I with Figures 4 C and 4F); however, it is not obvious how such a change would lead to a decrease in pore aperture under ambient conditions. Epidermal cells surrounding stomata are expected to exert a mechanical advantage [], so, if anything, weakening of these supporting cells might lead to an increased pore aperture for any given guard cell turgor pressure, the opposite of the phenotype observed. Decreased pressure within the guard cells under ambient conditions would obviously lead to a decreased pore aperture, but it is not apparent why this would be a primary outcome following treatment with polygalacturonase (and which was not observed after cellulase treatment). A final possibility is that the loss of polar stiffening observed after polygalacturonase treatment ( Figure 4 K; Figure S4 B) underpins the shift in stomatal dynamics. Considering the pore aperture response to altered turgor pressure depicted for the baseline and fixed pole models ( Figure 2 D), it is clear that, above 1 MPa, a loss of polar stiffening leads to a large decrease in aperture for any given pressure as the stomatal dynamics shift from the “fixed poles” to the “baseline” curve. This would account for the decreased aperture under ambient conditions recorded in stomata treated with polygalacturonase ( Figure 4 B). It should be noted that our model predicts that after loss of polar stiffening and consequent shift to the baseline model, stomata are still able to open, but the final aperture is expected to be smaller than in the fixed poles model. The experimental data in Figure 4 B support this prediction. Cellulase treatment of stomata led to results intermediate between control and polygalacturonase-treated samples ( Figure 4 B). There was no evidence of decreased aperture under ambient conditions, but the maximal aperture obtained under conditions favoring opening was lower than control. We suspect that this decrease in maximal aperture might reflect a gradual loss in tissue integrity after cellulase treatment, as previously observed []. Overall, our observations are consistent with the proposal that polar stiffening, mediated at least in part by localized accumulation of de-esterified pectin, plays a role in stomatal function. Stiffening of guard cell poles limits stomatal complex extension under opening conditions, leading to a mechanical system that shows a greater response in pore aperture per change in guard cell pressure.

17 McAusland L.

Vialet-Chabrand S.

Davey P.

Baker N.R.

Brendel O.

Lawson T. Effects of kinetics of light-induced stomatal responses on photosynthesis and water-use efficiency. 26 Franks P.J.

Farquhar G.D. The mechanical diversity of stomata and its significance in gas-exchange control. 20 Amsbury S.

Hunt L.

Elhaddad N.

Baillie A.

Lundgren M.

Verhertbruggen Y.

Scheller H.V.

Knox J.P.

Fleming A.J.

Gray J.E. Stomatal function requires pectin de-methyl-esterification of the guard cell wall. 29 Lawson T.

Blatt M.R. Stomatal size, speed, and responsiveness impact on photosynthesis and water use efficiency. 30 Franks P.J.

W Doheny-Adams T.

Britton-Harper Z.J.

Gray J.E. Increasing water-use efficiency directly through genetic manipulation of stomatal density. Such a system would be expected to be evolutionarily advantageous. Plants adapt stomatal aperture to changing environments, and limits in the rapidity with which they can do this leads to inefficiencies []. Indeed, it has been proposed that one of the reasons for the evolutionary success of some plant groups is that their stomata have evolved to be able to respond more rapidly to changing environment []. Whether the structure of the guard cell wall in the stomatal poles has played an evolutionary role in improving stomatal efficiency awaits elucidation, but our work sets the foundation for this future research. Due to the importance of stomata in plant water relationships, a deeper understanding of the properties of guard cell walls in setting the mechanical response to external triggers may also help in the selection and engineering of improved crops [].

In conclusion, the results reported here negate a widely held view on the importance of radial guard cell wall thickening in stomatal opening, provide an alternative view on the importance of guard cell geometry in dissipating cell wall stress gradients, and identify polar stiffening of stomata as a potentially widespread phenomenon that leads to improved stomatal response to altered guard cell turgor pressure.