The mechanical diversity of stomata and its significance in gas-exchange control

Abstract

Given that stomatal movement is ultimately a mechanical process and that stomata are morphologically and mechanically diverse, we explored the influence of stomatal mechanical diversity on leaf gas exchange and considered some of the constraints. Mechanical measurements were conducted on the guard cells of four different species exhibiting different stomatal morphologies, including three variants on the classical "kidney" form and one "dumb-bell" type; this information, together with gas-exchange measurements, was used to model and compare their respective operational characteristics. Based on evidence from scanning electron microscope images of cryo-sectioned leaves that were sampled under full sun and high humidity and from pressure probe measurements of the stomatal aperture versus guard cell turgor relationship at maximum and zero epidermal turgor, it was concluded that maximum stomatal apertures (and maximum leaf diffusive conductance) could not be obtained in at least one of the species (the grass Triticum aestivum) without a substantial reduction in subsidiary cell osmotic (and hence turgor) pressure during stomatal opening to overcome the large mechanical advantage of subsidiary cells. A mechanism for this is proposed, with a corollary being greatly accelerated stomatal opening and closure. Gas-exchange measurements on T. aestivum revealed the capability of very rapid stomatal movements, which may be explained by the unique morphology and mechanics of its dumb-bell-shaped stomata coupled with "see-sawing" of osmotic and turgor pressure between guard and subsidiary cells during stomatal opening or closure. Such properties might underlie the success of grasses.

Figure 1.

Images of stomata (closed) from each of the four species examined. A, H. prolifers; B, N. exaltata; C, T. virginiana; D, T. aestivum. All are in epidermal peels, bathed in 25 mm MES, pH 6.5, 1.0 mm KCl, 0.1 mm CaCl₂. Scale bar = 20 μm; all images to same scale.

Figure 2.

Cross section of H. prolifera stoma sampled by snap freezing an intact leaf. A, Closed; B, open under full sunlight, very high humidity. Note absence of guard cell mechanical interaction with adjacent epidermal cells. Cryo-SEM. Scale bar = 20 μm. G, Guard cell; E, epidermal cell.

Figure 3.

Cross section of N. exaltata stoma sampled by snap freezing an intact leaf. A, Closed; B, open under full sunlight, very high humidity. Note absence of guard cell mechanical interaction with adjacent epidermal cells. Cryo-SEM. Scale bar in B = 20 μm. Both images are to the same scale. G, Guard cell; E, epidermal cell.

Figure 4.

Cross section of T. virginiana stoma sampled by snap freezing an intact leaf. A, Closed; B, open under full sunlight, very high humidity. Note in B the strong guard cell mechanical interaction with adjacent subsidiary cells. Cryo-SEM. Scale bar in A = 20 μm. Both images are to the same scale. G, Guard cell; S, subsidiary cell.

Figure 5.

Cross section of T. aestivum stoma sampled by snap freezing an intact leaf. A, Closed; B, open under full sunlight, very high humidity. Note in B the strong guard cell mechanical interaction with adjacent subsidiary cells. Cryo-SEM. Scale bar in A = 10 μm. Both images are to the same scale. G, Guard cell; S, subsidiary cell.

Figure 6.

Relationship between a and P_g for the four species at full P_e and zero P_e (mean ± se). Note the massive impediment to stomatal opening (mechanical advantage) by P_e in T. virginiana and T. aestivum compared to little or no effect in H. prolifera and N. exaltata.

Figure 7.

A visual impression of the difference, across the four stomatal types, in a_max per unit a_sc. This ratio, a_max/a_sc, sets the upper limit for stomatal conductance per unit leaf area. A, H. prolifera; B, N. exaltata; C, T. virginiana; D, T. aestivum. Images were traced from stomata in which P_g was held at approximately 4 MPa with zero P_e. Scale bar = 20 μm.

Figure 8.

A, Maximum stomatal pore area; B, ratio of maximum stomatal pore area to projected total stomate area (pore plus guard cells); C, ratio of maximum stomatal pore area to projected guard cell area. All measurements were conducted on stomata in which P_g was held at approximately 4 MPa with zero P_e (mean ± se).

Figure 9.

A, Simulation of the effect of D on the rate and magnitude of stomatal opening in a plant having stomatal mechanical characteristics similar to T. virginiana (high epidermal mechanical advantage) but no mechanism to actively compensate for the effect. B, Comparison of the opening sequence of T. virginiana stomata at D = 1 kPa and D = 2 kPa, following a change in photosynthetically active radiation from 0 to 1,000 μmol m⁻² s⁻¹. Plant well watered; leaf temperature 25°C. C, As for B, with T. aestivum. Arrows indicate the direction of the difference in g_sw at D = 2 kPa relative to D = 1 kPa.

Figure 10.

A, Simulated stomatal opening sequence; and B, associated changes in guard cell (Π_g) and epidermal (Π_e) osmotic pressures for a plant with (solid lines) a mechanism by which epidermal osmotic pressure declines in association with increasing guard cell osmotic pressure during stomatal opening (see “Methods and Materials” for model details). All other stomatal functional characteristics, as well as environmental conditions, are constant. Dotted lines correspond to the condition in which Π_e remains constant during stomatal opening. A decline in Π_e during stomatal opening could allow the epidermal mechanical advantage to be overcome under conditions of low D, allowing the observed maximum a to be achieved at low D. Note also that not only does the mechanism allow g_ws to reach its full potential, but the rate of opening is also greatly increased.

Figure 11.

Schematic of the proposed osmotic see-saw (combined opposite changes in both osmotic and turgor pressure between guard and subsidiary cells) in T. aestivum and other grass-type stomata. The mechanism reduces the large mechanical advantage that would otherwise prevent stomatal opening under high humidity, even if maximum turgor were generated in guard cells. A corollary is that the mechanism also greatly accelerates the rate of stomatal opening, helping to explain why T. aestivum has rates of stomatal opening more than an order of magnitude faster than any of the other species (Fig. 12). A, Cross-section of guard cells (thick dark walls) and subsidiary cells (thin walls) of closed stoma; B, open stoma, showing highly displaced subsidiary cells which have transferred most of their potassium to the guard cells, undergoing a significant reduction in turgor as a consequence. Compare SEM images in Figure 5.

Figure 12.

Maximum rate of increase in stomatal conductance to water vapor (dg_sw/dt), quantified in relation to (A) leaf area, (B) individual stomata, or (C) projected area of the inflated guard cell pair, as a proxy for guard cell size. The grass T. aestivum was substantially faster in all categories. (Mean ± se. See “Methods and Materials” for an explanation of the units.)