Figure 4.

Polar Cell Wall Structure Plays a Role in Stiffening and Stomatal Function

(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 CO₂ after all treatments, but the stomatal aperture attained after PGase treatment is significantly smaller, both at ambient and low CO₂, 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).

(D) Distribution of E_a across 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).

(E) Distribution of E_a around the circumference (as shown in schematic in Figure 1) of the stomatal complex shown in (C). Two main peaks of E_a are 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.

(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 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 Figure S4). Scale bars in (A), (C), (F), and (I), 10 μm.

See also Figures S3 and S4.