Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe

Abstract

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.

Figure 1

Confocal images of the lower one-half of a spherical polystyrene bead containing a fluorescent dye in the outer layer. Image a is distorted in the z-plane by differences in refractive index among mounting materials. In image b, the pixel spacing in the z-plane has been corrected to make the image of the bead spherical (see text for discussion). The distances R (= 10.36 μ m) and r (= 7.25 μ m) represent the uncorrected and corrected radii, respectively. The image of the top one-half of the bead was further distorted by the light passing through the bead itself, and this portion of the image has been cropped for clarity.

Figure 2

Composite figure showing medial paradermal (x-y plane) and transdermal (x-z plane) images of guard cells as affected by turgor pressure. The pressure probe was inserted through the top of the left cell and into the right cell, and this caused both cells to fill with oil (see “Materials and Methods” for details). Scale bar = 10 μm. Movies showing three-dimensional reconstructions of guard cells can be found at http://bioweb.usu.edu/kmott or www.plantphysiol.org.

Figure 3

Measurements of overall guard cell length and stomatal one-half aperture as a function of guard cell turgor pressure for three guard cells.

Figure 4

Guard cell volume (V_g, μm³) as a function of guard cell turgor pressure (P_g, MPa) for three guard cells. Symbols represent direct measurements of V_g (from confocal images) and P_g (with the pressure probe); the lines show the power functions that were fit to the data.

Figure 5

Moduli of elasticity (ε_g = V_g × dP_g/dV_g) for the three guard cells with P_g-V_g curves shown in Figure 4. ε_g for each cell was determined by differentiation of the power function that was fit to that cell's V_g and P_g measurements.