Measurement of diffusion within the cell wall in living roots of Arabidopsis thaliana

Abstract

To quantify the diffusion constant of small molecules in the plant cell wall, fluorescence from carboxyfluorescein (CF) in the intact roots of Arabidopsis thaliana was recorded. Roots were immersed in a solution of the fluorescent dye and viewed through a confocal fluorescence microscope. These roots are sufficiently transparent that much of the apoplast can be imaged. The diffusion coefficient, D(cw), of CF in the cell wall was probed using two protocols: fluorescence recovery after photobleaching and fluorescence loss following perfusion with dye-free solution. Diffusion coefficients were obtained from the kinetics of the fluorescent transients and modelling apoplast geometry. Apoplastic diffusion constants varied spatially in the root. In the elongation zone and mature cortex, D(cw)=(3.2+/-1.4)x10(-11) m(2) s(-1), whereas in mature epidermis, D(cw)=(2.5+/-0.7)x10(-12) m(2) s(-1), at least an order of magnitude lower. Relative to the diffusion coefficient of CF in water, these represent reductions by approximately 1/15 and 1/195, respectively. The low value for mature epidermis is correlated with a suberin-like permeability barrier that was detected with either autofluorescence or berberine staining. This study provides a quantitative estimate of the permeability of plant cell walls to small organic acids-a class of compounds that includes auxin and other plant hormones. These measurements constrain models of solute transport, and are important for quantitative models of hormone signalling during plant growth and development.

Differential vascularization of nematode-induced feeding sites

“…Correspondingly, following application of CF to tomato leaves, CF fluorescence should appear in roots at 1-2 hours if the dye is transported via the phloem and symplastically released into the GCs. The very long time of 2 days is sufficient to allow CF diffusion to the RK in the apoplast (33). Even though CF is relatively membrane-impermeant, incorporation of this dye across membranes does take place at slow rates (33,34).…”

“…The very long time of 2 days is sufficient to allow CF diffusion to the RK in the apoplast (33). Even though CF is relatively membrane-impermeant, incorporation of this dye across membranes does take place at slow rates (33,34). The observed accumulation of CF in GCs can alternatively be explained by the enhanced incorporation associated with the huge surface of these cells.…”

Multiscale modelling of auxin transport in the plant-root elongation zone

“…Recent experimental work (Kramer et al 2007) has confirmed apoplastic diffusion to be slower than that in the cytoplasm, and found that D * a = 3.2 × 10 −11 m 2 s −1 . The remaining parameter values, pH c , pH w , pK and V * , are well characterised, and we use the representative values given in Swarup et al (2005); these parameters are used to calculate the constants (2.1, 2.4).…”

“…1). The model includes both passive and active auxin transport across the cell membranes, and auxin diffusion within the apoplast [and thus allows for the possibility that auxin travels relatively large distances through the apoplast before entering a cell cytoplasm (Kramer et al 2007)]. In contrast to the cylinder of cells in Swarup et al (2005), we consider for simplicity a two-dimensional configuration with each layer represented by a row of N cells, the cells in each row being labelled by i = 1, 2, 3, .…”

“…As the apoplastic diffusion coefficient is well characterised (Kramer et al 2007), we choose the time scale based on the rate at which the apoplastic auxin diffuses the length of the tissue. We nondimensionalise the cytoplasm lengths on the typical one, l * T , the auxin concentrations on the upstream concentration, C * , and the fluxes on the typical apoplastic diffusive flux, (2.6).…”