Diffusion of anionic and neutral GFP derivatives through plasmodesmata in epidermal cells of Nicotiana benthamiana

Plasmodesmata (Pd) are trans-wall membrane channels that permit cell-to-cell transport of metabolites and other small molecules, proteins, RNAs, and signaling molecules. The transport of cytoplasmic soluble macromolecules is a function of the electrochemical gradient between adjacent cells, the number of Pd per interface between adjacent cells, Stokes radius (R(S)), area of the cytoplasmic annulus, and channel length. The size of the largest molecule that can pass through Pd defines the Pd size exclusion limit. However, since the shape and size of a molecule determines its capacity to diffuse through pores or tubes, R(S) is a better measure. Relatively small changes in R(S) can cause large differences in the mobility of molecular probes, particularly if the pore size is close to that of the probe. In addition, as the dimensions of a macromolecule approach that of the channel, membrane charge effects may become important. We employed quantitative tools and molecular modeling to measure the apparent coefficient of conductivity of Pd, C(Pd), for the non-targeted transport of macromolecules. This method allowed us to examine the influence of protein charge and R(S) on C(Pd) in Nicotiana benthamiana. The C(Pd) of modified green fluorescent proteins (GFPs) of different sizes but with the same charge as native GFP and of a more negatively charged derivative were determined. We found that the C(Pd) of cytoplasmic soluble GFP and cytoplasmic forms of modified GFP were the most strongly correlated with R(S) and that the apparent aberrant increase in C(Pd) of a negatively charged GFP derivative was, at least in part, the result of the charge effect on R(S).