Effects of tissue-preparation-induced callose synthesis on estimates of plasmodesma size exclusion limits

Plasmodesmata are often characterised by their size exclusion limit (SEL), which is the molecular weight of the largest dye, introduced by microinject ion, that will move from cell to cell. In this study we investigated whethe r commonly used techniques for isolation and manipulation of tissues, and m icroinjection of fluorescent dyes, affected the SEL, and whether any such e ffects could be ameliorated by inhibiting callose deposition. We examined y oung root epidermal cells of Arabidopsis thaliana and staminal hair cells o f Tradescantia virginiana, two tissues often used in experiments on symplas tic transport. Transport in root tips dissected from the main plant body an d in stamen hairs removed from the base of the stamen filament was compared with transport in undissected roots and stamen hairs attached to the base of the filament, respectively. Tissues were microinjected with fluorescent dyes (457 Da to >3 kDa) with or without prior incubation in the callose dep osition inhibitors 2-deoxy-D-glucose or aniline blue fluorochrome. In both tissues, dissection reduced the SEL, which was largely prevented by prior i ncubation in 2-deoxy-D-glucose but not by incubation in aniline blue fluoro chrome. Thus, standard methods for tissue preparation can cause sufficient callose deposition to reduce cell-to-cell transport, and this needs to be c onsidered in studies employing microinjection. Introduction of the dyes by pressure injection rather than iontophoresis decreased the SEL in A. thalia na but increased it in T. virginiana, showing that these two injection tech niques do not necessarily give identical results and that plasmodesmata in different tissues may respond differently to similar experimental procedure s.