Regulation Of Plasmodesmata In Leaf Development And Phloem Loading In Arabidopsis Leaves

Plasmodesmata are channels with important role in molecular transport and intercellular communication in plants.Previous studies demonstrated the strong influence of plasmodesmata function on organ development and photoassimilate transport.The permeability of plasmodesmata is mainly regulated by deposition and degradation of callose in cell wall surrounding the channel's collars.At present,there are data on plasmodesmata ultrastructure and data for inter-cellular transport of proteins.But the data on in vivo permeability of plasmodesmata for small molecules is absent.The data is very important because both the hormones that control all developmental processes and sugars that are produced by photosynthesis are small molecules.In this project,photoactivation microscopy on a laser-scanning confocal microscope was used to measure plasmodesmata mediated cell wall permeability.First,permeability was measured in leaves of the model plant Arabidopsis thaliana at three developmental stages.These experiments revealed a decreased permeability of individual plasmodesmata,but increased permeability per cell-cell-interface due to the increased number of plasmodesmata per interface during development.Moreover,a directionality in the permeability of plasmodesmata in epidermal cells above the midrib and petiole was discovered,with much higher cell coupling in the longitudinal direction compared to the radial direction.The dependence of the changes in permeability could be ascribed to callose deposition by analysis of the callose synthase mutant.Furthermore,the impact of directionality on hormone transport was demonstrated by application of a drop of auxin to the leaf tip,which triggered a reaction at the leaf base that depended more on directionality than on absolute permeability.In a second part,confocal microscopy combined with photobleaching of a small,mobile dye was used to measure cell coupling in leaves of tobacco and cucumber plants.It was found that plasmodesmata permeability is related to the mechanism of sugar export from leaves.In tobacco,which represents active apoplastic export type,cell coupling between mesophyll cells and bundle sheath cells was higher than cell coupling between bundle sheath and the vascular cells of the phloem.In cucumber,which represents active symplasmic export,the opposite was found to be true.Furthermore,correlation of permeability with structural parameters of plasmodesmata,determined by transmission electron microscopy was tested.Results show that structural features not visible on standard transmission electron microscopy images must influence plasmodesmata permeability.In the third part,a beta-galactosidase responsible for callose degradation was over-expressed in tobacco leaf cells.Photoactivation microscopy showed the expected increase in plasmodesmata permeability.It was tested if this has an influence on the export of sugars from the leaf,but no difference was found.