CALCIUM AND ACIDIC PECTIN DISTRIBUTION IN FLAX CELL-WALLS - EVIDENCE FOR DIFFERENT KINDS OF LINKAGES IN THE CELL JUNCTION AND MIDDLE LAMELLA OF THE CORTICAL PARENCHYMA OF FLAX HYPOCOTYL

We used three different sample preparation methods (conventional gluta raldehyde-fixation, pyroantimonate precipitation method and a cryo-met hod) to investigate the distribution of calcium by secondary ion mass spectrometry in the cortical cell walls of flax seedling hypocotyl. Th e ionic images of calcium distribution were compared to the transmissi on electron microscopy images of the distribution of pectic polysaccha rides. For pectic acid localization both a direct labelling with gold- labelled endopolygalacturonase (endoPG, EC 3.2.1.15), and an indirect subtractive method using the same enzyme as a specific extracting agen t were used. Our results show that, whatever the method used to prepar e the samples, the calcium signal was lower in the cell walls of the c ortical parenchyma than in the cell walls of the epidermal and subepid ermal layers especially in the tricellular junctions. In the cortical cell walls, acidic pectins were mainly located in the middle lamella, in the corner of the intercellular spaces and the wall area surroundin g air-spaces. The removal of pectic polysaccharides from these wall-mi crodomains observed when samples were treated with the endoPG was stro ngly inhibited in the presence of calcium ions. Thus, these cations we re able to prevent the endoPG-induced disorganization of the cortical parenchyma of flax hypocotyl. This finding indicates that at this step of flax growth (a) the amount of calcium loaded in these wall areas w as too low to promote the formation of calcium bridges and (b) the pec tic polysaccharides were linked to the cell wall through different kin ds of linkages depending to the wall areas, and most importantly throu gh covalent linkages.