AFFINODETECTION OF THE SITES OF FORMATION AND OF THE FURTHER DISTRIBUTION OF POLYGALACTURONANS AND NATIVE CELLULOSE IN GROWING PLANT-CELLS

The object of the present paper is to complement the cytochemical detection of the polysaccharides of the plant cell wall and of its precursors, taking benefit of two kinds of affinity methods: the enzyme-gold technique and immunocytochemistry. Cellobiohydrolase (CBH 1, EC 3.2.1.91) was used to target native crystalline cellulose and two monoclonal antibodies, JIM 5 and JIM 7, were used to target homogalacturonan sequences with various degrees of esterification. Observations were performed at the light microscope level (UV epifluorescence, enzyme-gold silver staining) and at the electron microscope level. Two types of biological specimens, both in steady state of growth, were chosen: in vitro cultures of melon cells (thin, unidirectional primary walls, loosely associated cells), and elongating zone of mung bean hypocotyl (thick walled and tightly associated cells). The following points were examined successively: the labelling at the histological level, the detection of cellulose and of polygalacturonan components in muro, the visualization of the emerging sites of the polymers along the endomembrane flow and their post-synthetic modifications (crystallization and methylation respectively). JIM antibodies showed the early labelling of homogalacturonans on the bulging margins of the dictyosomes. The labelled vesicles appeared as sites of polymerization, cytoplasmic transport and beginning of molecular maturation with likely an early action of methyl transferases. The first labelling of cellulose occurred only on the outer face of the plasma-membrane. Later on, CbH 1-gold complexes remained distributed throughout the width of the growing wall, despite the surface expansion and the dispersion of the ordered framework. No significant change of the cellulose crystallinity was noticed. A co-localization of polygalacturonan and cellulose markers was seen from the assembly to the deassembly of the cell wall. In complement, subtractive cytochemistry was performed using PATAg in association with an endopolygalacturonase to split the pectic chains or chelators (EDTA, EGTA, oxalate) to solubilize the calcium-connected polyuronic acid chains. All the attacks exposed the individual microfibrils of the cellulose framework revealing uniformly the helicoidal organization and confirming that cellulose and polygalacturonans remain closely associated spatially during growth.