Use of purified endopolygalacturonase for a topochemical study of elongating cell walls at the ultrastructural level.

Endopolygalacturonase, a fungal enzyme purified by Albersheim and his group which specifically degrades the galacturonosyl linkages of pectic polysaccharides, was used at the ultrastructural level on intact tissues from differentiated organs. Specimens were taken from the elongating zone of mung bean (Vigna radiata) hypocotyls. Incubation with the enzyme solution was performed en bloc, prior to embedding and to ultrastructural cytochemistry (PATAg test for polysaccharides), or by flotation of ultrathin frozen sections. From the morphological viewpoint, the images obtained are sharp and reproducible. They support the plywood model for the organization of expanding walls. The ordering of walls appears to be built up very early, at the very beginning of elongation, in the upper part of the hypocotyl (hook). The specificity of the enzyme allows a topochemical study of the wall. Data indicate an uneven distribution of polysaccharides of pectic type across a single wall and among the different cells. Outer and inner wall areas are highly resistant to extraction by endopolygalacturonase. In the middle lamella the insolubility of amorphous components probably indicates a local concentration of highly methyl-esterified carboxyl groups not susceptible to endopolygalacturonase attack; conversely, adjacent parts of the middle lamella are regularly extracted, indicating a high concentration of galacturonans. An intense extraction occurs in the bow-shaped zone, revealing the occurrence of a massive embedding of unesterified pectic polysaccharides around the fibrillar subunits responsible for the twisted patterns. In the inner and recent part of the wall, the ordering of fibrillar subunits seems progressive and possibly related to the peptic embedding. This incrusting material could play a role in the morphogenesis of the ordered wall by means of specific assembly and interconnections of wall subunits.