DYNAMIC CHANGES IN CELL-SURFACE MOLECULES ARE VERY EARLY EVENTS IN THE DIFFERENTIATION OF MESOPHYLL-CELLS FROM ZINNIA-ELEGANS INTO TRACHEARY ELEMENTS

The Zinnia mesophyll cell system consists of isolated leaf mesophyll c ells in culture that can be induced, by auxin and cytokinin, to transd ifferentiate semi-synchronously into tracheary elements (TEs). This sy stem has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for i n cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with t he transition from primary to secondary cell wall formation. At very e arly stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the cultu re medium were found. Changes in the secretion of pectic polysaccharid es, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recog nize these three classes of cell-wall molecule, respectively. Twenty-f our hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thicken ings of mature TEs but not in their surrounding primary walls. Methyl- esterified pectic polysaccharides are present in all cell walls and ar e secreted into the culture medium throughout the time course of diffe rentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively un branched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amo unt of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control m edium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from induc tive medium 6 h before any secondary thickenings are visible in the ce lls. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, alt hough methyl-esterified pectin is present. All three epitopes are expr essed in the vascular bundles, and the AGP is specifically localized i n the xylem cells. Fucosylated xyloglucan is also present in the epide rmal tissue, and the AGP is present in guard cells. The dynamic behavi our of these specific cell-wall molecules is tightly correlated with d ifferentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate det ermination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.