MONOCLONAL ANTIBODIES TO ARABINOGALACTAN-PROTEINS (AGPS) RELEASED BY GYMNOCOLEA INFLATA WHEN LEAF AND BRANCH DEVELOPMENT IS DESUPPRESSED

A panel of six McAbs were developed against arabinogalactan-proteins (AGPs) produced by a leafy liverwort, Gymnocolea inflata (Huds.) Dum. Five of these specify AGPs that are only released into the nutrient culture medium when increased cell proliferation correlated with an increase in the size and number of leaves and branches is experimentally induced. Because of this, we hypothesize that the AGP(s) specified by one or more of these McAbs function to help suppress cell proliferation, especially in certain leaf and branch primordia. The sixth McAb specifies AGPs that are released into the culture medium whether or not an increase of cell proliferation and organ initiation is induced. This panel of McAbs should prove to be a valuable asset for identifying, affinity purifying, and chemically characterizing one or more AGPs that appear to mediate the temporal and spatial suppression of cell proliferation in developing organ primordia. Some time ago, it was found that whenever ammonium ion (NH4?) was a component of any of several macronutrient formulations used to prepare media for the axenic culture of Gymnocolea inflata (Huds.) Dum., there was a profound change from their normal pattern of development (Basile & Basile 1980). Specifically, plants that typically produce only two rows of leaves (i.e., exhibit ? phyllotaxy) and rarely branch, developed three rows of leaves (1/3 phyllotaxy) and branched much more frequently than when cultured in the absence of NH4? or when growing in their natural habitats. Because the changes in phyllotaxy and branching pattern were a consistent and characteristic effect on liverwort morphogenesis produced by antagonists of the normal synthesis of Hyp-protein (see Basile & Basile 1982 for review), it was hypothesized that the presence of NH4? was somehow antagonizing normal Hyp-protein synthesis/function in this plant. In support of this interpretation was the finding that there was significantly less peptide-bound hydroxyproline in the "cell wall fractions" from plants cultured in NH4+-containing media (Basile & Basile 1980). The nature of the Hyp-protein being altered in correlation with changes in leaf and branch development was not known at the time. Subsequent studies, however, suggested that the Hyp-protein being altered belongs to a large class of Hyp-containing glycoproteins and proteoglycans, the arabinogalactan-proteins (AGPs) (Basile et al. 1986; Basile & Basile 1987, 1990, 1993). When the AGPs extracted from G. inflata plants grown in the absence and presence of NH4? were compared by cesium chloride (CsC1) gradient analysis, there was a statistically significant difference in the buoyant density range of AGPs. The AGPs extracted from the plants cultured in the absence of NH4? had a buoyant density range extending from 1.50 to 1.66, while those cultured in the presence of NH4? had a range extending only to 1.62. In short, there was an apparent loss or release of higher buoyant density AGPs from the cultured plants correlated with the NH4+-induced desuppressed leaf and branch development. When the aqueous culture media in which the plants had been growing were similarly analyzed, it appeared that the "lost" AGPs had been released from the plants into the culture medium (Basile & Basile 1993). In an attempt to better identify, isolate, and characterize the AGP(s) that appear to be released from plants correlated with desuppressed cell proliferation, we undertook to obtain monoclonal antibodies (McAbs) that were monospecific for it/them. We report here the successful identification of five McAbs that bind to AGPs that only appear in the culture medium when the culture conditions are changed so as to induce increased cell proliferation in plants correlated with an experimentally induced increase in the size and number of leaves and