Subcellular distribution of arabinogalactan proteins in pollen grains and tubes as revealed with a monoclonal antibody raised against stylar arabinogalactan proteins

Abstract

Summary. Monoclonal antibody PCBC3, raised against stylar extracts from Nicotiana alata flowers, was deduced from enzymelinked immunosorbent assays and inhibition of immuno-gold labelling on tissue sections to bind specifically to carbohydrate epitopes on arabinogalactan proteins (AGPs) but not to other arabinosecontaining cell wall polysaccharides. When pollen grains of N. tabacum were hydrated in fixative, PCBC3 bound to vesicles in the vicinity of the endoplasmic reticulum but, when grains were hydrated for 20 rain in culture medium before fixation, binding was restricted to the plasma membrane. The generative-cell plasma membrane was also labelled in grains of Lycopersicon peruvianum. In pollen tubes of N. tabacum grown in liquid culture, the AGPs detected by PCBC3 were located in several regions, including the plasma membrane, tubular-vesicular structures (plasmalemmasomes) at and under the plasma membrane, and multilamellar bodies within vacuoles, features generally associated with endocytosis. Labelling was not evident in secretory vesicles or the plasma membrane at the pollen-tube tip. The AGPs detected with PCBC3 were also present in pollen-tube walls, near the interface between the inner, callosic layer and the outer, fibrillar, pectic layer. Pollen tubes of N. tabacum grown in medium lacking added CuSO4 produce a wall with an abnormally thickened fibrillar layer, and this layer was uniformly labelled with PCBC3. The disposition of wall AGPs thus changes in pollen tubes of different morphologies. Keywords: Arabinogalactan protein; Endocytosis; Nicotiana;Pollen; Pollen tube.Abbreviations: AGP arabinogalactan protein; c~-L-Araf c~-L-arabinofuranose; ELISA enzyme-linked immunosorbent assay; MAb monoclonal antibody; PBS phosphate-buffered saline.

Distribution of cell wall components in Sphagnum hyaline cells and in liverwort and hornwort elaters

“…The plasma membrane AGP epitope identified by JIM8 has been found in cells of the embryo, the sexual organs of flowers, and pre-lignified xylem vessels, all of which have an altered cell wall composition (Pennell et al 1991). AGPs identified by JIM13 have been detected in the cell walls of growing pollen tubes (Li et al 1992;Ferguson et al 1999) and implicated in the development of root tissue Rae et al 1991) andtracheary elements (McCann 1997;Dolan and Roberts 1995;Schindler 1995). JIM13 labeling in Megaceros secondary walls indicates the presence of AGPs and is consistent with observations in higher plants, particularly in tracheary elements, where it is believed to play a functional role.…”

“…CCRC-M7 [which recognizes arabinosylated (1 fi 6)-linked b-galactan] and CCRC-M1 [terminal a-(1 fi 2)-linked fucosyl residue of XG] were a gift from the Complex Carbohydrate Research Center, Athens, Ga, LM5 [which recognizes (1 fi 4)-b-galactan] was purchased from PlantProbes, Leeds, UK, and anti-(1 fi 3)-b-glucan antibody was purchased from Biosupplies, Australia. Immunogold labeling was performed as described by Ferguson et al (1998Ferguson et al ( , 1999. Primary antibodies omitted from the labeling procedure acted as a control.…”

Immunolocalization of LM2 arabinogalactan protein epitope associated with endomembranes of plant cells

“…Very recently several AGP epitopes have been found in vesicles located in the vicinity of ER (Ferguson et al 1999) or in Golgi apparatus (Vicr6 et al 1998, Winicur et al 1998) of some dicotyledonous species. Ferguson et al (1999) used monoclonal antibody PCBC3 which recognizes both arabinosyl and galactosyl residues in relatively large (0.4 gm) electron-lucent vesicles near the ER of tobacco cells. Vicr6 et al (1998) have reported that antibody Gal4 against ~-(1---~6)-D-galactan labels trans-type cisternae and vesicles of Golgi apparatus within flax meristematic root cells.…”

Pollen and pistil in the progamic phase

“…Observation of in vitro-cultured pollen tubes of tobacco, petunia and lily suggest that at least part of the primary wall is lost during growth (Derksen et al 1995b(Derksen et al , 1999b. Deposition of the probably more persistent, cellulosic part of the primary wall occurs at the tip and continues into the tube, where the secondary wall is formed (Derksen et al 1995a(Derksen et al ,b, 1999bFerguson et al 1999). Cellulose of both the primary and secondary walls and callose of the secondary wall are supposedly deposited by synthases in the plasmamembrane that originally must have been delivered in the tip by the SV.…”