The location of (1?3)-?-glucans in the walls of pollen tubes of Nicotiana alata using a (1?3)-?-glucan-specific monoclonal antibody

Abstract

The location of the (1→3)-β-glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (1→3)-β-glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (1→3, 1→4)-β-glucan-BSA conjugate. Binding was inhibited by (1→3)-β-oligoglucosides (DP, 3-6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (1→3)-β-linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 10(4)M(-1). When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (1→3)-β-glucan in the inner wall layer of thin sections of the N. alata pollen tubes.

The glucans extracted with warm water from diatoms are mainly derived from intracellular chrysolaminaran and not extracellular polysaccharides

“…In the present study, a specific monoclonal antibody raised to laminaran (Meikle et al, 1991) localized the 1,3-b-D-glucan to vacuoles in P. tricornutum, C. fusiformis, and T. pseudonana (Fig. 2).…”

“…The blocks of C. fusiformis cells prepared by freeze substitution were a kind gift of Drs Nicole Poulsen and Nils Kro¨ger of the University of Regensburg, Germany. The primary antibody used for immunolocalization was a monoclonal anti-1,3-b-D-glucan antibody raised to laminaran (Meikle et al, 1991) from Biosupplies (Melbourne, Australia, product #400-2). The secondary antibody was a goat anti-mouse antibody conjugated to 18 nm gold particles from British Biocell International (Cardiff, Wales, product #EM.GAM18).…”

Pattern of Deposition of Cell Wall Polysaccharides and Transcript Abundance of Related Cell Wall Synthesis Genes during Differentiation in Barley Endosperm

“…Grain samples from 10 to 28 DAP were prepared for microscopy and changes to the endosperm were monitored by taking transverse sections of the developing grains. The distribution and relative abundance of polysaccharides in the differentiating grain were detected on sections using monoclonal antibodies raised against specific polysaccharide epitopes including (1→3)-b-D-glucan (Meikle et al, 1991), xyloglucan LM15 (Marcus et al, 2008), (1→3,1→4)-b-D-glucan (Meikle et al, 1994), hetero-(1→4)-b-Dmannan (Pettolino et al, 2001), and arabino-(1-4)-b-D-xylan LM11 (McCartney et al, 2005). Sections for light microscopy (1 mm) were either stained in toluidine blue to highlight tissue architecture or processed for immunogold labeling.…”

Immunolocalization and possible roles of pectins during pollen growth and callose plug formation in angiosperms

“…Immunoelectron microscopy is useful for identifying the localization of pollen wall polysaccharides; researchers have used various antibodies recognizing a-L-arabinofuranosyl residues (Anderson et al 1987), callose (Meikle et al 1991, Geitmann et al 1995a, Hasegawa et al 1996, Roy et al 1997, Ferguson et al 1998, and pectins (Li et al 1994, Geitmann et al 1995a, Geitmann et al 1995b, Li et al 1995, Jauh & Load 1996. Using JIM5 against de-esteri®ed pectin or JIM7 against esteri®ed pectin, researchers have reported the localization of pectins in pollen of Arabidopsis thaliana (Van Aelst & Van Went 1992), Nicotiana tabacum (Geitmann et al 1995b, Li et al 1995, Brugmansia suaveolens (Geitmann et al 1995a), and Lilium longi¯orum (Jauh & Load 1996).…”