Immunogold Labeling of Rosette Terminal Cellulose-Synthesizing Complexes in the Vascular Plant Vigna angularis

Abstract

The catalytic subunit of cellulose synthase is shown to be associated with the putative cellulose-synthesizing complex (rosette terminal complex [TC]) in vascular plants. The catalytic subunit domain of cotton cellulose synthase was cloned using a primer based on a rice expressed sequence tag (D41261) from which a specific primer was constructed to run a polymerase chain reaction that used a cDNA library from 24 days postanthesis cotton fibers as a template. The catalytic region of cotton cellulose synthase was expressed in Escherichia coli , and polyclonal antisera were produced. Colloidal gold coupled to goat anti-rabbit secondary antibodies provided a tag for visualization of the catalytic region of cellulose synthase during transmission electron microscopy. With a freeze-fracture replica labeling technique, the antibodies specifically localized to rosette TCs in the plasma membrane on the P-fracture face. Antibodies did not specifically label any structures on the E-fracture face. Significantly, a greater number of immune probes labeled the rosette TCs (i.e., gold particles were 20 nm or closer to the edge of the rosette TC) than did preimmune probes. These experiments confirm the long-held hypothesis that cellulose synthase is a component of the rosette TC in vascular plants, proving that the enzyme complex resides within the structure first described by freeze fracture in 1980. In addition, this study provides independent proof that the CelA gene is in fact one of the genes for cellulose synthase in vascular plants. INTRODUCTIONCellulose is the most abundant biopolymer on earth and is the major constituent of the plant cell wall (Franz and Blaschek, 1990;. Cellulose is a biopolymer consisting only of ␤ -1,4-glucans. Approximately 40 ␤ -glucan chains are synthesized from a multimeric enzyme complex, and these chains associate immediately upon synthesis to form the crystalline entity known as a microfibril . Crystalline cellulose is defined by its various allomorphs, with cellulose I denoting the most abundant native crystalline form .One of the great enigmas in plant biology is the biosynthesis of cellulose. During the past 50 years, the site of cellulose synthesis has been investigated intensively, but until now, there has been no direct proof for the existence of a multimeric enzyme complex located in the plasma membrane of vascular plant cells. Roelofsen (1958) first suggested that cellulose might be assembled by a large enzyme complex at the growing tip. As early as 1972, Dobberstein and Kiermayer (1972) had visualized ordered particle complexes within "f-vesicles" of the Golgi apparatus in the green alga Micrasterias denticulata. These particles were implicated in the biosynthesis of cellulose. This work is significant historically, because the first observation of what was later to be beautifully imaged by freeze fracture was from sectioned material. Thus, the ordered granule complex, first postulated by Preston (1964), was found only eight years later, but it was not until the freeze-fracture techniq...

Biogenesis of Cellulose Nanofibrils by a Biological Nanomachine

“…In 1999, immunolabeling in conjunction with FF-TEM showed that the rosette CSC contained CesA protein (6). The rosette region of the CSC is about 25 nm in diameter and contains six intramembrane particles.…”

Cell Wall Polysaccharide Synthesis

“…The rosettes have been shown to be cellulose synthase by immunological methods (12). The only known components of cellulose synthase in higher plants are the CESA proteins.…”

Cellulose synthesis: mutational analysis and genomic perspectives using Arabidopsis thaliana

“…2). Predictions [24,77] and now experimental evidence [79] place this central region facing the cytoplasm so that only the small regions between transmembrane domains 1 and 2, 3 and 4, 5 and 6, and 7 and 8 contact the wall. A highly conserved cysteine-rich sequence precedes the first two transmembrane domains.…”

“…Proteins immunologically related to the central catalytic domain of a cotton CesA occur in characteristic clusters of six particles on the cytoplasmic face of the Vigna angularis plasma membrane [79] where they could use cytoplasmic UDP-Glc. Whether any other proteins occur in the same structures remains to be determined.…”

“…The catalytic domains of the CesA proteins and of the type III endo-1,4-b-glucanase are exposed at the inner [79] and outer faces of the plasma membrane [80], respectively. If synthesis proceeds from cytoplasmic substrate (UDP-Glc) to external product (cellulose), the endo-1,4-b-glucanase-dependent step will therefore follow the glycosyltransferase-dependent step.…”