Molecular and biochemical studies of cellulose and callose synthase
Cotton fibers consist of almost pure cellulose in the secondary wall stage. To isolate a cellulose synthase gene from cotton, the catalytic region of cellulose synthase in Aceobacter xylinum was used to search the EST database. The 5’-RACE was performed to obtain the full-length cellulose synthase gene, GhCeSA3, from a cotton cDNA library of the secondary wall stage of cotton fibers. The deduced amino acid sequences of the cDNA fragment showed the conserved residues (D, D, D) and QXXRW motif indicating that the cDNA fragment encodes the catalytic region of cellulose synthase. Its 3.2 kb coding region encodes 1067 amino acids with the predicted molecular mass of 119 kDa, eight transmembrane helices and seven N-glycosylation sites. The conserved domains of the GhCeSA3 include a cysteine rich region at the N-terminus, hypervariable regions, and plant specific and conserved regions. Initial RT-PCR results indicate that the GhCeSA3 may be expressed minimally at 7 days of post anthesis (DPA), more at 14 and 21 DPA and less again at 28 DPA. Western analysis showed the presence of cellulose synthase in cotton fibers, Arabidopsis and mung bean. When the solubilized enzyme was incubated with UDP-glucose, microfibrils were synthesized in vitro. Specific antibody localization indicated that the cellulose synthase remained tightly associated with the cellulose microfibrils. The average diameter of microfibrils and cellulose synthase aggregates was estimated to be 2.49±0.36 nm and 30±8.02 X 36.88±7.47 nm, respectively. Immobilized anti-GhCeSA3 antibodies in a column were able to adsorb β-glucan synthase activity indicating that the antibodies specifically retained active cellulose synthases. Loading the solubilized enzymes onto the anti-GhCeSA3 antibody column and supplying with UDP-glucose, cellulose I and II decorated with CBH-I gold were observed. The diameter of the in vitro synthesized cellulose microfibril aggregate was measured to be 2.05±0.30 nm. The cellulose synthesizing protein aggregates were found at the ends of cellulose microfibrils which were labeled specifically with the anti-GhCeSA3 antibodies. They were observed in different shapes such as doughnuts, globular, and tooth-shaped with the estimated diameter of 30.47±7.97 X 38.21±7.70 nm. The estimated diameter is larger than the rosette TC found in freeze-fracture replica indicating that the proteins observed were on the cytoplasmic side of the proteins deeply projecting into the cytoplasm away from the plasma membrane. No callose synthase was found in the anti-GhCeSA3 antibody column. Anti-callose synthase antibodies immobilized in a column were also able to adsorb β-glucan synthase activity indicating that the antibodies specifically retained active callose synthase. Callose synthase and potential associated polypeptides were then eluted from the anti-callose synthase antibody-protein A beads. Conversely, no cellulose synthase was detected. These results indicated that cellulose and callose synthase are definitely different proteins located in different enzyme complexes. Identification of the cellulose synthase gene and their products specifically retained in the cellulose synthase antibody column have greatly advanced our understanding of cellulose biosynthesis in vascular plants.