Abstract
Cotton fibers are an excellent model for understanding of cellulose biosynthesis in higher plants. In this study, we determined a high cellulose biosynthesis activity in vitro by optimizing biochemical reaction conditions in cotton fibers. By adding a commercial cellulase enzyme into fibers extraction process, we extracted markedly higher levels of GhCESA1 and GhCESA8 proteins and observed an increase in β-1,4-glucan and β-1,3-glucan products in vitro. LC-MS/MS analysis of anti-GhCESA8-immunoprecipitated proteins showed that 19 proteins could be found in three independent experiments including four CESAs (GhCESA1,2,7,8), five well-known non-CESA proteins, one callose synthase (CALS) and nine novel proteins. Notably, upon the cellulase treatment, four CESAs, one CALS and four novel proteins were measured at relatively higher levels by calculating total peptide counts and distinct peptide numbers, indicating that the cellulase-aid-extracted proteins most likely contribute to the increase in β-glucan products in vitro. These results suggest that the cellulase treatment may aid to release active cellulose synthases complexes from growing glucan chains and make them more amenable to extraction. To our knowledge, it is the first time report about the functional identification of the potential proteins that were associated with plant cellulose and callose synthases complexes by using the cellulase-aided protein extraction.
Highlights
2, 7, 8) are involved in cellulose biosynthesis of secondary cell wall and GhCESA3, 5, 6, 9, 10 are associated with primary cell wall formation in cotton[2,17,18,19]
We could demonstrate a powerful approach for functional identification of the components that are associated with cellulose and callose synthases complexes by performing a proteomic profiling of the cellulase-aid-extracted proteins in cotton fibers
We found that approximately 90% of the total β-1,4-glucan product could be effectively digested by β-1,4-glucanases, and the remaining pellet was defined as crystalline cellulose
Summary
2, 7, 8) are involved in cellulose biosynthesis of secondary cell wall and GhCESA3, 5, 6, 9, 10 are associated with primary cell wall formation in cotton[2,17,18,19]. It has been proposed that wound-induced callose is produced by cellulose synthase as a result of a loss of accessory proteins required for cellulose biosynthesis or a change of conformation during protein extraction[41]. This hypothesis is under debate because a plant callose synthase (designated CALS) has been identified that does not show any significant similarity to plant CESAs42,43. We could demonstrate a powerful approach for functional identification of the components that are associated with cellulose and callose synthases complexes by performing a proteomic profiling of the cellulase-aid-extracted proteins in cotton fibers
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