Study On The Elongation Characteristics Of The Cell Wall Of The Stalk Of Coprinus Cinerea And The Function Of The Cell Wall Protein

Basidiomycete stipe growth is similar to the elongation growth of plants,and cell growth needs to get rid of the shackles of stipe cell wall,but the cell wall extension still lack of further research.At present,there are two kinds of hypotheses on cell wall extension,one is hydrolysis of wall polymers by enzymes,the other is disruption of hydrogen bonding of wall polymers exclusively by turgor pressure stress.In contrast,the theory of plant cell wall extension is expansin proteins extend cell walls by a hydro lysis-free process that disrupts hydrogen bonding between cell wall polysaccharides.However,it is unknown if this mechanism is operative in mushrooms because the chemical composition in mushroom cell walls is different from that of plant cell walls.Previously,we observed an acid-induced short-term wall extension in Flammulina velutipes apical stipes during a 15 min period after been switched from a neutral to an acidic pH.However we failed to further study the characterization and mechanism of stipe elongation of the F.velutipes because of its limited 1 mm fast elongation region.In this study,we report that Coprinopsis cinerea stipes possess a 9 mm fast elongation apical region,which is suitable as a model material for wall extension studies.The elongating apical stipe shows two phases of acid-induced wall extension,an initial quick short-term wall extension during the first 15 min and a lower and gradually decaying long-term wall extension over the subsequent 2 h.After heating,apical stipes lose the long-term wall extension,retaining a lower short-term wall extension.We defined the short-term wall extension as heat-insensitive wall extension,while the long-term wall extension as heat-sensitive wall extension.In contrast,no matter heat or not,the non-elongating basal stipes only show a weaker short-term wall extension.After treated with protease or protein denaturation,apical stipes still only retaining heat-insensitive wall extension.Therefore we propose that the long-term heat-sensitive wall extension is a protein-mediated process involved in stipe elongation,whereas the short-term heat-insensitive wall extension is non-protein mediated process not involved in stipe elongation.Because of difference wall extension activity in different regions of C.cinerea stipes,we find some differentially expressed genes and proteins by comparative transcriptome and proteomics,which can provide the certain basis for the research of wall extension related genes and proteins.In order to further prove the heat-sensitive wall elongation is a protein-mediated process,we purified exogenous expansin-like protein from snail stomach juice using ammonium sulfate precipitation,ion-exchange chromatography and molecular-exclusive chromatography,which can reconstitute heat-inactivated stipe wall extension without any hydrolytic activity.Then we try to isolate and purify endogenous expansin-like protein from C.cinerea stipes.The preliminary results showed that the endogenous expansin-like protein does exist.However we finally did not obtain the purified protein because of various reasons.Moreover characterization and mechanism of the heat-sensitive wall extension and the heat-insensitive wall extension were further study.We speculate that the two types of wall extension may be related to the hydrogen bond between the cell wall polysaccharide components.And the speculation was turned out to be true by hydrogen bond damage agent related experimental.Our results do not support the previous hypotheses that hydrolysis of wall polymers by enzymes,or disruption of hydrogen bonding of wall polymers exclusively by turgor pressure stress is involved in stipe wall extension.We suggest that stipe wall extension may be mediated by endogenous expansin-like proteins that facilitate cell wall polymer slippage by disrupting hydrogen bonding between glucan chains or chitin chains,rather than cleaving covalent bonds.Afterward an extracellular ?-glucosidase was purified from cell walls of C.cinerea elongating stipes,which can not reconstitute heat-inactivated stipe wall extension.And the function of the enzyme in the stipe elongation was further research.The P-glucosidase did not degraded modified-end various ?-glucans,while hydrolyzed various p-glucans with free end and relative oligosaccharides with ?-1,3-,or ?-1,4-,or ?-1,6-linkage.Though this ?-glucosidase possesses glycosyltransferase activity for laminarioligosaccharides,it did not transfer glucose residue from laminaritriose to ?-glucan from stipe cell wall to produce larger ?-glucan molecules,instead,it decrease the molecular size of the stipe wall ?-glucan and produce glucose.Because the molecular size of the wall ?-glucans in the elongating apical stipe is smaller than that of the the non-elongating basal stipes,this ?-glucosidase expressed more highly in the elongating apical stipe and did not induce wall extension of stipe.Our results do not support the previous hypotheses that glycoside hydrolases may function on the branching or elongating of(3-1,3-glucan in stipe elongation.We propose the ?-glucosidase functions on trimming or cutting the ?-glucan side-chains in P-1,3-glucan backbone to prevent them from formation of longer branches in?-1,3-glucan backbone,keeping the wall in a plastic status for wall diffuse growth.