Study On The Cell Wall Structure, Composition And Cell Wall Remodeling Enzymes Of Coprinus Cinerea Stalk

Macrofungi usually have significant fruiting bodies with umbrella-type appearance,typically like basidiomycetes mushroom Coprinopsis cinerea whose fruiting body autolysis in a very short time after the umbrella's opening and releases basidiospores.One of the most interesting phenomena in this morphogenesis is that the stipe increases many times in length in a very short time.Previous studies have shown that the main growth region of the stipe is the part under the pileus.This way of growth is similar to the coleoptile growth of plants which mainly depends on the growth of cell elongation.On the one hand,the growth of stipe cells need to get rid of the restriction of the cell wall,so it is necessary for the cell walls to have enough plasticity to make sure this new cell wall components can be embedded into existing cell wall in order to achieve elongation;on the other hand,the cell wall to provide sufficient mechanical strength to maintain integrity of the cell shape,which also means that the cell wall should be rigid enough.Thus,Stipe cell wall undergoes significant reconstruction in the elongation process of the fruiting body.The previous studies on different regions of C.cinerea stipe elongation and cell wall extension focus on 27 mm long fruiting bodies.In this paper,we study the remodeling of cell wall construction in stipe elongation growth on cell wall ultrastructure of C.cinerea elongating stipe and cell wall molecular organization.Moreover,we cloned the enzymes may participate in cell wall remolding during this process and heterologous recombined them,identified their related biological function.In order to attain the distinct differences in every stipe region,the 60mm-lengthed C.cinerea fruiting body were selected in this study.The stipe is divided into six regions from the top to the base,taking 1-10 mm from the upper part,20-30 mm from the middle,40-50 mm the basal and 50-60 mm swollen plectenchyma,so we can get a clear comparison of the differences in microcosmic structure.By using field emission scanning electronic microscopy(FESEM),a large amount of granular protrusions can be found on the outer cell wall surfaces in both elongating and non-elongating stipe regions,but the granular protrusions on inner cell wall surfaces can only be found in non-elongating stipe regions.Using alkali to remove granular protrusions,amorphous materials overlying on both the inner and outer cell wall surfaces were found in the non-elongating stipe regions.?-1.3-glucanase treatment can not only remove those granular protrusions and underlying amorphous materials on the wall surfaces but also remove wall matrices embedding chitin microfibrils on the cell walls of most stipe regions,except for the outer cell wall surfaces of the non-elongating stipe regions where most of the wall matrices remained.The chitin microfibrils were closely and transversely arranged on both the inner and outer cell wall surfaces in the elongating apical stipe region,whereas they were loosely and transversely arranged on the inner cell wall surfaces and further became sparser and even randomly arranged on the outer cell wall surface in the non-elongating stipe regions.We propose that the surface deposition of granular protrusions and amorphous materials and the change of microfibril architecture and wall matrices may cause the loss of wall plasticity and cease of stipe elongation.The alkali-insoluble cell wall(3-1,3-glucan molecular organization of the stipe upper and basal region are investigated.The oligosaccharide fragments of the alkali-insoluble cell wall hydrolysis with endo-1,3-beta-glucanase analyzed by high performance anion exchange chromatography with pulsed amperometric detector coupled to mass spectrometry(HPAEC-PAD-MS),there are fewer ?-1,6 branch points in the ?-1,3-glucan chains of elongated upper region,while there are more ?-1,6 branching points in the ?-1,3-glucan chains of non-elongated basal region.There is no difference in the ?-1,4-linkages between upper region and basal region.Obviously,the increased branching points may cuase more crosslink between the various glucan and cell wall matrix could cease of stipe elongation.There are significantly more glucanse-resistant components in the basal region than in the upper region of stipe.The dissertation has a further research on the Pichia pastoris reconstruction expression of the related cell wall reconstruction enzymes which are involved in the process of stipe enlongation.The enzymes include chitinases,chitin deacetylases andendo-l,3-beta-glucanosyltransferases.The chitinases can soften cell wall in the process of stipe enlongation and growth,which can make the cell wall have some plasticity.The enzymatic characteristics of recombined chitinase Chi? showed that the Chi? is a novel chitinase,both with endochitinase activity and exochitinase activity.With a low degree of oligomer of chitin oligosaccharide as a substrate,such as(GlcNAc)3-5,Chi? having exochitinase activity and the release of N-acetylglucosamine monomer from(GlcNAc)3-5;When the chitin oligosaccharide degree of polymerizationismore than five,it having an endochitinaseactivity and the release different lengths of chitin oligosaccharides from(GIcNAc)6-7.In the previous study,qPCR analysis shows that this enzyme has significant expression during the growth of C.cinerea.It's suggested that Chi? may participates in the cell wall softening in process of stipe enlongation and degration of the cell wall in the process of auto lysis of the fruiting body.ChiEn1 which contains multi-enzyme activities,including exo-N,N'-diacetyl-chitobiohydrolase activity,transglycosylation activity,and endochitinase activity.Notably,these three activities of ChiEnl shift relative to each other with the degree of polymerization of chitin oligosaccharides.ChiEnl released(GIcNAc)2 residues from the non-reduced end of(GlcNAc)3-5 via exo-N,N'-diacetylchitobiohydrolase activity,whereas it cleaved(GlcNAc)3 and relative chitin oligosaccharides from(GlcNAc)6-8 via endochitinase activity.ChiEnl also transferred a(GlcNAc)2-residue between chitin oligosaccharides(GlcNAc)4-6,but not between chitin trisaccharides(GlcNAc)3,to produce longer chitin oligosaccharides,and the latter was consequently converted to(GlcNAc)3.Finally,(GlcNAc)3 was degraded to(GlcNAc)2 and GlcNAc.ChiEn1 cleaved off(GlcNAc)3 only from the non-reduced end of(GlcNAc)8,but can hardly hydrolyse the unsoluble chitin substrates.We propose that although ChiEn1 does not possess ?-N-acetylhexosaminidase activity or monomer cleaving exochitinase activity,it converts soluble chitin oligosaccharides to GlcNAc monomers via a new hydrolysis mechanism-the synergistic action of its three endogenous enzyme activities.The gene has more expression in the process of pileus expansion and autolysis,but has less expression in stipe,which is in accordance with its enzyme features when with high concentration of ChiEn1,it exhibits strong hydrolysis capacity,while with a low concentration,it exhibits strong transglycosylation capacity.It can be speculated that the enzyme is involved in the extension of chitin chains in the process of stipe enlongation and cooperate with other chitinases degraded cell wall chitin components in pileus expansion and autolyzed hydrolysis process of C.cinerea.Chitin is deacetylated by the enzyme chitin deacetylase to form chitosan and acetate.The chemical and physical properties of chitin and chitosan are very different.It can be speculated that chitin deacetylase may participate in the change of reconstruction and modification of physics and chemistry properties of cell wall in the process of stipe maturation.The activity of recombined Cdal was detected and analyzed by HPAEC-PAD with chitin oligosaccharides Studies have shown that it may be a degree of polymerization of 5 or more chitin oligosaccharides polymerization from 1 to 6.The product is patially deacetyled chitin oligosaccharides and it have no effect on N-acetyl-glucosamine.By the quantitative PCR analysis of cda1,we found that the cdal expression level following with the stipe cell wall maturation degree.The Cdal may participate in the reconstruction and modification of chitin in the process of cell wall maturation.The enzyme endo-1,3-beta-glucanosyltransferases splits a ?-1,3-glucan molecule internally and transfers the newly generated reducing end to the non-reducing end of another ?-1,3-glucan molecule,resulting in the elongation of the glucan chain.There are just two endo-1,3-beta-glucanosyltransferases in the genome of C.cinerea,Gltl and Glt2,with highly homologous sequences and close protein molecular weight size.They are strictly the pH dependent enzyme with the same activity and only active in acidic conditions which used laminarinhexaose as a substrate can produce laminarinoligosaccharides with a degree of polymerization from 2 to 23.While the polymerization degree less than or equal to four,there is no transglycosylation products.It found that rglt1 and rglt2 had higher expression in upper stipe by the quantitative PCR analysis.It is suggested that Gltl and Glt2 may participate in the glucan chains elongation in process of stipe elongation growth.