Evolutionary Pattern Of Structure And Type Of Agrocybe Cylindracea Polysaccharide During The Developmental Process And Preliminary Study On Its Synthetic Pathway

The growth and development of edible fungi is a complex life process,and polysaccharides play an important role in this process.Studies indicated that the growth process of edible fungi can affect the physicochemical properties and structural characteristics of their polysaccharides significantly.Agrocybe cylindracea is a famous edible fungus in China,which has abundant chemical components.As one of the important biological macromolecules of A.cylindracea,polysaccharides contained various bioactivities.While the structural elucidation of A.cylindracea polysaccharides can contribute to explore their health functional roles in depth.However,the structural characterization of A.cylindracea polysaccharides primarily focused on the fruiting body of A.cylindracea at the mature stage,while few studies have been reported on their changes during the growth process.This hinders the application of A.cylindracea polysaccharides in the food industries and the development of related health products.Therefore,the present study selected the A.cylindracea fruiting bodies of four developmental stages as a research subject,including tide-turning stage,immature stage,mature stage and pileus expansion stage.Firstly,the microscopic morphology,texture characteristics,chemical composition and monosaccharide composition of A.cylindracea at the different stages were compared and comprehensively evaluated.Secondly,the strauctural chracterisitics of polysaccharides from A.cylindracea water extract/alkali extract at four developmental stages were systematically studied.And the possible biosynthetic pathways of polysaccharides during development were explored by omics technology.The main research contents and findings are summarized as:(1)The microscopic morphology,texture characteristics and chemical components of A.cylindracea at different stages were comprehensively compared.With the growth of A.cylindracea,the stipe cells were larger gradually and the intercellular space widened significantly.Meanwhile,their hardness,elasticity,chewiness and cohesion remarkably increased.In addition,the total sugar and glucose contents of A.cylindracea also showed an increasing trend,reaching the highest at the pileus expansion stage.Their contents were 45.8% and 23.8%,respectively.However,the contents of total protein and amino acid decreased significantly.Compared with the tide-turning stage of A.cylindracea,the total amino acids at the pileus expansion stage decreased by 50.7%,mainly including aspartic acid,glutamic acid,phenylalanine,lysine and arginine.(2)A.cylindracea water-extracted polysaccharides at different developmental stages were prepared by hot water extraction,as well as its α-glucan was further characterized.The water-extracted polysaccharide from A.cylindracea increased significantly at the pileus expansion stage and the increased polysaccharide was α-glucan.Structural identification revealed that this α-glucan might be a glucose polymer(glycogen).This polysaccharide was further prepared using gradient ethanol-precipitation method with a yield of 30.0% and an α-glucan content of 79.8%.The results of transmission electron microscopy and iodine staining confirmed that the polysaccharide was glycogen,which had typical glycogen morphology of β particles.This glycogen had a molecular weight of 1375 k Da.Its main chains were comprised of →4)-α-D-Glcp-(1→ and →4,6)-α-D-Glcp-(1→ residues at a ratio of 5:1,and α-D-Glcp-(1→ residue attached to the O-6 as the side chain.In addition,the glycogen and natural starch exhibited the entirely distinct Maltese cross characteristic and unique crystalline structure.(3)A.cylindracea water-extracted polysaccharides were treated using α-amylase to obtain the heteroglycans from water extract and their structures were further characterized.The yields of heteroglycans exhibited a significant reduction with the growth of A.cylindracea,but their structural characteristics were similar.Four water-soluble heteroglycans,ACPM-Et50-I ACPM-Et50-II,ACPM-Et60 and ACPMEt80 were isolated from A.cylindracea at each maturity stage using the combination of gradient ethanol precipitation and gel column.The results showed that the chemical composition,monosaccharide composition and structural features of ACPM-Et50-I ACPM-Et60 and ACPM-Et80 had no significantly change at all four stages.Among them,ACPM-Et50-I was a branched heterogalactan with →6)-α-D-Galp-(1→ as main chain,branched at the O-2 position.The side chains were consisted of α-L-Fucp-(1→and α-D-Galp-(1→ residues.In contrast,ACPM-Et60 and ACPM-Et80 were branched heteroglucans with backbone of →6)-β-D-Glcp-(1→.However,ACPM-Et50-II was a glucuronofucogalactoglucan at the tide-turning stage but a glucuronofucoglucogalactan at the pileus expansion stage.Their main chains were composed of →6)-β-D-Glcp-(1→,→3,6)-β-D-Glcp-(1→,→4)-α-D-Glc Ap-(1→,→6)-α-D-Galp-(1→ and →2,6)-α-D-Galp-(1→.Their ratios were 3:1:1:1:1 and2:1:1:3:2,respectively.The side chains were α-L-Fucp-(1→,β-D-Glcp-(1→ or →3)-β-D-Glcp-(1→ residues.This work is first to report a glucuronofucogalactoglucan in A.cylindracea and reveals its dynamic evolution signatures during maturation.(4)Alkaline-extracted polysaccharides were obtained from the residue after water extraction by using sodium hydroxide solution extraction,as well as their structures were further characterized.Their yields,chemical composition and monosaccharide composition varied obviously in different developmental stages.The yields and protein content of alkaline-extracted polysaccharide declined with the growth of A.cylindracea,while the neutral sugar and glucose contents increased significantly,reaching 68.5% and 55.8% respectively at the pileus expansion stage.In contrast,alkali-extracted polysaccharides had similar structural characteristics at the four stages.From each growth stage,four polysaccharide subfractions were isolated by gradient ethanol precipitation,including JACP-Et30,JACP-Et50,JACP-Et60 and JACP-Et70.JACP-Et30 from four stages and JACP-Et50 from initial three stages were identified heteroglucans with→3)-β-D-Glcp-(1→ and →6)-β-D-Glcp-(1→residues as main chains respectively,whereas other subfractions can be considered as β-(1→6)-Dglucans containing minor glucuronic acid.These subfractions were predominantly replaced by Glcp residues at the O-3 and O-6 positions.(5)The relevant genes of A.cylindracea polysaccharides synthesis was annotated and mined by the whole genome and transcriptome sequencing of A.cylindracea at different stages,as well as their biosynthetic pathway was further preliminarily analyzed.The genome size of this A.cylindracea was 68.1 Mb,with a total length of 32760279 bp,containing a total of 18123 coding genes.Among them,there were 694 carbohydrate-related CAZyme genes.Transcriptomic analysis indicated that the tide-turning stage and pileus expansion stage had significant genes expression differences,while the differences between immature stage and mature stage were the minimum.The results were consistent with the chemical composition analysis.KEGG enrichment found that the upregulated differential expression genes in the pileus expansion stage were mainly involved in the pathways of carbohydrate metabolism compared with the tide-turning stage,including fructose and mannose metabolisms,pentose phosphate pathway,glycolysis/gluconeogenesis and the amino sugar and ribose metabolisms.According to the changes of polysaccharides at the different developmental stages,22 key enzymes of polysaccharide synthesis were identified.On the basis,the possible synthetic pathway of polysaccharide was predicted.In addition,the mechanism of glycogen synthesis may be mainly through the synergistic regulation of glycogen synthesis and degradation pathways to promote glycogen accumulation at the pileus expansion stage.In summary,the polysaccharides from A.cylindracea mainly showed the changes of yield rather than the differences of structural types during the developmental process.The yield of water-extracted polysaccharides from A.cylindracea increased significantly(8.5%)at the pileus expansion stage,and the increased polysaccharide could be α-glucan.This α-glucan was identified as glycogen.The heteroglycan of water-extracted polysaccharides can be obtained by enzymolysis with α-amylase.Its yield decreased significantly with the growth of A.cylindracea,and the highest yield(5.7%)was presented in the tide-turning stage.In addition,the yield of alkali-extracted polysaccharides also decreased significantly.However,the purified fractions of polysaccharides at different stages had the same structural types,mainly including glucans and heteroglucans.Glucans include glycogen and β-(1→6)-D-glucans containing minor glucuronic acid.Heterodextran includes heterogalactan and heterodextran.Among them,most polysaccharides possessed an overall similarity in structure type and glycosidic bonds proportions during the growth process,but the structural type of some heteroglucans had changed.It was identified as a glucuronofucogalactoglucan at the tide-turning stage but a glucuronofucoglucogalactan at the pileus expansion stage.In general,A.cylindracea mainly showed the significant increase of glycogen during the pileus expansion stage,which may be related to the expression levels of related enzymes and their encoding genes of glycogen synthesis and degradation.