Analysis Of Active Compounds From Chestnut Shell And Mechanism Of Starch Accumulation In Kernels Of Castanea Mollissima

Chinese chestnut(Castanea mollissima Blume)belonging to the family of Fagaccac and chestnut genus is a kind of economic arbor plant.It is called the king of dried fruit owing to its abundant starch.Moreover,the chestnut shell contains a variety of effective active ingredients.On the one hand,the present work aimed to extract and separate the active ingredients from chestnut shell,and evaluate their biological activity to identify compounds with high activity.On the other hand,the different developmental Chinese chestnut kernels were used as experimental materials for the analysis of termporal alterations on growth properties and starch accumulations in this work.Based on the preliminary results,the Chinese chestnut kernels at key developmental stages were selected as samples for mRNA transcriptome sequencing analysis by Illumina-Solexa.The key genes encoding the functional enzymes and transcriptional factors involved in starch biosynthesis were identified,and further confirmed with the results of qRT-PCR in developing Chinese chestnut kernels.The obtained results were summarized as follows:1.The crude alcoholic extract of chestnut shell was extracted by ethyl acetate and n-butyl alcohol successively,and then their antioxidant activities were determined by DPPH radical scavenging,ABTS radical scavenging and ferric-reducing antioxidant power(FRAP)assays.The results showed that both ethyl acetate and n-butyl alcohol extracts of Chestnut shell exhibited remarkable DPPH radical scavenging activity,ABTS radical scavenging activity and reducing power in vitro.Between the two extracts,the n-butyl alcohol extract possessed higher antioxidant activity in all three assays.The alpha glycosidase enzymes inhibitory activity of ethyl acetate and n-butyl alcohol extracts from chestnut shell was measured by PNPG method.It turned out that both the two kind of extracts showed high inhibitory activity of alpha-glycosidase enzymes.Furthermore,the inhibitory activity of n-butanol extract was also higher than that of ethyl acetate.Qualitative analysis was performed by UPLC-QTOF-MS/MS for analysis of major active components in n-butyl alcohol extracts,and thirteen compounds were tentatively identified in current research.The compounds were mainly phenolic compounds,which could be divided into ellagic acid,gallic acid and caffeoylshikimic acid derivatives.Others,including organic acids,sugars and terpenoids,were also found in n-butyl alcohol extracts,in which quinic acid,cinncassiol A 19-O-beta-D glucoside and glaucarubol-15-O-beta-D-glucopyranoside were identified for the first time in chestnut shells.Another unknown ellagic acid derivative(C23H31O10)may be discovered for the first time which needed to be further studied.2.The Chinese chestnut kernels from ten different developmental stages were used as the experimental materials for the measurement of fresh weight,fruit index,sucrose,starch,amylose,amylopectin and other nutrient content as well as microscopic observation on the accumulation of starch granules.It was found that at 10-40 DAF,the seed structure(kernel)had not developed yet.Periodic acid-Schiff(PAS)staining showed that the cotyledons began to stretch 50 DAF later.The fresh weight increased very slowly during 10-50 DAF followed by a much faster growth rate.In particular,the fresh weight increased almost linearly during 70-90 DAF,and then maintained steady,indicating that the growth of Chinese chestnut kernels was mainly during 50-90 DAF.Also,PAS staining demonstrated that the critical period for starch granule accumulation happened during the same period of 70-90 DAF.The starch content displayed a notable accumulation from 70(58.29 ± 3.26 mg/g)to 90 DAF(390.88 ±7.45 mg/g).Additionally,the ratio of amylose to amylopectin was approximately 2.41:1 in mature chestnut kernel.3.Based on the dynamic patterns of growth properties,starch contents and the microscopic observation results of starch granule accumulation,six key Chinese chestnut kernels developing stages,including 50,60,70,80,90 and 100 DAF,were used as experimental materials for mRNA sequencing analysis by Illumina-Solexa.The resulting approximately 40 millions valid reads were generated in each sample,and 38,146 transcripts were obtained after blasting with reference genome.Using 50 DAF as a control,489,350,382,396 and 521 up-regulated,and 1,479,1,847,1,746,1,559 and 1,466 down-regulated transcripts were characterized specifically at 60,70,80,90 and 100 DAF with DESeq software.According to the Venn diagram analysis,a total of 3,885 differential genes were found to be expressed in all the six developmental stages,while 459,150,109,82 and 541 differential genes were specifically expressed in the 60,70,80,90 and 100 DAF of Chinese chestnut kernel,respectively.4.Combined with mRNA transcriptome sequencing analysis and qRT-PCR detection,it was speculated that during the stages of 50-60 DAF,the sucrose transporter(SUC2)unloaded sucrose from sieve into apoplast,where the sucrose was catalyzed by cwINV1 into glucose and fructose and then transported to the sink tissue through HEX6.While during the period of 60-100 DAF,the unloading of sucrose in chestnut kernel was carried out mainly by transfering into the companion cells through plasmodesma and then transported to the sink cells through plasmodesma.Also,it was found that the genes including cSUSY2,pFK4 and UGP related with the metabolism of sucrose were up-regulated at 50-70 DAF.Moreover,several genes(including PFK,GPI,FBA,TPI,PGK,PK and GAPC)involving in the plastid and cytosolic glycolysis pathways were all up-regulated,indicating a carbon flux into starch accumulation via plastid and cytosolic glycolysis in developing Chinese chestnut kernels.And then it proved that the glycolysis provided G-6-P as well as ATP for starch biosynthesis in amyloplast.Due to the production of the pyruvate,the end product of glycolysis entering into tricarboxylic acid cycle was completely oxidized and ATP was generated simultaneously.5.By a combination of the mRNA transcriptome sequencing analysis and qRT-PCR detection,a total of twelve genes(including SUSY2,FK,UGP,GPI2,GPT,AGP3,AGP2,GBSS1,SS1,SS3,SBE3 and PYG)with up-regulated transcript was identified to be related with starch biosynthesis pathway,as was consistent with termporal alterations of starch of Chinese chestnut kernels.It illuminated that they were closely related with starch accumulation in the development process of chestnut kernels.Also with the same analytical method,several transcription factors(including ABI4,WRI1,WRKY19,MYC2,NAC,AIL1 and AIL6)were found in the development process of chestnut kernels,which maybe regulated the starch accumulation.All the study above could lay an important foundation for comprehensive utilization of chestnut resources,and may also provide some reference for molecular improvement of starchy crops or woody starch plants.