| Tuber indicum Cooke&Massee,a typical Chinese black truffle,is the most commercial member of Tuber among over sixty species in China due to their highest production and widest distribution.Besides in southwest China(i.e.,Yunnan and Sichuan province),it was also found in northeastern China in recent years.Tuber spp.have a long growth period,and the mature time ranges from the beginning of winter to February or March of the following year.However,immature truffle ascocarps were usually sold early in July every year in the local market.The unscientific collection behavior not only leads to a serious resource waste,but also blocks the transmission of the genetic materials and species.Thus,it is urgent to detect and comparatively analyze the volatile compounds,metabolites,and their molecular mechanisms of truffle with harvest time for quality evaluation and resource conservation of Chinese truffle.In this study,T.indicum ascocarps regularly collected in truffle natural production areas in China from five harvest times were used to explore the changes of gene expression,metabolite accumulation,chemical components,and related activities using HS-SPME-GC-MS,UPLC-MS/MS,RNA-seq and so on.The main results are as follows:1.Volatile organic compound(VOCs)of T.indicum ascocarps from different harvest times were analyzed.Tota1 315 compounds were analyzed and 1-octen-3-ol had the highest content in all analyzed samples(32.29-68.38%).Although sulfur-containing compounds were detected in the samples collected from every month,only ascocarps from August had the highest content of S-containing compounds(2.48%),which was significantly higher than that of other months.By stepwise discriminant analysis,the following five VOCs could effectively distinguish the T.indicum ascocarps in different harvest times:acetaldehyde,1-pentanol,4-ethyl-2-methoxy-phenol,5-methyl-2-phenyl2-hexenal,and 1-tridecene.This statistical model established was effective in discrimination the ascocarps of T.indicum from different geographical sources.2.Medium and large polar metabolites in T.indicum ascocarps from different harvest times were identified.Using UPLC-MS/MS,663 medium and large polar metabolites were detected in T.indicum ascocarps.According to the types of compounds,they were divided into 9 categories,including 120 lipids,90 phenolic acids,78 organic acids,71 alkaloids,115 amino acids and their derivatives,67 nucleotides and their derivatives,12 flavonoids,2 terpenoids and 108 other compounds.Some compounds were detected only in the ascocarps at a certain stage of maturity,for example,2,4dichlorophenoxyacetic acid was only detected in completely immature ascocarps collected in July,and p-aminoanisole,6-methoxyquinoline-4-carboxylic acid,1-Ogentisoyl-D-glucoside,protocatechuic acid-4-O-glucoside,vanilloloside and 3(hydroxymethyl)phenol were only detected in completely mature ascocarps collected in October and November.The Fold Change value and VIP value of OPLS-DA model were used to screen the differential metabolites in T.indicum ascocarps from five harvest times and found that the changes of metabolite content with the maturation of T.indicum ascocarps were inconsistent.Compared to the immature ascocarps,there were 81 up-regulated and 179 down-regulated differential metabolites in the mature ascocarps,including 54 lipids and 27 nucleotides and their derivatives down-regulated.The relative content of sphingolipids increased first and then decreased during the maturation and the relative content of two terpenoids,betulin and 11-keto-ursolic acid,decreased with the maturation.The content of amino acids,saccharides and flavones varied with the maturation,depending on the specific compound.3.Gene expression pattern of T.indicum ascocarps from different harvest times was analyzed.RNA-seq was performed on T.indicum ascocarps from different harvest times based on Illumina sequencing platform.659738640 clean reads were obtained.with above 5.9G clean bases and 88%Q30 for each sample.Further screening the differentially expressed genes of T.indicum ascocarps in adjacent harvest time showed that there was the largest number of differentially expressed genes between August and September,with 570 up-regulated and 21 7 down-regulated genes,suggesting that August to September was the critical period for the development of T.indicum ascocarps.GO functional classification significantly enriched with differentially expressed genes of T.indicum ascocarps from adjacent harvest time was membrane,ribonucleoprotein complex biogenesis,ribosome biogenesis,organonitrogen compound biosynthetic process and sulfur compound metabolic process.KEGG pathways,including 2-oxocarboxylic acid metabolism,biosynthesis of secondary metabolites,pantothenate and CoA biosynthesis,biosynthesis of amino acids,sulfur metabolism,ribosome biogenesis in eukaryotes and nitrogen metabolism,were significantly enriched with differentially expressed genes.88 genes related to the VOCs synthesis and metabolism were screened from four pathways including sulfur metabolism and the synthesis of S-containing VOCs,Ehrlich pathway and the synthesis of aldehydes and alcohols,synthesis from fatty acids and synthesis of isoprenoids,and the expression level of these genes inconsistently changed with the maturation,which preliminarily revealed the molecular mechanism of VOCs change with the maturation.The biosynthesis of secondary metabolites and the biosynthesis of amino acid were simultaneously significantly enriched with a large number of differentially expressed genes and differential metabolites.4.Chemical composition and antioxidant activities of T.indicum ascocarps from different harvest times were preliminary explored.Using Folin-Ciocalteu method,Al(NO3)3-NaNO2-NaOH colorimetry method,DPPH assay,ABTS assay and FRAP assay,total phenolic content,total flavonoid content and antioxidant activities of T.indicum ascocarps collected from five harvest times were analyzed.The results showed that T.indicum ascocarps collected in July possessed the highest amount of flavonoids(3.89 mg/g dw)and the highest ABTS·+ scavenging activity(EC50=3.73 mg/mL).In addition,those ascocarps collected in August contained the highest phenolics content(4.78 mg/g dw),the highest DPPH· radical scavenging activity(EC50=3.73 mg/mL)and ferric reducing activity power(243.63 μmol FeSO4/g).The study reveals T.indicum ascocarps collected in early time of maturity did yield significantly higher content of phenolics and flavonoids and possessed stronger antioxidant activity than those collected in other months.This study contributes to preliminary understanding the influence of harvest time on the types and contents of the VOCs and metabolites of T.indicum ascocarps and provided a theoretical basis for the identification of truffle in different harvest times.This study will be helpful for the chemical composition and quality assessment of Chinese black truffle and will also provide a basis theory for the development of pharmaceutical production and functional food on T.indicum. |
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