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Transcriptome and Metabolome Analyses Reveal Differences in Terpenoid and Flavonoid Biosynthesis in Cryptomeria fortunei Needles Across Different Seasons

ABSTRACT:

Cryptomeria fortunei (Chinese cedar) has outstanding medicinal value due to its abundant flavonoid and terpenoid contents. The metabolite contents of C. fortunei needles differ across different seasons. However, the biosynthetic mechanism of these differentially synthesized metabolites (DSMs) is poorly understood. To improve our understanding of this process, we performed integrated non-targeted metabolomic liquid chromatography and gas chromatography mass spectrometry (LC-MS and GC-MS), and transcriptomic analyses of summer and winter needles. In winter, the C. fortunei needle ultrastructure was damaged, and the chlorophyll content and Fv/Fm were significantly (p < 0.05) reduced. Based on GC-MS and LC-MS, we obtained 106 and 413 DSMs, respectively; based on transcriptome analysis, we obtained a total of 41.17 Gb of clean data and assembled 33,063 unigenes, including 14,057 differentially expressed unigenes (DEGs). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these DSMs/DEGs were significantly (p < 0.05) enriched in many biosynthesis pathways, such as terpenoids, photosynthates, and flavonoids. Integrated transcriptomic and metabonomic analyses showed that seasonal changes have the greatest impact on photosynthesis pathways, followed by terpenoid and flavonoid biosynthesis pathways. In summer Chinese cedar (SCC) needles, DXS, DXR, and ispH in the 2-methyl-pentaerythritol 4-phosphate (MEP) pathway and GGPS were highly expressed and promoted the accumulation of terpenoids, especially diterpenoids. In winter Chinese cedar (WCC) needles, 9 genes (HCT, CHS, CHI, F3H, F3'H, F3'5'H, FLS, DFR, and LAR) involved in flavonoid biosynthesis were highly expressed and promoted flavonoid accumulation. This study broadens our understanding of the metabolic and transcriptomic changes in C. fortunei needles caused by seasonal changes and provides a reference regarding the adaptive mechanisms of C. fortunei and the extraction of its metabolites.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse phase, Gas Chromatography MS - positive, Liquid Chromatography MS - positive - reverse phase

SUBMITTER: Yingting Zhang

PROVIDER: MTBLS4099 | MetaboLights | 2022-08-12

REPOSITORIES: MetaboLights

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Project description:Radix bupleuri is a traditional Chinese crude drug and it is still widely used in contemporary medicine. Bupleurum chinense DC. and B. scorzonerifolium Willd. are officially recorded as the source species of Radix bupleuri in Pharmacopoeia of the PeopleM-bM-^@M-^Ys Republic of China. The obvious difference in chemical active components and pharmacological action of Radix bupleuri derived from the two species has caused attention. But to date, no definite criterions and regulates are issued based on plant species sources to direct the standard uses of Radix bupleuri for their extracts and traditional decoction pieces in medical market. Using 454-FLX pyrosequencing with one half-plate run for each cDNA library, a total of 419,576 and 416,112 reads, among those 313,483 and 342,263 high quality ESTs were obtained for B. chinense and B. scorzonerifolium, respectively. The average read length was 279 bp and 310 bp, respectively. The assembled 28,897 and 30,912 unigenes for B. chinense and B. scorzonerifolium were successively annotated based on sequence similarity by major public nucleotide/protein databases, Nt, Nr, SwissProt, KEGG, COG, Interpro and GO. Based on the alignments, 71.9% (20,787) and 75.8% (23,426) of the total unigenes for B. chinense and B. scorzonerifolium could be matched to nucleotides from NCBI Nt database. Certain percent from 27.1% (7,820) to 70.8% (20,461) of the total unigenes for B. chinense, and from 26.4% (8,145) to 72.1% (22,289) of the total unigenes for B. scorzonerifolium, could be matched to proteins from different databases. For the same database, similar percent of the total unigenes for B. chinense and B. scorzonerifolium were annotated. A total of 17,117 (59.2%) and 19,416 (62.8%) unigenes for B. chinense and B. scorzonerifolium could be found homologous genes in opposite dataset. According to KEGG annotation, 3,637 and 3,821 unigenes were assigned relating to metabolism in datasets of B. chinense and B. scorzonerifolium, respectively. Carbohydrate metabolism, energy metabolism and amino acid metabolism were the highest three groups in metabolism category and followed by lipid metabolism group for B. chinense. In comparison, lipid metabolism group had most unigenes for B. scorzonerifolium and it had much more unigenes than the same group for B. chinense (827 to 562). A total of 70 and 52 unigenes were related to metabolism of terpenoids and polyketides in two separate datasets, which seems agree with the situation of higher content of terpenoid saikosaponins in B. chinense than B. scorzonerifolium in some extent. To compare the biosynthesis of main bioactive components in roots of B. chinense and B. scorzonerifolium, putative genes which may participate in the biosynthesis of terpenoid, triterpenoid, sterol, ligan and flavonoid were searched according to unigenes annotation. Most lignan and flavonoid biosynthesis related genes were found in both datasets of B. chinense and B. scorzonerifolium, while some terpenoid or triterpenoid related genes were dominantly found in the dataset of B. chinense. This reflected in some extent that these genes expressed in low levels and relative metabolites were biosynthesized in lower quantities in roots of B. chinense than those of B. scorzonerifolium. Besides abovementioned comparison on two transcriptome, the expression of some genes involved in biosyntheses of main medicinal secondary metabolites were analyzed by qRT-PCR in roots of the two species. These molecular biological data will make a useful clue and arouse more studies on the reasonable application of the two Bupleurum species. The fine roots of two-year old plants of B. chinense and B. scorzonerifolium planted in the same place at the same time were collected at the usual harvest time of medicinal materials of Bupleurum. Those roots from the two species plants were used for 454 sequencing.

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Transcriptome and Metabolome Analyses Reveal Differences in Terpenoid and Flavonoid Biosynthesis in Cryptomeria fortunei Needles Across Different Seasons

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