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Genome-Wide Identification of Circular RNAs Potentially Involved in the Biosynthesis of Secondary Metabolites in Salvia miltiorrhiza.

ABSTRACT: Circular RNAs (circRNAs) play various roles in cellular functions. However, no studies have been reported on the potential involvement of circRNAs in the biosynthesis of secondary metabolites in plants. Here, we performed a genome-wide discovery of circRNAs from root, stem and leaf samples of Salvia miltiorrhiza using RNA-Seq. We predicted a total of 2,476 circRNAs with at least two junction reads using circRNA_finder and CIRI, of which 2,096, 151 and 229 were exonic, intronic and intergenic circRNAs, respectively. Sequence similarity analysis showed that 294 out of 2,476 circRNAs were conserved amongst multiple plants. Of the 55 predicted circRNAs, 31 (56%) were validated successfully by PCR and Sanger sequencing using convergent and divergent primer pairs. Alternative circularisation analysis showed that most parental genes produced two circRNAs. Functional enrichment analyses of the parental genes showed that the primary metabolism pathways were significantly enriched, particularly the carbon metabolism. Differential expression analysis showed that the expression profiles of circRNAs were tissue-specific. Co-expression analysis showed 275 circRNAs, and their parental genes had significantly positive correlations. However, 14 had significantly negative correlations. Weighted gene co-expression network analysis showed that nine circRNAs were co-expressed with four modules of protein-coding genes. Next, we found 416 exonic circRNAs with miRNA-binding sites, suggesting possible interactions between circRNAs and miRNAs. Lastly, we found six validated circRNAs, namely, SMscf2473-46693-46978, SMscf3091-29256-29724, SMscf16-111773-112193, SMscf432-13232-13866, SMscf7007-10563-10888 and SMscf1730-1749-2013, which were originated from the genes involved in the biosynthesis of secondary metabolites. Their parental genes were acetyl-CoA C-acetyltransferase 1 (SmAACT1), 1-deoxy-d-xylulose-5-phosphate synthase 2 (SmDXS2), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase 1 (SmHDR1), kaurene synthase-like 2 (SmKSL2), DWF4 and CYP88A3, respectively. In particular, the correlation coefficient of SMscf2473-46693-46978 and SmDXS2 gene was 0.86 (p = 0.003), indicating a potential interaction between this pair of circRNA and its parent gene. Our results provided the first comprehensive catalogue of circRNAs in S. miltiorrhiza and identified one circRNA that might play important roles in the biosynthesis of secondary metabolites.

SUBMITTER: Jiang M

PROVIDER: S-EPMC8602197 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Terpenoids are the largest class of plant secondary metabolites and have attracted widespread interest. Salvia miltiorrhiza, belonging to the largest and most widely distributed genus in the mint family, is a model medicinal plant with great economic and medicinal value. Diterpenoid tanshinones are the major lipophilic bioactive components in S. miltiorrhiza. Systematic analysis of genes involved in terpenoid biosynthesis has not been reported to date. Searching the recently available working draft of the S. miltiorrhiza genome, 40 terpenoid biosynthesis-related genes were identified, of which 27 are novel. These genes are members of 19 families, which encode all of the enzymes involved in the biosynthesis of the universal isoprene precursor isopentenyl diphosphate and its isomer dimethylallyl diphosphate, and two enzymes associated with the biosynthesis of labdane-related diterpenoids. Through a systematic analysis, it was found that 20 of the 40 genes could be involved in tanshinone biosynthesis. Using a comprehensive approach, the intron/exon structures and expression patterns of all identified genes and their responses to methyl jasmonate treatment were analysed. The conserved domains and phylogenetic relationships among the deduced S. miltiorrhiza proteins and their homologues isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as 1-deoxy-D-xylulose 5-phosphate synthase, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, hydroxymethylglutaryl-CoA reductase, and geranylgeranyl diphosphate synthase, are encoded by multiple gene members with different expression patterns and subcellular localizations, and both homomeric and heteromeric geranyl diphosphate synthases exist in S. miltiorrhiza. The results suggest the complexity of terpenoid biosynthesis and the existence of metabolic channels for diverse terpenoids in S. miltiorrhiza and provide useful information for improving tanshinone production through genetic engineering.

Project description:Plant natural products have been co-opted for millennia by humans for various uses such as flavor, fragrances, and medicines. These compounds often are only produced in relatively low amounts and are difficult to chemically synthesize, limiting access. While elucidation of the underlying biosynthetic processes might help alleviate these issues (e.g., via metabolic engineering), investigation of this is hindered by the low levels of relevant gene expression and expansion of the corresponding enzymatic gene families. However, the often-inducible nature of such metabolic processes enables selection of those genes whose expression pattern indicates a role in production of the targeted natural product.Here, we combine metabolomics and transcriptomics to investigate the inducible biosynthesis of the bioactive diterpenoid tanshinones from the Chinese medicinal herb, Salvia miltiorrhiza (Danshen). Untargeted metabolomics investigation of elicited hairy root cultures indicated that tanshinone production was a dominant component of the metabolic response, increasing at later time points. A transcriptomic approach was applied to not only define a comprehensive transcriptome (comprised of 20,972 non-redundant genes), but also its response to induction, revealing 6,358 genes that exhibited differential expression, with significant enrichment for up-regulation of genes involved in stress, stimulus and immune response processes. Consistent with our metabolomics analysis, there appears to be a slower but more sustained increased in transcript levels of known genes from diterpenoid and, more specifically, tanshinone biosynthesis. Among the co-regulated genes were 70 transcription factors and 8 cytochromes P450, providing targets for future investigation.Our results indicate a biphasic response of Danshen terpenoid metabolism to elicitation, with early induction of sesqui- and tri- terpenoid biosynthesis, followed by later and more sustained production of the diterpenoid tanshinones. Our data provides a firm foundation for further elucidation of tanshinone and other inducible natural product metabolism in Danshen.

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Genome-Wide Identification of Circular RNAs Potentially Involved in the Biosynthesis of Secondary Metabolites in Salvia miltiorrhiza.

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