Comparative Transcriptomics Unveil the Crucial Genes Involved in Coumarin Biosynthesis in Peucedanum praeruptorum Dunn

Abstract

Peucedanum praeruptorum Dunn is a commonly used traditional Chinese medicine that is abundant in furano- and dihydropyrano coumarins. When P. praeruptorum reaches the bolting stage, the roots gradually lignified, and the content of coumarins declines rapidly. Non-bolting has always been a decisive factor for harvesting the P. praeruptorum materials. To evaluate the amount of coumarin components in unbolted and bolted P. praeruptorum, the variations of praeruptorin A, praeruptorin B, praeruptorin E, peucedanocoumarin I, and peucedanocoumarin II were determined. Additionally, 336,505 transcripts were obtained from the comparative transcriptome data. Among them, a total of 1,573 differentially expressed genes were screened out. To identify the critical genes involved in coumarin biosynthesis, comparative transcriptomics coupled with co-expression associated analysis was conducted. Finally, coumarin biosynthesis-related eighteen candidate genes were selected for the validation of qPCR. Additionally, a phylogenetic tree and the expression profile of ATP-binding cassette (ABC) transporters were constructed. To clarify the main genes in the regulation of coumarin biosynthesis, the interaction network of the co-expression genes from thirteen modules was constructed. Current results exhibited the significant increment of praeruptorin A, praeruptorin B and praeruptorin E in the bolted P. praeruptorum. Although, peucedanocoumarin I and peucedanocoumarin II were slightly increased. Besides the content of coumarins, the essential genes involved in the coumarin biosynthesis also exhibited an overall downward trend after bolting. Three peroxidases (PRXs) involved in the production of lignin monomers had been demonstrated to be downregulated. PAL, C4H, HCT, COMT, CCoAOMT, and some ABC transporters were dramatically downregulated at the bolting stage. These results indicated that the downregulation of coumarin biosynthetic genes in the bolted P. praeruptorum ultimately reduced the formation of coumarins. However, the mechanism through which bolting indirectly affects the formation of coumarin still needs extra functional verification.

Genome sequencing-based transcriptomic analysis reveals novel genes in Peucedanum praeruptorum

“…First, the raw data files acquired from the previous high-throughput sequencing were subjected to base calling analysis to convert them into original reads [ 5 , 13 ]. Secondly, we removed low-quality and adapter sequences to obtain clean reads.…”

“…Our previous studies had shown that most of the carbon and nitrogen sources in the roots would gradually transfer to the apical tissues for reproductive growth after bolting [ 13 ]. The expression of numerous crucial enzyme genes implicated in the biosynthesis of coumarin, including PAL , C4H , HCT , COMT , and CCoAOMT , exhibited a decrease, but some ABC transporters involved in transmembrane transport demonstrated an up-regulation [ 5 ]. Genes involved in transmembrane transport, ion channels, cytokinin synthesis, hexose phosphate metabolism, inositol phosphate metabolism, DNA-binding, phosphatidylinositol signaling, fatty acid metabolism, the peroxisome, and autophagy were significantly enriched in this study.…”

“…These genes may be involved in the methylation modification of phenylpropanoids during the bolting [ 24 ]. Our previous studies showed that the expression of apoptosis-related genes and peroxidases may be related to secondary wall thickening and root lignification after bolting [ 5 ]. At this time, part of the coumarin is degraded and part is transferred to the ground for the growth of vegetative organs.…”

“…Some key genes involved in the biosynthesis and transport of P. praeruptorum coumarins have been successfully identified using transcriptome and metabolome techniques. It was believed that CYP450 family genes and MDR transporters were involved in the biosynthesis and transport of coumarins [ 5 , 6 ]. Many studies have proved that PAL , 4CL , C2’H , BMT , COMT , AS , PS , etc.…”

Transcriptome Profiling Reveals Molecular Responses to Salt Stress in Common Vetch (Vicia sativa L.)

“…Lignin staining was conducted according to a previous method [ 29 ]. The 3 cm root tip samples were cut from seedlings after 3 d of salt treatment and immediately placed in a fixation solution containing 5% formaldehyde, 5% acetic acid, and 50% ethanol for 24 h at room temperature, followed by dehydration in a stepwise manner with 20% ethanol–water mixtures at 60 min intervals until 100% ethanol was reached.…”