Integrative analysis of the metabolome and transcriptome provides insights into the mechanisms of lignan biosynthesis in Herpetospermum pedunculosum (Cucurbitaceae).

BACKGROUND: Herpetospermum pedunculosum (Ser.) C. B. Clarke is a traditional Chinese herbal medicine that heavily relies on the lignans found in its dried ripe seeds (Herpetospermum caudigerum), which have antioxidant and hepatoprotective functions. However, little is known regarding the lignan biosynthesis in H. pedunculosum. In this study, we used metabolomic (non-targeted UHPLC-MS/MS) and transcriptome (RNA-Seq) analyses to identify key metabolites and genes (both structural and regulatory) associated with lignan production during the green mature (GM) and yellow mature (YM) stages of H. pedunculosum. RESULTS: The contents of 26 lignan-related metabolites and the expression of 30 genes involved in the lignan pathway differed considerably between the GM and YM stages; most of them were more highly expressed in YM than in GM. UPLC-Q-TOF/MS confirmed that three Herpetospermum-specific lignans (including herpetrione, herpetotriol, and herpetin) were found in YM, but were not detected in GM. In addition, we proposed a lignan biosynthesis pathway for H. pedunculosum based on the fundamental principles of chemistry and biosynthesis. An integrated study of the transcriptome and metabolome identified several transcription factors, including HpGAF1, HpHSFB3, and HpWOX1, that were highly correlated with the metabolism of lignan compounds during seed ripening. Furthermore, functional validation assays revealed that the enzyme 4-Coumarate: CoA ligase (4CL) catalyzes the synthesis of hydroxycinnamate CoA esters. CONCLUSION: These results will deepen our understanding of seed lignan biosynthesis and establish a theoretical basis for molecular breeding of H. pedunculosum.

Transcriptome Analysis of CYP450 Family Members in Fritillaria cirrhosa D. Don and Profiling of Key CYP450s Related to Isosteroidal Alkaloid Biosynthesis.

Fritillaria cirrhosa D. Don (known as Chuan-Bei-Mu in Chinese) can synthesize isosteroidal alkaloids (ISA) with excellent medicinal value, and its bulb has become an indispensable ingredient in many patented drugs. Members of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating steroidal alkaloids biosynthesis. However, little information is available on the P450s in F. cirrhosa. Here, we performed full-length transcriptome analysis and discovered 48 CYP450 genes belonging to 10 clans, 25 families, and 46 subfamilies. By combining phylogenetic trees, gene expression, and key F. cirrhosa ISA content analysis, we presumably identify seven FcCYP candidate genes, which may be hydroxylases active at the C-22, C-23, or C-26 positions in the late stages of ISA biosynthesis. The transcript expression levels of seven FcCYP candidate genes were positively correlated with the accumulation of three major alkaloids in bulbs of different ages. These data suggest that the candidate genes are most likely to be associated with ISA biosynthesis. Finally, the subcellular localization prediction of FcCYPs and transient expression analysis within Nicotiana benthamiana showed that the FcCYPs were mainly localized in the chloroplast. This study presents a systematic analysis of the CYP450 gene family in F. cirrhosa and provides a foundation for further functional characterization of the CYPs involved in ISA biosynthesis.

[Cloning and prokaryotic expression of FcLEA-D29 gene of Fritillaria cirrhosa].

Fritillaria cirrhosa, the most valuable source of the precious Chinese medicinal material Fritillariae Cirrhosae Bulbus, suffers from various stresses during growth which influence the yield and quality of the medicinal part. This study aims to explore the genes related to stress resistance in this medicinal species. To be specific, based on the transcriptome data of F. cirrhosa, a gene encoding the late embryogenesis abundant(LEA) protein was obtained, which was named as FcLEA-D29. The gene sequence and protein structure were analyzed with bioinformatics methods and qRT-PCR was used to detect the expression of the gene in different tissues and in response to temperature stress. The low-temperature tolerance of FcLEA-D29 was verified by the prokaryotic expression system. The results showed that FcLEA-D29 contained an open reading frame of 777 bp, encoding 258 amino acids. Multiple sequence alignment revealed that FcLEA-D29 protein belonged to LEA-D29 subfamily of LEA3 family. qRT-PCR results showed that FcLEA-D29 was specifically expressed in bulbs and responded to low temperature. The strain with the recombinant plasmid demonstrated better growth status than the control strain in the instance of low temperature stress, suggesting that FcLEA-D29 may play a role in bulb development and low temperature response of F. cirrhosa. This study laid a basis for further research on the role of FcLEA-D29 in the accumulation of secondary metabolites in F. cirrhosa, especially alkaloids, under low temperature and provided evidence for the low-temperature adaptation of F. cirrhosa.