Metabolite and transcriptomic changes reveal the ripening process in Sinopodophyllum hexandrum fruit

Sinopodophyllum hexandrum is a perennial herb that produces an aryl-tetralin-type lignan, podophyllotoxin (PPT) isolated from rhizomes and used as a tumor inhibitor. Fruit from the plant, also called Himalayan Mayapple, is used as well in traditional Chinese medicine with biologically active metabolites including lignans, flavonoids, phenolics, and polysaccharides. During fruit maturation, a significant alteration in metabolite composition occurs. To characterize and monitor formation and turnover of metabolites during ripening, solvent-extractions of fruit were analyzed via UPLC-ESI-MS/MS during four developmental stages: young- (S1), expansion- (S2), mature- (S3) and softened-fruit (S4). To study enzymatic processes involving fruit ripening, Mayapple transcripts were monitored by RNA-seq and validated by qRT-PCR. PPT content significantly decreased while total flavonoid and polysaccharide content increased during ripening. Twenty-five differentially accumulated flavonoids (DAFs) and 1775 differentially expressed genes (DEGs) were observed during one or more of the developmental stages. Based on functional annotation, there were 116 and 82 DEGs associated with phenylpropanoid metabolism (i.e., PPT and flavonoids) and glycometabolism (e.g., starch, sucrose, and glucose), respectively. Additionally, differential expression of transcription factors (TFs) (e.g., MYB, bHLH, and WRKY) in fruits supported the phenylpropanoid metabolism and glycometabolism. Gene expression levels associated with PPT biosynthesis (e.g., DIRs, PLRs, and SDHs), flavonoids (e.g., CHSs, F3′Hs, and F3′5′Hs), and polysaccharides (e.g., INV, GOLS2, and AGLU), as well as TFs (e.g., MYB1, BLH2, and WRKY65) were consistent with changing levels of metabolites detected during ripening. These findings track dynamic alterations in ripening from bitter to sweet during fruit maturation. Moreover, these results allow for the prediction of metabolite occurrence and abundance during Mayapple fruit maturation.