Abstract
Alkaloids are diverse group of secondary metabolites generally found in plants. Opium poppy (Papaver somniferum L.), the only commercial source of morphinan alkaloids, has been used as a medicinal plant since ancient times. It produces benzylisoquinoline alkaloids (BIA) including the narcotic analgesic morphine, the muscle relaxant papaverine, and the anti-cancer agent noscapine. Though BIAs play crucial roles in many biological mechanisms their steps in biosynthesis and the responsible genes remain to be revealed. In this study, expressions of 3-hydroxy-N-methylcoclaurine 4′–methyltransferase (4′OMT) and reticuline 7-O-methyltransferase (7OMT) genes were subjected to manipulation to functionally characterize their roles in BIA biosynthesis. Measurements of alkaloid accumulation were performed in leaf, stem, and capsule tissues accordingly. Suppression of 4′OMT expression caused reduction in the total alkaloid content in stem tissue whereas total alkaloid content was significantly induced in the capsule. Silencing of the 7OMT gene also caused repression in total alkaloid content in the stem. On the other hand, over-expression of 4′OMT and 7OMT resulted in higher morphine accumulation in the stem but suppressed amount in the capsule. Moreover, differential expression in several BIA synthesis genes (CNMT, TYDC, 6OMT, SAT, COR, 4′OMT, and 7OMT) were observed upon manipulation of 4′OMT and 7OMT expression. Upon silencing and overexpression applications, tissue specific effects of these genes were identified. Manipulation of 4′OMT and 7OMT genes caused differentiated accumulation of BIAs including morphine and noscapine in capsule and stem tissues.
Highlights
Most plants synthesize different kinds of natural products possessing commercial value such as secondary metabolites in response to various environmental or developmental factors
Different types of benzylisoquinoline alkaloids (BIA) like protoberberine, morphinan, and others share the early common steps in the biosynthetic pathway. (S)-reticuline is the central intermediate of opium BIA ramification and its formation needs a series of enzymes including norcoclaurine synthase (NCS; Lee and Facchini, 2010), norcoclaurine 6-Omethyltransferase (6OMT; Morishige et al, 2000), coclaurine Nmethyltransferase (CNMT; Choi et al, 2002), and 3-hydroxyN-methylcoclaurine 4′-O-methyltransferase (4′OMT; Morishige et al, 2000)
To measure the effect of 4′OMT suppression on the other selected transcripts involved in BIA biosynthesis, qRT-PCR assay was carried out and the results showed that these genes were differentially expressed in different tissues
Summary
Most plants synthesize different kinds of natural products possessing commercial value such as secondary metabolites in response to various environmental or developmental factors. The opium poppy (Papaver somniferum L.), belongs to the Papaveraceae family and has been used as a medicine or drug for a long time (Schiff, 2002) It produces a number of BIAs including the narcoticanalgesic morphine, the cough suppressant codeine, the muscle relaxant papaverine, and the Functions of 4′OMT and 7OMT Genes anti-microbial sanguinarine (Allen et al, 2004; Desgagné-Penix et al, 2010; Gurkok et al, 2015). (S)-reticuline is the central intermediate of opium BIA ramification and its formation needs a series of enzymes including norcoclaurine synthase (NCS; Lee and Facchini, 2010), norcoclaurine 6-Omethyltransferase (6OMT; Morishige et al, 2000), coclaurine Nmethyltransferase (CNMT; Choi et al, 2002), and 3-hydroxyN-methylcoclaurine 4′-O-methyltransferase (4′OMT; Morishige et al, 2000). Albeit a lot of enzymes cloned, BIA biosynthesis, and regulation has not been fully discerned yet
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