Systematic silencing of benzylisoquinoline alkaloid biosynthetic genes reveals the major route to papaverine in opium poppy

• Published in: The Plant journal : for cell and molecular biology 2012-10, Vol.72 (2), p.331-344
• Main Authors: Desgagné‐Penix, Isabel, Facchini, Peter J
• Format: Article
• Language: English
• Subjects: Alkaloids; Alkaloids - metabolism; benzylisoquinoline alkaloids; Benzylisoquinolines - metabolism; Biological and medical sciences; Biosynthesis; Biosynthetic Pathways; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Dehydrogenation; Enzymes; functional genomics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Plant; Gene Silencing; genes; Genetics; Latex - chemistry; Latex - isolation & purification; metabolic engineering; Methyltransferases - genetics; Methyltransferases - metabolism; Muscle relaxants; N-Methyltransferase; Narcotics; opium poppy; Papaver - chemistry; Papaver - enzymology; Papaver - genetics; Papaver - metabolism; Papaver somniferum; papaverine; Papaverine - metabolism; Parasympathetic nervous system; Plant biology; Plant physiology and development; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Stems - chemistry; Plant Stems - enzymology; Plant Stems - genetics; Plant Stems - metabolism; Real-Time Polymerase Chain Reaction; RNA, Messenger - genetics; RNA, Plant - genetics; secondary metabolism; Transcription; Vasodilator Agents - metabolism; Vasodilators; virus‐induced gene silencing
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/j.1365-313x.2012.05084.x

Papaverine, a major benzylisoquinoline alkaloid in opium poppy (Papaver somniferum), is used as a vasodilator and antispasmodic. Conversion of the initial intermediate (S)‐norcoclaurine to papaverine involves 3′‐hydroxylation, four O‐methylations and dehydrogenation. However, our understanding of papaverine biosynthesis remains controversial more than a century after an initial scheme was proposed. In vitro assays and in vivo labeling studies have been insufficient to establish the sequence of conversions, the potential role of the intermediate (S)‐reticuline, and the enzymes involved. We used virus‐induced gene silencing in opium poppy to individually suppress the expression of six genes with putative roles in papaverine biosynthesis. Suppression of the gene encoding coclaurine N‐methyltransferase dramatically increased papaverine levels at the expense of N‐methylated alkaloids, indicating that the main biosynthetic route to papaverine proceeds via N‐desmethylated compounds rather than through (S)‐reticuline. Suppression of genes encoding (S)‐3′‐hydroxy‐N‐methylcoclaurine 4‐O‐methyltransferase and norreticuline 7‐O‐methyltransferase, which accept certain N‐desmethylated alkaloids, reduced papaverine content. In contrast, suppression of genes encoding N‐methylcoclaurine 3′‐hydroxylase or reticuline 7‐O‐methyltransferase, which are specific for N‐methylated alkaloids, did not affect papaverine levels. Suppression of norcoclaurine 6‐O‐methyltransferase transcript levels significantly suppressed total alkaloid accumulation, implicating (S)‐coclaurine as a key branch‐point intermediate. The differential detection of N‐desmethylated compounds in response to suppression of specific genes highlights the primary route to papaverine.