Gene transcript and metabolite profiling of elicitor-induced opium poppy cell cultures reveals the coordinate regulation of primary and secondary metabolism

Abstract

Elicitor-induced sanguinarine accumulation in opium poppy (Papaver somniferum) cell cultures provides a responsive model system to profile modulations in gene transcripts and metabolites related to alkaloid biosynthesis. An annotated expressed sequence tag (EST) database was assembled from 10,224 random clones isolated from an elicitor-treated opium poppy cell culture cDNA library. The most abundant ESTs encoded defense proteins, and enzymes involved in alkaloid metabolism and S-adenosylmethionine-dependent methyl transfer. ESTs corresponding to 40 enzymes involved in the conversion of sucrose to sanguinarine were identified. A corresponding DNA microarray was probed with RNA from cell cultures collected at various time-points after elicitor treatment, and compared with RNA from control cells. Several diverse transcript populations were coordinately induced, with alkaloid biosynthetic enzyme and defense protein transcripts displaying the most rapid and substantial increases. In addition to all known sanguinarine biosynthetic gene transcripts, mRNAs encoding several upstream primary metabolic enzymes were coordinately induced. Fourier transform-ion cyclotron resonance-mass spectrometry was used to characterize the metabolite profiles of control and elicitor-treated cell cultures. Principle component analysis revealed a significant and dynamic separation in the metabolome, represented by 992 independent detected analytes, in response to elicitor treatment. Identified metabolites included sanguinarine, dihydrosanguinarine, and the methoxylated derivatives dihydrochelirubine and chelirubine, and the alkaloid pathway intermediates N-methylcoclaurine, N-methylstylopine, and protopine. Some of the detected analytes showed temporal changes in abundance consistent with modulations in the profiles of alkaloid biosynthetic gene transcripts.

Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

“…The time course for the induction of NCS transcripts and protein in elicitor-treated opium poppy cell cultures is also in agreement with the transcriptional activation profiles of all other known enzymes involved in sanguinarine biosynthesis (Zulak et al, 2007). The localization of NCS to sieve elements of the phloem in all opium poppy organs was demonstrated by coimmunolocalization in serial sections of the sequential enzyme 6OMT, which was previously shown to reside in this cell type using a variety of cellular markers (Samanani et al, 2006).…”

Molecular Cloning and Characterization of Tetrahydroprotoberberine cis-N-Methyltransferase, an Enzyme Involved in Alkaloid Biosynthesis in Opium Poppy

“…7) reflects the coordinated temporal induction of all other known genes involved in sanguinarine biosynthesis in opium poppy (8). Moreover, the accumulation profiles of N-methylstylopine (41), other benzophenenthridine alkaloid pathway intermediates (41), and sanguinarine (41,60) are in agreement with the temporal induction of TNMT activity in elicitor-treated opium poppy cell cultures.…”

“…Nucleic Acid Isolation and Analysis-The synthesis and sequencing of an elicitor-treated opium poppy cell culture cDNA library and the assembly of the corresponding expressed sequence tag (EST) data base were described previously (41). A full-length cDNA encoding TNMT was identified by screening the EST data base in silico using the tBLASTn algorithm (42) in DNATools 6.0.…”

Functional analyses of genetic pathways controlling petal specification in poppy

“…1) and so are well-placed for comparative studies. P. somniferum is one of the oldest cultivated medicinal plants, and its continuing pharmaceutical importance as the source of a variety of morphinan alkaloids has made it one of the best-studied Papaver species (Chitty et al, 2006;Kapoor, 1995;Millgate et al, 2004;Zulak et al, 2007). Furthermore, functional genetic analyses can be rapidly carried out in P. somniferum using virus-induced gene silencing (Hileman et al, 2005).…”