Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

Abstract

Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of L-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type-specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type-specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types.

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

“…The substantial amino acid sequence identity among known biosynthetic enzymes suggests a monophyletic origin for BIA metabolism in plants belonging to the Papaveraceae, Ranunculaceae, Berberidaceae, and related families (Liscombe et al, 2005;Samanani et al, 2005). Proteins in the PR10/Bet v1 family that possess NCS activity have now been isolated from two members of the Papaveraceae (P. somniferum and P. bracteatum) and two members of the Ranunculaceae (T. flavum and C. japonica), all of which produce BIAs (Samanani et al, 2004;Liscombe et al, 2005;Minami et al, 2007; Figure 7).…”

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

“…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. CNMT cDNAs from P. somniferum (8), T. flavum (29), and C japonica (6) were used as query sequences. Total RNA was isolated using the method of Cheng and Seemann (43).…”

“…The recently reported molecular cloning of cDNAs encoding CNMT from P. somniferum (8), T. flavum (29), and C. japonica (6) provided query sequences to identify a full-length cDNA encoding opium poppy TNMT in an EST data base by in silico mining. We previously used this approach to identify cDNAs encoding opium poppy NCS isoforms with T. flavum NCS as the query sequence (4).…”

“…1). CNMT has been purified (28), and corresponding cDNAs have been isolated from Coptis japonica (6), Thalictrum flavum (29), and P. somniferum (8). TNMT protein has been isolated from Eschscholzia californica and Corydalis vaginans cell cultures (30) and purified from Sanguinaria canadensis cell cultures (31).…”

Two B-type ATP-binding cassette (ABC) transporters localize to the plasma membrane in <i>Thalictrum minus</i>

“…This similarity suggests the possibility that TmABCBs might function in influx or efflux of berberine, or perhaps both, similar to AtABCB21. In the rhizome of T. flavum, a similar species of T. minus, the presence of berberine importer and exporter was suggested, because berberine probably moves from the apical meristem, the biosynthesis site, to the pith and cortex, the accumulation sites (Samanani et al 2005). In intact T. minus plants, although rhizome was not examined due to the small size of analyzed plants, berberine accumulates to high levels in root tissues (Figure 4).…”