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.

Figure 1.

The Corresponding cDNAs for Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis Have Been Isolated. TYDC, tyrosine/dopa decarboxylase; 6OMT, (S)-norcoclaurine-6-O-methyltransferase; CNMT, (S)-coclaurine N-methyltransferase; CYP80B, (S)-N-methylcoclaurine-3′-hydroxylase; 4′OMT, (S)-3′-hydroxy-N-methylcoclaurine-4′-O-methyltransferase; BBE, berberine bridge enzyme; SOMT, (S)-scoulerine-9-O-methyltransferase; CYP719A, (S)-canadine synthase.

Figure 2.

Protoberberine Biosynthetic Gene Transcripts Are Present in Various Organs of T. flavum. RNA gel blot hybridization analysis was performed using 15 μg of total RNA, which was fractionated, transferred to a nylon membrane, and hybridized at high stringency to ³²P-labeled cDNAs. Gels were stained with ethidium bromide before blotting to ensure equal loading.

Figure 3.

Protoberberine Alkaloid Biosynthetic Gene Transcripts Are Localized to the Immature Endodermis and Surrounding Tissues in T. flavum Roots. (A) to (R) In situ hybridization using DIG-labeled antisense probes for TYDC ([A] and [B]), NCS ([C] and [D]), 6OMT ([E] and [F]), CNMT ([G] and [H]), CYP80B ([I] and [J]), 4′OMT ([K] and [L]), BBE ([M] and [N]), SOMT ([O] and [P]), and CYP719A ([Q] and [R]) performed on cross ([A], [C], [E], [G], [I], [K], [M], [O], and [Q]) and longitudinal ([B], [D], [F], [H], [J], [L], [N], [P], and [R]) root sections. (S) and (T) In situ hybridization using a DIG-labeled sense probe for CYP719A performed on cross (S) and longitudinal (T) root sections. Bar = 50 μm for (A) to (T).

Figure 4.

Protoberberine Alkaloid Biosynthetic Gene Transcripts Are Localized to the Protoderm of Leaf Primordia in T. flavum Rhizomes. (A) Rhizome longitudinal section stained with toluidine blue O. (B) to (K) In situ hybridization using DIG-labeled antisense probes for TYDC (B), NCS (C), 6OMT ([D] and [K]), CNMT (E), CYP80B (F), 4′OMT (G), BBE (H), SOMT (I), and CYP719A (J) performed on longitudinal ([B] to [J]) and cross (K) rhizome sections. (L) In situ hybridization using a DIG-labeled sense probe for CYP719A performed on a longitudinal rhizome section. Yellow and red asterisks in (A) show the location of leaf primordia and axillary buds, respectively. Bar = 250 μm in (A) and 50 μm in (L). Bar in (L) also applies to (B) to (K).

Figure 5.

Protoberberine Alkaloids Accumulate in the Mature Endodermis of Roots, and in the Pith and Cortex of Rhizomes, in T. flavum. (A) to (D) Accumulation of protoberberine alkaloids in a fresh whole root (A) shown by light microscopy and in 0.5-mm cross sections of fresh roots at various stages of development ([B] to [D]) revealed by epifluorescence microscopy. (E) and (F) Accumulation of protoberberine alkaloids in fresh longitudinal sections (E) and cross sections (F) of a rhizome shown by light microscopy. ap, apical meristem; co, cortex; en, endodermis; ep, epidermis; ex, exodermis; pe, petiole; pi, pith; ro, root; va, vascular tissue. Bars = 500 μm in (A), 40 μm in (B) to (D), and 500 μm in (E) and (F).

Figure 6.

The Cell Type–Specific Localization of Protoberberine Alkaloid Gene Transcripts and Product Accumulation Are Temporally and Spatially Distinct in T. flavum Roots and Rhizomes, Respectively. (A) Model showing the relationship between the sites of protoberberine alkaloid biosynthetic gene expression (blue) and alkaloid accumulation (yellow) in a root. (B) Model showing the relationship between the sites of protoberberine alkaloid biosynthetic gene expression (blue) and alkaloid accumulation (yellow) in a rhizome. ap, apical meristem; co, cortex; en, endodermis; pi, pith; va, vascular tissue.