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Multicellular Compartmentation of Catharanthus roseus Alkaloid Biosynthesis Predicts Intercellular Translocation of a Pathway IntermediateBenoit St-Pierrea, Felipe A. Vazquez-Flota1,a, and Vincenzo De Luca2,aa Institut de Recherche en Biologie Végétale, Département de Sciences Biologiques, Université de Montréal, 4101 rue Sherbrooke est, Montréal, Québec H1X 2B2, Canada Correspondence to: Vincenzo De Luca, vincenzo.de.luca{at}umontreal.ca (E-mail), 514-872-9406 (fax)
In situ RNA hybridization and immunocytochemistry were used to establish the cellular distribution of monoterpenoid indole alkaloid biosynthesis in Madagascar periwinkle (Catharanthus roseus). Tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), which are involved in the biosynthesis of the central intermediate strictosidine, and desacetoxyvindoline 4-hydroxylase (D4H) and deacetylvindoline 4-O-acetyltransferase (DAT), which are involved in the terminal steps of vindoline biosynthesis, were localized. tdc and str1 mRNAs were present in the epidermis of stems, leaves, and flower buds, whereas they appeared in most protoderm and cortical cells around the apical meristem of root tips. In marked contrast, d4h and dat mRNAs were associated with the laticifer and idioblast cells of leaves, stems, and flower buds. Immunocytochemical localization for TDC, D4H, and DAT proteins confirmed the differential localization of early and late stages of vindoline biosynthesis. Therefore, we concluded that the elaboration of the major leaf alkaloids involves the participation of at least two cell types and requires the intercellular translocation of a pathway intermediate. A basipetal gradient of expression in maturing leaves also was shown for all four genes by in situ RNA hybridization studies and by complementary studies with dissected leaves, suggesting that expression of the vindoline pathway occurs transiently during early leaf development. These results partially explain why attempts to produce vindoline by cell culture technology have failed.
The organs forming the plant body consist of several different cell types that are organized in relation to each other and that confer specific functions to the resulting organ. Each cell type emerges from an undifferentiated meristem according to a sophisticated and partially understood developmental program (
In addition to morphogenesis, developmental processes result in biochemical specialization of cells for the biosynthesis and/or accumulation of secondary metabolites, such as phenylpropanoids (
Vindoline biosynthesis in Catharanthus roseus also appears to be under this type of developmental control (
More than 100 C. roseus alkaloids that have been identified share many common biosynthetic steps. The early stages of alkaloid biosynthesis in C. roseus involve the formation of tryptamine from tryptophan and its condensation with secologanin to produce the central intermediate strictosidine, the common precursor for the monoterpenoid indole alkaloids (Figure 1). The enzymes catalyzing these two reactions are tryptophan decarboxylase (TDC) and strictosidine synthase (STR1), respectively (reviewed in
It has been suggested that certain tissues are required for alkaloid biosynthesis and/or accumulation. In this study, we have used in situ RNA hybridization and immunocytochemistry to localize the cell-specific expression of tdc, str1, d4h, and dat in different tissues of C. roseus plants. The results suggest that the early (TDC and STR1) and late (D4H and DAT) reactions of vindoline biosynthesis occur in different cells.
Previous studies with C. roseus have illustrated that genes involved in indole alkaloid biosynthesis are preferentially expressed in young actively growing tissues. Very young leaves were fixed, embedded, sectioned, and prepared for in situ RNA hybridization studies to localize transcripts of tdc, str1, d4h, and dat, which catalyze early (tdc and str1) and late (d4h and dat) steps in vindoline biosynthesis, respectively (Figure 1). The longitudinal section of a young leaf (Figure 2, diagram at top) shows that the basal end is curled and suggests that cell expansion leading to leaf uncurling has yet to be completed because the bases of angiosperm leaves are developmentally younger than are the more distal parts.
C. roseus has simple, elliptical mesomorphic leaves (
Cell-Specific Distribution of tdc, str1, d4h, and dat Transcripts in Developing Leaves Similar in situ RNA hybridization studies with older leaves also were performed (data not shown). Expression of all four genes decreased significantly in older leaves, but the same epidermis-, idioblast-, and laticifer-specific expression as in younger tissues was observed.
Laticifer- and Idioblast-Specific Expression of DAT
A comparison of longitudinal leaf sections hybridized with antisense dat probe (Figure 3A) to the hand-cut sections shows a clear correlation between the expression of dat and the greenish yellow fluorescence that is characteristic of laticifer and spongy mesophyll–associated idioblast cells (Figure 3E). This correlation also was demonstrated for palisade mesophyll–associated idioblast cells; their characteristic fluorescence is best viewed on the adaxial faces of a whole mount (Figure 3F). Additional in situ RNA hybridization studies were conducted with paradermal sections prepared by starting from the adaxial side of the leaves. Palisade mesophyll idioblasts and vasculature-associated laticifer cells are visualized first (Figure 3B), whereas cross-connecting laticifer cells become apparent in the paradermal section 20 µm further into the leaf (Figure 3C). The palisade mesophyll idioblasts (Figure 3B) can be distinguished from other palisade cells because of their larger size (
Cell-Specific Distribution of TDC, D4H, and DAT Proteins in Developing Leaves
Differential Accumulation of TDC, STR1, D4H, and DAT in the Leaf Blade To confirm this general trend, we dissected immature leaves (Figure 5, lower right) into base, middle, and tip areas to determine the levels of these four transcripts and of TDC, D4H, and DAT enzymes. RNA and protein were extracted separately from whole leaves or their components, as shown in Figure 5. RNA gel blot analysis (Figure 5, left) clearly showed the basipetal distribution of all four transcripts, with the highest, intermediate, and lowest levels occurring in the basal, middle, and tip regions of the leaf, respectively. However, immunoblot analysis (Figure 5, right) revealed that the levels of the three proteins were almost equivalent in the basal and middle regions of the leaves. Their levels declined only in the tip region of the leaf, suggesting that these proteins were more stable than were their corresponding mRNAs during leaf maturation. These results, which clearly suggest that the pathway leading to vindoline biosynthesis appears very early in leaf development, corroborate the qualitative differences in the levels of the corresponding transcripts as observed by in situ RNA hybridization of C. roseus leaves (Figure 2).
Sites of Indole Alkaloid Biosynthesis in Flower Primordia, Stems, and Roots Tips
Serial longitudinal sections of root tips (Figure 6C, Figure 6F, Figure 6I, and Figure 6L) also were studied for the presence of each of these transcripts. Expression of both tdc and str1 is restricted to protodermal and cortical cells along most of the longitudinal sections, but it appears to decrease in older developmental stages (Figure 6C and Figure 6F). The root cap and the stele do not appear to express either of these mRNAs. It is not surprising that neither d4h (Figure 6I) nor dat (Figure 6L) transcripts are expressed in root tips, because roots do not express the late stages of vindoline biosynthesis. The cellular distribution of TDC, D4H, and DAT antigens also was studied in flower buds, stems, and root sections (data not shown). The results obtained clearly corroborate those of the in situ RNA hybridization studies reported in Figure 6.
We have demonstrated the cell type–specific localization of enzymes involved in steps in indole alkaloid biosynthesis in C. roseus that are common to all C. roseus alkaloids (TDC and STR1) and in steps comprising the vindoline-specific branch of the pathway (D4H and DAT). One striking observation was the coordinate regulation observed for both the common and vindoline-specific branch of the pathway. For instance, the mRNA distribution patterns for both tdc and str1 were identical within different tissues and developmental stages. Expression of tdc and str1 was restricted to the epidermis of leaves, stems, and flowers (Figure 2A to 2H, Figure 6A, Figure 6B, Figure 6D, and Figure 6E). In contrast, expression of tdc and str1 in roots was restricted to the cortical and protodermal cells of the root apex (Figure 6C and Figure 6F). It was shown previously that the distribution in various organs and induction kinetics of tdc and str1 mRNAs under various conditions were strictly coordinated (
The genes representing the late stage of vindoline biosynthesis also were expressed, at least at the protein level, in the same cell type. D4H and DAT proteins were associated with laticifers and idioblasts of leaves (Figure 4C to 4F), stems, and flowers (results not shown). d4h and dat mRNAs also were colocalized in idioblast and laticifer cells of leaves, stems, and flower buds (Figure 2I to 2P, Figure 6G, Figure 6H, Figure 6J, and Figure 6K), and there was no detectable expression of either gene in roots (Figure 6I and Figure 6L). However, ectopic accumulation of d4h mRNA was observed in upper epidermal cells and in some spongy mesophyll cells in the vicinity of idioblast cells (Figure 2I to 2L) without the concomitant accumulation of D4H protein (Figure 4C and Figure 4D). Whereas coordinate regulation of d4h and dat was expected from many lines of evidence, particularly from their induction kinetics and light-dependent regulation in developing seedlings, as well as their distribution patterns in mature plants, some data suggested specific mechanism of regulation for d4h. For instance, d4h mRNAs (
The in situ RNA hybridization and immunocytochemical localization studies presented in this report revealed that expression of the vindoline pathway follows a basipetal distribution in young expanding leaves (Figure 2 and Figure 4). These results were confirmed by immunoblot and RNA blot analysis of dissected leaf segments (Figure 5). This expression pattern suggests activation of the vindoline pathway genes in young immature leaf tissues and rapid downregulation with tissue maturation. In dicots, cell division frequently occurs until the lamina has reached ~90% of its final leaf area ( The studies also revealed a spatial separation of the vindoline pathway within particular cells in leaf, stem, and flower tissues (Figure 2, Figure 4, and Figure 6). These results indicate that the early vindoline pathway is restricted to the epidermal layers of immature leaves and stems, whereas the late steps committed exclusively to vindoline formation are located in laticifers and idioblasts. This raises questions about the number of pathway steps occurring in the epidermis and how specific intermediates are mobilized into laticifers and idioblasts for elaboration into vindoline.
Tabersonine and post-tabersonine intermediates have been detected in C. roseus roots ( The role of laticifers and idioblasts in the terminal steps of vindoline biosynthesis raises the question of where the biochemical coupling of vindoline and catharanthine to make highly toxic indole alkaloid dimers occurs. It is conceivable that these specialized cells are required to accommodate and sequester the toxic dimers from normal cellular processes.
The participation of separate organs and/or cells in the synthesis and accumulation of secondary metabolites appears to be a common characteristic in plants. For example, nectaries, trichomes, and other secretory structures that originate from epidermal cells (
It is not clear if a similar root-to-shoot alkaloid transport system exists in C. roseus. Catharanthine and advanced precursors of vindoline biosynthesis, such as tabersonine, may be synthesized in roots to be transported to laticifers and idioblasts in leaves and stem, for elaboration into dimeric alkaloids and vindoline, respectively. However, the ability of in vitro leaf cultures, which have no roots, to make vindoline suggests that roots are not absolutely required to make this possible (
The clear differential cell localization of the early and late stages of vindoline biosynthesis in leaves strongly suggests that undetermined poststrictosidine compounds are mobilized from the epidermis to laticifers and idioblasts, where at least the last two reactions of vindoline biosynthesis take place. Differential cell-specific expression and intercellular transport of metabolites also have been suggested for the phenylpropanoid pathway (
However, the locations of the pathways leading to the formation of flavonoids or furanocoumarins were mutually exclusive. Flavonoids and furanocoumarins are two major products derived from different branches of the phenylpropanoid pathway, downstream from 4-coumaryl–CoA. chs, which is involved specifically in flavonoid biosynthesis, was restricted to the epidermis, spongy mesophyll, and oil duct surrounding cells. S-adenosyl-L-methionine:bergaptol O-methyltransferase, which is involved in furanocoumarin biosynthesis, occurred in the vascular bundle, palisade parenchyma, and the oil duct epithelial cells ( A similar cellular distribution to those of leaves also was found for TDC, STR1, and D4H (F.A. Vazquez-Flota, B. St-Pierre, and V. De Luca, manuscript in preparation) in cotyledons of developing seedlings. No differences in the cellular distribution of D4H were found in the cotyledons of dark- or light-grown seedlings. The presence of inactive D4H protein in idioblasts and laticifers of etiolated seedlings indicates that this isoform of D4H is expressed properly in laticifers and idioblasts from the early stages of seedling development. The results suggest that light activates d4h and dat expression (F.A. Vazquez-Flota, B. St-Pierre, and V. De Luca, manuscript in preparation) rather than inducing the production of particular cells, such as idioblasts or laticifers. It remains to be established, however, whether light triggers the formation/proliferation of particular subcellular structures required for vindoline biosynthesis. Because no clear physiological roles for vindoline have been described, this alkaloid could be considered both as a final product as well as a precursor in the formation of the dimeric alkaloids. In the latter event, the suggested spatially separate origin of the catharanthine subunit may play a critical role in regulating the formation of dimers, which have been shown to be toxic when applied to cell suspensions of C. roseus (V. De Luca, unpublished results).
The distribution of tdc and str1 in actively growing shoots and roots of C. roseus plants (Figure 2 and Figure 6;
Tissue Fixation and Embedding
In Situ RNA Hybridization Rehydrated sections were prepared for in situ hybridization by treatment with proteinase K (2 µg/mL in 100 mM Tris-HCl and 50 mM EDTA, pH 8.0) for 30 min at 37°C, followed by two rinses with TBS buffer (150 mM NaCl and 10 mM Tris-HCl, pH 7.5), by blocking of proteinase K with glycine (2 mg/mL in TBS) for 2 min, and by two rinses in TBS buffer. Sections were postfixed with 3.7% formaldehyde in PBS buffer for 20 min and washed in TBS for 5 min. Finally, sections were acetylated with acetic anhydride (0.25% in 0.1 M triethanolamine–HCl, pH 8.0) for 10 min, washed with TBS, dehydrated in an ethanol series, and air dried. For hybridization, portions of hybridization mixture (60 µL) were dispersed on a cover slip (22 x 50 mm), and the slides were inverted onto the droplet of probe. Hybridization mixture included 200 ng/mL of hydrolyzed digoxigenin–labeled RNA transcripts, 40% formamide, 10% dextran sulfate, 1 mg/mL yeast tRNA, 0.5 mg/mL polyadenylic acid, 0.3 M NaCl, 0.01 M Tris-HCl, pH 6.8, 0.01 M Na phosphate, pH 6.8, 5 mM EDTA, and 40 units per mL RNasin ribonuclease inhibitor (Promega). Hybridization was for 16 to 18 hr at 50°C in an atmosphere of 50% formamide. Cover slips were then detached by soaking in 2 x SSC at 37°C (1 x SSC is 0.15 M NaCl and 0.015 M sodium citrate). Slides were treated with RNase A (50 µg/mL in 0.5 M NaCl, 10 mM Tris-HCl, pH 7.5, and 1 mM EDTA) for 30 min at 37°C and then washed in 2 x SSC for 1 hr, in 1 x SSC for 1 hr, and in 0.1 x SSC for 1 hr at 65°C. For immunolocalization of hybridized transcripts, slides were washed in TBST (0.1 M Tris-HCl, pH 8.0, 0.15 M NaCl, and 0.3% Triton X-100) for 10 min and blocked with 2% BSA fraction V (Boehringer Mannheim) in TBST for 16 hr at 4°C. Portions (60 µL) of sheep anti-digoxigenin–alkaline phosphatase conjugate (Boehringer Mannheim) at a 1:200 dilution in a solution of 1% BSA in TBST were dispensed onto cover slips, and the slides were inverted onto the droplet. After a 2-hr incubation at room temperature, the unbound conjugates were washed twice for 15 min with TBST and twice for 10 min with AP buffer (0.1 M Tris-HCl, pH 9.5, 0.1 M NaCl, and 10 mM MgCl2). For color development, slides were immerged in 175 µg/mL 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and 350 µg/mL nitro blue tetrazolium chloride in AP buffer for 8 to 10 hr at 22°C. After development, slides were washed in water and mounted with 50% glycerol, 7% gelatin, and 1% phenol and covered with a cover slip.
Immunocytochemical Localization of TDC, D4H, and DAT
Fluorescence Microscopy
Protein and RNA Gel Blot Analysis
Procedures for the isolation, electrophoresis, and blotting of RNA, as well as the conditions for hybridization, posthybridization washes, and autoradiography, have been reported elsewhere (
1 Current address: Unidad de Biologia Experimental, Centro de Investigacion Cientifica de Yucatan, Apdo. Postal 87 Cordemex 97310 Merida, Yucatan, Mexico.
We thank Dwight Beebe for access to the Olympus BH-2 microscope. We also thank Sylvain Lebeurier for maintenance of plants. This work was supported by the Natural Sciences and Engineering Research Council of Canada and Le Fonds pour la Formation de Chercheurs et l'Aide à la Recherche. Received November 30, 1998; accepted March 9, 1999.
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