CatharanthusProc Indian Natn roseus Sci Acad alkaloids 74 No.3 pp. 97-109 (2008) 97

Research Paper Predominance of the Serpentine Route in Monoterpenoid Indole Alkaloid Pathway of Catharanthus roseus

DIGVIJAY SINGHa, SANJAY KUMAR RAIa, SHASHI PANDEY-RAIa, SUCHI SRIVASTAVAa, RAGHVENDRA KUMAR MISHRAa, SRIKANT SHARMAb, AND SUSHIL KUMARa* aNational Institute for Plant Genome Research (NIPGR), Post Box. No 10531, aJawaharlal Nehru University Campus, Aruna Asaf Ali Marg, New Delhi-110 067 and bRegional Research Laboratory, Bhubaneshwar-751013, India

(Received 24 April 2008; Accepted 03 November 2008)

Single runs of RP-HPLC procedure were used for simultaneous and organ-wise quantification of the terpenoid indole alkaloids (TIAs) serpentine, ajmalicine, vindoline, catharanthine, vincristine and vinblastine in whole adult plants of

Catharanthus roseus, sampled from 81 accessions and F2 generation of a cross. The average total TIA content was 159 mg distributed among stem, root, leaf and inflorescence organs in 13:8:3:1 proportion. The root and stem barks, leaf-petiole and corolla petals were several fold TIA-richer than root and stem woods, leaf–lamina and rest of flower organs, respec- tively. TIAs were undetectable in seed but present in the seedling. Quantitatively, serpentine (+ ajmalicine), serpentine and catharanthine, vindoline, serpentine, catharanthine and vinblastine (+ vincristine) were the principal TIAs detected in stem, root, leaf and inflorescence organs, respectively. Significant amounts of vinblastine, vindoline and catharanthine were found present in the non-chlorophyllous corolla petals. Correlation studies showed that contents of catharanthine and serpentine in roots, catharanthine, vindoline and vinblastine in leaves, serpentine in roots and stems and stems and leaves were interdependent. Serpentine could be involved in suppressing the accumulation of other TIAs in leaves. The observed inter- and intra- organ contents of end-products from serpentine, catharanthine and vindoline TIA branch path- ways and correlations between them showed predominance of the serpentine branch. Key Words: Catharanthus roseus; Medicinal alkaloids of Catharanthus roseus; Terpenoid indole alkaloids; Organ-wise alkaloid compartmentation; Serpentine, catharanthine and vindoline biosynthetic routes; Serpentine route predominance.

Introduction from agriculturally produced herbage of C. roseus [11]. Plants in the course of their life cycle synthesize a myriad The cost of production of the pharmaceutically important of secondary metabolites in the form of phenolics, C. roseus alkaloids is high because yields are low and terpenoids and alkaloids [1]. The principal roles of differential extraction processes are tedious. secondary metabolites have been shown to include, Considerable progress has been made in enunciation mobilization of nutrients from soil, facilitation of of the complexity of TIA biosynthetic pathway of C. pollination and seed dispersal, toleration of salinity, roseus (Fig. 1) in terms of the identity of intermediates drought and inclement temperature and protection and enzymes along with the characteristics of structural against diseases and pests [2-6]. Many of the secondary and regulatory genes as well as intra- and inter-cellular metabolites are fine chemicals, pigments and drugs that sites of their expression [12-25] and interaction of are used in food-, cosmeticeutical- and pharmaceutical- environmental conditions and genetic control industry. The periwinkle plant species Catharanthus mechanisms [25,26-28]. All the TIAs are derived from roseus synthesizes more than 120 terpenoid indole the intermediate strictosidine, which is formed by the alkaloids (TIAs), some of which are in clinical use. conjugation of secologanin [14,29,30], a product of Among these, vindoline and vincristine are potent plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) anticancer agents while serpentine and ajmalicine are pathway [18,20,21], and tryptamine, a product of used in the treatment of hypertension arrhythmia and shikimate pathway [31]. Three branches beyond the other vascular diseases [7-9]. The industrial production central metabolite strictosidine are responsible for of vincristine and vinblastine involves semisynthetic vindoline, catharathine and ajmalicine production. coupling of their natural precursors vindoline and Serpentine is the catabolic end product of ajmalicine catharanthine [10]. In the absence of any reliable while catabolic dimerization of vindoline and biotechnological production systems, vindoline, catharanthine produces their end products as vinblastine catharanthine, ajmalicine and serpentine are extracted and vincristine [13,15,22,32-38]. The shoot of C. roseus

*Address for correspondence: 3NIPGR, JNU Campus, Post Box 10531, New Delhi–110067, India. e-mail: [email protected] 98 Digvijay Singh et al.

Plastidic non-mevalonate pathway Shikimate pathway Pyruvate + Glyceraldehyde-3-phosphate Chorismate

Geranyl diphosphate and

meristem Indole

Geraniol meristem

10-Hydroxy geraniol and parenchyma Tryptophan

7-Deoxyloganin Tryptamine Loganin

cells and cells of adjoining tissues

Shoot epidermal cells and root Secologanin

Cells of epidermis and root root and epidermis of Cells root and shoot phloem phloem shoot and root adjoining tissues

Strictosidine

Tabersonine

Root and/or shoot Ajamalicine

idioblasts other and Desacetoxyvindoline and other kinds of cells laticifers

Catharanthine idioblasts Serpentine , Deacetylvindoline ,

other kinds of cells cells of kinds other

Laticifers kinds of cells in root and shoot and root in cells of kinds Shoot laticifers, idioblasts and

Vindoline Vinblastine Vincristine

Fig 1. Biosynthetic pathway for terpenoid indole alkaloids (TIAs) in Catharanthus roseus. The results of the present study and earlier studies [19,22,24,34] have been used to depict the cell type- and organ- wise compartmentation of biosynthesis and storage of TIA metabolites has been shown to accumulate vindoline, catharanthine, organs has been considered a pre-requisite [58] for the vinblastine, vincristine, ajmalicine and serpentine, in genetic construction of C. roseus to obtain high yields varying concentrations [37,39-44]. The leaves contain of specific TIAs in identified plant organs. With this vindoline in much higher concentrations than the stem objective, in the present investigation the presence of and flower parts of shoot, where the vindoline six TIAs was quantified simultaneously in adult plant concentrations are reported to be marginal [42,45,46]. organs taken from 81 accessions (genotypes), including

Ajmalicine, serpentine and catharanthine occur in an Indian cultivar, and individual F2 generation plants exclusion of the other TIAs in the root system [42,43,47- of a cross between parents differing in TIA expression, 53]. The concentrations of ajmalicine, serpentine and in single runs of a reversed-phase high performance catharanthine in roots are much higher than those in liquid chromatographic (RP-HPLC) system. The leaves [54]. Development of medicinal cultivars in which resulting quantitative description of organ-wise stocks one or more of the pharmaceutically important TIAs are of TIAs and known characteristics of organs reveal that accumulated in increased amounts is an important inter-organ reticulation of TIAs and dominance of objective of genetical studies on C. roseus [55-57]. serpentine route in the distal end of TIA pathway are Knowledge about correlated quantitative distribution two of the determinants of differential storage of TIAs of TIA metabolites and of relative sizes of TIA rich in organs. Catharanthus roseus alkaloids 99

Materials and Methods accession-wise. For each replication, the accessional Plant Material organ pools were sampled for TIA analyses. In this experiment flowers were collected accession-wise in Genetic Resources and Growth Conditions each replication daily for one week before harvest. The In the three experiments reported here, a set of 81 flowers were dried in paper bags in an incubator at 0 accessions, an F2 population and the Indian cultivar 37 C, weighed and utilized for TIA analysis. In the Nirmal of C. roseus were used. The accessions used are experiment -2, 20 weeks old plants were harvested. The a part of the genetic resources of C. roseus being root, stem, leaves, flower buds, flowers and siliquae were maintained by our group at the Centre. The F2 population separated individual plant-wise. In the root and stem belonged to the cross gsr-8 x Delhi Pink, where gsr-8 is samples, the bark was separated by peeling it free of a salinity tolerant mutant in the Glycophytic Salinity wood. The leaves were separated into petioles and Response (GSR)-8 gene [56] and Delhi Pink an accession laminae. Thus 9 organ samples were derived from each collected in New Delhi [59,60]. The two parents differ plant. The plant-wise individual organs were placed in in a number of morphological characters and TIA profile paper bags and dried in an incubator at 39°C until such that gsr-8 has leaf-less white flowered inflorescence samples achieved constant weight. For the experiment - with TIA-poor roots and leaves and Delhi Pink has 3, flowers and siliquae were harvested at noon-time from normal pink flowered inflorescence with TIA-rich roots one hundred 22 weeks old plants. Pedicel, calyx, corolla and leaves. The cultivar Nirmal is rich in TIAs and has tube, petals, anthers, stigma and style and carpel and white flowered normal inflorescence [60]. nectary organs were dissected out from individual flowers The plants were grown in the experimental field plot and pooled to obtain 7 flower organ samples. Likewise of the Centre during the years 2002 and 2003. Seeds seeds were separated from fruits and embryos from seed were sown in February in earthen trays of 20 cm diameter coat to obtain 3 samples. A set of mature seeds was sown and 5 cm girth filled with soil: farm yard manure :: 2:1 on wet filter paper in petridishes and incubated in growth mixture. The seedlings were transplanted to field in April. room at 25°C with 16 h : 8 h :: light : dark cycle to Before transplantation, field plot had been solarized and obtain seedlings. The samples of flower and fruit organs applied with N, P and K at 80, 40 and 40 kg ha-1, and whole seedlings were placed in separate paper bags respectively. The experiment-1 where genetic resources and dried as above and weighed. were evaluated was conducted twice, in 2002 and 2003, Determination of Indole Alkaloids with 4 replications. Field plot used was the same in both The dried organ samples were stored in dessicators, the seasons. The accessions were randomized in each powdered and extracted at room temperature. The replication. Five seedlings/genotype were transplanted extraction procedure reported and deployed earlier [60- in a 1.5 m row/replication; the row to row distance was 62] was used. Each sample was assayed by a RP-HPLC kept at 50 cm. The experiment-2 on an F population 2 method with photo-diode array detection to separate and was performed in the 2003 season. Here 250 F progeny 2 quantify simultaneously the TIAs serpentine, ajmalicine, plants were grown 30 cm x 50 cm apart in field. For the catharanthine, vindoline, vinblastine and vincristine [62]. experiment-3, a large population of Nirmal plants was Figure 2 presents representative HPLC profiles for the grown in field in 2003, as mentioned above. The nursery extracts of seeds, flowers, roots, leaves and stems of C. pans and field plots were irrigated as and when required. roseus plants. The plants were applied dithane M-45 to prevent appearance of fungal disease(s). Anatomical Localization of Cells Stained by Harvest and Preparation of Plant Material for Dragendorff Reagent in Sections of Various Organs Chemical Analysis Various organs and/or their portions, excised from freshly harvested 22 weeks old C. roseus cv Nirmal plants, were Whole plants were dug out in the experiment-1 and –2, placed in petridishes containing water in preparation of with effort to recover maximum of the root system. Roots sectioning. Organs were cut transversely using a hand were washed free of soil by exposing them to running held blade to obtain their sections. All the organs studied water. In the experiment-1, the plants were harvested were cut in their central regions. The sections were when they had achieved an age of 32 weeks. Each stained with Dragendorff reagent [63] and examined harvested plant was placed in a paper bag. The plants in immediately using a compound binocular microscope. paper bags were dried in a shed circulated with stream The cells which have been previously shown to contain of air at room temperature. Dried plant was separated alkaloids and called idioblasts, laticifers and specialized into its root, stem, and leaf components. The harvested parenchyma took up red stain of varying intensities organs were weighed plant-wise. The root, stem, and leaf [22,34,64]. Number of each kind of stained cells was organs from the five plants of a replication were pooled 100 Digvijay Singh et al.

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Fig 2. Sample RP-HPLC chromatograms of six pharmaceutically important terpenoid indole alkaloids (TIAs: S, serpentine; C. catharanthine; A. ajamalicine; VCR, vincristine; VLB, vinblastine; and V, vindoline) in an artificial mixture of these (standard) and in the extracts of root, stem, leaf, flower and seed of periwinkle Catharanthus roseus. The mobile phase used was linear gradient from 90:10

to 35:65 in 30 min of 0.01M phosphate buffer (pH 4.9) and acetonitrile. The stationary phase used was Phenomenex C18 (250 x 4.6 mm, 5¼m) column. The separated TIAs identified here were detected at 220 nm wavelength (more procedural details are given in the experimental section [62])

counted section wise. It is known that cells rich in TIAs accumulated 47, 28 and 25% respectively of all the get intensely stained by Dragendorff reagent, but the alkaloids present in the material. Of the 1227 mg of total reagent is not TIA specific. extracted alkaloidal material present in an average plant, about 13% or 158.8 mg comprised the six TIAs quantified Statistical Analysis in this study. Serpentine was observed to be the principal The analysis of variance and covariance were carried stored alkaloid; it was accumulated in 121.8 mg amount out by following the statistical procedures described by in the whole plant, about 69% of it was in stem, 28% in Cochran and Cox [65] and Panse and Sukhatme [66]. root and 3% in leaves. Ajmalicine, an intermediate in the serpentine branch, was accumulated in 8.8 mg Results amount, most of it in roots. The total plant content of Whole Plant TIA Profile catharanthine was 8.2 mg and roots and leaves contained The experiment-1 (Table 1 and 2) and experiment-2 86 and 14% of it, respectively. A 13.8 mg amount of (Table 2 and 3) provide information on the pattern of vindoline was located in leaves. The end products of distribution of TIAs among the major organs of C. roseus vindoline and catharanthine branches, vincristine and plant. It can be seen from the Table 1 that in plants of 32 vinblastine were stored in leaves in 0.6 mg amount. The weeks age the stem, leaves and root, which accounted observations given in the Table 3 (and Table 2) revealed for about 63, 26 and 11% of 141.2 g of plant weight, tissue -wise location of the TIAs in different plant organs. Catharanthus roseus alkaloids 101

Table 1. Contents of the pharmaceutically important terpenoid indole alkaloids (TIAs) in root, stem and leaf organs of 32 weeks old plants of 81 accessions of Catharanthus roseus

S.No. Parameter Whole plant Roots Stem Leaves 1 Dry weight (g) 141.2±8.3 15.3±0.7 88.4±5.4 36.5±2.6 2 All alkaloids (AA; mg) 1227±81 293±15 589±50 348±32 3 TIAs (mg) 158.8±10.5 50.6±4.6 84.1±6.9 18.7±1.4 4 Serpentine (S; mg) 121.8±5.3 34.4±2.0 84.1±6.9 3.3±0.4 5 Ajmalicine (A; mg) 8.8±0.6 8.8±0.6 Na N 6 Catharanthine (C ; mg) 8.20±0.35 7.09±2.69 N 1.07±0.19 7 Vindoline (V; mg) 13.8±1.4 N N 13.8±1.4 8 Vinblastine (VB)+ Vincristine (VC) (mg) 0.57±0.09 N N 0.57±0.09 a = N, not detected (< 0.1 mg /kg of material).

Table 2. Contents of the pharmaceutically important terpenoid indole alkaloids (TIAs) in various organs of 32 weeks old plants of the accession

gsr-8 and Pink Delhi, a part of the 81 accessions analyzed in the table 1 and parents of F2 generation plants analyzed in the Table 3. S.No. Parametera gsr-8 Pink Delhi Root Stem Leaves Root Stem Leaves 1 Dry weight (g) 12.1 ± 2.3 60.3 ± 5.1 44.6 ± 2.7 14.8 ± 2.7 78.5 ± 8.2 41.2 ± 2.9 2 Serpentine (S; mg) 37.8 ± 9.1 64.9 ± 8.0 3.3 ± 0.9 23.8 ± 4.5 80.4 ± 4.6 5.1 ± 1.4 3 Ajmalicine (A; mg) 10.1 ± 2.3 0.2 ± 0.1 N 10.5 ± 1.4 N N 4 Catharanthine (C; mg) 3.9 ± 1.1 N 1.7 ± 0.6 3.9 ± 1.0 N N 5 Vindoline (V; mg) N b N 11.7 ± 1.7 N N 42.6 ± 7.7 6 Vinblastine (VB)+ Vincristine (VC)(mg) N N 0.9 ± 0.2 N N 6.6 ± 1.8 a = Five individual plants from each of the replication of the experiment-1 (Year 2002) were analyzed; b = N, Not detected (< 0.1 mg/kg of material). (a) The barks of root and stem accumulated TIAs in larger Inter–organ Relationships in TIA Accumulation quantities than corresponding woods, although the The phenotypic correlations observed between percent biomass of barks was relatively smaller than that of contents of various TIAs within and between organs of woods in both root and stem. Serpentine, ajmalicine and 81 accessions (experiment-1) are presented in the Table catharanthine were accumulated in 12.3, 20.6 and 25.3 4. High level of positive correlation was noted between fold higher amounts in root bark as compared to root accumulation capacities for catharanthine and serpentine wood. Like-wise serpentine and ajmalicine were stored (ajmalicine included) in roots. The accumulation in 5.3 and 1.4 fold higher amount in stem bark than in capacities of catharanthine, vindoline and vinblastine stem wood. (b) The dimeric TIAs vinblastine and (vincristine included) in leaves were also interdependent. vincristine were accumulated in the leaf laminae but not Relationships, if any, between root serpentine and in the leaf petioles. Vindoline was accumulated in 32.1 catharanthine accumulation capacities on one hand and fold higher amounts in leaf laminae than leaf petioles. those for serpentine, catharanthine, vindoline and TIA Accumulation Capacity of Organs vinblastine in leaves on the other hand were not significant. However serpentine accumulation capacities The organ-wise percent concentrations of TIA alkaloids of root and stem on one hand and of stem and leaves on were measured in the experiment-1 (Table 1 and TIA the other hand were interdependent. An important content observations on flowers). Serpentine (ajmalicine observation was that in leaves the accumulation of included) was accumulated in the tissues of root, stem, serpentine interfered with that of catharanthine, vindoline leaf and flower organs; its content was relatively low in as well as vinblastine. shoot-leaves (10.0 ± 1.0 mg/100 g), flowers (19.4 ± 1.4 mg/100 g) and stem (92.0 ± 3.2 mg/100 g), as compared TIAs in Flower Organs to that in root where it was present in very high The experiment-3 was carried out to compare the TIA- concentrations (286.4 ± 10.0 mg/100 g). The contents accumulation patterns in roots, leaves, flower and siliqua of catharanthine were 21.1 ± 1.5 mg/100 g in root, 2.6 ± organs of C. roseus cv Nirmal plants (Tables 5-7). Table 0.4 mg/100 g in leaves, 0.3 ± 0.02 mg/100 g in flowers 5 shows that flowers share some of their TIA features and lower than the detection limit in stem. Vindoline with both roots and leaves. Table 6 gives the TIA was accumulated in leaves and flowers in concentrations concentrations in different organs of flowers. It is seen of 38.6 ± 3.1 and 7.3 ± 0.6 mg/100 g, respectively. that corolla- petals and –tubes that account for 52.5 and Vinblastine (vincristine included) was accumulated by 29.7% of flower by weight, respectively, are also the leaves in 1.5 ± 0.2 mg/100 g concentration and by flowers principal TIA bearing components of flowers. Of the 45.2 in 15 fold lower concentration. mg of TIAs present in 100 g of flowers, corolla petal 102 Digvijay Singh et al.

Table 3. Contents of the pharmaceutically important terpenoid indole alkaloids (TIAs) in various organs of 20 weeks old F2 generation plants from the cross between gsr-8 and Pink Delhi accessions in Catharanthus roseus

S.No. Parameter Whole plant Roots Stem Leaves Inflorescences Whole Bark Wood Whole Bark Wood Whole Petioles Laminae All Flower Flowers Siliquae components buds 1 Dry weight (g) 44.3 3.7 1.1 2.5 26.6 11.2 15.4 11.0 0.9 10.0 3.1 0.5 1.5 1.1 ±6.2 ±0.5 ±0.2 ±0.4 ±4.5 ±2.1 ±2.5 ±1.4 ±0.1 ±1.3 ±0.3 ±0.1 ±0.3 ±0.2 2 All alkaloids 1162 97 74 24 702 537 165 203 40 163 155 33 61 62 (AA; mg) ±175 ±15 ±12 ±4 ±133 ±104 ±34 ±28 ±6 ±23 ±20 ±6 ±11 ±13 3 TIAs (mg) 42.8 11.2 10.5 0.8 18.4 15.3 3.1 11.2 0.7 10.5 2.2 0.9 1.0 0.4 ±8.0 ±2.3 ±2.0 ±0.3 ±4.7 ±3.9 ±0.9 ±1.8 ±0.1 ±1.7 ±0.3 ±0.2 ±0.2 ±0.1 4 Serpentine 26.8 8.0 7.4 0.6 17.6 15.0 2.8 0.4 0.2 0.2 NSb NS NS NS (S; mg) ±6.6 ±1.8 ±1.6 ±0.2 ±4.8 ±3.9 ±0.9 ±0.1 ±0.1 ±0.1 5 Ajmalicine 3.576 2.701 2.577 0.124 0.710 0.417 0.293 0.005 0.005 N NS NS NS NS (A; mg) ±0.477 ±0.452 ±0.413 ±0.049 ±0.247 ±0.151 ±0.147 ±0.001 ±0.001 6 Catharanthine 1.085 0.552 0.531 0.021 N N N 0.558 0.172 0.382 0.005 0.001 0.002 0.001 (C; mg) ±0.136 ±0.098 ±0.121 ±0.010 ±0.060 ±0.010 ±0.050 ±0.001 ±0.001 ±0.001 +0.001 7 Vindoline 11.4 Na N N N N N 10.131 0.301 9.672 1.217 0.627 0.370 0.210 (V; mg) ±1.8 ±1.716 ±0.083 ±1.649 ±0.186 ±0.151 ±0.084 ±0.050 8 Vinblastine (VB)+ 0.094 N N N N N N 0.094 N 0.094 N N N N Vincristine (VC) (mg) ±0.013 ±0.013 ±0.013 a = N, not detected (< 0.1 mg/kg of material); b = NS, not shown..

Table 4. Coefficient of correlation between alkaloidal traits of 32 weeks old plants of 81 accessions of Catharanthus roseus

Plant Percent content Character Character designation organ of alkaloid(s) designation C2 C3 C4 C5 C6 C7 C8 C9 C10 Root All C1 +0.71**e +0.46**f -0.23* +0.03 +0.01 -0.18 +0.06 +0.19 +0.04 S + Aa C2 – +0.50** +0.05 +0.27* -0.04 -0.11 +0.02 +0.11 +0.01 Cb C3 – -0.19 +0.07 -0.03 -0.17 +0.05 +0.11 +0.04 Stem All C4 – +0.37** +0.10 +0.27* -0.10 -0.23* -0.10 S + A C5 – +0.09 +0.24* -0.08 -0.18 -0.06 Leaf All C6 – +0.39** +0.02 -0.01 -0.02 S + A C7 – -0.23* -0.38** -0.21* CC8 – +0.64** +0.98** Vc C9 – +0.60** VB + VCd C10 – a = Serpentine + ajmalicine; b = Catharanthine; c = Vindoline; d = Vinblastine+vincristine; e = Significant with 99% level of confidence; f = Significant with 95% probability. (pedicel), calyx, corolla, anthers (androecium) and style, Table 5. Relative concentration of some of the pharmaceutically stigma and ovary (gynoecium), albeit in different important terpenoid indole alkaloids (TIAs) in the root, leaf concentrations. The corolla petals had vindoline in and flower organs of 22 weeks old Catharanthus roseus cv highest concentration in flower, the petals contained 13.8 Nirmal plants mg of the total of 23.2 mg present in all floral organs. S.No. Alkaloid(s) Content (mg) in 100g of dry There was 87 fold more vindoline in corolla petals than roots leaves flowers in corolla tube. Vindoline concentration was very low in 1 Serpentine 95.2 0.5 12.7 anthers (0.2 mg), corolla tube (0.1 mg) and style and 2 Ajamalicine 86.8 Na 3.3 3 Catharanthine 37.8 38.6 4.2 stigma (0.1 mg). Serpentine content in corolla tube was 4 Vindoline Tb 24.7 21.7 relatively high (5.2 mg of a total of 12.7 mg/100 g of 5 Vincristine N 1.7 0.2 flowers). Bulk of the flower ajmalicine was also 6 Vinblastine N 15.8 3.1 concentrated in corolla tube (3.2 mg of a total of 3.3 mg/ Total 219.8 81.3 45.2 100 g flowers). Corolla petals accumulated the entire a = N, content not detected (< 0.1mg/kg of plant material); b = T, Trace amount present (< 1mg/kg of plant material). flower TIAs in high amounts, except ajmalicine: serpentine (2.9 mg), catharanthine (3.8 mg), vincristine and corolla tube accounted for 23.3 and 8.6 mg or 70.4% (0.2 mg) and vinblastine (2.5 mg). Gynoecium and of TIAs cumulatively. The calyx, anther and styles and corolla tube were low in dimeric alkaloids; vinblastine stigma are relatively smaller organs and respectively was however present in relatively higher concentrations accounted for 1.3, 0.9 and 0.2 mg or 5.3% of the total in the stalk (0.5 mg). Catharanthine was present in corolla flower TIAs. Vindoline and serpentine were the TIAs petals and ovary (0.4 mg). The pedicel, calyx and ovary, found present in all the organs of flowers studied-stalk which bear dark green colour of chlorophyll and are Catharanthus roseus alkaloids 103

Table 6. Relative contents of the pharmacetically important terpenoid indole alkaloids (TIAs) in various flower organs of 22 weeks old Catharanthus roseus cv Nirmal plants

S.No. Flower organ(s) Weight(g) in 100g dry flowersTotal alkaloid yield (mg)Yield or content (mg) of certain TIA alkaloids Serpentine Ajmalicine Catharanthine Vindoline Vincristine Vinblastine Altogether 1 Stalk (Pedicel)4.70 162 2.4 Na Tb 2.0 N 0.5 4.9 2 Calyx 2.12 129 0.2 N N 1.1 N T 1.3 3 Corolla petals 52.49 1102 2.9 N 3.8 13.8 0.2 2.5 23.2 4 Corolla tube 29.70 1057 5.2 3.2 T 0.1 T 0.1 8.6 5 Anthers 4.43 108 0.7 N T 0.2 N T 0.9 6 Style and stigma2.58 75 0.1 T T 0.1 T T 0.2 7 Ovary and nectaries 3.97 288 1.2 0.1 0.4 4.4 T T 6.1 8 Total 99.99 2921 12.7 3.3 4.2 21.7 0.2 3.1 45.2 a = N, not detectable (< 0.1 mg/kg of plant material); b = T, trace amount (< 1mg/kg of plant material).

Table 7. Relative content of the pharmaceutically important terpenoid indole alkaloids (TIAs) in various fruit organs of 22 weeks old Catharanthus roseus cv Nirmal plants

Fruit organ Weight (g) in Total alkaloid Yield or content (mg) of certain TIA alkaloids 100g dry siliquae yield (mg) serpentine vindoline catharanthine Siliqua capsule 78.8 3650 18.4 7.7 T b Seed coat 19.5 520 Na NN Embryo 1.7 52 N NN a = N, Not detectable (< 0.1mg/kg of plant material); b = T, Trace amount (< 1mg/kg of material). photosynthetically active flower organs, contained seedlings examined, the green cotyledons were seen serpentine and vindoline. emerging out of seed coats which were still stuck on the cotyledons. Serpentine, ajmalicine, vindoline and TIAs in Siliquae, Seeds and Seedlings catharanthine were present in 1.5, 0.4, 0.2 and 0.1 mg Table 7 summarizes the data on TIA contents of the amounts in 100 g nascent seedlings, respectively. siliquae organs of C. roseus cv. Nirmal (experiment-3). Organ-wise Variation in TIA Component of Total It is shown that neither seed coat nor embryo contained Alkaloids any of the TIAs examined. However, the siliqua capsules contained 18.4 mg of serpentine, 7.7 mg of vindoline The observations given in the tables 1, 3, 6 and 7 allow and 0.3 mg of catharanthine in 100 g siliquae of which the organs of C. roseus to be arranged in the following 21.2 g were seeds. Since seeds were found not to contain order of increasing relative concentrations of total TIAs, it was desired to find out if TIA accumulation alkaloid content in them: leaves (1.0) < stem (1.1) < roots occurred in very young seedlings. Table 8 shows that (1.6) < flowers (2.6) < seeds (2.7) < siliquae (3.3). The while both mature seeds and nascent seedlings (latter total alkaloid concentration varied in dry organs from grown in the presence of light) were rich in total about 29 mg/g in leaves to 98 mg/g in siliquae. On the alkaloids, only the seedlings had the TIAs. In the other hand, the percent content of the profiled

Table 8. Relative content of pharmaceutically important terpenoid indole alkaloids (TIAs) in seeds and seedlings of Catharanthus roseus cv Nirmal

Material Content (mg) in 100g of material all alkaloids Pharmaceutically important alkaloids serpentine ajmalicine vindoline catharanthine Seeds 2698 Nb NNN Seedlingsa 4100 1.5 0.4 0.2 0.1 a = seedlings were sampled at 120 h from the time of initiation of germination on filter paper soaked with water in petridishes incubated at 25°C in 16 h hight and 8 h dark conditions; b = N, content not detected (< 0.1mg/kg of material). 104 Digvijay Singh et al. pharmaceutically important alkaloids (TIAs) in total more densely populated with stained/alkaloid rich cells extracted alkaloids varied between 0.8% in siliquae to than other tissues of root and stem (Table 9). The stem 14.4% in roots. The following is the order of organs in was especially rich in seleridial idioblasts which were terms of the relative concentration of TIAs in total present in the inner cortex in patches of 15 to 20 cells in alkaloids: siliquae (1.0) < flowers (2.5) < leaves (6.7) < the form of ring along the periphery of outer phloem, stem (10.0) < roots (18.1). interrupted by bundles of blast fibers. The cortex tissues of leaf petioles and leaf laminae were rich in specialized Distribution of Dragendorff Reagent Stained Cells parenchymatous cells which occurred in small clusters in Tissues of Various Organs that were arranged on the two sides of the bean shaped In both root and stem, laticifers, idioblasts and/or vascular tissue called adaxial phloem here (Fig. 2). In specialized parenchymatous cells that took up intense the pith region of root and stem, such cells were present red stain were present in hypodermis, cortex and pith on the outer periphery of the tissue. The hypodermis of (Fig. 3; Table 9). The cortex of the root and stem were leaf petiole, palisade and spongy tissue of leaf laminae

A B a b a c b c d d e e f C D

d b a e b c d

E F

c a a

Fig 3 Sections of Catharanthus roseus organs stained with Dragondorff reagent. Parts of organ sections: (A) Transverse section (TS) of root; (B) TS of stem; (C) TS of leaf petiole; (D) Longitudinal section of stem; (E) TS of leaf lamina; and (F) TS of siliqua. The TS of leaf lamina in the mid vein region appeared similar to TS of petiole (E). A portion of carpel is seen inside the siliqua capsule in the TS of siliqua (F). Among the cells that took red stain of Dragondorff reagent intensely, the following labels were assigned: a = laticifer/ idioblast cell; b = bast fiber; c = inner cortex or adaxial phloem cell; d = outer phloem; e = xylem; f = pith. Catharanthus roseus alkaloids 105

Table 9. Relative distribution of the cells exhibiting putative alkaloid accumulation, in 22 weeks old plants of Catharanthus roseus cv Nirmal

S.No. Organa Tissue General featuresb Number of cells that took up dark red Dragendorff stainb 1 Root Cork layer Long compact cells in 3 to 7 layers, interspersed with 27 ± 1 large size laticifers/idioblast cells 2 Outer cortex Large cells loosely arranged in 4 to 7 layers 17 ± 1 3 Inner cortex Several layers of compactly arranged cells, interspersed with bundles of bast fibres 390 ± 61 4 Phloem 4 or 5 layers of small cells 212 ± 70 5 Pith Small in size, inner to xylem 2 ± 1 6 Stem Hypodermis Small cells, compactly arranged in 3 or 4 layers 18 ± 1 7 Outer cortex 5 to 7 layers of cells 9 ± 1 8 Inner cortex Several layers of cells interspersed with bundles of bast fibers 370 ± 40 9 Phloem Small compact cells arranged in 3 or 4 layers 312 ± 65 10 Pith Cortex like cells inner to xylem and internal phloem 415 ± 33 11 Leaf petiole Hypodermis 2 to 3 layers of cells 21 ± 1 12 Cortex Cells in 3 to 7 layers 3 ± 1 13 Adaxial phloem 1 or 2 layers 79 ± 10 14 Leaf lamina Pallisade 1 or 2 layers of large sized cell(s) 32 ± 7 15 Spongy parenchyma Small cells in 2 or 3 layers 11 ± 4 16 Flower pedicel Mesophyll Cells in 6 or 7 layers 12 ± 2 17 Adaxial phloem 1or 2 layer(s) 30 ± 12 18 Calyx Mesophyll Cells in 3 or 4 layers 20 ± 10 19 Corolla tube 0 20 Corolla petals 15 ± 1c 21 Siliqua 25 ± 2 a = Epidermal cells of root, stem, leaf, flower and siliqua took up intense yellowish stain; b = The data pertain to full transverse section of each organ studied; c = Relates to all the 5 petals.

and mesophyll of siliquae were densely packed with Absence of Serpentine, Vindoline and Catharanthine laticifers. The flower organs contained thin walled in Seeds idioblasts and specialized parenchymatous cells. The Like siliquae and flowers, the seeds were observed to be stained cells appeared to be highly mucilagenous in rich in their total alkaloid content. However, all the anthers and organs of gynoecium. A general positive profiled TIAs were found lacking from seed organs (seed correlation was noted between frequency and size of coat and embryo). The whole cotyledon stage seedlings, Dragendorff reagent stained cells and TIA concentrations bereft of seed coat, contained serpentine, ajmalicine, in organs, such that the organs could be arranged in the vindoline and catharanthine. This shows that the branches following order in terms of the two parameters: root and of TIA biosynthetic pathway responsible for serpentine, stem > leaf > flower petals, siliqua (Tables 1, 3, 6, 7 catharanthine and vindoline, which are blocked during and 9). seed development, get induced during seed germination. Discussion Previously, light exposed cotyledon stage seedlings have been shown to synthesize and accumulate ajmalicine, In this study, the root, stem, leaf, flower and fruit (siliqua) vindoline and catharanthine, with and without exposure organs of the dried adult plants of C. roseus [28] were to methyl jasmonate, the elicitor of TIA pathway [44,67]. investigated for their net content of the total alkaloids, serpentine, vindoline and catharanthine, the products of Presence of Serpentine, Vindoline, Catharanthine, three late branches of TIA pathway, and ajmalicine, the Vincristine and Vinblastine in Flower Petals immediate precursor of serpentine along with vincristine The whole flowers as well as flower petals were observed and vinblastine, the dimeric products of vindoline and to be rich in total alkaloids and to contain serpentine, catharanthine. The results of the study have adduced catharanthine, vindoline, vincristine and vinblastine. The evidence for the genetically regulated compartmentation results confirmed the earlier observation that roots of TIAs among C. roseus. Some of the important results contain serpentine and catharanthine but do not contain of the study are discussed below. vindoline, vincristine and vinblastine. Previous work has 106 Digvijay Singh et al. shown that while serpentine and catharanthine branches of TIA pathway are active in underground roots and etiolated dark grown seedlings, tabersonine, a major intermediate of the vindoline branch, is not converted into vindoline in the absence of exposure to light. This is because, of the six reactions known to be involved in the transformation of tabersonine to vindoline, at least one is catalyzed by the action of a thylakoid associated enzyme. In the first reaction tabersonine is hydroxylated by a microsomal tabersonine 16- hydroxylase enzyme [68]. The product is O-methylated by a cytosolic S- adenosyl-L-met, 16 hydroxyl- tabersonine O- methyltransferase enzyme [69]. An as yet unknown enzyme hydroxylates the 2, 3-double bond of the 16- methoxy compound. Next the intermediate product is N-methylated by a S-adenosyl-L-Met-5-Adenosyl-L- Met: 2, 3 dihydro-3-hydroxytabersonine enzyme which is associated with thylakoids, to produce desacetoxy vindoline [15]. The cytosolic enzyme 2-oxoglutatarate dependent dioxygenase (D4H) hydroxylates Fig 4 Predominance of serpentine synthetic route over vindoline desacetoxyvindoline at its 4 position [70]. Lastly, the and catharanthine routes in the accomplishment of the normal cytosolic enzyme desacetoxy-vindoline 4-O-acetyl terpenoid indole alkaloid biosynthetic pathway in Catharanthus roseus organs (root, R; stem, S; leaf L; and inflorescence, I). transformate (DAT) O-acetylates deacetylvindoline to produce vindoline [71]. The expression of D4H and DAT has been shown to be light dependent and it is also shown Recent work on the localization of tissues where TIA . roseus that D4H family proteins exist in seedlings grown in dark pathway is expressed in C has revealed that the [17,67]. Since the flower petals do not possess active central intermediate strictosidine is synthesized in thylakoid differentiated plastids (chloroplasts), presence epidermal cells. It has been shown for vindoline synthesis of vindoline in them suggests the following: (a) vindoline in leaves that reactions from strictosidine to 16- is transported into flower petals from other organs; methyltabersonine or its 2, 3-dihydro derivative also (b) some precursor of vindoline is transported which is occur in epidermis. These intermediates are transported converted into vindoline in flower petals; or (c) there to inner layers where in idioblast/laticferous cells the exists in flower petals a pathway of vindoline synthesis subsequent steps to make vindoline are completed that does not require intermediation of photosynthetically [23,24]. Although only partly demonstrated, it can be active chloroplasts. This hypothesized pathway may visualized that parallel transport of intermediates from require light for its operation, since roots do not produce epidermis in shoot organs and outer parenchyma vindoline. Further, our results demonstrate that reactions (protoderm and cortical cells) in root to inner layers of beyond vindoline and catharanthine that are involved in mesophyll/ parenchyma/ vasculature/ idioblasts/ synthesis of vincristine and vinblastine may not require laticifers must be responsible for the synthesis of active chloroplast participation. catharanthine and serpentine [19,22,34]. Indeed, in the present study bulk of serpentine and catharanthine in root Ubiquitous Presence of Serpentine and serpentine in stem were found to be present in the Among the TIAs profiled, serpentine was observed to bark tissues which include epidermis, cortex and phloem be a major component in all organs, except leaves and parenchyma tissue. flowers where vindoline was the main component. The stem has epidermal layer, parenchyma and Ajmalicine, the precursor of serpentine [72], was phloem cells where various steps of the TIA pathway accumulated in three to four fold lower concentrations are presumably accomplished, yet this organ mainly than serpentine in roots, flowers and siliquae and in much accumulates serpentine in the main. Understanding of lower concentrations in stem and leaves. Serpentine was the mechanisms that operate in stem to block the the principal alkaloid of stem, where from vindoline, expression of vindoline and catharanthine branches will catharanthine and dimeric alkaloids were absent. Among be crucial for the genetic engineering of C. roseus for various organs, the stem, which forms the bulk of C. increased yields of vindoline and catharanthine. Stem roseus plant, presents the extreme example of can also be the major source of ajmalicine if the compartmentation of TIA expression (Fig. 4). reaction(s) from ajmalicine to serpentine can be Catharanthus roseus alkaloids 107 genetically blocked. The fact that the vindoline and Acknowledgements catharanthine are accumulated at higher levels than Grateful thanks are due to CSIR for the award of serpentine in leaves indicates that blocking of serpentine Emeritus Scientistship and to INSA for Senior branch may be a practical means to increase the yield of Scientistship schemes to SK and Director, NIPGR for catharanthine in roots and catharanthine and vindoline the provision of various facilities. The work was in leaves. financially supported by the Department of Plant Phenotype(s) Favourable for High Yields of Biotechnology, Council of Scientific and Industrial Pharmaceutical TIAs Research (CSIR) and National Institute for Plant Genome Research (NIPGR), Government of India. Authors are In the TIA profiles of dissected organs of F population 2 grateful to Asis Dutta, Sabhyata Bhatia and late Jayanti plants of a cross between two hierarchically different Sen for their helpful cooperation. Help of Richa Pandey, parents, some morphological features that favoured high Swati Chaudhary, Sunil Kumar and Vinod Kumar is also yields of pharmaceutically active TIAs from root and thankfully acknowledged. leaf organs became apparent. Bulk of ajmalicine and catharanthine were in root bark and very small amounts References of these alkaloids were present in the woody tissues of 1. J Buckingham and H M Bradley Dictionary of Natural Products. Chapman & Hall, London, New York (1998) root. Therefore thick bark in root emerged as a selection 2. TJC Luizendijk, E van der Heijden and R Verpoorte criterion for high yields of TIAs from roots. The leaf Involvement of strictosidine as a defensive chemical in lamina was observed to be for richer in catharanthine, Catharanthus roseus. J Chem Ecol 22 (1996) 1355-1366 vindoline, and vindoline + vincristine than leaf petiole. 3. JK Lovett and AHC Hoult Allelopathy in plants. In: Robert Accordingly small size petiole appears to be a selection MF, Wink M (Eds), Alkaloids: Biochemistry, Ecology and criterion for high yields of leaf TIAs. These selection Medicinal Applications, Plenum Press, New York (1998) pp criteria are recommended to be used in addition to large 337-346 In: Robert MF, sizes of leaf and root organs and higher concentrations 4. R Verpoorte Antimicrobial active alkaloids. Wink M (Eds), Alkaloids: Biochemistry, Ecology and of specific TIAs in these organs. Medicinal Applications. Plenum Press, New York (1998) pp 397-426 Conclusion 5. M Wink Mode of action of alkaloids. In: Robert MF, Wink M Simultaneous quantification of several TIA-precursors, (Eds), Alkaloids Biochemistry, Ecology and Medicinal -intermediates and end products in various organs/sub- Applications. Plenum Press, New York, (1998) pp 301-325 organs of root, stem, leaf, flower, fruit and seed of 6. P Mishra and S Kumar Emergence of periwinkle genetically variant populations of C. roseus plants has Catharanthus roseus as a model system for molecular biology of alkaloids, phytochemistry, pharmacology, plant biology revealed the basic features of in-planta distributional and in vitro and in vivo cultivation. J Medicin Arom Plant Sci network compartments of TIA-metabolites. In the 22 (2000) 306-337 perennial C. roseus plant, whereas the abcissant organs, 7. D Leveque, J Wihlm, F Jehl Pharmacology of Catharanthus leaves and flowers, were found to contain the near-end roseus alkaloids. Bull Cancer 83 (1996) 176-186 products vindoline and catharanthine and end-products 8. T Schmeller and M Wink Utilization of alkaloid in modern serpentine, vinblastine and vincristine, the perennial medicine. In : Robert MF, Wink M (Eds), Alkaloids: Biochemistry, Ecology and Medicinal Applications. Plenum organs stem and root respectively contained only Press, New York (1998) 435-458 serpentine and serpentine and catharanthine. Expression 9. R Van der Heijden, DI Jacobs, W Snöeirjer, D Hallard and R of the products of serpentine branch appeared to suppress Verpoorte The Catharanthus alkaloids: pharmacognosy and the expression of the vindoline and catharanthine biotechnology. Curr Med Chem 11 (2004) 607-628 branches of TIA pathway in leaves. Non-chlorophyllous 10. N Langlois, F Gueritte, Y Langlois and P Potier Application corolla-petals contained vindoline as well as vinblastine of a modification of the Polonovski reaction to the synthesis and vincristine; transport of vindoline or occurrence of of vinblastine type alkaloids. J Am Soc 98 (1976) 7017-7024 an alternative pathway for vindoline is indicated. TIA 11. G Pasquali, DD Poto and AG Fett- Neto Metabolic engineering of cell cultures versus whole plant complexity pathway was latent in seeds; it got activated upon seed in production of bioactive monoteroene indole alkaloids: germination. An approach to obtain vindoline and recent progress related to the old dilemma. J Biosc Bioeng catharanthine rich plant type may be to develop a 101 (2006) 287-296 genotype in which the serpentine route gene functions 12. AI Scott Biosynthesis of indole alkaloids. Chem Res 3 (1970) have been knocked out. Thick root bark and small leaf 151–157 petiole are surmised as morphological features 13. V De Luca, J Balsevich, RT Tyler, U Eilert, BD Panchuk and WGW Kurz Biosynthesis of indole alkaloids: developmental favourable for TIA richness in root and leaf organs, regulation of the biosynthetic pathway from tabersonine to respectively. vindoline in Catharanthus roseus. J Plant Physiol 125 (1986) 147-156 108 Digvijay Singh et al.

14. V De Luca Enzymology of indole alkaloid biosynthesis. In: 28. M El-Sayed and R Verpoorte Methyljasmonate accelerates Lea, PJ (Ed), Methods in Plant Biochemistry, Enzymes of catabolism of monoterpenoid indole alkaloids in Secondary Metabolism, Academic Press, London (1993) pp Catharanthus roseus during leaf processing. Fitoterapia 76 345-368 (2005) 83-90 15. M Dethier and V De Luca Partial purification of a N- 29. TD McKnight, CA Roessner, R Devagupta, AT Scott and methyltransferase involved in vindoline in Catharanthus CL Nessler Nucleotide sequence of a cDNA encoding the roseus. Phytochemistry 32 (1993) 673–678 vacuolar protein strictosidine synthase from Catharanthus 16. A Contin, R Van der-Heijden, AWM Lefeber and R Verpoorte roseus. Nucleic Acids Res 18 (1990) 4939 The iridoid glucoside secologanin is derived from the novel 30. H Yamamoto, N Katano, A Ooi and K Inoue Secologanin triose-phosphate/pyruvate pathway in Catharanthus roseus synthase, which catalyzes the oxidative cleavage of loganin cell culture. FEBS Lett 434 (1998) 413–416 into secologanin, is a cytochrome P450. Phytochemistry 53 17. FA Vazquez Flota and V De Luca Developmental and light (2000) 7-12 regulation of desacetoxy vindoline 4-hydroxylase in 31. V De Luca, C Marineau, Brisson N Molecular cloning and Catharanthus roseus (L) G Don. Plant Physiol 117 (1998) analysis of cDNA encoding a plant tryptophan decarboxylase: 1351-1361 comparison with animal dopa decarboxylases. Proc Natl Acad 18. K Chahed, A Oudin, N Guivarch, S Hamdi, J-C Chenieux, Sci USA 86 (1989) 2582-2586 M Rideau and M Clastre 1-Deoxy-D-xylulose 5-phosphate 32. AE Goodbody, CD Watson, CCS Chapple, J Vukovic and M synthase from periwinkle: cDNA identification and induced Misawa Extraction of 3’,4’- anhydrovinblastin from gene expression in terpenoid indole alkaloid-producing cells. Catharanthus roseus. Phytochemistry 27 (1988) 1713-1717 Plant Physiol Biochem 38 (2000) 559-566 33. JV Shanks, R Bhadra, J Morgan, S Rijhwani and S Vani 19. S Irmler, G Schröder, B St-Pierre, NP Crouch, M Hotze, J Quantification of metabolites in the indole alkaloid pathway Schmidt, D Strack, U Matern and J Schröder Indole alkaloid of Catharanthus roseus: implications for metabolic biosynthesis in Catharanthus roseus : new enzyme activities engineering. Biotechnol Bioeng 58 (1998) 333-338 and identification of cytochrome P450 CYP72A1 as 34. B St-Pierre, FA Vazquez-Flota and V De Luca Multicellular secologanin synthase. Plant J 24 (2000) 797-804 compartmentation of Catharanthus roseus alkaloid 20. B Veau, M Courtois, A Oudin, J-C Chenieux, M Rideau and biosynthesis predicts intercellular translocation of a pathway M Clastre Cloning and expression of cDNA encoding two intermediate. Plant Cell 11 (1999) 887-900 enzymes of the MEP pathway in Catharanthus roseus. 35. V De Luca and B St-Pierre The cell and developmental Biophys Acta 1517 (2000) 159-163 biology of alkaloid biosynthesis. Trends Plant Sci 5 (2000) 21. G Collu, N Unver, AMG Peltenberg-Looman, R van der 349-364 Heijden, R Verpoorte, J Memelink Geraniol 10-hydroxylase, 36. A Geerlings, MM Ibanez, J Memelink, R van der Heijden a cytochrome P450 enzyme involved in terpenoid indole and R Verpoorte Molecular cloning and analysis of alkaloid biosynthesis. FEBS Lett 508 (2001) 215-220 strictosidine b-D-glucosidase, an enzyme in terpenoid indole 22. V Burlat, A Oudin, M Courtois, M Rideau and B St-Pierre alkaloid biosynthesis in Catharanthus roseus. J Biol Chem Co-expression of three MEP pathway genes and geraniol 10- 275 (2000) 3051-3056 hydroxylase in internal phloem parenchyma of Catharanthus 37. P Laflamme, B St-Pierre and V De Luca Molecular and roseus implieates multicellular translocation of intermediates biochemical analysis of a Madagascar periwinkle root- during the biosynthesis of monoterpene indole alkaloids and specific minovincinine-19-hydroxy-O-acetyltransferase. isoprenoid-derived primary metabolites. Plant J 38 (2004) Plant Physiol 125 (2001) 189-198 131-141 38. J Murata and V De Luca Localization of tabersonine-16- 23. J Murata and V De Luca Localization of tabersonine-16- hydroxylase and 16-OH tabersonine-16-O-methyltransferase hydroxylase and 16-OH tabersonine-16-O-methyltransferase to leaf epidermal cells defines them as a major site of to leaf epidermal cells defines them as a major site of precursor biosynthesis in the vindoline pathway in precursor biosynthesis in the vindoline pathway in Catharanthus roseus. Plant J 44 (2005) 581-594 Catharanthus roseus. Plant J 44 (2005) 581-594 39. GH Svoboda, DA Blake The phytochemistry and 24. S Mahroug, V Courdavault, M Thiersault, B St. Pierre and V pharmacology of Catharanthus roseus (L) G Don. In: Taylor, Burlat Epidermis is a pivotal site of atleast four secondary WI, Farnsworth, NR (Eds), Catharanthus alkaloids. Marcel metabolic pathways in Catharanthus roseus aerial organs. Dekker, NewYork, (1975) pp 45-124 Planta 223 (2006) 1191-1200 40. F Reda Distribution and accumulation of alkaloids in 25. H Rischer, M OresÇicÇ, TS Nen-Laakso, M Katajamaa, F Catharanthus roseus G Don during development. Pharmazie Lammertyn, W Ardiles-Diaz, MCE Van Montagu, D Inze, 33 (1978) 233-234 KM Oksman-Caldentey and A Goossens Gene-to-metabolite 41. OP Virmani, GN Srivastava and P Singh Catharanthus roseus networks for terpenoid indole alkaloid biosynthesis in – The tropical periwinkle. Ind Drugs 15 (1978) 231-252 Catharanthus roseus cells. Proc Nat Acad Sci USA 103 (2006) 5614-5619 42. P Westekemper, U Wieczorek, F Gueritte, N Langlois, Y Langlois, P Potier and MH Zenk Radioimmunoassay for the 26. M Kaltenbach, G Schroder, E Schmelzer, V Lutz and J determination of the indole alkaloid vindoline in Schroder Flavonoid hydroxylase from Catharanthus roseus Catharanthus roseus. Planta Med 39 (1980) 24-37 : cDNA hetrologous expression, enzyme properties and cell- type specific expression in plants. Plant J 19 (1999) 183- 43. B Deus-Neumann, J Stockigt and MH Zenk 193 Radioimmunoassay for the quantitative determination of catharanthine. Planta Med 53 (1987) 184-188 27. J Memelink Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. 44. M Magnotta, J Murata, J Chen and V De Luca Identification FEBS Lett 508 (2001) 215-220 of a low vindoline accumulating cultivar of Catharanthus Catharanthus roseus alkaloids 109

roseus (L.) G. Don by alkaloid and enzymatic profiling. 59. P Mishra, M Singh, S Dwivedi, S Kumar Descriptores of Phytochemistry 67 (2006) 1758-1764 periwinkle Catharanthus roseus. J Medicin Arom Plant Sci 45. S Mandal and ML Maheshwari High pressure liquid 22 (2000) 268-272 chromatographic determination of vindoline, catharanthine, 60. P Mishra, GC Uniyal, S Sharma and S Kumar Pattern of vincaleucoblastine and vincristine in periwinkle leaf. Ind J diversity for morphological and yield related traits among Pharm Sci 49 (1986) 205-209 the periwinkle Catharanthus roseus accessions collected from 46. BG Mersey and AJ Cutler Differential distribution of specific in and around Indian subcontinent. Genetic Res Crop Evol. indole alkaloids in leaves of Catharanthus roseus. Can J Bot 48 (2001) 273-286 64 (1986) 1039-1045 61. YN Shukla, A Rani and S Kumar Effect of temperature and 47. S El-Deeb, MS Karawia and H Abushady Vinca rosea Linn: pH on the extraction of total alkaloids from Catharanthus II Isolation of ajmalicine (d-yohimbine) from the root. Proc roseus leaves. J Medicin Arom Plant Sci 19 (1997) 430-431 Pharm Soc Egypt Sci. 39 (1957) 201-204 62. DV Singh, S Pandey-Rai, S Srivastava, SK Rai, R Misra and 48. CPN Nair and P Pillay Lochnericine: a new alkaloid from S Kumar Simultaneous quantification of some pharmaceutical Lochnera rosea. Tetrahedron 6 (1959) 89-91 Catharanthus roseus leaf and root terpenoid indole alkaloids 49. GH Svoboda, AT Oliver and DR Bedwell Alkaloids of Vinca and their precursors in single runs by reversed-phase liquid rosea (Catharanthus roseus): Extraction and characterization chromatography. JAOAC Int 87 (2004) 1287-1296 of root alkaloids. Lloydia 26 (1963) 141-153 63. Merck Index. Merck and Co, Rohway, New Jersey (1940) 50. A Levy, J Mile, A Ashri and D Palevitch Heterosis and 64. LR Yoder and PG Mahlberg Reactions of alkaloid and correlation analysis of the vegetative components and histochemical indicators in laticifers and specialized ajmalicine content in the roots of the medicinal plant - parenchyma cells of Catharanthus roseus (Apocynaceae). Am Catharanthus roseus (L) G Don. Euphytica 32 (1983) 557- J Bot 63 (1976) 1167-1173 564 65. WG Cochran and GM Cox Experimental Designs. Wiley, 51. S Mandal, VK Srivastava and ML Maheshwari Estimation London (1957) of ajmalicine and serpentine in Catharanthus roseus G Don. 66. VG Panse and PV Sukhatme Statistical Methods for Ind J Pharm Sci 45 (1983) 23-26 Agricultural Workers, Indian Council of Agricultural 52. B Ganapathi and F Kargi Recent advances in indole alkaloid Research, New Delhi (1985) production by Catharanthus roseus (periwinkle). J Exp Bot 67. Vázquez-Flota, M Carrillo-Pech, Y Minero-García and MH 41 (1990) 259-267 María de Lourdes Alkaloid metabolism in wounded 53. M Monforte-Gonzalez, T Ayora-Talavera, IE Maldonado- Catharanthus roseus seedlings. Plant Physiol Biochemi 42 Mendoza and VM Loyola-Vargas Quantitative analysis of (2004) 623-628 serpentine and ajmalicine in plant tissues of Catharanthus 68. St-Pierre and V De Luca A Cytochrome P-450 roseus and hyoscyamine and scopolamine in root tissues of Monooxygenase Catalyzes the First Step in the Conversion Datura stramonium by densitometry in thin layer of Tabersonine to Vindoline in Catharanthus roseus. Biochem chromatography. Phytochem Anal 3 (1992) 117-121 and Enzym 109 (1995) 131-139 54. M Sevastre-Rigouzzo, C Nef-Campa, A Ghesquiere and H 69. J Balsevich, V De Luca and WGW Kurz Altered alkaloid Chrestin Genetic diversity and alkaloid production in pattern in dark grown seedlings of Catharanthus roseus. The Catharanthus roseus and C trichophyllus and their hybrids. isolation and characterization of 4-desacetoxyvindoline: A Euphytica 66 (1993) 151-159 novel indole alkaloid and proposed precursor of vindoline. 55. RN Kulkarni, K Baskavan, RS Chandrashekhara and S Kumar Heterocycles 24 (1986) 2415-2421 Inheritance of morphological traits of periwinkle mutants with 70. E De Carolis and V De Luca Purification, characterization modified contents and yields of leaf and root alkaloids. Plant and kinetic analysis of a 2-oxoglutarate – dependent Breed 118 (1999) 71-74 dioxygenase involved in vindoline biosynthesis from 56. S Pandey-Rai, R Luthra and S Kumar Salt tolerant mutants Catharanthus roseus. J Biol Chem 268 (1993) 5504-5511 in glycophytic response (GSR) genes in Catharanthus roseus. 71. R Power, WG Kurz and V De Luca Purification and Theoret Appl Genet 106 (2003) 221-230 characterization of acetylcoenzyme A: deacetylvindoline 4- 57. S Gupta, S Pandey-Rai, S Srivastava, SC Naithani, M Prasad O-acetyltransferase from Catharanthus roseus. Arch Biochem and S Kumar Construction of primary genetic linkage map Biophys 279 (1990) 370–376 of medicinal plant Catharanthus roseus. J Genet 86 (2007) 72. N Misra, R Luthra, S Kumar Enzymology of indole alkaloid 259-268. biosynthesis in Catharanthus roseus. Ind Biochem Biophys 58. RA Dixon Engineering of plant natural product pathways. 3 (1996) 261-273 Curr Opi Plant Biol 8 (2005) 329-336