Planta

, Volume 223, Issue 6, pp 1191–1200 | Cite as

Epidermis is a pivotal site of at least four secondary metabolic pathways in Catharanthus roseus aerial organs

  • Samira Mahroug
  • Vincent Courdavault
  • Martine Thiersault
  • Benoit St-Pierre
  • Vincent Burlat
Original Article

Abstract

Catharanthus roseus produces a wide range of secondary metabolites, some of which present high therapeutic values such as antitumoral monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine, and the hypotensive MIA, ajmalicine. We have recently shown that a complex multicellular organisation of the MIA biosynthetic pathway occurred in C. roseus aerial organs. In particular, the final steps of both the secoiridoid–monoterpene and indole pathways specifically occurred in the epidermis of leaves and petals. Chorismate is the common precursor of indole and phenylpropanoid pathways. In an attempt to better map the spatio-temporal organisation of diverse secondary metabolisms in Catharanthus roseus aerial organs, we studied the expression pattern of genes encoding enzymes of the phenylpropanoid pathway (phenylalanine ammonia-lyase [PAL, E.C. 4.3.1.5], cinnamate 4-hydroxylase [C4H, E.C. 1.14.13.11] and chalcone synthase [CHS, E.C. 2.3.1.74]). In situ hybridisation experiments revealed that CrPAL and CrC4H were specifically localised to lignifying xylem, whereas CrPAL, CrC4H and CrCHS were specifically expressed in the flavonoid-rich upper epidermis. Interestingly, these three genes were co-expressed in the epidermis (at least the upper, adaxial one) together with three MIA-related genes, indicating that single epidermis cells were capable of concomitantly producing a wide range of diverse secondary metabolites (e.g. flavonoïds, indoles, secoiridoid–monoterpenes and MIAs). These results, and data showing co-accumulation of flavonoids and alkaloids in single cells of C. roseus cell lines, indicated the spatio-temporal feasibility of putative common regulation mechanisms for the expression of these genes involved in at least four distinct secondary metabolisms.

Keywords

Catharanthus Epidermis Flavonoids In situ hybridisation Microscopy Monoterpenoid indole alkaloids 

Abbreviations

MIA(s)

Monoterpenoid indole alkaloid(s)

PAL

Phenylalanine ammonia-lyase

C4H

Cinnamate 4-hydroxylase

CHS

Chalcone synthase

MEP

2C-methyl-d-erythritol 4-phosphate

DXS

1-deoxy-d-xylulose 5-phosphate (DXP) synthase

DXR

DXP reductoisomerase

MECS

2C-methyl-d-erythritol 2,4-cyclodiphosphate (MEC) synthase

G10H

Geraniol 10-hydroxylase

SLS

Secologanin synthase

TDC

Tryptophan decarboxylase

STR

Strictosidine synthase

D4H

Desacetoxyvindoline 4-hydroxylase

DAT

Deacetylvindoline 4-O-acetyltransferase

2,4-D

2,4-dichlorophenoxyacetic acid

MM

Maintenance medium

PM

Production medium

FAA

Formaldehyde acetic acid ethyl alcohol

2-APB

2-aminoethyldiphenyl borinate

AP

Alkaline phosphatase

BCIP

5-Bromo-4-chloro-3-indolyl phosphate

NBT

Nitro blue tetrazolium chloride

ORCA

Octadecanoid-responsive Catharanthus AP2 transcription factor

References

  1. Achnine L, Blancaflor EB, Rasmussen S, Dixon RA (2004) Colocalization of L-phenylalanine ammonia-lyase and cinnamate 4-hydroxylase for metabolic channeling in phenylpropanoid biosynthesis. Plant Cell 16:3098–3109PubMedCrossRefGoogle Scholar
  2. Andi S, Taguchi F, Toyoda K, Shiraishi T, Ichinose Y (2001) Effect of methyl jasmonate on harpin-induced hypersensitive cell death, generation of hydrogen peroxide and expression of PAL mRNA in tobacco suspension cultured BY-2 cells. Plant Cell Physiol 42:446–449PubMedCrossRefGoogle Scholar
  3. Arvy MP, Imbault N, Naudascher F, Thiersault M, Doireau P (1994) 2,4-d and alkaloid accumulation in periwinkle cell suspensions. Biochimie 76:410–416PubMedCrossRefGoogle Scholar
  4. Bevan M, Shufflebottom D, Edwards K, Jefferson R, Schuch W (1989) Tissue- and cell-specific activity of a phenylalanine ammonia-lyase promoter in transgenic plants. EMBO J 8:1899–1906PubMedGoogle Scholar
  5. Brown S, Renaudin JP, Prévot C, Guern J (1984) Flow cytometry and sorting of plant protoplasts: technical problems and physiological results from a study of pH and alkaloids in Catharanthus roseus. Physiol Vég 22:541–554Google Scholar
  6. Burlat V, Oudin A, Courtois M, Rideau M, St-Pierre B (2004) Co-expression of three MEP pathway genes and geraniol 10 hydroxylase in internal phloem parenchyma of Catharanthus roseus implicates multicellular translocation of intermediates during biosynthesis of monoterpene indole alkaloids and isoprenoid-derived primary metabolites. Plant J 38:131–141PubMedCrossRefGoogle Scholar
  7. Cacace S, Schröder G, Wehinger E, Strack D, Schmidt J, Schröder J (2003) A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations. Phytochemistry 62:127–137PubMedCrossRefGoogle Scholar
  8. Courdavault V, Thiersault M, Courtois M, Gantet P, Oudin A, Doireau P, St-Pierre B, Giglioli-Guivarc’h N (2005) CaaX-prenyltransferases are essential for expression of genes involved in the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells. Plant Mol Biol 57:855–870PubMedCrossRefGoogle Scholar
  9. Czichi U, Kindl H (1977) Phenylalanine ammonia-lyase and cinnamic acid hydroxylase as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: cooperation and subcellular distribution. Planta 134:133–143CrossRefGoogle Scholar
  10. Deus B, Zenk MH (1982) Exploitation of plant cells for the production of natural compounds. Biotechnol Bioeng 24:1965–1974CrossRefGoogle Scholar
  11. van der Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289:295–297PubMedCrossRefGoogle Scholar
  12. Gamborg OL, Miller RRA, Ojima K (1968) Nutriment requirements of suspension cultures of soybean root cells. Exp Cells Res 50:151–158CrossRefGoogle Scholar
  13. Gantet P, Memelink J (2002) Transcription factors: tools to engineer the production of pharmacologically active plant metabolites. Trends Pharmacol Sci 23:563–569PubMedCrossRefGoogle Scholar
  14. van der Heijden R, Jacobs DI, Snoeijer W, Hallard D, Verpoorte R (2004) The Catharanthus alkaloids: pharmacognosy and biotechnology. Curr Med Chem 11:607–658CrossRefGoogle Scholar
  15. Hisiger S, Jolicoeur M (2005) Plant cell culture monitoring using an in situ multiwavelengh fluorescence probe. Biotechnol Prog 21:580–589PubMedCrossRefGoogle Scholar
  16. Hotze M, Schröder G, Schröder J (1995) Cinnamate 4-hydroxylase from Catharanthus roseus, and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli. FEBS Lett 374:345–350PubMedCrossRefGoogle Scholar
  17. Hrazdina G, Jensen RA (1992) Spatial organization of enzymes in plant metabolic pathways. Annu Rev Plant Physiol Plant Mol Biol 43:241–267CrossRefGoogle Scholar
  18. Hutzler P, Fischbach R, Heller W, Jungblut TP, Reuber S, Schmitz R, Veit M, Weissenbock G, Schnitzler JP (1998) Tissue localization of phenolic compounds in plants by confocal laser scanning microscopy. J Exp Bot 49:953–965CrossRefGoogle Scholar
  19. Irmler S, Schröder G, St-Pierre B, Crouch NP, Hotze M, Schmidt J, Strack D, Matern U, Schröder J (2000) Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase. Plant J 6:797–804CrossRefGoogle Scholar
  20. Jackson D (1992) In situ hybridisation in plants. In: Gurr SJ, McPherson MJ, Bowles DJ (eds) Molecular plant pathology: a practical approach. IRL Press, Oxford, pp 163–174Google Scholar
  21. Kaltenbach M, Schröder G, Schmelzer E, Lutz V, Schröder J (1999) Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants. Plant J 19:183–193PubMedCrossRefGoogle Scholar
  22. Kao YY, Harding SA, Tsai CJ (2002) Differential expression of two distinct phenylalanine ammonia-lyase genes in condensed tannin-accumulating and lignifying cells of quaking aspen. Plant Physiol 130:796–807PubMedCrossRefGoogle Scholar
  23. Kolb CA, Käser MA, Kopecký J, Zotz G, Riederer M, Pfündel EE (2001) Effects of natural intensities of visible and ultraviolet radiation on epidermal ultraviolet screening and photosynthesis in grape leaves. Plant Physiol 127:863–875PubMedCrossRefGoogle Scholar
  24. Kutchan TM (2005) A role for intra- and intercellular translocation in natural product biosynthesis. Curr Opin Plant Biol 8:292–300PubMedCrossRefGoogle Scholar
  25. Lee BK, Park MR, Srinivas B, Chun JC, Kwon IS, Chung IM, Yoo NH, Choi KG, Yun SJ (2003) Induction of phenylalanine ammonia-lyase gene expression by paraquat and stress-related hormones in Rehmannia glutinosa. Mol Cells 16:34–39PubMedGoogle Scholar
  26. Levee V, Seguin A (2001) Inducible expression of the heterologous PAL2 promoter from bean in white pine (Pinus strobus) transgenic cells. Tree Physiol 21:665–672PubMedGoogle Scholar
  27. Li J, Ou-Lee TM, Raba R, Amundson RG, Last RL (1993) Arabidopsis flavonoid mutants are hypersensitive to UV-B irradiation. Plant Cell 5:171–179PubMedCrossRefGoogle Scholar
  28. Maruyama T, Kanaji T, Nakade S, Kanno T, Mikoshiba K (1997) 2APB, 2-aminoethoxydiphenyl borate, a membrane-penetrable modulator of Ins(1,4,5)P3-induced Ca2+ release. J Biochem (Tokyo) 122:498–505Google Scholar
  29. Memelink J, Verpoorte R, Kijne JW (2001) ORCAnization of jasmonate-responsive gene expression in alkaloid metabolism. Trends Plant Sci 6:212–219PubMedCrossRefGoogle Scholar
  30. Mérillon JM, Ouelhazi L, Doireau P, Guillot A, Chenieux J-C, Rideau M (1986) Indole alkaloid accumulation and tryptophan decarboxylase activity in Catharanthus roseus cells cultured in three different media. Plant Cell Rep 5:23–26CrossRefGoogle Scholar
  31. Neumann D, Krauss G, Hieke M, Gröger D (1983) Indole alkaloid formation and storage in cell suspension cultures of Catharanthus roseus. Planta Med 48:20–23PubMedCrossRefGoogle Scholar
  32. Osakabe Y, Nanto K, Kitamura H, Kawai S, Kondo Y, Fujii T, Takabe K, Katayama Y, Morohoshi N (1996) Immunocytochemical localization of phenylalanine ammonia-lyase in tissues of Populus kitakamiensis. Planta 200:13–19PubMedCrossRefGoogle Scholar
  33. Peer WA, Brown DE, Tague BE, Muday GK, Taiz L, Murphy AS (2001) Flavonoid accumulation patterns of transparent testa mutants of Arabidopsis. Plant Physiol 126:536–548PubMedCrossRefGoogle Scholar
  34. Rasmussen R, Dixon RA (1999) Transgene-mediated and elicitor-induced perturbation of metabolic channeling at the entry point into the phenylpropanoid pathway. Plant Cell 11:1537–1552PubMedCrossRefGoogle Scholar
  35. Richard S, Lapointe G, Rutledge RG, Seguin A (2000) Induction of chalcone synthase expression in white spruce by wounding and jasmonate. Plant Cell Physiol 41:982–987PubMedCrossRefGoogle Scholar
  36. Saslowsky D, Winkel-Shirley B (2001) Localization of flavonoid enzymes in Arabidopsis roots. Plant J 27:37–48PubMedCrossRefGoogle Scholar
  37. Schröder G, Wehinger E, Lukacin R, Wellmann F, Seefelder W, Schwab W, Schröder J (2004) Flavonoid methylation: a novel 4’-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases. Phytochemistry 65:1085–1094PubMedCrossRefGoogle Scholar
  38. Schmelzer E, Kroeger-Lebus S, Hahlbrock K (1989) Temporal and spatial patterns of gene expression around sites of attempted fungal infection in parsley leaves. Plant Cell 1:993–1001PubMedCrossRefGoogle Scholar
  39. Schmid J, Doerner PW, Clouse SD, Dixon RA, Lamb CJ (1990) Developmental and environmental regulation of a bean chalcone synthase promoter in transgenic tobacco. Plant Cell 2:619–631PubMedCrossRefGoogle Scholar
  40. Schnitzler J-P, Jungblut TP, Heller W, Köfferlein M, Hutzler P, Heinzmann U, Schmelzer E, Ernst D, Langebartels C, Sandermann H Jr (1996) Tissue localization of UV-B-screening pigments and of chalcone synthase mRNA in needles of scots pine seedlings. New Phytol 132:247–258CrossRefGoogle Scholar
  41. St-Pierre B, Vazquez-Flota FA, DeLuca V (1999) Multicellular compartmentation of Catharanthus roseus alkaloid biosynthesis predicts intercellular translocation of a pathway intermediate. Plant Cell 11:887–900PubMedCrossRefGoogle Scholar
  42. Subramaniam R, Reinold S, Molitor EK, Douglas CJ (1993) Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa × Populus deltoides) phenylalanine ammonia-lyase genes. Plant Physiol 102:71–83PubMedCrossRefGoogle Scholar
  43. Ye ZH (1996) Expression patterns of the cinnamic acid 4-hydroxylase gene during lignification in Zinnia elegans. Plant Sci 121:133–141CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Samira Mahroug
    • 1
    • 2
  • Vincent Courdavault
    • 1
  • Martine Thiersault
    • 1
    • 2
  • Benoit St-Pierre
    • 1
    • 2
  • Vincent Burlat
    • 1
    • 2
  1. 1.Université François-Rabelais de ToursEA 2106 “Biomolécules et Biotechnologies Végétales” UFR Sciences et TechniquesToursFrance
  2. 2.Université François-Rabelais de ToursUnité sous Contrat reconnue par l’INRA “Facteurs de transcription et ingénierie métabolique végétale”ToursFrance

Personalised recommendations