Plant Cell Reports

, Volume 28, Issue 8, pp 1215–1234 | Cite as

Optimization of the transient transformation of Catharanthus roseus cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase

  • Grégory Guirimand
  • Vincent Burlat
  • Audrey Oudin
  • Arnaud Lanoue
  • Benoit St-Pierre
  • Vincent Courdavault
Original Paper


The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring.


Biolistic Catharanthus roseus Geraniol 10-hydroxylase (CYP76B6) GFP Methyl erythritol phosphate pathway Monoterpene indole alkaloids 


C. roseus

Catharanthus roseus




Differential interference contrast


Dimethylallyl diphosphate


1-Deoxy-d-xylulose 5-phosphate reductoisomerase


1-Deoxy-d-xylulose 5-phosphate synthase


Endoplasmic reticulum


Green fluorescent protein


Geraniol 10-hydroxylase (CYP76B6)


Hydroxymethylbutenyl 4-diphosphate synthase


Internal phloem-associated parenchyma


Isopentenyl diphosphate


Methyl-d-erythritol 2,4-diphosphate synthase


Methyl-d-erythritol 4-phosphate


Monoterpene indole alkaloids


Naphthalene acetic acid


Organized smooth ER


Class III peroxidase isoenzyme


Strictosidine β-d-glucosidase


Secologanin synthase (CYP72A1)


ER targeting signal peptide and anchoring transmembrane helix of G10H


Strictosidine synthase


Tryptophan decarboxylase


Transmission electron microscopy


Yellow fluorescent protein


Truncated variant of G10H lacking the ER anchoring transmembrane helix



This research was financially supported by the Ministère de l’Enseignement Supérieur et de la Recherche (MENRT) and by the Ligue contre le Cancer (Comité du Loir et Cher). G. Guirimand was financed by a MENRT fellowship. We thank Dr. Chris Hawes (Oxford Brookes University) who kindly provided the ER-YFP marker. We thank Marie-Antoinette Marquet, Evelyne Danos and Céline Melin for help in maintaining cell cultures. We also thank Dr. B. Arbeille and the staff of the Electron Microscopy Platform (CHU Bretonneau, Tours, France) for providing excellent working conditions for ultramicrotomy and TEM studies.

Supplementary material

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Supplemental data 1 (JPG 1,046 kb)

299_2009_722_Fig9_ESM.jpg (2.1 mb)

Supplemental data 2 (JPG 2.11 mb)


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Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Grégory Guirimand
    • 1
  • Vincent Burlat
    • 1
  • Audrey Oudin
    • 1
  • Arnaud Lanoue
    • 1
  • Benoit St-Pierre
    • 1
  • Vincent Courdavault
    • 1
    • 2
  1. 1.Université François Rabelais de Tours, EA 2106 “Biomolécules et Biotechnologies Végétales”, IFR 135 “Imagerie fonctionnelle”ToursFrance
  2. 2.Université François Rabelais de Tours, EA 2106 “Biomolécules et Biotechnologies Végétales”, UFR des Sciences et TechniquesToursFrance

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