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JXB Advance Access published online on February 28, 2005
Journal of Experimental Botany, doi:10.1093/jxb/eri116
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Journal of Experimental Botany © Society for Experimental Biology 2005; all rights reserved
Received October 14, 2004
Accepted January 14, 2005

RESEARCH PAPER

Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus

Diane Leménager 1, Lazhar Ouelhazi 2, Samira Mahroug 3, Bertrand Veau 3, Benoit St-Pierre 3, Marc Rideau 3, Jone Aguirreolea 4, Vincent Burlat 3, and Marc Clastre 3*

1 EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France; Departamento de Fisiologia Vegetal, Universidad de Navarra, c/Irunlarrea s/n, 31008 Pamplona, Spain
2 EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France; Laboratoire de Biochimie Végétale et Symbiotes, Institut National de la Recherche Scientifique et Technique, BP 95, 2050 Hammam-Lif, Tunisie
3 EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France
4 Departamento de Fisiologia Vegetal, Universidad de Navarra, c/Irunlarrea s/n, 31008 Pamplona, Spain

* To whom correspondence should be addressed.
Marc Clastre, E-mail: marc.clastre{at}univ-tours.fr


  Abstract

Identification of molecular markers of monoterpenoid indole alkaloid (MIA) accumulation in cell-suspension cultures of Madagascar periwinkle (Catharanthus roseus (L.) G. Don) was performed by two-dimensional polyacrylamide gel electrophoresis. Comparison of the protein patterns from alkaloid-producing and non-producing cells showed the specific occurrence of a 28 kDa polypeptide restricted to cells accumulating MIAs. The polypeptide was purified by preparative two-dimensional gel electrophoresis, digested with trypsin, and microsequenced by the Edman degradation method. Cloning of the corresponding cDNA revealed that the protein which has been named CrPS (Catharanthus roseus Protein S) is a member of the {alpha}/{beta} hydrolase superfamily. Time-course expression studies by northern blot analysis confirmed that CrPS gene expression was associated with MIA accumulation in cell suspension cultures. In the whole plant, multicellular compartmentation is required for alkaloid biosynthesis. In situ mRNA hybridization on developing leaves revealed that CrPS mRNA and transcripts encoding the first enzymes of the MIA pathway were co-localized in internal phloem parenchyma cells. The possible implication of the alkaloid-accumulation associated protein CrPS in the signal transduction pathway leading to MIA production is discussed.

Keywords: {alpha}/{beta} hydrolase; Catharanthus roseus, in situ RNA hybridization; monoterpenoid indole alkaloid; phytohormone; two-dimensional gel electrophoresis.
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