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JXB Advance Access originally published online on February 28, 2005
Journal of Experimental Botany 2005 56(414):1221-1228; doi:10.1093/jxb/eri116
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.

RESEARCH PAPER

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

Diane Leménager1,2, Lazhar Ouelhazi1,3 {dagger}, Samira Mahroug1, Bertrand Veau1, Benoit St-Pierre1, Marc Rideau1, Jone Aguirreolea2, Vincent Burlat1 and Marc Clastre1,{ddagger}

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

{ddagger} To whom correspondence should be addressed at Laboratoire de Biologie Moléculaire Végétale, Faculté des Sciences Pharmaceutiques, 31 Avenue Monge, F-37200 Tours, France. Fax: +33 247 27 66 60. E-mail: marc.clastre{at}univ-tours.fr

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}/ß 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.

Key words: {alpha}/ß hydrolase, Catharanthus roseus, in situ RNA hybridization, monoterpenoid indole alkaloid, phytohormone, two-dimensional gel electrophoresis


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