Purification, molecular cloning, and cell-specific gene expression of the alkaloid-accumulation associated protein CrPS in Catharanthus roseus.
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.
Leménager
Diane
D
EA2106 Biomolécules et Biotechnologies Végétales, Université de Tours, 31 Avenue Monge, F-37200 Tours, France.
Ouelhazi
Lazhar
L
Mahroug
Samira
S
Veau
Bertrand
B
St-Pierre
Benoit
B
Rideau
Marc
M
Aguirreolea
Jone
J
Burlat
Vincent
V
Clastre
Marc
M
D000470:Alkaloids / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D030008:Catharanthus / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D015180:Electrophoresis, Gel, Two-Dimensional; D018506:Gene Expression Regulation, Plant; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D000470:Alkaloids; D017931:DNA Primers; D010940:Plant Proteins; D018749:RNA, Plant