Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase.
The Madagascar periwinkle (Catharanthus roseus) produces the well-known and remarkably complex anti-cancer dimeric alkaloids vinblastine and vincristine that are derived from the coupling of vindoline and catharanthine monomers. This study describes the novel application of a carborundum abrasion (CA) technique for large-scale isolation of leaf epidermis-enriched proteins in order to purify to apparent homogeneity 16-hydroxytabersonine-16-O-methyltransferase (16OMT), which catalyses the second of six steps in the conversion of tabersonine into vindoline, and to clone the gene. Functional expression and biochemical characterization of recombinant 16OMT demonstrated its very narrow substrate specificity and high affinity for 16-hydroxytabersonine. In addition to allowing the cloning of this gene, the CA technique clearly showed that 16OMT is predominantly expressed in Catharanthus leaf epidermis. The results provide compelling evidence that most of the pathway for vindoline biosynthesis, including the O-methylation of 16-hydroxytabersonine, occurs exclusively in the leaf epidermis, with subsequent steps occurring in other leaf cell types.
Department of Biological Sciences, Brock University, St Catharines, Ontario, L2S 3A1, Canada.
Dylan
D
Levac
Jun
J
Murata
Won S
WS
Kim
Vincenzo
V
De Luca
D030008:Catharanthus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D026121:Indole Alkaloids / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D019441:Plant Epidermis / Q000201:enzymology*; D018515:Plant Leaves / Q000201:enzymology*; D011804:Quinolines / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D014747:Vinblastine / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism; D014748:Vinca Alkaloids / Q000302:isolation & purification / Q000378:metabolism
D026121:Indole Alkaloids; D011804:Quinolines; D011994:Recombinant Proteins; D014748:Vinca Alkaloids; C009373:tabersonine; C009667:vindoline; D014747:Vinblastine; C527828:16-methoxytabersonine methyltransferase, Catharanthus roseus; D008780:Methyltransferases; C017836:catharanthine