Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase.

Abstract	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.
Authors	Dylan Levac, Jun Murata, Won S Kim, Vincenzo De Luca
Journal	The Plant journal : for cell and molecular biology (Plant J) Vol. 53 Issue 2 Pg. 225-36 (Jan 2008) ISSN: 0960-7412 [Print] England
PMID	18053006 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References	Indole Alkaloids Quinolines Recombinant Proteins Vinca Alkaloids tabersonine vindoline Vinblastine 16-methoxytabersonine methyltransferase, Catharanthus roseus Methyltransferases catharanthine
Topics	Catharanthus (enzymology, genetics) Cloning, Molecular Indole Alkaloids (isolation & purification, metabolism) Kinetics Methyltransferases (genetics, isolation & purification, metabolism) Molecular Sequence Data Plant Epidermis (enzymology) Plant Leaves (enzymology) Quinolines (isolation & purification, metabolism) Recombinant Proteins (metabolism) Vinblastine (analogs & derivatives, isolation & purification, metabolism) Vinca Alkaloids (isolation & purification, metabolism)

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