Present address: Nara Institute of Science and Technology, Graduate School of Biological Sciences, 8916-5, Takayama Ikoma, Nara 630-0101, Japan.
Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase
Article first published online: 4 OCT 2007
The Plant Journal
Volume 53, Issue 2, pages 225–236, January 2008
How to Cite
Levac, D., Murata, J., Kim, W. S. and De Luca, V. (2008), Application of carborundum abrasion for investigating the leaf epidermis: molecular cloning of Catharanthus roseus 16-hydroxytabersonine-16-O-methyltransferase. The Plant Journal, 53: 225–236. doi: 10.1111/j.1365-313X.2007.03337.x
- Issue published online: 4 OCT 2007
- Article first published online: 4 OCT 2007
- Received 14 August 2007; accepted 18 September 2007.
- 16-hydroxytabersonine 16-O-methyltransferase (16OMT);
- Catharanthus roseus (Madagascar periwinkle);
- carborundum abrasion;
- enrichment of leaf epidermal proteins and mRNA;
- gene expression;
- cellular localization
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.