Our long-term goal is to understand the mechanisms by which Camptotheca acuminata regulates the biosynthesis, transport, and degradation of camptothecin (CPT), an antitumor and antiviral alkaloid. This work should provide insight into the coordinate regulation of other complex secondary product pathways and may suggest biotechnology strategies for increasing the production of CPT and other alkaloids. Our first specific aim is to investigate regulation at the molecular genetic level by identifying and characterizing genes that encode specific enzymes of the CPT pathway, including two previously isolated genes encoding tryptophan decarboxylase and 3-hydroxy-3-methylglutary CoA reductase, as well as two new genes encoding geraniol-10- hydroxylase from the terpenoid side of the pathway and CPT 10 -hydroxylase, an enzyme that occurs very late in the pathway. Characterization of the developmental and environmental conditions required for the expression of these genes will elucidate the overall regulation of the pathway. Our second specific aim is to investigate the regulation of this pathway at the intracellular and intercellular levels. We discovered that CPT accumulates to high levels in very young leaves, but it is not made there. Because CFT is insoluble in aqueous Solutions, it is likely that a water.soluble precursor is transported to young leaves where the final steps in the pathway are carried out. We will identify the initial sites of Synthesis by asking which tissues can incorporate radiolabeled precursors into intermediates in the CPT pathway. Transported intermediates will be identified by feeding labeled precursors to the sites of synthesis and examining labeled products in the vascular system of the plant The metabolic fate of CPT, including its likely conversion into 10-hydroxy-CPT, will be examined also. Our third specific aim is to develop procedures for the transformation and regeneration of Camptotheca. We have expressed foreign genes in transgenic callus and suspension culture cells, and we have regenerated multiple shoots from stem explants. We will develop methods to combine these two capabilities so that transgenic trees can be produced to allow facile analysis of alkaloid genes and over-expression of the CPT pathway.
Showing the most recent 10 out of 11 publications
