Micro-analysis and localization of camptothecin in Camptotheca acu

Abstract

Studies are underway to increase production of camptothecin (CPT), a monoterpene alkaloid with proven anticancer properties in its natural source, Camptotheca acuminate, a tree indigenous to China. Several semi-synthetic analogs of CPT are currently marketed for cancer treatment creating an increased demand for CPT and threatening the future of the endangered tree. Despite the full chemical synthesis of CPT, extensive extraction from C. acuminate continues. The long term goal of this project is to identify naturally high producers of CPT in the available C. acuminate germplasm and to maximize production of CPT through molecular and cultural practices. The specific objectives of this study were to develop a rapid micro-assay to screen C. acuminate leaf tissue for atypical CPT production and determine the tissue and subcellular sites of CPT accumulation. A rapid micro-assay employing thin-layer chromatography and imaging software was developed. Tissue treatment and extraction solvent markedly influenced the yield of CPT. The best results were obtained with lyophilized tissue extracted with dichloromethane. Using this method, two different aged leaves from five clonally propagated C. acuminate trees were screened for CPT content. Younger leaves (10 cm) consistently exhibited greater concentrations of CPT on a dry weight basis and a single clone stood out as a high producer when older leaves (20 cm) were screened. To aid in the subcellular localization of CPT, a procedure was developed to isolate protoplasts from C. acuminate leaf tissue. Removing background cell wall fluorescence and exploiting the blue autofluorescence of CPT, the vacuole was identified as the site of CPT accumulation. To confirm this, leaf tissue was immunolabeled with anti-CPT. Secondary metabolite leaching during fixation was ruled out and vacuolar localization of CPT was confirmed. The amount and distribution of anti-CPT label varied with leaf development beginning with the mesophyll and subpalisade layer of small leaves and increasing and redistributing to the palisade layer in addition to the subpalisade in larger leaves. Chemical analysis of CPT supported the immunolabeling observations. Equipped with a screening procedure and knowledge of the CPT accumulation sites, we can move towards the goal of engineering C. acuminate for maximum CPT production.