Transport of camptothecin in hairy roots of Ophiorrhiza pumila

Abstract

We have investigated the subcellular accumulation and transport of camptothecin (CPT), a monoterpene indole alkaloid, in hairy roots of Ophiorrhiza pumila. This hairy root produces high amounts of CPT and excretes it into the culture medium. When the hairy roots were exposed to UV radiation, autofluorescence emitted from CPT showed subcellular localization of CPT in the vacuole. Treatment with several inhibitors suggested that CPT excretion is a transporter-independent passive transport controlled by the concentration gradient of the compound. Interestingly, the hairy roots treated with brefeldin A, a vesicle transport inhibitor, showed increased CPT excretion. This could be explained by an increased transport rate of CPT from the endoplasmic reticulum (ER) to the cytoplasm when transport of CPT to the vacuole is blocked. The much higher concentration of CPT in the cytoplasm resulted in the increased excretion rate. This result indicates that CPT is biosynthesized at the ER and transported to accumulate in the vacuole by the same machinery that is used for vacuolar protein sorting. How O. pumila is insensitive to CPT is discussed.

Mechanisms of resistance to self-produced toxic secondary metabolites in plants

“…Camptothecin has been reported to be accumulated in the glandular trichomes of leaves and stems of C. acuminata (Li et al 2002). Subcellular localization studies showed that camptothecin is accumulated in the vacuole, which is considered to be a site for avoiding toxicity (Pasqua et al 2004;Sirikantaramas et al 2007c). Interestingly, in the hairy roots of O. pumila (Saito et al 2001), camptothecin is partially accumulated in the vacuole and partially excreted to the culture medium (Sirikantaramas et al 2007c).…”

Identification of the ligand of Pru p 3, a peach LTP

“…Based on the pattern described for ceramides (Shin et al 2014) and camptothecin (Zhao 2010 The chemical identification of the ligand of Pru p 3 was also confirmed by its capacity to emit blue light when excited with UV radiation (254 nm; Fig. 3a), a feature also shown by camptothecin (Sirikantaramas et al 2007). Moreover, camptothecin is well known for its ability to inhibit the catalytic activity of topoisomerase I, the enzyme responsible for relaxing supercoiling DNA (Wright et al 2015).…”

“…In many cases, secondary metabolites and their pathway intermediates are phytotoxic. Plants have evolved several defence mechanisms to avoid self-toxicity from secondary metabolites (Sirikantaramas et al 2007). A basic strategy to avoid self-toxicity is tight regulation of biosynthesis in terms of temporal/spatial gene expression and localization.…”

Improving Secondary Metabolite Production in Tissue Cultures

“…Since it possesses topoisomerase I poisoning properties, its semi-synthetic derivatives, topotecan and irinotecan, have been developed to be clinically used as anticancer drugs. Previously, we have established a hairy root culture of O. pumila which has already been shown to be a desirable experimental system to study the biosynthesis of camptothecin, since the culture produces a high level of CPT and excretes it into the culture medium (Sirikantaramas et al 2007 ).…”