Polyketide synthase enzymes and genes in Plumbago indica

Abstract

Studies on the enzyme and gene of a polyketide synthase in Plumbagin indica have been performed in order to understand the enzymatic formation of plumbagin in plant. Tissue cultures of P. indica were successfully established in forms of callus, root culture and in vitro plantlets. Callus cultures derived from young stem explants were generated on MS medium supplemented with 1.0 g/l 2.4-dichlorophenoxyacetic acid (2.4-D) and 0.1 mg/l 6-benzylaminopurine (BA). Root cultures were established from young leaf segments on Gamborg’s B5 (B5) medium supplemented with 1.0 mg/l α-napthaleneacetic acid (NAA) and 0.1 mg/l kinetin. Induced roots were cultured in Murashige and Skoog (MS) liquid medium without growth regulators for root proliferation. Plantlets were regenerated from nodal segments and maintained on Linsmaier and Skoog (LS) medium also without growth regulators. Plumbagin content present in these P.indica tissues were determined using high performance liquid chromatography (HPLC). The analysis revealed that the content of plumbagin in the aerial parts and roots of the micropropagated plantles was significantly higher than that in callus and root cultures. By using a radiolabelled compound of malonyl-CoA as substrate, a standard enzyme assay was established to detect plant polyketide synthase activities in crude protein extracts prepared from the various tissue cultures of P. indica. The formation of plumbagin was, however, not detected in the established enzyme assay conditions. The technique of molecular cloning was, therefore, introduced to express and characterize the enzyme. A cDNA encoding a polyketide synthase, PinPKS, was isolated from a cDNA library prepared from the RNA of P. indica roots. The recombinant activity with acetyl-CoA as a starter molecule and malonyl-CoA as a co-substrate. Analysis of the resulting reaction mixture revealed that there was enzymatic formation of various sizes of pyrone products. These included the pyrones of triketide, formation of various sizes of pyrone products. These included the pyrones of triketide, tetraketide, pentaketide (one each) and three hexaketides. Addition of a crude protein extract of P. indica tissues into the reaction mixture could lead to an unknown compound which has not been identified. With many attempts, no structures of naphthoquinones formed from the hexaketide level have been found in the enzyme assay. Required factors for plumbagin formation in vitro remain to be discovered.