BIOCHEMICAL AND MOLECULAR ANALYSES OF PHYTOALEXIN BIOSYNTHESIS IN APPLE CELL CULTURES

Abstract

Apple scab disease caused by the fungus Venturia inaequalis is the most devastating disease of apple in India and worldwide. Most of the commercial apple cultivars are susceptible to scab disease. Till now, metabolites conferring resistance to scab-disease in apple are largely unknown. Identifying metabolites associated with scab-resistance and their underlying biosynthetic genes may provide novel targets to breed for enhanced scab-resistance. The first aim of this thesis work is to decipher bioactive anticancer metabolites produced in the cell suspension culture of apple cv. Florina upon elicitor-treatment. Yeast-extract treated apple cell culture showed enhanced accumulation of phenolics acids, which is preceded by the enhancement of the activity of phenylalanine ammonia-lyase (PAL) enzyme. The elicited extract showed significant anticancer activity against human cervical (HeLa cells) and breast (MCF-7 cells) cancer cell lines using MTT assay. The second aim of this thesis work was to apply metabolomics tool to identify the metabolites responsible for scab-disease resistance in apple and their regulation during host-pathogen interactions. Comparative gas-chromatography-mass spectrometry based metabolomics of Venturia inaequalis (VIE)-treated cell cultures showed enhanced accumulation of phenolics with formation of three new biphenyl-dibenzofuran phytoalexins, namely, aucuparin, noraucuparin and eriobofuran. The third aim of this thesis work was to detect salicylaldehyde synthase (SAS) activity from Venturia inaequalis elicitor (VIE)-treated cell suspension cultures of apple (Malus domestica ‘Florina’). SAS catalyzes non-oxidative C2-side chain cleavage of 2-coumaric acid to form salicylaldehyde (SALD) in the presence of a reducing agent such as cysteine. Elicitor treatment further resulted in an 8.7-fold increase in the activity of the phenylalanine ammonia-lyase (PAL) enzyme that preceded the peak of SAS activity and total SA accumulation, suggesting the involvement of the phenylpropanoid pathway in SA metabolism. The preferred substrate for SAS was 2-coumaric acid (Km = 0.35 mM), with cysteine being the preferred reducing agent. The fourth aim of this thesis work was to clone and functionally characterize a biphenyl phytoalexin biosynthetic gene, 3,5-dihydroxybiphenyl O-methyltransferase (MdfOMT) from elicitor-treated cell suspension culture of apple cv. Florina. MdfOMT is involved in biphenyl biosynthesis. The coding sequence of MdfOMT was functionally expressed in E. coli. MdfOMT catalyzed regiospecific O-methylation of 3,5-dihydroxybiphenyl at 5´- position. The enzyme showed absolute substrate preference for the 3,5-dihydroxybiphenyl. Although MdfOMT product was not detected in the cell cultures the level of noraucuparin, aucuparin and eriobofuran were significantly uplifted upon elicitor-treatment. MdfOMT fused with N- and C- terminal yellow fluorescent protein showed cytoplasmic localization in the epidermis of Nicotiana benthamiana leaves.