MOLECULAR ANALYSIS OF CINNAMATE CoA-LIGASE FROM PHYTOALEXIN PRODUCING APPLE CELL CULTURES

Abstract

Apple is an important fruit crop, grown mainly in the temperate region of the world. Apple fruits have high nutritional value and tremendous health protective properties. Routine consumption of apples is linked with low risk of life-threatening diseases. Despite huge economic importance, apple production is badly affected by scab disease. Except for few wild apple cultivars, most of the commercial apple cultivars are susceptible to the apple scab-disease, caused by the fungus Venturia inaequalis. To date, the scab-resistance mechanisms in apples are not well understood. The present study investigates the biochemical and molecular basis of scab-resistance mechanisms in apple. First, cell cultures of moderately scab-susceptible apple cultivar cv. Golden Delicious were developed as a model system to understand apple -Venturia (scab) interactions, both at metabolite and molecular levels. A validated HPLC-mass spectrometry based analytical method was developed to rapidly separate and quantify biphenyl and dibenzofurans, the scab-induced phytoalexins of apple. Further this thesis investigates the comparative metabolomics profile of elicitor-treated cell culture of apple cv. Golden Delicious to identify defense responsive metabolites. Gas chromatography–mass spectrometry (GC-MS) coupled with multivariate analysis were applied to analyze the metabolite profiles of elicited cell cultures. Using non-targeted comparative metabolomics a total of 43 differentially accumulating metabolites were identified in Golden Delicious cell culture treated with Venturia-elicitor, out of which phenolics and biphenyl accumulation was significantly enhanced upon elicitor-treatment. Furthermore, using a genome database of Rosaceae and sequence information from plant secondary metabolism- specific CoA ligase, a cDNA encoding cinnamate:CoA ligase (CNL) was cloned and functionally characterized from the VIE-treated cell cultures of cv. Golden Delicious. This enzyme channels carbon flux from the phenylpropanoid pathway towards benzenoid-metabolism and finally towards biphenyl phytoalexin biosynthesis. MdCNL preferred cinnamic acid as a substrate but failed to accept benzoic acid. MdCNL activity was found to be strictly dependent on the presence of K+ and Mg2+ ions in the assay buffer at optimum concentrations of 100 and 2.5 mM, respectively. Coordinated increase in the phenylalanine ammonia-lyase (PAL) and MdCNL transcript levels preceded accumulation of biphenyl phytoalexin noraucuparin and aucuparin in VIE-treated cell cultures of apple cv. Golden Delicious. When greenhouse-grown apple plants of cv. Shireen (scab-resistant cultivar) and cv. Golden Delicious (moderately scab-susceptible cultivar) were infected with the scab fungus V. inaequalis, up-regulation of MdCNL transcript levels was observed in the ii internodal region with accumulation of aucuparin and noraucuparin phytoalexins. Both phytoalexin levels and MdCNL transcript levels were significantly higher in the cultivar Shireen than that of Golden Delicious. No phytoalexins were detected in the leaves. MdCNL contained a C-terminal type 1 peroxisomal targeting signal consisting of SRL tri-peptide, which directed an Nterminal reporter fusion (YFP-CNL) to the peroxisomes. Together, the data suggest that MdCNL catalyzed cinnamoyl-CoA formation is required for biphenyl phytoalexin biosynthesis in apple.