title: A mechanistic insight into hydrogen peroxide-mediated elicitation of bioactive xanthones in Hoppea fastigiata shoot cultures.
abstract: Elicitation of xanthones is mediated by ROS where Ca (2+) mediated generation of H 2 O 2 activates the shikimate pathway, a key regulator in early steps of xanthone biosynthesis in H. fastigiata. Shoot cultures of Hoppea fastigiata upon treatment with yeast extract (YE) accumulate an enhanced amount of 1,3,5-trihydroxy-8-methoxy xanthone. We demonstrated that YE treatment was followed by a rapid burst of reactive oxygen species (ROS, O2 (-) and H2O2) and subsequent increase in xanthone contents. The antioxidant enzymes (NADPH oxidase, superoxide dismutase (SOD), peroxidase and catalase) followed a similar kinetics as that of ROS, depending on their role in production or degradation. It was observed that shikimate dehydrogenase (SKDH) and shikimate kinase (SK) activities enhanced after 8 h, benzophenone synthase activity continued to rise after elicitation and peaked at 18 h. Activities of phenylalanine ammonia-lyase and 4-hydroxycinnamoyl-CoA ligase remained suppressed and unaffected, respectively, after elicitation. This suggests a possible phenylalanine-independent biosynthesis of xanthones. Successive treatment of shoots cultures with a NADPH-oxidase inhibitor diphenylene iodide and a ROS-scavenger dihydrolipoic acid showed inhibition in ROS (O2 (-) and H2O2) accumulation. These treatments were also shown to decrease the activities of SKDH and SK, leading to a suppressed amount of xanthones formation. Although O2 (-) showed continuous increase upon treatment with a SOD inhibitor diethyldithiocarbamic acid, the contents of H2O2 and xanthones were decreased, which correlates well with the reduced activities of SKDH and SK. Treatments with calcium antagonists, such as, lanthanum chloride and EGTA were also shown to block the activities of SKDH, SK, NADPH-oxidase and SOD, and consequently leading to suppressed accumulation of ROS (O2 (-) and H2O2) and xanthones.
authors.affiliation: Agricultural and Food Engineering Department, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur, Kharagpur, 721 302, India.
authors.forename: Utkarsh Ravindra
authors.initials: UR
authors.lastname: Moon
authors.affiliation: Agricultural and Food Engineering Department, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur, Kharagpur, 721 302, India. adin@iitkgp.ac.in.
authors.forename: Adinpunya
authors.initials: A
authors.lastname: Mitra
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D001688:Biological Products / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D019735:Carbon-Carbon Ligases / Q000378:metabolism; D002374:Catalase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003066:Coenzyme A Ligases / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D029965:Gentianaceae / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000378:metabolism*; D019255:NADPH Oxidases / Q000378:metabolism; D009195:Peroxidase / Q000378:metabolism; D010650:Phenylalanine Ammonia-Lyase / Q000235:genetics / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D018520:Plant Shoots / Q000187:drug effects / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D013482:Superoxide Dismutase / Q000378:metabolism; D046509:Tissue Culture Techniques; D044004:Xanthones / Q000378:metabolism*
chemical_list: D001688:Biological Products; D017382:Reactive Oxygen Species; D044004:Xanthones; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase; D002374:Catalase; D009195:Peroxidase; D013482:Superoxide Dismutase; D019255:NADPH Oxidases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022642:shikimate kinase; D010650:Phenylalanine Ammonia-Lyase; D003066:Coenzyme A Ligases; C019943:4-coumarate-CoA ligase; D019735:Carbon-Carbon Ligases; C098887:benzophenone synthase; D002118:Calcium
keywords: Hoppea; Reactive oxygen species; Shikimate; Shoot culture; Xanthone