Hypericin biosynthesis in Hypericum hookerianum Wight and Arn: Investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization

Published Date

October 2014, Vol.337(10):571–580, doi:10.1016/j.crvi.2014.08.002

Plant biology and pathology/Biologie et pathologie végétales

Title 

Hypericin biosynthesis in Hypericum hookerianumWight and Arn: Investigation on biochemical pathways using metabolite inhibitors and suppression subtractive hybridization

• Author 

• Padmesh P. Pillai ^a,,
• Aswati R. Nair ^b

• aBiotechnology and Bioinformatics Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695 562, India
• bSchool of Biotechnology, National Institute of Technology, Calicut 673 601, India

Received 25 February 2014. Accepted 5 August 2014. Available online 16 September 2014.

Abstract

The biochemical pathway to hypericin biosynthesis is presumed to be polyketide synthase (PKS) mediated, but it has not been experimentally validated, and no alternate route (chorismate/o-succinylbenzoate pathway) has been analyzed. We report here our earlier developed auxin inducible culture systems of Hypericum hookerianum as a model, to study the metabolic pathway to hypericin synthesis. Inhibitors of the alternate pathway at varying concentrations showed steady synthesis of total hypericins with means of 2.80 ± 0.22, 18.75 ± 0.01; 16.39 ± 3.75, 29.60 ± 1.90 (mevinolin) 2.53 ± 0.10, 18.12 ± 0.56; 0.14 ± 0.01, 14.28 ± 1.11 (fosmidomycin) and 2.7 ± 0.35, 18.75 ± 0.61; 0.14 ± 0.01, 12.80 ± 1.09 mg g⁻¹ DW (glyphosate) in the control and auxin-induced shoot and shoot-forming callus cultures, respectively. SSH analysis classified the differentially expressed sequences into protein synthesis (38%), modification (20%), electron transport (9%) and remaining as unclassified (11%) and unknown proteins (22%). Functional annotation of sequences indicates the presence of additional protein components besides PKS activity. Our results demonstrate direct biochemical and molecular evidence of PKS hypothesis of hypericin biosynthesis for the first time.

Graphical abstract

Keywords

• Hypericum hookerianum
• Hypericin
• Mevinolin
• Fosmidomycin
• Glyphosate
• Type III PKS
• SSH

Abbreviations

• AQ, anthraquinone
• BPS, benzophenone synthase
• CHS, chalcone synthase
• DW, dry weight
• FW, fresh weight
• Kin, kinetin
• NAA, α-naphthaleneacetic acid
• OKS, octaketide synthase
• PGR, plant growth regulators
• PKS, polyketide synthase
• SSH, suppression subtractive hybridization

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