Mannitol Stress Directs Flavonoid Metabolism toward Synthesis of Flavones via Differential Regulation of Two Cytochrome P450 Monooxygenases in Coleus forskohlii.

Praveen Awasthi,Ram A Vishwakarma,Yashbir S Bedi,Ajai Prakash Gupta,Sumit G Gandhi

doi:10.3389/fpls.2016.00985

Abstract

Cytochrome P450 monooxygenases (CYP450s) are known to play important roles in biosynthesis of all secondary metabolites, including flavonoids. Despite this, few CYP450s have been functionally characterized in model plants and roles of fewer CYP450s are known in non-model, medicinal, and aromatic plants. Our study in Coleus forskohlii indicates that flavone synthase (CYP93B) and flavonoid 3′ monooxygenase (CYP706C) are key enzymes positioned at a metabolic junction, to execute the biosynthesis of different sub-classes of flavonoids (flavones, flavonol, anthocynanin, isoflavones etc.) from a common precursor. Such branch points are favored targets for artificially modulating the metabolic flux toward specific metabolites, through genetic manipulation or use of elicitors that differentially impact the expression of branch point genes. Genkwanin, the only flavone reported from C. forskohlii, is known to possess anti-inflammatory activity. It is biosynthesized from the general flavonoid precursor: naringenin. Two differentially expressed cytochrome P450 genes (CfCYP93B, CfCYP706C), exhibiting maximum expression in leaf tissues, were isolated from C. forskohlii. Mannitol treatment resulted in increased expression of CfCYP93B and decrease in expression of CfCYP706C. Metabolite quantification data showed that genkwanin content increased and anthocyanin levels decreased in response to mannitol treatment. Alignment, phylogenetic analysis, modeling, and molecular docking analysis of protein sequences suggested that CfCYP93B may be involved in conversion of naringenin to flavones (possibly genkwanin via apigenin), while CfCYP706C may act on common precursors of flavonoid metabolism and channel the substrate toward production of flavonols or anthocynanins. Decrease in expression of CfCYP706C and increase in accumulation of genkwanin suggested that mannitol treatment may possibly lead to accumulation of genkwanin via suppression of a competitive branch of flavonoids in C. forskohlii.

Highlights

Flavonoids are a diverse group of secondary metabolites with a ubiquitous presence in plants
These Cytochrome P450 monooxygenases (CYP450s) were named on the basis of sequence identity with other members of various CYP450 families as; CfCYP93B and CfCYP706C
CYP450s play an important role in the biosynthesis of secondary metabolites (Bak et al, 2011)

Summary

Introduction

Flavonoids are a diverse group of secondary metabolites with a ubiquitous presence in plants. Increased expression of a F3′5′H resulted in increased hydroxylation of anthocyanidins, thereby an increased blue hue in the flower colors (Tanaka, 2006) Genes such as FNS and F3′M which are placed at a metabolic junction, serve as promising candidates for modulation of the metabolic flux, through use of elicitors or by genetic engineering, for increasing the flux through a specific branch of the pathway (Stephanopoulos, 1999). In rice it has been shown that FNS I catalyzes the conversion of naringenin to apigenin using co-factors oxoglutarate, FeSO4, ascorbate, and catalase (Lee et al, 2008). FNS II, in contrast, is a membrane bound enzyme belonging to CYP93B family, which catalyzes the conversion of naringenin to apigenin, without the need of co-factors such as FeSO4, ascorbate, and catalase (Martens and Mithöfer, 2005). FNS II activity was first demonstrated in Antirrhinum majus L. (Plantaginaceae; Stotz and Forkmann, 1981) and osmotically stressed cell suspension culture of Glycine max (L.) Merr. (Fabaceae) (Kochs and Grisebach, 1987) and later was functionally characterized in wide range of plant species (Martens and Mithöfer, 2005)

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Conclusion