Article pubs.acs.org/JAFC
Cite This: J. Agric. Food Chem. 2019, 67, 7399−7409
Molecular and Functional Characterization of Oryza sativa Flavonol Synthase (OsFLS), a Bifunctional Dioxygenase Sangkyu Park, Da-Hye Kim, Bo-Ra Park, Jong-Yeol Lee, and Sun-Hyung Lim* National Institute of Agricultural Sciences, Rural Development Administration, JeonJu, 54874, Republic of Korea
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ABSTRACT: Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. We isolated OsFLS from the rice (Oryza sativa) cultivar “Ilmi” OsFLS includes highly conserved 2-ODD-specific motifs and FLSspecific regions. Recombinant OsFLS exhibited both FLS and flavanone 3β-hydroxylase (F3H) activities, converting dihydroflavonols into flavonols and flavanones into dihydroflavonols, respectively, and more efficiently used dihydrokaempferol than dihydroquercetin as a substrate. OsFLS was expressed in both nonpigmented and pigmented rice seeds and was developmentally regulated during seed maturation. Transgenic tobacco (Nicotiana tabacum) plants expressing OsFLS produced pale pink or white flowers with significantly increased levels of kaempferol-3-O-rutinoside and dramatically reduced levels of anthocyanin in their petals. Additionally, pod size and weight were reduced compared to the wild type. Several early and late biosynthetic genes of flavonoid were downregulated in the transgenic flowers. We demonstrated that OsFLS is a bifunctional 2ODD enzyme and functions in flavonol production in planta. KEYWORDS: flavonoid, flavonoid biosynthetic pathway, flavonol, flavonol synthase, kaempferol, quercetin, rice
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INTRODUCTION Flavonols, comprising the largest class of flavonoids in plants, are classified into three subclasses (kaempferol, quercetin, and myricetin) based on the hydroxylation patterns of their flavonoid B-rings. Most flavonols undergo various modifications, such as glycosylation and methylation, resulting in a large number of different molecules.1 Each subclass of flavonols shows different spatiotemporal distribution and accumulation patterns, which are influenced by environmental factors.2−5 Flavonols have various physiological functions in plants, such as UV protection, the regulation of auxin transport, male fertility,6−9 and flower pigmentation in combination with anthocyanins.10,11 Many studies have reported the various health-promoting effects of flavonols, due to their antioxidant, antiproliferative, antiangiogenic, and neuropharmacological properties,12,13 and thus interest in flavonol-rich foods is increasing. Flavonols are synthesized via the flavonoid biosynthetic pathway (Figure 1). The first committed step of this pathway is catalyzed by chalcone synthase (CHS), which synthesizes chalcone from 4-coumaroyl-CoA and three molecules of malonyl-CoA. Chalcone isomerase (CHI) then catalyzes the conversion of chalcone to the flavanone naringenin, a universal precursor in this pathway. Flavanone naringenin can be hydroxylated at the C-3 position by flavanone 3β-hydroxylase (F3H), resulting in dihydroflavonol, or it can be desaturated at the C-ring by flavone synthase I or II (FNSI or FNSII), resulting in flavone. Flavonols are synthesized through desaturation of the C-ring of dihydroflavonols by flavonol synthase (FLS). During this step, dihydroflavonol 4-reductase (DFR) competes with FLS for dihydroflavonols and generates leucoanthocyanidins, which are converted to anthocyanidins by anthocyanidin synthase (ANS). Of the enzymes in the flavonoid pathway, F3H, FNSI, FLS, and ANS have similar enzymatic properties and have been © 2019 American Chemical Society
classified into the 2-oxoglutarate-dependent dioxygenase (2ODD) superfamily. The 2-ODDs are nonheme iron-containing cytosolic proteins that require 2-oxoglutarate and molecular oxygen as a cosubstrate and ferrous iron as a cofactor to catalyze a variety of oxidation reactions, including hydroxylations, desaturations, and oxidative ring closures.14 Among these four enzymes, FLSs share similarity with ANSs at the amino acid sequence level (50−60% similarity), and F3Hs are highly similar to FNSIs (80%). However, FLSs and ANSs share a lower level of sequence similarity with F3Hs and FNSIs (
