Phytochem Rev (2016) 15:363–390 DOI 10.1007/s11101-015-9426-0

Methoxylated flavones: occurrence, importance, biosynthesis

Anna Berim . David R. Gang

Received: 13 March 2015 / Accepted: 20 July 2015 / Published online: 28 July 2015 Ó Springer Science+Business Media Dordrecht 2015

Abstract Lipophilic flavones with several methoxyl Keywords Flavonoids Á Lipophilic Á O-methylation Á residues occur in various clades of land plants, from Bioactivity Á Biosynthetic network liverworts to core eudicots. Their chemodiversity is mediated by the manifold combinations of oxygena- Abbreviations tion and methoxylation patterns. In the Lamiaceae, CHS Chalcone synthase Asteraceae, and Rutaceae, (poly)methoxylated fla- FNS Flavone synthase vones are thought to be produced by secretory tissues FOMT Flavonoid O-methyltransferase and stored externally or in oil cavities. They exhibit an F(digit)OMT Flavonoid (digit)-O- array of bioactivities in vitro and in vivo, and may methyltransferase constitute part of the plants’ chemical defense mech- 2-ODD 2-Oxoglutarate-dependent anisms and represent promising natural lead mole- dioxygenase cules for the development of potent antiproliferative, PMF Polymethoxylated flavones antidiabetic, or anti-inflammatory drugs. The biosyn- PTC52 Protochlorophyllide a oxygenase thesis of (poly)methoxylated flavones in sweet basil RO Rieske-type oxygenase (Ocimum basilicum L.) has been largely elucidated in the past few years. The knowledge obtained in those studies can be used for enzymatic semi-synthesis of these flavones as well as for further cell biological and physiological studies of basil trichome metabolism. In addition, these findings create an excellent starting Introduction point for investigations into (poly)methoxylated flavone metabolism in more and less distantly related Flavonoids are a large and diverse group of specialized taxa, which would shed light on the evolution of this metabolites comprising, according to recent accounts, biosynthetic capacity. over 9000 distinct chemical units (Ferrer et al. 2008). The basic skeleton of a flavonoid is a C6–C3–C6 structure formed by the stepwise condensation of a & & A. Berim ( ) Á D. R. Gang ( ) phenylpropenoyl-CoA starter molecule, mostly p- Institute of Biological Chemistry, Washington State University, 100 Dairy Road, Pullman, WA 99164, USA coumaroyl-CoA, with three malonyl-CoA units, each e-mail: [email protected] of which undergoes decarboxylation, and by the D. R. Gang subsequent cyclization of the polyketide chain to form e-mail: [email protected] a phloroglucinol ring (Fig. 1). Compounds with this 123 364 Phytochem Rev (2016) 15:363–390

OH O S-CoA 3 4 OH Enz OH 2 OH 3´ O HO 4´ 2´ OH 5 HO O O O CHS O S β CHS 6 1´ α + 3x HO S-CoA 5´ 6´ OH O OH O -3xCO2 O O OH p-coumaroyl-CoA malonyl-CoA tetraketide chalcone aurone intermediate 3´ 4´ OH OH OH OH 8 B HO 7 O 2 5´ HO OH HO O HO O A C 6 3 4 OH OH 5 OH O OH O OH O OH O flavonol dihydroflavonol flavanone dihydrochalcone FNS I or FNS II

OH OH HO O HO O HO O+

OH OH O OH O OH OH flavone isoflavone anthocyanin

Fig. 1 Biosynthetic origin and types of flavonoids. Depicted is dependent dioxygenases, or FNS II, cytochrome P450-depen- the first committed step of flavonoid biosynthesis, catalyzed by dent monooxygenases) are responsible for the formation of chalcone synthase (CHS), and types of flavonoid backbones flavones. The backbone numbering is indicated on chalcone and formed by downstream cyclizations and further core structure flavanone structures modifications. Flavone synthases (FNS I, 2-oxoglutarate-

parental bicyclic C6–C3–C6 structure are called chal- compounds reported as of 2005 (Martens and Mithofer cones, and the type III polyketide synthase catalyzing 2005), with their numbers steadily increasing. About this first committed step of flavonoid biosynthesis is 60 new flavones were reported between 2007 and 2009 designated chalcone synthase (Abe and Morita 2010; (Veitch and Grayer 2011). Like in all natural product Winkel 2006). To give rise to the tricyclic phenylchro- groups, their chemical diversity is achieved by man- mane backbone typical for the majority of flavonoids, ifold modifications of the flavone backbone sub- chalcones are further transformed by chalcone iso- stituents. These modifications lead to differing merase (Ferrer et al. 2008). Based on modifications to chemical properties of the individual flavone entities. the structure of this phenylchromane skeleton, flavo- Conjugations with saccharides, i.e., glycosylations noids are subdivided into several groups, such as make the molecule more hydrophilic. Among glyco- flavanones, flavonols, dihydroflavonols, flavones, iso- sylated flavones, both C- and O-glycosylations are flavones, anthocyanins, etc. (Fig. 1). The accumula- found. Other modifications, such as prenylations or tion of flavonoids of some type or other is ubiquitous methylations, render the molecule more lipophilic. It in land plants (Winkel-Shirley 2001). is relevant to mention at this point that C-methylation Flavones, the subject of this review, differ from the of flavonoids in Pinus strobus has been shown to flavonols by the lack of a hydroxyl residue at position originate during the scaffold formation step catalyzed 3 of the C ring. However, they are not intermediates en by a specialized chalcone synthase that utilizes an route to flavonols and are formed via a separate branch unusual substrate (methylmalonyl-CoA), resulting in a of the greater flavonoid biosynthetic network (Fig. 1). C-methyl moiety at positon 6 (Schroder et al. 1998), Flavones are one of the most frequently occurring sub- rather than being introduced by C-methyltransferases classes of flavonoids, with more than 500 distinct as happens, e.g., in the biosynthesis of sterols

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(Nes 2003). However, there are currently no data other algae concerning the analogous reaction in non-gym- “viridiplantae” green algae nosperms. Prenylated lipophilic flavonoids have been land plants liverworts Marchantiales reported from a number of species and exhibit strong mosses hornworts cytotoxic potential, as recently reviewed by Smejkal vascular plants lycophytes (2014). In the present review, we will focus on lipophilic ferns Polypodiales seed plants gymnosperms

O-methylated (or methoxylated) flavones. The desig- angiosperms basal angiosperms Laurales nation ‘‘polymethoxylated flavonoids’’ has traditionally magnoliids Piperales been reserved for compounds with four and more monocots Poales Sapindales methoxyl residues. The occurrence of polymethoxy- Gentianales lated flavones (PMFs) sensu stricto is rather restricted. eudicots Lamiales Asterales In addition, even plant species accumulating PMFs Fabales Fagales fitting this definition tend to additionally accumulate sets of flavones with fewer ‘‘decorations’’,which belong Fig. 2 Occurrence of (poly)methoxylated flavones in land to the same pathway or metabolic network and probably plants. Depicted are the major clades of the land plants. Orders represent biosynthesis intermediates or alternative end- for which the occurrence of (poly)methoxylated flavone occurrence has been documented are shown next to branches. points. We therefore describe the topic of this review as The list of orders is not comprehensive for the eudicots (poly)methoxylated flavones, and will try to present an overview of their occurrence in the plant kingdom, their importance for plants and humans, and the current status broad conclusions concerning the occurrence of of knowledge concerning the biochemistry and genetics (poly)methoxylated flavones must await comprehen- of their production. Special attention will be paid to sive and systematic studies. Nevertheless, it is already flavones with additional hydroxylations at positions 6 obvious that the ability to biosynthesize and 8 of the phenylchromane scaffold (Fig. 1), whose (poly)methoxylated flavones is widespread in land biosynthesis we have recently studied in detail. plants from liverworts and ferns to core eudicots (Fig. 2). This biosynthetic capability thus appears to have emerged early on after plants invaded terrestrial The distribution and diversity environments, if not sooner. Indeed, certain ferns, such of (poly)methoxylated flavones in the plant as selected Cheilanthes and Nothoelana species, kingdom Pteridaceae (Wollenweber and Schneider 2000) and liverworts, such as Monoclea, Monocleaceae (Kraut While a great and growing number of phytochemical et al. 1992), Marchesinia, Lejeuneaceae (Nagashima studies reporting already known and novel compounds et al. 1999) and Asterella, Aytoniaceae (Neves et al. are published every year, the majority of these reports 1998) accumulate compounds identical to those found refer to investigations in angiosperms, which receive in angiosperms. Remarkably, an overview of plant the largest share of scientific attention. The angios- families accumulating (poly)methoxylated flavonoids perm families in which the production of modified at positions 6 and 8 (including flavones and (poly)methoxylated flavones has been very well other types of flavonoids) reveals there are very few or documented include the Lamiaceae, Asteraceae and no reports on the occurrence of these compounds in Rutaceae. A large number of species from each of gymnosperms. Only a few occurrences have been those families have been studied by experts in this reported for monocots, such as the grass Gynerium field, such as Eckhard Wollenweber, Renee Grayer, sagittatum (Benavides et al. 2007). And from the Jeffrey Harborne, and Tom Mabry. The findings from magnoliid clade, a few members of the Laurales those studies were used in chemotaxonomic research (Leong et al. 1998; Tanaka et al. 2006) and Piperales prior to the advent of molecular biological tools. Less (Cheng et al. 2003; Felippe et al. 2008) accumulate intensive investigations have been conducted with (poly)methoxylated flavones (Fig. 2). Molecular elu- selected species from further families, such as the cidation of these compounds’ biosynthesis across Rosaceae, Plantaginaceae, Juglandaceae, and Pteri- different lineages is required to clarify the evolution- daceae. Given this current status of understanding, ary origins of this biosynthetic capacity. 123 366 Phytochem Rev (2016) 15:363–390

Meticulous cataloguing of new flavonoids and their been reported from individual species belonging to occurrence has been continuously carried out by other families such as the Asteraceae (Praxelis Harborne and Baxter (1999), Harborne and Williams clematidea; de Azevedo Maia et al. 2011), Lauraceae (2000, 2001), and Williams and Grayer (2004). A (Laurus nobilis; Tanaka et al. 2006), and Moraceae recent review covering the period after 1992 system- (Ficus ssp. hirta; Lansky et al. 2008), is the methy- atically listed the updates on the occurrence of 309 lation of the hydroxyl moiety at position 5. theoretically possible flavone aglycones and their The 5-, 7-, and 40-OH residues are derived from the methyl ethers (Valant-Vetschera and Wollenweber flavonoid scaffold formation step and are thus present 2006). Further updates on newly reported flavonoids, in most flavonoids. Nevertheless, unusual flavones including flavones, have been prepared by Veitch and lacking the hydroxyl moieties at positions 40 and/or 7, Grayer (2008; 2011). We will thus refrain from listing such as zapotin (5,6,20,60-tetramethoxyflavone, Fig. 3) individual compounds and will only try to briefly have been reported from Casimiroa species, Rutaceae summarize the trends recognized so far. (Garratt et al. 1967), Struthiola argentea, Malvaceae The arrays of flavonoids accumulated by a genus (Ayers et al. 2008), and from Primula veris, Primu- frequently display certain common signature features laceae (Budzianowski et al. 2005). Primula species are that appear dominant and are shared by a number of known for their farinose exudates that are in part species in the genus, and sometimes by numerous comprised of highly unusual, partly methoxylated genera in the family. One such feature is the accumu- flavones lacking the typical oxygenations, some of lation of both flavones and flavonols. Flavones seem to them bearing merely a single hydroxyl or methoxyl be the major group of methoxylated flavonoids moiety (Valant-Vetschera and Wollenweber 2006). produced by the Lamiaceae, with flavonols only being The occurrence of such compounds raises the question reported in a few instances, such as in Scutellaria spp. of whether their backbone results from utilization of (Tomas-Barberan and Wollenweber 1990) and two unusual substrates by chalcone synthase, e.g., cin- species from the clade Coleus of the genus Plectran- namoyl-CoA as starter, or from the polyketide scaffold thus (Grayer et al. 2010). In the Asteraceae and modification, e.g., by reduction of the 7-OH moiety. Rutaceae, the accumulation of methoxylated flavonols Primula veris also produces flavonoids with the with substitution patterns analogous to flavones in the methylenedioxy bridge substructure (Budzianowski same species is quite common (Valant-Vetschera and et al. 2005). Flavones substituted at positions 6 and 8 Wollenweber 2006). The pattern analogies include the but lacking a hydroxyl moiety at position 5 have also additional hydroxylations or methoxylations at posi- been reported, e.g., from Euryops arabicus, Aster- tions 6 and/or 8 of the flavonoid backbone. These aceae (Alarif et al. 2013). The formation of 5-deoxy recurring patterns suggest that the enzymes involved flavonoids in the course of isoflavone biosynthesis in in backbone modifications may be able to accept both the Fabaceae has been shown to be catalyzed by a flavone and flavonol substrates. Another structural chalcone reductase acting on the p-coumaroyl-trione detail worth mentioning is that in the vast majority of intermediate prior to its aromatization (Bomati et al. Lamiaceae species investigated to date, all compounds 2005), a mechanism that might be utilized in other oxygenated at position 8 are also oxygenated at plant families as well. Overall, the comprehensive position 6, suggesting that the introduction of the overview by Valant-Vetschera and Wollenweber latter residue occurs before the former in the course of (2006) suggests that no substitution pattern can be biosynthesis (Tomas-Barberan and Wollenweber reliably viewed as being unnatural, as there are reports 1990; Valant-Vetschera and Wollenweber 2006). In of the most unexpected compounds isolated from contrast, members of the Rutaceae and selected various sources. members of the Asteraceae have been reported to The different modifications increasing lipophilicity accumulate flavones such as isoscutellarein-5,7,8,40- are not mutually exclusive in flavones. Flavones tetramethyl ether and isosinensetin (Fig. 3), where decorated with both prenyl and methoxyl residues only position 8 is oxygenated, alongside flavones with have been reported from Neoraputia paraensis (Ru- methoxylated 6- and 8-positions. A structural feature biaceae) (Souza et al. 1999), and flavones featuring of the lipophilic flavonoids that is prevalent in Citrus both O-methyl and C-methyl residues were found species and other Rutaceae members, but has also in Leptospermum scoparium (Myrtaceae) 123 Phytochem Rev (2016) 15:363–390 367

R1 R1 OR2 OCH3 R5 R2 R4O O H3CO O

R3O H3CO R3 OH O OCH3O Examples of (poly)methoxylated flavones Citrus lipophilic flavonoids

R13 =R =H,R 24 =R =CH 35 , R = OCH 3 : pebrellin R13 =R =H,R 2 =OCH 3 : tangeretin

R12 =R =H,R 34 =R =CH 35 ,R =OCH 3 : xanthomicrol R12 =R =OCH 33 , R =H: nobiletin

R123 =R =R =H,R 4 =CH 35 ,R =OCH 3 : thymusin R1323 =OCH ,R =R =H: sinensetin

R1 =OH,R 234 =R =R =CH 35 ,R =OCH 3 : gardenin D R12 =R =OCH 33 ,R =H: hexamethoxyflavone

R15 =R =OCH 3234 ,R =R =R =CH 3 : 5-demethylnobiletin R123 =R =R =OCH 3 : heptamethoxyflavone

R125343 =OH, R =R =H,R =R =CH : cirsiliol OCH3 R1 =OCH 325 ,R =R =H,R 34 =R =CH 3 : cirsilineol OCH3

R1 =OH,R 234 =R =R =CH 35 ,R =H: eupatorin H3CO O R1323345 =OCH ,R =R =CH ,R =R =H: eupatilin OCH3

R1 H3CO OR2 OR5 OH O R4O O umuhengerin

H3CO

OR3 O O

Examples of 8-, but not 6-oxygenated methoxylated flavones OCH H CO 3 R1 =OCH 32345 ,R =R =R =R =CH 3 : isosinensetin 3 OCH3O R1 =H,R 2345 =R =R =R =CH 3 : isoscutellarein tetramethyl ether OCH3 zapotin OCH3 R2O O

H3CO OR1 O Nevadensin glycosides found inLysionotus pauciflorus : β R12 = -D-glucose, R = H: 5-O -glucoside β RR12= H, = -D-glucose : 7-O -glucoside ββ R1 = 6-O - -D-glucosyl- -D-glucosyl,R2 = H: 5-O -gentiobioside αβ RR12= H, = 6-O - -L-rhamnosyl- -D-glucosyl: 7-O -rutinoside Fig. 3 Structures of selected (poly)methoxylated flavones mentioned in the text

(Haberlein et al. 1994). Even though infrequently, limited occurrence appears to be very isolated. For glycosidated forms of highly methoxylated flavones example, no methoxylated flavones have been are occasionally found (e.g., in Helichrysum melaleu- reported from the Apiaceae except for from Dorema cum, Asteraceae (Gouveia and Castilho 2010), Becium aucheri (Wollenweber et al. 1995), which accumu- grandiflorum, Lamiaceae (Grayer and Veitch 1998), lates salvigenin (Fig. 4) and other 6-substituted Salvia verticillata, Lamiaceae (Lu and Foo 2002), lipophilic flavones. The occurrence of exudate flavo- Limnophila aromatica, Plantaginaceae (Bui et al. noids including flavones is scattered across distantly 2004)). Four different glycosides of nevadensin related genera in the Rosaceae (Wollenweber and (Fig. 3) have been reported from Lysionotus pauci- Doerr 2008). In several genera of the Lamiaceae, such florus, Gesneriaceae (Liu et al. 1996, 1998). Such as Thymus, Salvia and Ocimum (Grayer et al. 2001;Lu flavones combine hydrophilic and lipophilic structural and Foo 2002; Tomas-Barberan and Wollenweber features, and could be either stored externally (see 1990), all investigated species appear to accumulate below), or sequestered into vacuoles as is usual for (poly)methoxylated flavones. In other families, other flavonoid glycosides (Zhao and Dixon 2010). methoxylated flavones are produced sporadically in Their isolation using extended (24 h–1 week) some species of the same genus. Several Antirhhinum, methanolic or aqueous-ethanolic extraction of dried Anarrhinum, and Calceolaria species (Plantagi- plant powder does not allow conclusions or even naceae) accumulate methoxylated flavones, while speculations regarding their localization at this point other analyzed species in the same genera did not in time. accumulate these compounds to detectable levels In some plant families, methoxylated flavones have (Wollenweber et al. 2000). Very marked qualitative been reported from only individual species, but this differences in methoxylated flavone accumulation 123 368 Phytochem Rev (2016) 15:363–390

OCH3 OCH3 OCH3 OH OCH3 HO O HO O H3CO O ObF8OMT-1 ObPFOMT-1 H3CO H3CO OH O OH O OH O ObFOMT1 acacetin pilosin gardenin B ObFOMT2 ObF7ODM1 ObF7ODM1 ObFOMT3 ObF8OMT-1 ObFOMT5 OCH3 OH OH OCH3 OCH3 OH OCH3

H3CO O HO O H3CO O H3CO O HO O

H3CO H3CO H3CO OH O OH O OH O OH O OH O apigenin-7,4´-dimethyl ether apigenin cirsimaritin 8-hydroxysalvigenin nevadensin ObFOMT3 ObFOMT1 OH ObFOMT4 OH OCH ObFOMT2 ObFOMT6 ObF8H-1 3 ObFOMT3 H3CO O H3CO O H3CO O

CYP82D33 HO H3CO OH O OH O OH O genkwanin scutellarein-7-methyl ether salvigenin ObFOMT4 ObFOMT3 ObFOMT6 ObFOMT5 OCH3

H3CO O

CYP82D33 HO OH O ladanein

Fig. 4 Biosynthesis of (poly)methoxylated flavones in sweet conversion not occurring in the plant based on biochemical basil. Depicted are steps of lipophilic flavone biosynthesis in data. Enzyme designations have been published previously: sweet basil as elucidated recently. Bold arrows indicate major ObFOMT1-6 (Berim et al. 2012), flavone 6-hydroxylase pathways, thinner arrows indicate routes to less abundant CYP82D33 (Berim and Gang 2013b), ObF8OMT-1 and flavones that are expected to occur based on current knowledge ObPFOMT-1 (Berim and Gang 2013a), flavone 8-hydroxylase status, dotted arrows indicate conversions that are biochemi- ObF8H-1 (Berim et al. 2014), flavone 7-O-demethylase cally possible, but have not been sufficiently confirmed to occur, ObF7ODM1 (Berim et al. 2015) blunt arrow indicates product inhibition, crossed arrow a were even reported for different subspecies of the between the different species. For example, nevaden- same genus, Ononis natrix, Fabaceae (Wollenweber sin has been isolated from the fern Cheilanthes et al. 2003). However, those results did not originate argentea, Pteridaceae (Wollenweber and Roitman from the same study in the same research group and 1991), from several Ocimum species, Lamiaceae may need to be reproduced in one and the same (Grayer et al. 2001), Rosa centifolia, Rosaceae laboratory for verification. In other families, only (Wollenweber et al. 1993), and Esenbeckia almaw- isolated investigations in a certain genus, but no illia, Rutaceae (Barros-Filho et al. 2007). Umuhen- systematic surveys have been conducted. A prominent gerin (Fig. 3) has been reported from Lantana trifolia, example is the family Rutaceae, where numerous Verbenaceae (Rwangabo et al. 1988), Chromolaena Citrus and some Casimiroa species have been ana- arnottiana, Asteraceae (Degutierrez et al. 1995), lyzed, but less attention has been devoted to other Cardiospermum corindum, Sapindaceae (Silva et al. genera (estimated 160). 2014), and Gardenia spp., Rubiaceae (Gunatilaka From the evolutionary and biosynthetic point of et al. 1982). Elucidation of the biosynthetic pathways view it is important to reiterate that while certain and the molecular basis for the accumulation of these highly decorated compounds occur scattered in dis- compounds in different phyla will help conclude tantly related species, the routes to these structures whether the biosynthetic capacity originated once or might be different there, as the sets of accumulated multiple times. flavones, some of which are expected to represent In most reports, the designation exudate or external pathway intermediates, only seem to overlap partially flavonoids has been adopted based on isolation

123 Phytochem Rev (2016) 15:363–390 369 method, such as brief or extended rinses in diethyl in undifferentiated tissue, at least in those species. ether, acetone, or n-hexane. These methods have been Such is the case in sweet basil (Berim and Gang, used as a rough means to assess the near-surface unpublished). However, that combined evidence may localization of lipophilic flavonoids, which may be be the result of the targeted approaches used to identify secreted by epidermal cells or trichomes, or could be the biosynthetic machinery in those species where stored under the cuticle (Tomas-Barberan and Wol- cell-specific production was likely, and thus the most lenweber 1990). The actual storage site(s) of flavones promising approach (as has been demonstrated as and the site(s) of their biosynthesis thus remain to be indicated herein) for those species was to use fully clarified in almost all plant species. Direct evidence differentiated, relatively easily isolatable distinct cell for subcuticular storage of flavones has been procured types as the biological source for gene discovery. by analysis of essential oil collected using glass Contrary to this notion, Liu et al. (1992) isolated microcapillaries directly from trichomes for pepper- several methoxylated flavones from Artemisia annua mint Mentha 9 piperita (Voirin et al. 1993) and sweet suspension cultures. Studies with further species are basil Ocimum basilicum (Berim et al. 2012). Enrich- necessary to determine what the more common ment of transcripts coding for proteins involved in scenario is. (poly)methoxylated flavone biosynthesis in isolated Concluding this section, it might also be prudent to trichomes as compared to whole leaf suggested that emphasize that the separation techniques and detec- the production of these flavones is indeed limited to tion abilities, such as sensitivity and deconvolution trichomes in basil (Berim et al. 2014). The occurrence capacities of analytical instruments, have made of polymethoxylated flavones in the essential oil tremendous advances in the last decades. The original cavities of grapefruit Citrus 9 paradisi has also been studies in the 80s and 90s of the twentieth century had shown by direct collection of the oil using glass to rely on chromatographic studies (thin layer and capillaries (Voo et al. 2012). Microarray analysis of high-pressure) coupled with color reactions or UV transcripts from essential oil-secreting epithelial cells analysis. NMR studies were frequently only feasible and from parenchymal cells revealed strong enrich- for selected metabolites isolated in sufficient amounts, ment of several transcripts related to flavonoid and were often precluded by the paucity of plant biosynthesis in the former cell type, supporting the material. A re-investigation of numerous species enhanced if not exclusive production of citrus PMFs might therefore bring new insights into the occurrence by these cells. In some other cases, histochemical of (poly)methoxylated flavones including minor com- analysis of secretory tissues was used to assess the ponents of the flavone profile. A further factor to be composition of secretions (Bisio et al. 1999; Goepfert considered might be the origin and growth conditions et al. 2006; Liu and Liu 2012). Due to low specificity of the plant material. In an investigation into of histochemical staining, those results should be methoxylated flavone aglycones in the Lamiaceae, interpreted with caution. The exclusiveness of flavone Tomas-Barberan and Wollenweber (1990) observed storage under the subcuticular space of basil trichomes that two Scutellaria species produce lipophilic fla- appeared plausible based on the disproportionally high vones in their native habitat but did not accumulate flavone amounts found in trichomes, sufficing to any when grown in the greenhouse. account for the total amount in the leaf (Berim et al. 2012), but has not been demonstrated by direct means, e.g., by tissue imaging as of today. Notably, in ferns, Proposed physiological roles of (poly)methoxylated lipophilic methoxylated flavonoids are also thought to flavones be synthesized and secreted by trichomes, but there are also non-glandular species among fern flavonoid Flavonoids play various roles in physiological pro- producers (Wollenweber and Schneider 2000). cesses ranging from defense against the UV-light and Because all available evidence suggests that the antioxidant activities to plant reproduction and fertil- production of flavones is restricted to highly special- ity (Ferreyra et al. 2012; Mouradov and Spangenberg ized, fully differentiated secretory cells in species 2014). The methylation of free hydroxyl moieties in where it has been investigated, it could be expected (poly)methoxylated flavones decreases their antioxi- that the underlying biosynthetic pathways are inactive dant potential, yet increases their stability and their 123 370 Phytochem Rev (2016) 15:363–390 ability to permeate membranes but does not affect UV- Early studies also noted a correlation between the absorption capacity. The occurrence of methoxylated occurrence of external methoxylated flavones in the flavones in both modern plants and ancestral lineages Lamiaceae and the habitat of the species (Tomas- suggests that their original physiological role might be Barberan and Wollenweber 1990), with species grow- universal and related to challenges of terrestrial life. ing in xeric and alpine semi-arid climates being likely Yet to date, very few facts have been reliably to produce these compounds. This observation sug- established regarding the actual physiological role in gested some role in adaptation to ecological environ- planta for the vast majority of these compounds. ment. However, as mentioned above, more detailed When considering the possible physiological roles and comprehensive analyses using modern analytical of metabolites, their localization in the organism and in instrumentation may reveal a much broader apparent the cell has to be taken into account. The fact that distribution of these compounds within the Lamiaceae (poly)methoxylated flavones are often stored exter- and other plant families and across ecological niches. nally indicates that they might fulfill a function on or Polymethoxylated flavones of citrus are enriched in near the surface of the plant. The associated functions flavedo tissue, suggesting that they might be important could include UV protection, antimicrobial activity, or for defense. Citrus PMFs exhibited antibacterial activity herbivore feeding deterrent effects (Gould and Lister when tested on several pathogens (Johann et al. 2007) 2006). Interestingly, only the trichomes of Phillyrea and exerted antifungal effect on Colletotrichum gloeos- latifolia (Oleaceae) growing under high solar radiation poroides (Almada-Ruiz et al. 2003), the causal agent of accumulated flavonoid glycosides in their subcuticular anthracnose in tropical fruit. PMFs have been proven cavities, while the trichomes of the plants of the same efficient for reducing the growth of the green mold- species growing in a shady habitat produced phenyl- causing necrotrophic fungus Penicillum digitatum and propanoid acid derivatives (Tattini et al. 2000). for causing morphological changes in fungal hyphae Unfortunately, no analogous evidence exists for (Ortuno et al. 2006). Also, increased accumulation of (poly)methoxylated flavones. Prior to their secretion, PMFs in citrus fruit inoculated post-harvest with lipophilic flavones may traverse the intracellular Phytophthora citrii (Del Rio et al. 2004)andP. membranes and be temporarily or permanently digitatum spores has been shown (Ballester et al. enriched in subcellular locations. Flavonoids have 2013a, b). Ortuno et al. (2011) observed an inverse previously been localized to vacuoles (Zhao and Dixon relationship between susceptibility to fungal infection 2010) and chloroplasts (Agati et al. (2007) and and accumulation of PMFs and flavanones. In citrus references therein), where they might participate in fruit infected with C. gloeosporoides, PMF concentra- photoprotection (Agati et al. 2013). Furthermore, in tions did not increase significantly as compared to several species flavonoids have been localized in control (Jeong et al. 2014). In contrast, in citrus fruit nuclei, where they are hypothesized to play regulatory from citrus greening (huang-long-bin)-infected trees, roles (Saslowsky et al. (2005) and references therein). significantly, but not dramatically increased levels of Flavonols in Chrysosplenium americanum, which are polymethoxylated flavones were observed (Massenti both methoxylated and glycosylated and are thus et al. 2015). amphiphilic, appear to accumulate as cell wall deposits Allelopathy is another inter/intraspecies interaction in the mesophyll and epidermis (Ibrahim 2005). The type where flavonoids are considered to be involved electron-transfer inhibitory effects of flavonoids on (Weston and Mathesius 2013). Early studies in ferns isolated plant mitochondria membranes were studied suggested that components of farinous exudates in in the 1980s–1990s (Ravanel et al. 1990) and refer- Pityrogramma calomelanos, such as mono- and ences therein), but those studies have not been repeated dimethoxy(dihydro)chalcone, autoinhibited spore ger- and deepened since then. The conducted structure– mination and produced inhibition zones around indi- activity correlation studies indicated that higher poten- vidual thriving plants in colonies in field studies (Star tial for inhibition of the external NADH dehydroge- 1980). For a dimethoxylated flavone, tricin from rice, nase correlated with higher lipophilicity and discussed allelopathic effects have been demonstrated (Kong the better membrane permeability for more lipophilic et al. 2004). Notably, tricin is exuded from roots in the compounds as the possible reason. The physiological form of glycosides, which are subsequently cleaved to relevance of these findings remains to be clarified. release the bioactive aglycone (Kong et al. 2007). The 123 Phytochem Rev (2016) 15:363–390 371 allelopathic potential of rice has been known for a Bioactivities of (poly)methoxylated flavones while, and further compounds, such as momilactones A and B appear to be critical for suppressing the As abundant and common constituents of herbal growth of major weeds such as Echinochloa crus-galli medicines, flavonoids have been the subject of various (Xu et al. 2012). Tricin released from seed hulls of rice in vitro and in vivo bioactivity studies and have been also significantly reduced the spread of root rot found to exert numerous pharmacologically relevant mediated by soil-borne fungi (Kong et al. 2010). effects ranging from antioxidant to antiproliferative It remains to be tested whether the disruption of (Kumar and Pandey 2013). One of the frequently cited (poly)methoxylated flavone accumulation results in a considerations concerning the translation of data pro- visible phenotype and/or has far-reaching conse- duced in vitro into physiological relevance is the poor quences on trichome or whole plant physiology and bioavailability of rather hydrophilic flavonoid glyco- metabolism. Remarkably, intact flavonoid biosynthesis sides, and the extensive conjugation of polyphenolic has recently been shown to be necessary for terpene flavonoid aglycones in phase II of xenobiotic metabo- production in tomato (Solanum lycopersicum) (Kang lism (Viskupicova et al. 2008). While the bioavailability et al. 2014). Terpene content was significantly lower in might be expected to be better for (poly)methoxylated the af mutant lacking functional chalcone isomerase flavones due to their increased lipophilicity and protec- and thus deficient in anthocyanins in all of its organs tion of hydroxyl moieties by methyl groups, their poor and in rutin in trichomes. The decrease in terpene water solubility may arise as a dose-limiting factor accumulation was found to be due to a dramatic (Walle 2007b). In addition, some of the typical decrease of both trichome density and terpene biosyn- polyphenol-associated effects, such as antioxidant thesis in individual trichomes. Because the proposed activity, are directly dependent on free hydroxyl site of methoxylated flavone biosynthesis in the Lami- residues, which may be blocked in methylated com- aceae, Asteraceae, and Rutaceae coincides with terpene pounds. As flavonoids have been shown to undergo O- production, the possibility that such an unexpected demethylation by human liver microsomes (Koga et al. relationship also exists in species from these families 2011; Xiao and Hoegger 2013), the possibility that should not be dismissed. Another possibility is that (poly)methoxylated flavones might in fact act as pro- flavonoids act synergistically with terpenes, e.g., drugs and undergo bioactivity-enhancing demethylation enhancing the insect deterrent effects. Synergism after resorption has been considered. The mounting between different classes of specialized metabolites is evidence for this hypothesis has recently been summa- being actively discussed (War et al. 2012). In addition, rized by Arroo et al. (2014). it continues to be a matter of debate concerning plant (Poly)methoxylated flavones have been tested in an specialized metabolism in general whether the com- array of bioassays and disease models and proven to pounds belonging to one class of metabolites share a exhibit potent bioactivities that in part surpass those of common role, or whether individual compounds per- unmethylated compounds (Walle 2007a, 2009). Much form dedicated functions (Firn and Jones 2009; Pich- attention has been dedicated to citrus flavones that are ersky et al. 2006). It has recently been shown that present in small amounts in orange juice (Barreca et al. various stress conditions induce changes in flavonoid 2013; Pupin et al. 1998) and are enriched in flavedo patterns, causing unequal increase in accumulation of and in cold-pressed citrus peel oil, the byproduct of specific individual compounds (Kovinich et al. 2014). juice production (Meiyanto et al. 2012; Uckoo et al. These accumulation patterns were mediated by unequal 2013). A large number of in vitro and some in vivo and stress-specific changes in expression of genes studies (that are not limited to citrus polymethoxylated underlying the production of respective anthocyanins, flavones) focus on several highly valuable potential suggesting this was a result of a regulated process. To application areas. One of these is cancer therapy and the best of our knowledge, actual studies addressing the chemoprevention. The growth of various cancer cell (poly)methoxylated flavone profile changes in response types has been shown to be inhibited by methoxylated to biotic or abiotic stress, or the feeding-deterrent flavones (Alarif et al. 2013; Du and Chen 2010; Park effects of such flavones are currently lacking. Such et al. 2014; Saito et al. 2015; Seito et al. 2011) in vitro studies are urgently needed to shed light on the true and in vivo. In most cases, the cytotoxic or cytostatic physiological roles of these compounds. effects were not equally pronounced against all cancer 123 372 Phytochem Rev (2016) 15:363–390 types, which may allow for the designation ‘‘selec- Sundaram et al. (2014) fed tangeretin to streptozo- tive’’, however, in many studies no direct comparison tocin-induced diabetic rats and found its effects on a with non-cancerous cells was included. Important number of parameters, such as activity of carbohy- observations were made in a study that tested the drate metabolic enzymes and blood insulin, glucose, cytotoxicity of flavones from Dracocephalum kotschyi and glycogen values, to be fully comparable with

(Lamiaceae) (Moghaddam et al. 2012). While the IC50 those of glibenclamide. Another study with mice fed values of methoxylated flavones were consistently a high-fat diet revealed that nobiletin was efficient in higher than those of more hydrophilic flavones preventing hyperglycemia and hyperinsulinemia, apigenin and luteolin with all five tested cancer cell with test periods stretching to 8 or 26 weeks types, the antiproliferative activity of methoxylated (Mulvihill et al. 2011). Activation of hepatic fatty flavones was selective towards malignant cells. In acid oxidation was discussed as one of the potential contrast, apigenin and luteolin were equally efficient mechanism (Mulvihill and Huff 2012). Feeding mice in inhibiting the growth of non-cancerous cells. In on a high-fat diet with a supplement enriched in cases where the potential mechanism was investi- several citrus PMFs as well as other flavonoids (rutin, gated, increased expression of granzyme B and hesperidin) reduced the weight gain and serum levels activation of the p38 MAPK pathway (Saito et al. of total cholesterol and triglycerides (Kang et al. 2015), antioxidant and S-phase shortening (Alarif 2012). There are so far only a few clinical trials in et al. 2013), or an antiangiogenic effect, either via G0/ humans. In double-blind, placebo-controlled clinical G1 cell cycle arrest (Lam et al. 2011, 2012) or via trials with the citrus PMF-enriched dietary supple- inhibition of VEGF expression (Abbaszadeh et al. ment DiabetinolÒ, patients taking the PMF prepara- 2014), were identified. Antitumor activity is also tion had significantly lower blood LDL and suggested to be in part mediated by suppression of cholesterol levels after 84 days of the supplement inflammation in an induced mouse skin cancer model intake (Evans et al. 2012). Another small clinical trial (Ma et al. 2014) and in interleukin-stimulated Caco-2 addressing the bioavailability of PMFs co-adminis- cells (During and Larondelle 2013). tered with tocotrienols (SytrinolÒ) showed that Aside from carcinogenesis-related models, anti- tangeretin and nobiletin were well resorbed and inflammatory properties were shown in an in vivo detectable in plasma (Evans et al. 2012). dermatitis model in mice for eupatilin, a flavone from There are further, less well explored areas of Artemisia species (Giangaspero et al. 2009), as well as (poly)methoxylated flavone application. Bioassay- for jaceosidin and related flavones from Eupatorium guided isolation of antiviral agents from supercritical arnottianum (Clavin et al. 2007). Polymethoxylated fluid extract of C. reticulata identified tangeretin and flavones were also found to be the constituents mainly nobiletin as the components active against the respi- responsible for the anti-inflammatory effect of Ju-Zhi- ratory syncytial virus, with efficacy comparable to that Jiang-Tang, a Traditional Chinese Medicine prepara- of the positive control ribavirin (Xu et al. 2014). A tion (Wang et al. 2014). recent study of methoxylated flavones from Stachys An intensively studied application area for citrus glutinosa (Lamiaceae) revealed that some flavones PMFs is as a nutraceutical supplement or as a exert unanticipated antagonistic effects on nociceptive therapeutic agent in pre-diabetic and metabolic system (Ruiu et al. 2015). Of the several structurally syndrome conditions (Nakajima et al. 2014). related flavones tested for their ability to bind opioid

In vitro, nobiletin and tangeretin suppress triglyc- receptors, xanthomicrol (Fig. 3) exhibited lowest Ki eride accumulation by adipocytes and improve the values in vitro and efficiently reduced the morphine- balance between insulin-sensitizing and insulin-re- induced antinociceptive effect in vivo. Another appli- sistance factor secretion (Miyata et al. 2011). Animal cation area is related to cell growth inhibitory studies have been conducted to assess the in vivo properties of citrus PMFs discussed above. Nobiletin efficacy of citrus PMF. Several studies indicated that and its synthetic derivatives were shown to inhibit the nobiletin, fed at different concentrations, could proliferation of eye lens epithelial cells, suggesting a improve hyperglycemia in obese diabetic ob/ob mice possible use in prevention of posterior capsular (Lee et al. 2010) and hyperglycemia and insulin opacification upon cataract surgery (Miyata et al. resistance on a high fat diet (Lee et al. 2013). 2013). 123 Phytochem Rev (2016) 15:363–390 373

Some structure–activity relationship studies have cytotoxic effects of artemisinin (reviewed by Ferreira been conducted to identify residues important for the et al. (2010)). Methoxylated flavones from Praxelis bioactivity of flavonoids, for instance for their positive clematidea did not exhibit antibacterial activity on effects on ocular blood flow (Park et al. 2004), and for their own on Staphylococcus aureus SA-1199B cells neuroprotective (Dajas et al. 2013; Echeverry et al. expressing the NorA efflux pump gene, but reduced 2010), GABA-binding (Kahnberg et al. 2002), and the minimal inhibitory concentrations of norfloxacin antimicrobial (Wu et al. 2013) activities. These studies and ethidium bromide when co-administered with mostly do not include compounds oxygenated at either drug (de Azevedo Maia et al. 2011). The positions 6 and 8 or comparisons of methylated versus suggested mechanism behind this effect is the inhibi- unmethylated compounds. An analysis of anti-fungal tion of drug efflux from resistant bacteria by the NorA activities of citrus flavonoids on Aspergillus niger, pump. methoxyl residues at positions 8, 5 and 3 were All the above described studies indicate that identified as those enhancing the monitored effect (poly)methoxylated flavones possess an array of (Liu et al. 2012). Antiproliferative activity of PMFs relevant pharmacological properties that should be towards leukemic HL60 cells increased with higher explored and may lead to development of new degrees of methoxylation of the A-ring and in the pharmacophores. However, a large portion of these presence of a 30-methoxyl residue (Kawaii et al. 2012). investigations do not address several critical concerns, In a side-by-side comparison of 5-demethylated such as specificity or selectivity of cytotoxic effects tangeretin (gardenin B) and tangeretin, the former towards malignant cells as compared to non-cancerous was more active in inhibiting skin tumorogenesis tissue, as well as unrealistically high inhibition in vivo (Ma et al. 2014) and exhibited higher cytostatic concentrations, which can only be reached over a activity on non-small lung cancer cells (Charoensin- short time period if at all in plasma. These consider- phon et al. 2013). The latter study assessed the ations must be taken into account prior to undertaking respective uptake rates of the two compounds, and further studies. In addition, even for the best-studied found that the intracellular concentrations of gardenin (poly)methoxylated flavones from Citrus, clinical B were 2.7–4.9 times higher than tangeretin. Thus, the trials are very scarce and need to be repeated and observed stronger effects may be in part due to better extended before final conclusions regarding the ben- bioavailability, be it by better uptake or cellular efits of these compounds for human health can be permeability for gardenin B. In this context, efforts to drawn. improve the delivery of (poly)methoxylated flavones, e.g., by ‘‘packaging’’ them into nanoemulsions (Li et al. 2012; Zheng et al. 2014) should be mentioned. Biosynthesis of (poly)methoxylated flavones These experiments seek to stabilize highly lipophilic compounds in the oil phase of the oil-in-water The biosynthesis of the simplest flavone, apigenin, has nanoemulsion, preventing their crystallization and been studied in detail. Apigenin is derived from the enabling the delivery of more concentrated formula- flavanone naringenin by the action of flavone synthase tions. Smaller nanoemulsion particle proved to be (FNS, Fig. 1). The flavone synthase step can be carried beneficial for the intracellular uptake of gardenin B by out by two divergent plant oxygenase classes, the intestinal cancer cells (Zheng et al. 2014). In a 2-oxoglutarate dependent dioxygenases (2-ODDs) comparison of chrysin to its 5,7-O-methylated deriva- that are classified as FNS I enzymes, and cytochrome tive, the more lipophilic compound exhibited stronger P450 dependent monooxygenases belonging to the anti-inflammatory effects in interleukin-stimulated CYP93 family of P450s, designated FNS II (Martens Caco-2 cells (During and Larondelle 2013). and Mithofer 2005). FNS II is believed to be the In addition to actively exerted pharmacological enzyme type involved in apigenin formation in most effects, synergistic action with other components of other plant species, although this certainly needs to be herbal extracts as well as the potential co-administra- verified. FNS I typically mediates the flavone synthase tion with therapeutic drugs has been discussed and step in Apiaceae species, yet its occurrence is not explored. Methoxylated flavonoids present in Artemi- restricted to this family, as was originally believed. An sia annua appear to potentiate the anti-plasmodial and FNS I functional in vitro has been isolated from rice 123 374 Phytochem Rev (2016) 15:363–390

(Lee et al. 2008). More recently, an FNS-like protein following a long tradition of plant natural products was found in the liverwort Plagiochasma appendic- chemists. Last but not least, a set of flavonoid O- ulatum (Han et al. 2014). The products of the methyltransferases was isolated from peppermint recombinant protein’s reaction with naringenin using a peppermint trichome expressed sequenced in vitro are both the flavone apigenin and the tag (EST) database, and tested with common flavo- 2-hydroxyl naringenin, formed by the initial step of noids such as luteolin and quercetin in a biotechno- the flavone synthase reaction. In addition, FNS I logical approach to flavonoid biodiversification activity is present in protein extracts from the horsetail (Willits et al. 2004). The use of mixed cultures of Equisetum arvense (Bredebach et al. 2011). E. coli expressing the isolated enzymes afforded Downstream of apigenin, the biosynthetic routes to in vivo production of di- and trimethylated flavonoids. various flavones might share some or numerous Aside from those studies, the natural biosynthesis of features across the accumulating species, or might be (poly)methoxylated flavones including the additional family-, genus- or species-specific. The biosynthesis oxygenations at positions 6 and 8 has been elucidated of (poly)methoxylated flavonoids has been studied on in the past few years in sweet basil, a representative of different levels in several species. A detailed investi- the mint family. An EST database constructed using gation focused on Chrysosplenium americanum (Sax- cDNA from peltate trichomes of four basil chemo- ifragaceae), which accumulates flavonols with up to types (Gang et al. 2001) provided the transcriptome five methoxyl residues at positions 3,6,7,20,40 and information for these studies. Isolated peltate glandu- additional glycosylations, but no flavones (Ibrahim lar trichomes also served as an enriched source of 2005). Those very extensive and meticulous studies enzymes and transcripts involved in flavone biosyn- were mostly conducted in an era when molecular thesis for all the necessary biochemical and gene biology tools were not readily available to plant cloning experiments. The isolated trichome model natural products chemists and biochemists, and thus system enabled the identification of functional cata- focused on isolation and characterization of native lysts leading to all major flavones in sweet basil. These plant enzymes, while the molecular (gene identifica- studies revealed numerous unpredictable and unprece- tion) data for many, in fact, most of the steps are still dented insights into both flavonoid metabolism and missing. A recent study in Solanum trichomes plant specialized metabolism in general. How the addressed the O-methylation processes leading to the same or analogous compounds are produced in other accumulation of the 3,7,30,40,50-pentamethylated fla- species of the mint family and in other plant families vonol myricetin (Schmidt et al. 2011, 2012). It remains unclear. However, the knowledge of the identified three flavonoid O-methyltransferases that molecular basis for flavone biosynthesis in one species are sufficient to introduce all of the necessary methyl (sweet basil) can now be used as a foundation to study residues. The mechanisms of B-ring 30- and 50- the analogous or perhaps homologous biosynthetic oxygenations are well understood (Halbwirth 2010; networks in other species. Below, we summarize these Seitz et al. 2007) and did not need to be re- latest findings concerning the different types of investigated. By contrast, molecular-level studies of modifications of the flavone backbone. A ring oxygenations are very scarce, as detailed below. Two 6-oxygenases belonging to two different oxygenase classes have been identified in the context Flavone O-methylations of flavonol (Anzellotti and Ibrahim 2004) and isoflavone biosynthesis (Latunde-Dada et al. 2001). Flavonoid O-methylation is a common reaction. Both No flavonoid 8-oxygenases have been isolated at the major classes of small molecule O-methyltransferases molecular level. Based on metabolic profile changes (cation-dependent and cation-independent) (Noel over a growth season, del Bano et al. (2004) proposed et al. 2003) are known to be involved in regioselective a biosynthesis scheme for major flavonoids found in methylation of flavonoids (Lam et al. 2007). As Rosmarinus officinalis, yet no biochemical or genetic methyltransferases share high levels of identity and data were obtained. As only the major flavonoids were conserved signature motifs across the plant kingdom, monitored, the authors acknowledged that the pro- identification of candidate flavonoid O-methyltrans- posed biosynthetic pathway was purely hypothetical, ferases (FOMTs) can be based on similarity searches 123 Phytochem Rev (2016) 15:363–390 375 against databases of relevant genes. This strategy was used to search the trichome EST collection from peppermint, and express six of 33 discovered putative FOMTs in E. coli (Willits et al. 2004). Analysis of those FOMTs afforded their classification according to regioselectivities, which were invariable, independent of the flavonoid substrate offered. Even though there were earlier reports of recombinantly produced flavonoid 7- (Christensen et al. 1998), 30- (Gauthier et al. 1996; Muzac et al. 2000), and 30-/50-OMTs (Cacace et al. 2003) this was the first systematic study that isolated FOMTs specific for positions 7,8,40 and 30 from the same species. As the goal of the study was to diversify flavonoids by regioselective or regiospecific methylation, compounds used as substrates were common commercially available flavonoids, and no conclusions regarding the biosynthetic routes in peppermint have been made. We subsequently used the sequences of FOMTs isolated from peppermint to identify candidate FOMTs in the basil trichome EST database. Our first study of flavone O-methylation processes in basil focused on 6-, 7-, and 40-O-methylations (Berim et al. 2012). The use of isolated enzymes to generate Fig. 5 Catalytic efficiencies of basil FOMTs and flavone commercially unavailable substrates, such as scutel- 6-hydroxylase. Catalytic efficiencies of ObFOMT1-6 (Berim larein 6-, 7- and 40-monomethyl ethers, allowed a et al. 2012), basil flavone 6-hydroxylase CYP82D33 (Berim and detailed analysis of each FOMT’s substrate prefer- Gang 2013b), and candidate basil F8OMTs (Berim and Gang 2013a). Data shown have been previously published in listed ences. As a result, it became clear that 7-O-methylated -1 -1 original papers. Units are s mM for ObF8OMT-1 and scutellarein, a compound not reported to accumulate ObPFOMT-1 and s-1 lM-1 for all other enzymes. Values in basil and not readily detectable in extracts from its determined for CYP82D33 have been divided by 10 to fit the leaves, must serve as in important pathway interme- scale diate, as it was readily converted into ladanein and cirsimaritin, two dimethylated flavones accumulated route to salvigenin. Similar conclusions have been by basil (Fig. 4). In contrast, both scutellarein 6- and drawn earlier from the investigation of O-methyl- 40-O-methyl ethers were poor substrates for all transferases involved in polymethoxylated flavonol isolated FOMTs. Both ladanein and cirsimaritin were, biosynthesis in C. americanum (Ibrahim 2005). In in turn, readily converted into salvigenin by respective those studies, chromatographically separated native regioselective FOMTs (Fig. 5) (Berim et al. 2012). enzymes were found to display not only stringent FOMTs are frequently characterized as multifunc- regiospecificity, but also very strong preferences for tional or promiscuous enzymes that accept a number selected partially methylated, physiologically relevant of more or less structurally-related substrates in vitro substrates (De Luca and Ibrahim 1985a, b; Khouri and in vivo (Lam et al. 2007). The detailed analysis of et al. 1988). A comprehensive study of their substrate the set of FOMTs from basil contradicted the possible preferences allowed a strictly defined flavonol methy- prediction that this presumed promiscuity would lation order in Chrysosplenium to become clear and enable a random methylation order in the conversion conclusive (Ibrahim 2005). In contrast, the substrate of scutellarein to salvigenin. Instead, it revealed that preferences of the three S. habrochaites FOMTs were all major basil FOMTs had very pronounced substrate not prominent enough to warrant the definition of a preferences (Fig. 5), and suggested that 7-O-methyla- certain methylation order based on their kinetic tion must precede the other O-methylations on the properties alone (Schmidt et al. 2012). 123 376 Phytochem Rev (2016) 15:363–390

The four FOMTs from basil that could be designated Fig. 6 Neighbor-joining tree of characterized and selected c F40OMTs based on similarity to the peppermint FOMT uncharacterized non-cation dependent OMTs. Relationships of selected non-cation dependent OMTs were inferred using with that regiospecificity (Fig. 6) share high levels of neighbor-joining method and its stability tested by bootstrap- identity of more than 87 % between one another (Berim ping (1000 replicates). Only bootstrap values [50 % are et al. 2012). However, experimental data revealed presented. Bootstrap values next to branch label refer to the stringent preference of a flavone 6-OH-residue by two of node at the base of a compressed sub-tree. Scale is in substitutions per amino acid. Stars next to a branch indicate the four enzymes (ObFOMT4 and ObFOMT6). The F7OMTs, circles F40OMTs. Other FOMTs are indicated as other two enzymes (ObFOMT3 and ObFOMT5) could function next to branch label. Branch labels ObFOMT1-6 and function both as F6OMTs and as F40OMTs, depending ObF8OMT-1 refer to originally published designations for basil on the substrate offered, but preferred the 40-OH moiety FOMTs (Berim et al. 2012; Berim and Gang 2013a). Branch labels beginning with ‘‘Ciclev’’ refer to C. clementina FOMT- with flavones expected to be part of metabolic pathway like proteins as designated in the Phytozome database. Other (Figs. 4, 5)(Berimetal.2012), such as ladanein and branch labels are abbreviated genus and species names, scutellarein-7-methyl ether. The high protein identity of followed by GenBankTM accession number of the protein, functionally divergent FOMTs presented an opportunity followed by function where fits the space. Known functions not listed in the figure for space reasons: CjBAC22084: colum- to study the structural basis for the different regioselec- bamine 2-OMT, LnABJ88947: coniferyl alcohol 9-OMT, tivities, which seem to be determined by just a few CrABR20103: 16-hydroxytabersonine 16-OMT. Ob: Ocimum amino acid residues. The facile switch of substrate basilicum, Mp: Mentha 9 piperita, Pp: Prunus pyrifolia, Pa: P. preference has been well documented in OMTs, but the armeniaca, Pd: P. dulcis, Hl: Humulus lupulus, Sh: Rosa hybrid, Rc: R. chinensis, Ln: Linum nodiflorum, Sc: Secale cereale, Sh: regioselectivity remained unchanged in those instances Solanum habrochaites, Cr: Catharanthus roseus, Vv: Vitis (Frick and Kutchan 1999;Gangetal.2002;Scallietetal. vinifera, Ec: Eschscholzia californica, At: Arabidopsis thaliana, 2008; Wang and Pichersky 1999). Homology modeling Ms: Medicago sativa, Hv: Hordeum vulgare, Cj: Coptis of selected basil FOMTs using isoflavone 40-OMT as japonica, Ge: Glycyrrhiza echinata, Mc: Mesembryanthemum crystallinum, Am: Ammi majus, Ls: Liquidambar styraciflua, template (Zubieta et al. 2001) allowed educated guesses Ca: Chrysosplenium americanum. Outgroup JQ1393ODPO is regarding the position of the divergent amino acids an O-demethylpuromycin OMT from Streptomyces anulatus. relative to the active site. Site-directed mutagenesis of The accession numbers of proteins within compressed branches: these selected residues revealed that the conversion of IOMTs (isoflavonoid OMTs): AAC49856, BAC58011- 0 BAC58013, AAC49926, AAB88294, Q29U70; alkaloid OMTs: 4 -O-methylating activity of ObFOMT3 into F6OMT AAP45313-AAP45315, ACN88562, BAB08004, BAB08005, activity was very facile and efficient (Berim et al. 2012). AAU20768, AAU20765; Psychotria OMTs: BAI79243- The reciprocal mutations in the F6OMT (ObFOMT4) BAI79245 did increase the 40-O-methylating capability, but left the ladanein 6-OMT activity the dominant one in this enzyme. Notably, none of the peppermint FOMTs was 6-OH of quercetagetin, a flavonol accumulating in reported to possess F6OMT activity, even though iceplant (Mesembryanthemum crystallinum)uponUV- scutellarein was tested as substrate (Willits et al. irradiation (Vogt et al. 1999). The possibility that 2004). As peppermint accumulates 6-O-methylated different classes of OMTs methylate this position in flavones (Jullien et al. 1984; Voirin and Bayet 1992), a different genera is further suggested by the character- re-investigation of the regioselectivities of those ization of the flavonol 6-OMT from C. americanum, FOMTs with partially methylated flavones that are which also required magnesium ions for activity (De likely to be part of the metabolic pathway may point out Luca and Ibrahim 1985a). the F6OMT in peppermint. Experimental data for An example of highly similar FOMTs exhibiting further FOMTs from Lamiaceae are necessary in order different regioselectivities has also been reported from to estimate whether the knowledge produced by the Solanum habrochaites (Schmidt et al. 2012), which studies in basil suffices to predict the regioselectivities accumulates 3,7,30,40,50-O-methylated myricetin (Sch- as flavonoid 6- or 40-OMTs in other mint family species, midt et al. 2011). A protein designated ShMOMT3 and whether flavonoid 6-O-methylation is catalyzed by regiospecifically methylates position 3-OH of myrice- the same class of O-methyltransferases in this family. tin and shares 88 % identity with ShMOMT1, which The only other flavonoid 6-O-methyltransferase identi- acts as a flavonol 30,50-OMT. Substrate-dependent fied at the molecular level is a cation-dependent OMT change of regioselectivity has been documented sev- (Ibdah et al. 2003), which methylates positions 30-and eral times in FOMTs. S. habrochaites ShMOMT 2 123 Phytochem Rev (2016) 15:363–390 377

68 90 IOMTs 99 C. clementina OMTs (7) 92 ObF8OMT-1 99 Ocimum& Mentha MpFOMT5 F8OMTs MpFOMT2 99 ObAAL30423 chavicol OMT 70

ObAAL30424 eugenol OMT m “COMTs” 99 Ciclev10015762m 73 99 Ciclev10015630m PpBAA86059 unknown 71 PaAAB71213 unknown Ciclev10008718m gent fro 85 99 Ciclev10015708m 99 Ciclev10015705m Ciclev10012033m PdCAA11131 unknown 86 50 HlABZ89567 unknown 60 RhAAM23005 orcinol OMT2 99 RhAAM23004 orcinol OMT1

LnABJ88947 enetically diver 98 ScAAO23335 unknown HvCAA54616 ShMOMT2 ADZ76434 99 Psychotria OMTs (3)

s

96 CrAAM97497 (F3´,5´OMT) d phylog 96 CrAAM97498 unknown

s 99 CrAAR02422 unknown 51 76 CrABR20103

OMT

58 CrAAR02419 tharanthu 59 CrAAR02418 unknown CjBAC22084 Ca 96 96 Alkaloid OMTs 99 VvAGN70871 hydroxy-isobutylpyrazine Ciclev10028731m functionally an 99 ObFOMT1 79 99 ObFOMT2 MpFOMT1A 99 MpFOMT1B 79 OMTs

99 MpFOMT4 & 99 ObFOMT5 99 ObFOMT3

ObFOMT4 (F6OMT) 6,7,4´-F

cimum Mentha 56 “non-COMTs” -

97 ObFOMT6 (F6OMT) O 99 Pinus COMTs/AEOMT EcBAE79723 reticuline 7-OMT 94 HlABZ89565 desmethylxanthohumol OMT 77 81 Ciclev10005309m 97 Ciclev10012087m 56 65 Ciclev10015993m POMT-7 P. deltoides

99 Ciclev10006704m ) Ciclev10005297m HlABZ89566 chalcones and other substrates

RcBAC78828 COMT -like” 81 VvADJ668 active with hydroxypyrazines 99 VvADJ66851 active with hydroxypyrazines 99 AtCAB64217 COMT-like MsCAB65279 COMT HvAAC18643 COMT 99 ShMOMT1 ADZ76433 (F3´,5´OMT) s (”COMT 64 ShMOMT3 AGK26768 (F3OMT) 94 PaACL13527 anol/isoeugenol OMT CjBAA06192 scoulerine 9-OMT 99 GeBAA13683 chalcone 2´-OMT 69 MsAAB48059 chalcone 2´-OMT 99 VpABD61227 unknown, active with gallates VpABD61228 unknown, active with gallates 99 Ciclev10020782m

96 Ciclev10020818m and similar OMT

95 RcBAC78826 eugenol OMT s 73 Ciclev10020880m N. tabacum COMTs 99 Ciclev10017683m 98 RcBAD18975 phloroglucinol OMT ) OMT 92 Ciclev10001555m McAAA33032 myo-inositol OMT MpFOMT3 (F3´OMT) feate 96 Monocot COMTs (incl. tricetin3´,4´,5´ OMT) af 66 AmAAR24906 bergaptol 5-OMT COMTs 99 51 52 Thalictrum tuberosum COMTs

McAAC18863 COMT hol (or c 71 LsAAD48913 COMT 99 CaAAA86982 (COMT) 99 CaP59049 (F3´OMT) catec 84 CaAAA80579 (F3´OMT) AtQ9FK25 (F3´OMT) Ciclev10024037m COMTs incl. functionally divergent OMTs PpCAC21601 COMT SaJQ1393O -demethylpuromycin OMT

0.1

123 378 Phytochem Rev (2016) 15:363–390 catalyzes 40-O-methylation of a substrate lacking other pathways, and highlighted the possible critical role oxygenations of ring B, but targets the 7-OH residue of of metabolite transport for the flavone profiles found in substrates with 50- and/or 30-hydroxyl moieties (Sch- basil. Such transport processes would impact the midt et al. 2011). Moreover, for an F7OMT from poplar reaction equilibrium, in this particular example with- (Populus deltoides) (POMT-7, Fig. 6) displaying 30- drawing the inhibitor from the site of biosynthesis. OMT activity with certain substrates (Kim et al. 2008), Not surprisingly, of the functionally characterized a single residue effecting this switch in regioselectivity OMTs, basil FOMTs are phylogenetically closest to was identified (Joe et al. 2010). An example of a cation- peppermint FOMTs (Berim et al. 2012). Less pre- dependent FOMT switching regioselectivity in a dictably, basil 7-, and 6-/40-FOMTs are also more substrate-dependent manner has been reported from closely related to each other than to FOMTs with the Arabidopsis (Wils et al. 2013). Again, a change in just same regioselectivity, but from a different species, one amino acid (G46Y) was sufficient to revert its e.g., S. habrochaites F7/40OMT (ShMOMT2) or P. regioselectivity from the unusual para-position of ring deltoides F7OMT (POMT-7) (Fig. 6). While B to the meta-position typical for this type of FOMT. ShMOMT2 is allocated to one cluster with some other The 8-hydroxyl residue of a number of FOMTs, such as Hordeum vulgare F7OMT, and (poly)methoxylated flavones is also often methylated. FOMTs from Catharanthus roseus, POMT-7 appears The characterization of peppermint FOMTs identified to be only distantly related to all other FOMTs an enzyme with such regioselectivity (Willits et al. identified so far. Basil and peppermint F8OMTs also 2004). The basil EST database contained a homolog of cluster together, but in a separate clade of non-cation peppermint F8OMT designated ObF8OMT-1 (Fig. 6), dependent OMTs (Fig. 6). which was cloned and tested with available substrates Basil also accumulates 30-methoxylated flavones (Berim and Gang 2013a). The kinetic analysis of such as cirsilineol (Fig. 3), even though they consti- ObF8OMT-1 could not be carried out with the tute only a minor portion of the total lipophilic proposed native methylation targets pilosin and 8-hy- flavones in investigated species (Grayer et al. 2001). A droxysalvigenin (Fig. 4), which were unavailable at search of the basil EST database for homologs of the time of analysis, and remain unavailable in peppermint F30OMT (Fig. 6) did not return any amounts necessary for extensive assay series to date. promising matches. As members of another class of As it is active with their synthetic analogues, 7,8- methyltransferases, the cation-dependent OMTs, also dihydroxyflavone and 8-hydroxy-7-methoxyflavone catalyze flavonoid 30-O-methylation (Ibdah et al. (Fig. 5), it is likely to also accept both of the possible 2003), candidate contigs belonging to this class of natural substrates mentioned above. Basil F8OMT OMTs were considered as potential catalysts for this does methylate 8-hydroxysalvigenin in vivo when reaction. The most abundantly represented transcript expressed in a yeast strain producing this flavone was cloned and the encoded protein designated (Berim et al. 2014). In addition, kinetic analysis of phenylpropanoid flavonoid O-methyltransferase ObF8OMT-1 in the presence of selected natural (ObPFOMT-1) (Fig. 7) was biochemically character- flavones revealed very strong product inhibition by ized (Berim and Gang 2013a). Surprisingly, its gardenin B, which would result from 8-O-methylation regioselectivity was not limited to the 30-OH moiety of 8-hydroxysalvigenin (Fig. 4), but not by nevaden- of flavones such as luteolin. Instead, that enzyme sin, the product of the possible pilosin 8-O-methyla- could rather be described as specific for OH residues tion (Berim and Gang 2013a). Whether product vicinal to another hydroxyl moiety. Depending on the inhibition by gardenin B becomes relevant in tri- substrate, it would therefore methylate the 8-, the 6- chomes depends on the affinity of ObF8OMT-1 for the and also the 5-OH moiety, the latter activity being corresponding substrate, 8-hydroxysalvigenin, and on probably irrelevant in basil where 5-O-methylated the concentrations of both substrate and the product, flavones are not known to occur. The enzyme’s gardenin B, in the cell. Both of those depend in part on requirement for vicinal hydroxyl moieties lets pilosin, the rate of secretion into the catalytically inactive but not 8-hydroxysalvigenin serve as a natural subcuticular storage space of glandular trichomes. The substrate and be 8-O-methylated. The catalytic effi- product inhibition feature of ObF8OMT-1 added ciency of ObPFOMT-1 was higher with 7,8,40-hy- complexity to the interpretation of biosynthetic droxyflavone than with the 30-O-methylated substrates 123 Phytochem Rev (2016) 15:363–390 379

99 AAN61072Mc luteolin and cirsiliol (Fig. 5). Based on the screen of Q43161Sl potential substrates and pseudo-substrates accepted by ObPFOMT-1 ObF8OMT-1, that enzyme should also be active and 60 NP_567739At s 100 CICLE v10009335 possibly even more efficient with pilosin as compared 90 CICLE v10009342 FOMT to 8-hydroxysalvigenin (Fig. 5) (Berim and Gang

85 CBI27950Vv P 75 XP002330529Pt 2013a). The conundrum regarding the respective 90 XP002514167Rc ADZ76153Vp contributions of the two F8OMT candidates to the 68 81 NP564917At formation of 8-O-methylated products in basil can 89 ACO52469Vv XP001755011Ppp only be resolved by identification of appropriate 100 CICLE v10026344 99 CICLE v10012561 mutants, or by genetic manipulation of individual 99 CICLE v10026347 candidate genes. Notably, the only other characterized 100 Q9ZTT5Pt s) 90 ADV40957Pr T F8OMT that catalyzes the 8-O-methylation of 8-OH- 100 95 CBL95257Pp CAK18782Pa kaempferol and 8-OH-quercetin in Lotus corniculatus 100 AAT40111Am CoAOM flower buds requires magnesium ions for activity (Jay P28034Pc 100 97 O04899Nt et al. 1985). The gene sequence for that enzyme is not

AAM66108At (C OMTs yet available, but it represents an example of a cation- 53 Q8H9B6St 69 XP002282867Vv dependent FOMT specific for 8-OH moiety of the ObCCoAOMT-1 flavonoid backbone.

eoyl-CoA XP003607927Mt f Of the flavone O-methylations occurring most CICLE v10029158 Caf ACF48821Gh frequently, only that at position 5-OH has not been Q43095Pt O04854Eg clarified so far. Given the functional versatility of both Q7f8T6Osj cation-dependent and non-dependent FOMTs, only 83 Q9XGP7Osj 100 XP003574709Bd actual investigations, most likely using Citrus species, Q55813Syn ADZ76154Vp will enable the identification of these probably unique 96 XP001776141Ppp enzymes. Based on the low sequence identities across AAF86386Sh plant families, even the in silico prediction of citrus 0.1 FOMTs’ regioselectivities may prove ambiguous unless confirmed experimentally. BLAST searches Fig. 7 Neighbor-joining tree of characterized and selected uncharacterized cation-dependent OMTs. The tree was con- against C. sinensis and C. clementina genomes using structed using MEGA5 and tested by bootstrapping (1000 one of the basil F7OMTs (ObFOMT1) or F6OMTs replicates). Only bootstrap values greater than 50 % are (ObFOMT4) returned very similar results, with top presented. Scale is in substitutions per amino acid. FkbG matches being identical and displaying 36–41 % OMT from Streptomyces hygroscopicus (AAF86386Sh) was used as as outgroup to root the tree. The tree is subdivided into identity with either of basil enzymes. Despite this two major clades of cation-dependent OMTs, the PFOMTs and low identity level, one of *20 top matches from C. the CCoAOMTs. Branch labels ObPFOMT-1 and ObC- clementina clustered with basil and peppermint CoAOMT refer to studied basil cation-dependent OMTs (Berim FOMTs (Fig. 6). Six citrus OMTs were grouped in a and Gang 2013a). Branch labels beginning with CICLE denote C. clementina cation-dependent OMTs and contain Phytozome clade vicinal to F8OMTs from basil and peppermint. accession numbers for respective entries. All other branches are Another seven C. clementina OMTs clustered together TM labeled with NCBI GenBank accession number and the in a clade sister to isoflavonoid OMTs. Some of the initials of genus and species. Rc: Ricinus communis, Ptr: analyzed citrus OMTs were allocated to the catechol- Populus trichocarpa, Vv: Vitis vinifera, Sl: Stellaria longipes, Mc: Mesembryanthemum crystallinum, Vp: Vanilla planifolia, OMT (COMT) like clade of OMTs (Fig. 6), which At: Arabidopsis thaliana, Ppp: Physcomitrella patens subsp. includes F30OMTs from C. americanum (Gauthier patens, Mt: Medicago truncatula, Ptm: Populus tremuloides, et al. 1996), Arabidopsis (Muzac et al. 2000), and Eg: Eucalyptus gunnii, St: Solanum tuberosum, Nt: Nicotiana peppermint (Willits et al. 2004) together with OMTs tabacum, Pc: Petroselinum crispum, Am: Ammi majus, Gh: Gossypium hirsutum, Pr: Pinus radiata, Pt: Pinus taeda, Pp: displaying activity with other phenolic substrates. Pinus pinaster, Pa: Picea abies, Osj: Oryza sativa cv. japonica, Three citrus OMTs clustered together in a clade sister Bd: Brachypodium distachion, Syn: Synechocystis sp. (strain to poplar POMT-7, an F7- and F30OMT. The lack of PCC 6803)

123 380 Phytochem Rev (2016) 15:363–390 readily identifiable FOMTs in C. clementina prompted T. erecta (Asteraceae) was found to be catalyzed by a us to look for candidate cation-dependent OMTs P450 monooxygenase, as evidenced by cofactor involved in PMF biosynthesis in this species. The top requirements and biochemical properties of the hits of a BLAST search against the C. clementina activity (Halbwirth et al. 2004). Based on this genome shared up to 66 % identity with ObPFOMT-1 knowledge, it seemed possible that the flavone that was used as query (Fig. 7). The two putative citrus 6-hydroxylase in basil could belong to either class PFOMTs with highest identity to ObPFOMT-1 were of oxygenase. Biochemical properties of the flavone 90 % identical. Given the easy switch of function 6-hydroxylase in crude trichome protein extracts observed in this enzyme class, this high protein suggested the underlying protein was a cytochrome identity could still translate into different regio- or P450-dependent monooxygenase, and its substrate substrate specificities. It thus remains possible that was genkwanin rather than apigenin (Fig. 4) (Berim both cation-dependent and non-cation dependent and Gang 2013b). This substrate requirement FOMTs participate in flavone-O-methylation in Citrus revealed the 7-O-methylation of apigenin as the species. entry point to the accumulation of all higher substi- Overall, the analysis of methylation processes in tuted flavones in basil. In this respect, basil methoxy- basil allowed outlining of several steps of the pathway lated flavone biosynthesis is similar to and helped define the order of these steps. By (poly)methoxylated flavonol biosynthesis in C. amer- identifying scutellarein-7-methyl ether as an impor- icanum, where the 6-oxygenation also appears to tant pathway intermediate, the study of FOMTs occur preferentially after three initial methylations prepared the foundation for the search for the flavone (Anzellotti and Ibrahim 2000; Ibrahim 2005). The 6-hydroxylase. protein underlying the genkwanin 6-hydroxylation in basil was classified as CYP82D33, a P450 family not previously implicated in flavonoid biosynthesis Oxygenations of flavonoid A ring (Berim and Gang 2013b). The recombinant protein required the 5-hydroxy-7-methoxyflavone pattern for Additional oxygenations of the flavonoid A ring in activity, yet was also active with the corresponding the context of (poly)methoxylated flavone biosynthe- flavanone, naringenin-7-methyl ether (sakuranetin) sis have only been studied in sweet basil. However, (Fig. 5). To test whether the oxygenation at position flavonoids of different types with oxygenation at 6 might indeed occur at flavanone stage prior to position 6 are widespread in the plant kingdom. Prior flavone formation, we investigated the activity of to the studies in basil, two examples of flavonoid basil FNS II (CYP93B23) with sakuranetin and the 6-hydroxylases were known on genetic level. In the product of its 6-hydroxylation, carthamidin (6-hy- biosynthesis of the 6-oxygenated isoflavone glycitein droxynaringenin-7-methyl ether). While CYP93B2 in soybean (Glycine max), the introduction of the exhibited *25 % relative activity with sakuranetin, 6-hydroxyl moiety was found to occur prior to aryl no conversion was detectable with carthamidin, ring migration (Latunde-Dada et al. 2001). It was leading to the conclusion that flavone formation has catalyzed by a cytochrome P450 monooxygenase to take place before the introduction of the 6-hy- (CYP71D9) that preferred flavanones liquiritigenin droxyl moiety. The fact that the 7-O-methylation of and naringenin as substrates and showed negligible naringenin occurs at about 30 % relative turnover activity with flavones and isoflavones. The second rate as compared to apigenin 7-O-methylation (Berim isolated flavonoid 6-hydroxylase was a 2-ODD from et al. 2012) additionally supported the proposed route C. americanum (Anzellotti and Ibrahim 2004). That from naringenin to apigenin and further to scutel- enzyme strongly preferred the 3,7,40-O-methylated larein-7-methyl ether as shown in Fig. 4. The flavonol quercetin as substrate, but was also active homolog of basil genkwanin 6-hydroxylase from with other methylation products of quercetin and, at peppermint designated CYP82D62 was also isolated about 10 % of the maximum turnover rate, with and found to share 73 % amino acid identity with the quercetin itself (Anzellotti and Ibrahim 2000). In basil genkwanin 6-hydroxylase. It exhibited substrate addition, the 6-hydroxylation of quercetin in crude requirements similar to those of basil genkwanin protein extract from the petals of Tagetes patula and 6-hydroxylase (Berim and Gang 2013b). 123 Phytochem Rev (2016) 15:363–390 381

The second flavone A ring hydroxylation occurring an N-terminal fusion. This fact establishes precedence in basil trichomes is that at position 8. Even without for the partitioning of flavonoid biosynthesis into the previous biochemical knowledge regarding the plastids, and additionally highlights the importance of pathway it may have been possible to guess that this intracellular metabolite translocation. position is hydroxylated only after position 6, because The identification of salvigenin 8-hydroxylase as an all 8-substituted flavones in basil (and the majority of RO required us to determine whether the basil genome Lamiaceae species) are also oxygenated at position 6 contained a putative PTC52, and if so, how similar it (Grayer et al. 2001; Tomas-Barberan and Wollenwe- was to the RO involved in flavone metabolism. In ber 1990). The isolation of this enzyme from basil was addition to the two highly similar copies of salvigenin supported by the biochemical analysis of its properties 8-hydroxylase, we were able to isolate full or partial in crude trichome protein extracts. The search for basil sequences of three predicted PTC52 genes (Fig. 8) flavone 8-hydroxylase also benefitted from the bio- (Berim et al. 2014). The conserved gene exon–intron chemical and phytochemical knowledge we collected structure implied that the genes originated by dupli- previously, as it allowed us to narrow the list of cation. This finding drew our attention to the occur- possible substrates down to salvigenin (Fig. 4). The rence of several divergent copies of PTC52-like genes marked differences between the salvigenin 8-hydrox- and proteins in a number of sequenced plant genomes ylase and the genkwanin 6-hydroxylase, known to be present in the Phytozome database (Goodstein et al. catalyzed by the CYP82D33 identified earlier (Berim 2012), such as rice (Oryza sativa), common flax and Gang 2013b), suggested the enzyme mediating (Linum usitatissimum), and cucumber (Cucumis salvigenin 8-hydroxylation was not a cytochrome sativus), to name a few. It is quite possible that the P450 monooxygenase. The only earlier report con- catalytic functions of at least some of those orthologs cerning flavonoid 8-hydroxylase activity in crude protein extract from the petals of Chrysanthemum segetum (Asteraceae) described an NADPH- and FAD-dependent protein with characteristics partially matching those of P450 monooxygenases, yet partially distinct (Halbwirth and Stich 2006). Basil salvigenin 8-hydroxylase exhibited properties differing from the flavonol 8-hydroxylase activity in Chrysanthemum, and was finally identified as a membrane-bound, ferredoxin-dependent Rieske-type oxygenase (RO) sharing some 50 % identity with putative protochloro- phyllide a oxygenases (PTC52) from numerous species (Berim et al. 2014). All other members of this RO subfamily functionally characterized to date were involved in chlorophyll metabolism (Bartsch et al. Fig. 8 Genomic organization and homologs of salvigenin 2008; Gray et al. 2004), and this was the first time that 8-hydroxylase in the basil genome. a Hybridization of a full- the recruitment of this class of enzyme for plant length salvigenin 8-hydroxylase (ObF8H-1) cDNA probe to specialized metabolism had been reported. The basil genomic DNA digested with HindIII (H) or EcoRV (EV) (both non-cutters) and separated on an agarose gel indicated recombinant purified enzyme was inactive with other more than one copy of this gene ([80 % identical with the offered flavones occurring in basil (cirsimaritin, probe) is present in the genome. Approximate fragment size is ladanein, scutellarein-7-methyl ether), and shared indicated on the left. White vertical line separates non-adjacent 94 % identity with the translated sequence of a second lanes. Fainter signals may represent PTC52-like genes in basil genome. b Conserved exon–intron structures of ObF8H-1 and abundantly expressed transcript in the basil EST partial or full-length PTC52 homologs isolated by homology database, whose substrate preferences remain to be cloning, which also match the splicing model of the minor examined. Like the other PTC52-like ROs in plants Arabidopsis PTC52 transcript support common origin of those (Gray et al. 2004), salvigenin 8-hydroxylase possesses genes. Only coding sequences are depicted. Grey rectangles represent exons, black lines represent introns, forked ends a plastid targeting peptide, which directs green indicate incomplete sequence. Scale is shown above. Figure re- fluorescent protein to chloroplasts when expressed as used from Berim et al. (2014) 123 382 Phytochem Rev (2016) 15:363–390 will also be in specialized metabolism. However, only significantly reduced the accumulation of nevadensin highly conserved copies of PTC52-like gene are and increased the relative abundances of gardenin B present in the genome of Citrus clementina, with and 8-hydroxysalvigenin, the two potential substrates encoded proteins sharing more than 85 % identity. As for 7-O-demethylation (Fig. 9) (Berim et al. 2015). the identity between basil’s salvigenin 8-hydroxylase The recombinant protein was active with both of these and basil’s putative PTC52 is only 60 % (Berim et al. flavones in vitro. Which of the two substrates is 2014), it appears unlikely that one of the known preferentially demethylated in vivo remains to be PTC52-like ROs in C. clementina will catalyze the clarified. In addition to its general novelty, we found flavone 8-oxygenation in that species. evidence that basil F7ODM might undergo alternative translation initiation and alternative transcription (Berim and Gang, unpublished). The discovery of an Flavone O-demethylation O-demethylation operating in flavonoid biosynthesis poses several questions. One is whether the route to Unlike O-methylations, O-demethylations are very nevadensin involves this unusual step in other species. uncommon in plant specialized metabolism. The only Another question is whether regiospecific flavone O- well-studied example is the sequential O-demethyla- demethylations are common in the Lamiaceae. For tion of thebaine to yield codeine and morphine in example, peppermint and Thymus species accumulate opium poppy, Papaver somniferum (Hagel and Fac- pebrellin and similar flavones lacking the 6-O-methyl chini 2010). The two underlying enzymes in that residue along with their 6-O-methylated counterparts. process are 2-ODDs that share less than 70 % identity with each other. An in planta and biochemical analysis of additional 2-ODDs from poppy revealed that O- demethylations and O-demethylenations of further alkaloids occurred, and identified a third 2-ODD from poppy as protopine O-dealkylase (Farrow and Fac- chini 2013). Based on this seemingly very rare and isolated occurrence of small molecule O-demethyla- tions, it came as a surprise that the methoxylated flavone biosynthesis in basil also includes a regiospecific O-demethylation of gardenin B and 8-hydroxysalvigenin, resulting in accumulation of nevadensin and pilosin (Fig. 4). The occurrence of an O-demethylation step was suggested by emerging knowledge concerning the biosynthetic pathway. Specifically, the fact that 7-O-methylation appeared to be a prerequisite for the introduction of 6-oxygena- tion in basil was in contrast to the occurrence of nevadensin in two of four studied basil lines (Berim and Gang 2013b). In addition, the rates of nevadensin 7-O-methylation by two major F7OMTs and by trichome protein extracts were very low, suggesting Fig. 9 Effect of prohexadione-calcium treatment on flavone that this reaction does not occur in the plant (Berim accumulation in basil. Accumulation of relevant flavones in et al. 2012). The flavone 7-O-demethylase activity was basil plants sprayed with prohexadione-calcium solution (sam- ples P1-4) or spraying solution lacking the active agent (samples demonstrated in crude trichome proteins extracts in C1-4) as determined in four consecutive leaf pairs starting with the presence of cofactors required by 2-ODDs, but not the youngest pair (denoted P1 and C1). Statistically significant by P450s (Berim and Gang 2013b). The involvement differences between the same leaf pairs of treated (P) and control of a 2-ODD in this step in vivo was further corrob- (C) plants are indicated above the bars of P-series as ***p \ 0.002, **p \ 0.01 or *p \ 0.02. Results are orated by exogenous treatment of basil plants with a mean ± SD (n = 5). See original publication (Berim et al. 2-ODD inhibitor, prohexadione-calcium, which 2015) for details on plant treatment and flavone analysis 123 Phytochem Rev (2016) 15:363–390 383

Even though no corresponding 6-O-demethylating flavonoid metabolon involves in part competitive activity was detected in peppermint trichome protein interactions between its members as has been shown extracts using gardenin B as substrate (Berim and for chalcone isomerase, flavonol synthase and dehy- Gang 2013b), this possibility should be further tested, droflavonol reductase in Arabidopsis (Crosby et al. e.g., under different conditions and with different 2011). In addition, alternative localizations have been flavones. In any case, the discovery of an O-demethy- suggested for some enzymes of the flavonoid pathway, lation step in flavonoid biosynthesis extends the such as nuclear (Saslowsky et al. 2005), vacuolar known occurrence range of this reaction type beyond (Tian et al. 2008), and plastidial (Tian et al. 2008). The alkaloid metabolism (Farrow and Facchini 2013; physiological relevance of these findings and the roles Hagel and Facchini 2010). of these enzymes in alternative cellular compartments remain to be elucidated. Given this current state of knowledge, it is intriguing to consider whether the Subcellular compartmentalization enzymes of lipophilic flavone network in basil of (poly)methoxylated flavone biosynthesis conform to their predicted localizations, whether they interact with each other, and how the expected The cellular organization of (poly)methoxylated compartmentation of part of the pathway into plastids flavonoid biosynthesis has not been extensively stud- affects the potential for metabolon formation. ied experimentally. In basil, all of the characterized FOMTs and the flavone 7-O-demethylase are pre- dicted to be cytosolic in silico. Flavone 6-hydroxylase, Insights and questions brought a CYP450 oxygenase, can be expected to be anchored up by the elucidation of the methoxylated flavone to the endoplasmic reticulum, and flavone 8-hydrox- biosynthesis in basil ylase is at least in part plastidial based on in planta targeting experiments, in silico prediction, and its As outlined above and as may be appropriate to phylogenetic origin (Berim et al. 2014). In addition, emphasize again here, our current knowledge of fractionation of trichome protein suggested that (poly)methoxylated flavonoid biosynthesis is unsuit- flavone 8-hydroxylase activity is membrane-bound. able for generalizations as it is derived from investi- The localization of the studied FOMTs and flavonol gations conducted in representatives of just a very few 6-hydroxylase of the polymethoxylated flavonol plant families. Multiple flavonoid O-methylation steps biosynthesis in American saxifrage has not been in context of one metabolic network have only been assessed, while the final products of this metabolic studied in detail in tomato (Solanaceae), sweet basil network and the glucosyltransferases involved in their (Lamiaceae), and American saxifrage (Saxifra- formation were shown to be associated with cell walls gaceae). The catalysts of ring A 6-oxygenations have and plasma membrane of epidermal cells (Ibrahim been investigated only in American saxifrage, soybean 2005). The current view regarding the organization of and sweet basil, and basil flavone 8-oxygenase is the general flavonoid biosynthesis is that its early steps only one with this regiospecificity currently identified occur on the cytoplasmic side of the endoplasmic at the molecular level. While any broad conclusions reticulum (Zhao and Dixon 2010), forming a multien- regarding (poly)methoxylated flavone and flavonoid zyme complex (metabolon) that may be associated metabolism must thus await findings from investiga- with the membrane by participating cytochrome P450 tions in other families such as Rutaceae and Aster- monooxygenases such as cinnamate 4-hydroxylase or aceae, our study of the flavone metabolic network in flavonoid 30- and 30,50-hydroxylases, and/or may rely basil trichomes led to recognition of several remark- on stable and dynamic protein–protein interactions in able features whose occurrence in other mint and non- the cytosol (Crosby et al. 2011; Laursen et al. 2015). mint species can now be tested. The biosynthesis of Organization of biosynthetic pathways in metabolons (poly)methoxylated flavones in basil is mediated by enhances their efficiency and is being documented for several novel enzymes and involves a Rieske oxyge- a growing number of plant metabolic processes nase as the flavone 8-hydroxylase and a flavone O- (Jorgensen et al. 2005; Lallemand et al. 2013; Laursen demethylation step. The stringent substrate require- et al. 2015; Li et al. 2015). The assembly of the ments of the oxygenases introducing the 6- and 123 384 Phytochem Rev (2016) 15:363–390

8-hydroxyl moieties entail the occurrence of several depend on availability of sufficient amounts of active obligate intermediates: scutellarein-7-methyl ether flavones. Elucidation of their natural biosynthesis in and 8-hydroxysalvigenin (and the respective sub- plants is a prerequisite for the reconstruction of the strates, genkwanin and salvigenin), which may be underlying pathway(s) in microorganisms, which may viewed as important hubs in the metabolic pathway. enable sustainable recombinant production of target The elucidated biosynthetic steps are compartmental- (poly)methoxylated flavones. Comprehensive knowl- ized between the cytosol, the cytoplasmic side of the edge of the natural biosynthetic mechanisms also enables endoplasmic reticulum, and plastids. In addition, analysis of the evolutionary relationships between the glandular trichomes secrete the products of their pathways in different plant genera and families, sheds metabolism out of the biosynthetically active cells. light on the physiology of the cells that produce them, and In the Lamiaceae and Asteraceae, a major portion of lays groundwork for studies of the cell biological secretions is stored under the elevated cuticle. The processes interacting with this metabolic network. Recent rates and specificities of the transport processes advances in delineation of (poly)methoxylated flavone underlying the necessary translocation of both essen- biosynthesis in sweet basil revealed a number of highly tial oil components and the methoxylated flavones, unusual biochemical mechanisms involved in this path- within and out of the secretory cells, are unknown. way in that species, and provided an excellent basis for Quantitative and qualitative investigations into these investigations into analogous, possibly homologous transport processes will help place the collected biosynthetic networks in other plants. biochemical and genetic knowledge into physiological context and shed light on the regulation and fine- Acknowledgments This work was in part supported by tuning of (poly)methoxylated flavone profiles, where Department of Energy Biological and Environmental Research program (Grant DE-SC0001728 to D.R.G.). We would also like intermediates of the biosynthesis, such as cirsimaritin, to thank the anonymous reviewers for helpful comments. ladanein, gardenin B, accumulate despite the high catalytic efficiency of the enzymes involved in their conversion. References

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