Phytochemistry 71 (2010) 10401049

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

Review

Multifunctional avonoid dioxygenases: Flavonol and anthocyanin biosynthesis in Arabidopsis thaliana L.

Stefan Martens a,*, Anja Preu b, Ulrich Matern a
a b

Institut fr Pharmazeutische Biologie, Philipps Universitt Marburg, Deutschhausstr. 17A, D-35037 Marburg/Lahn, Germany Fachgebiet Biomolekulare Lebensmitteltechnologie, TU Mnchen, Hochfeldweg 1, 85354 Freising, Germany

a r t i c l e

i n f o

a b s t r a c t
Flavonols and conditionally also anthocyanins, aside from avonols, are the predominant polyphenols accumulated in various tissues of the model plant Arabidopsis thaliana L. In vitro experiments suggested that the dioxygenases involved in their biosynthesis, avonol synthase and anthocyanidin synthase, are multifunctional enzymes showing distinct side activities. The in vivo relevance of the additional activities attributed to these enzymes, however, has remained obscure. In this review we summarize the most recent results and present nal proof of the complementing activities of these synthases for avonol and anthocyanidin formation in the model plant A. thaliana. The impact of their modication on the biosynthetic pathway and the pattern of avonoids in different plant tissues are discussed. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 29 January 2010 Received in revised form 14 April 2010 Available online 8 May 2010 Keywords: Flavonoid pathway Dioxygenases Flavonols Anthocyanins Arabidopsis thaliana

Contents 1. 2. 3. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiological function in plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Grid of flavonoid biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. General features of 2-oxoglutarate dependent dioxygenases (2-ODDs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Pivotal role of flavonol synthases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Formation of anthocyanidins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Flavonol and anthocyanidin biosynthesis in A. thaliana . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1040 1041 1042 1042 1043 1043 1044 1045 1047 1047 1047

4. 5.

1. Introduction Flavonoids represent a highly diverse class of polyphenolic secondary metabolites which are abundant in spermatophytic plants, but have also been reported from primitive taxa, such as liverworts (Conocephalum conicum L., Conocephalaceae; Feld et al., 2003) and horsetails (Equisetum arvense L., Equisetaceae; Oh et al., 2004).

* Corresponding author. Present address: Fondazione Edmund Mach, Istituto Agrario San Michele allAdige, IASMA, Centro Ricerca e Innovazione, Area Alimentazione, Via E. Mach 1, 38010 San Michele allAdige (TN), Italy. Tel.: +39 0461 615541; fax: +39 0461 615200. E-mail address: stefan.martens@iasma.it (S. Martens). 0031-9422/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2010.04.016

These polyphenols, although usually not essential for growth, have been accredited with particular functions during plant development and propagation ranging from pigmentation over ultraviolet screening to signaling interactions with insects and microbes (Harborne and Williams, 2000; Ververidis et al., 2007a). Most notably, they appear to be fundamental for environmental interactions, i.e. in the adaption to ecological niches or for coping with abiotic stresses. Flavonoids accumulate constitutively or may be induced upon challenge in owers, fruits, green tissues and roots. Based on the oxidation state and substitution pattern nine subgroups (Fig. 1) were distinguished: avanones (synonym dihydroavones), dihydroavonols (DHFs), avan-3,4-diols, anthocyanidins, avones, avonols, avan-3-ols, proanthocyanidins (PAs) and

S. Martens et al. / Phytochemistry 71 (2010) 10401049

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A
2' 1 8 7

3' 4'

B
2 3 6'

5'

A
6 5

C
4

flavonoid skeletal structure

B
O C O
2 3

O C O
2 3

O C O
2 3

O C OH O
2 3

O C
OH
2 3

OH OH

flavanones

flavones
O

dihydroflavonols

flavonols

flavan-3,4-diols

O C
2 3

2 3

O + C

2 3

OH

OH

flavan-3-ols

isoflavones

anthocyanidins

Fig. 1. Flavonoid structures. A. Flavan (C6C3C6) skeleton of avonoids. B. The oxidation status and saturation of the C-heterocycle dene the subgroups of avonoids.

isoavonoids. Overall, about 10,000 avonoids have been recorded which represent the third largest group of natural products following the alkaloids (12,000) and terpenoids (30,000) (Tahara, 2007; Ziegler and Facchini, 2008; Degenhardt et al., 2009). However, the number of avonoid structures to be expected in the plant kingdom might reach astronomical values assuming that twelve of the avan carbons (Fig. 1A) can be modied by a range of different substituents (Williams and Grayer, 2004). With the exception of avan-3-ols (catechins) these compounds accumulate mainly in form of their glycosides and are often concentrated in the upper epidermal cells of leaves and fruit skins. It is common that several types of avonoids are present in a single plant species. Signicant quantities of avonoids are consumed as part of the daily human diet, and their prospective health-promoting effects provide the rationale for further respective food supplements. Nevertheless, some concern was also raised about potentially deleterious effects of a high avonoid diet, but this has been revoked as largely unfounded (Okamoto, 2005). Cardioprotective, anti-oxidative, anti-angiogenic, anti-inammatory or neuroprotective properties and even putative anticancer potential have been ascribed particularly to avonols, e.g. quercetin (Harborne and Williams, 2000; Ross and Kasum, 2002; Havsteen, 2002). Rutin (quercetin 3-O-rutinoside), the major avonol in Citrus species (Rutaceae) or buckwheat (Fagopyrum esculentum Moench., Polygonaceae) and many other plants, has been shown to be an efcient nitric oxide scavenger, a property that averts nitric oxide-induced tissue damage (Haenen et al., 1997). These effects are in the focus of current investigations. Moreover, other benecial activities have also been documented, i.e. the inhibition of cyclooxygenase and/or 5-lipooxygenase activities involved in arachidonic acid metabolism as well as the inhibition of the acetyltransferase forming the platelet aggregating factor, and thus avonoids may act in a number of ways on blood components such as platelets, monocytes, or lowdensity lipoprotein (Harborne and Williams, 2000; Martens and

Mithfer, 2005; Ververidis et al., 2007a). Much less is known about the free-radical scavenging and pharmacological potentials of anthocyanins (Williams and Grayer, 2004) which impress mostly by their range of great colours. Accordingly, there is considerable interest to enhance the level of bioactive avonols in plants which are commonly grown for human consumption (Luo et al., 2008).

2. Physiological function in plants Colourless avonols are among the most abundant avonoids found in plants, usually conjugated in form of mono-, di- or triglycosides (Bhm et al., 1998). Their physiological relevance presumably differs across different plant species, but the absorption maximum at 280320 nm predicts a major function in tissue-protection from UV radiation (Stracke et al., 2007 and references therein). This can be inferred, for example, from soybean cultivars (Glycine max L., Fabaceae) with enhanced tolerance to UVB irradiation which accumulated signicantly more avonols than sensitive cultivars, and the expression of avonol biosynthesis was induced upon UV treatment (Reed et al., 1992; Kim et al., 2008). Sunlight also enhanced the avonol accumulation in grape vine (Vitis vinifera L., Vitaceae) (Fujita et al., 2006). Alternatively, avonols may act as ultraviolet ower pigments of attractive and defensive functions for insects (Gronquist et al., 2001). Furthermore, avonols possess a planar ring-architecture and may act as copigments sandwiched between anthocyanin molecules to shift the colour of owers and fruits (Yoshitama et al., 1992; Nielsen et al., 2002). Flavonols might be essential for male fertility, i.e. pollen germination and tube growth of Zea mays L. (maize, Poaceae) and Petunia hybrida Hort. ex Vilm. (petunia, Solanaceae), but not of Arabidopsis thaliana (L.) Heynh. (mouseear cress, Brassicaceae) or Eustoma grandiorum Grise. (lisianthus, Gentianaceae) (Mo et al., 1992; van der Meer et al., 1992; Burbulis et al., 1996; Nielsen et al., 2002). They were also proposed to be involved in auxin

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metabolism and transport as well as in the defense against microbes (Stafford, 1991; Stracke et al., 2007 and references therein). Other functions attributed to avonols concern the inuence on light and gravity responses of plants or the repression of bud outgrowth affecting lateral branching. These effects have been analyzed in mutants only (Stracke et al., 2007 and references therein; Owens et al., 2008a). Anthocyanin pigments of pink, red, orange, scarlet, purple, blue or blue-black and also yellow appearance have been reported (Davies et al., 2003), and the intensity and hue of tissue colour depends largely on the hydroxylation and glycosylation patterns as well as on the co-pigmentation with other avonoids, e.g. avonols. The bright colour of these pigments is often a critical factor for insect-mediated pollination of plants, and the pigments are implicated in further processes such as modulation of hormone responses, UVB protection, and photo-perception of autumn foliage (Reddy et al., 2007 and references therein). Anthocyanins have been reported not only from ower petals, but also from leaves, stems, roots, tubers, fruits and seeds (Williams and Grayer, 2004). In many instances colourless plant tissues are capable of accumulating anthocyanins under stress conditions such as low availability of nitrogen and phosphate, wounding, pathogen infection, treatment with methyl jasmonate, water stress or irradiation by ultraviolet, visible and far-red light (Stewart et al., 2001). Low temperature also induces anthocyanin synthesis in numerous plant species including A. thaliana and maize (Rowan et al., 2009).

3. Biosynthesis 3.1. Grid of avonoid biosynthesis The principles of avonoid biosynthesis, i.e. the biochemistry and molecular genetics, have been thoroughly studied over the last decades, and avonoids belong to the most intensively investigated plant secondary metabolites (Davies and Schwinn, 2007; Ververidis et al., 2007b). Advanced technologies including the recombinant expression enabled the in vitro classication of individual enzymes and, in particular, the determination of substrate and product specicities. The basal structure of a avanone (Fig. 1), consisting of a substituted 2,3-dihydro-2-(4-hydroxyphenyl)-4H1-benzopyran-4-one as in naringenin, is composed from p-coumaroyl-CoA contributing nine carbons forming the B- and C-rings, while the six carbons of the A-ring are added by three cycles of a polyketide chain elongation reaction requiring malonyl-CoA as
Isoflavones 3 x malonylCoA
IFS/HID CHS CHI FHT

the co-substrate. p-Coumaroyl-CoA, supplied by the consecutive action of the shikimate and general phenylpropanoid pathways, serves as a starter substrate for condensation which is catalyzed by a type III polyketide synthase known as chalcone synthase (CHS). The cyclization of the A-ring yielding the chalcone concomitantly with the polycondensation is a peculiar feature of CHS which is the rst enzyme committed to avonoid biosynthesis. Subsequently, chalcone isomerase (CHI) catalyzes the stereospecic intramolecular cyclization (Michael type addition) of the chalcone to the respective (2S)-avanone, although the nonstereospecic cyclization occurs readily at room temperature. (2S)-Flavanone is the common substrate for the various avonoid branch pathways which rely on three major reactions: aryl migration, oxidation or hydroxylation. Isoavones emerge from (2S)-avanones by C-2 to C-3 aryl migration concomitant with C2 hydroxylation. This reaction is catalyzed by isoavone synthase (IFS; synonym 2-hydroxyisoavanone synthase, 2HIS) which has been characterized in detail from several legume plants as a membrane-bound cytochrome P450 (Cyt P450) monooxygenase (Hagmann and Grisebach, 1984; Davies and Schwinn, 2007). Dehydration of the IFS product delivers the isoavone (Fig. 1). The oxidation of (2S)-avanones introducing a double bond at C2/C-3 to yield avones is accomplished by avone synthase I or II (FNS I and II) (Figs. 1B and 2). FNS I was characterized as a 2-oxoglutarate-dependent dioxygenase (2-ODD) and has been reported from species of the Apiaceae family only (Martens et al., 2001; Gebhardt et al., 2005, 2007) except for one recent report from rice (Lee et al., 2008). In contrast, the Cyt P450 monooxygenase FNS II was expected to be more widely distributed than FNS I (Martens and Mithfer, 2005) and has been described from various plant families. Dihydroavonols (Fig. 1B and 2) arise from (2S)-avanones by the action of avanone 3-hydroxylase (FHT, synonym F3H), another member of the 2-ODD class of enzymes. FHT occupies a key position in avonoid metabolism, competing with FNS I or II and controlling the ux of avanones into branch pathways for end products of distinct physiological functions (Owens et al., 2008b) (Fig. 2). Colourless or yellowish avonols (Fig. 2) may be formed from dihydroavonols by the action of avonol synthase (FLS) which again belongs to the 2-ODD class of enzymes. In competition with FLS, jointly acting dihydroavonol 4-reductase (DFR) and leucoanthocyanidin dioxygenase (LDOX; synonym anthocyanidin synthase, ANS) may utilize dihydroavonols for the formation of anthocyanidins (Fig. 2) or proanthocyanidins (Davies et al., 2003; Davies and Schwinn, 2007). Flavonoids and particularly anthocyanidins may be glucosylated by UDP-glucose:avonoid

DFR

LDOX

FGT

Chalcones

Flavanones

Dihydroflavonols

Leucoanthocyanins

Anthocyanidins

Anthocyanins

p-coumaroyl CoA

F2H/ HFD

FNSI&II

FLS

LAR

ANR

Flavones

Flavonols

transFlavan-3-ols

cisFlavan-3-ols

Proanthocyanidins
Fig. 2. General outline of the avonoid pathway. CHS, chalcone synthase; CHI, chalcone isomerase; FHT, avanone 3-b-hydroxylase; DFR, dihydroavonol 4-reductase; LDOX (ANS), anthocyanidin synthase; FGT, avonoid glycosyltransferase; FNS, avone synthase; FLS, avonol synthase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; IFS, isoavone synthase; HID, 2-hydroxyisoavanone dehydratase, F2H, avanone 2-hydoxylase; HFD, 2-hydroxyavanone dehydratase.

S. Martens et al. / Phytochemistry 71 (2010) 10401049

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O-glucosyltransferases (UFGT) for stable storage of pigments (Davies and Schwinn, 2007). Modications of the avonoid skeleton enable the formation of numerous metabolites. These modications require precisely regulated and coordinated hydroxylation, oxidoreduction, methylation, acetylation or glycosylation reactions. For example, avonoid 30 and 30 , 50 -hydroxylases (F30 H; F30 50 H) which both belong to the Cyt P450 family catalyze the single or double hydroxylation of the B-ring (Fig. 1A) of avanones or other avonoids, which significantly affect the physiological quality of avonols or anthocyanins as the end products of the branch pathways (Davies and Schwinn, 2007). Multiple activities are necessary to synthesize the full spectrum of natural avonoids, but only a few of these enzymes have so far been thoroughly examined at the biochemical and molecular level. It has become obvious, however, that genes selected by mutational studies or homology cloning require the functional expression and biochemical characterization of the polypeptides for unequivocal annotation. The marginal difference of only 10% in the DNA sequences of parsley FHT and FNS I is a striking example, emphasizing that base alignments are insufcient to prove functionality (Gebhardt et al., 2005, 2007). Unfortunately, a thorough biochemical characterization of enzymes attributed to avonoid metabolism has often been neglected. 3.2. General features of 2-oxoglutarate dependent dioxygenases (2-ODDs) A plethora of 2-ODDs is known from bacteria, fungi, plants or vertebrates, which catalyze diverse reactions including aliphatic hydroxylation, epoxidation, desaturation and desaturating cyclization, respectively. Most of these enzymes are involved in biosynthetic processes leading to collagen or other modied polypeptides and amino acids, alkaloids, ethylene, gibberellins and -lactam antibiotics, i.e. penicillins and cephalosporins (Prescott, 1993; DeCarolis and DeLuca, 1994; Prescott and John, 1996; Schoeld and Zhang, 1999; Prescott and Lloyd, 2000). Accordingly, studies on these dioxygenases were inspired by the medicinal and industrial relevance and focused initially on the architecture and mode of action of microbial and human 2-ODDs, but in recent years considerable insight has also been achieved into the functional organisation of plant 2-ODDs. A single plant may express numerous putative 2-ODDs, as was revealed by sequence analysis of the A. thaliana genome coming up with about 100 isogenes (Wilmouth et al., 2002). The activities of 2-ODDs depend on ferrous iron which is required for the reduction of molecular oxygen. Two electrons are provided in this process by the decarboxylation of a cosubstrate which is most commonly 2-oxoglutarate. The stabilization of iron(II) in the active site of these labile soluble enzymes is brought about by the unique conformation of the enzyme polypeptide. Eight amino acid residues are strictly conserved in all of these dioxygenases which include two histidines (His221, His277; numbers refer to AtFLS1 sequence) and one acidic amino acid (Asp223) as well as one arginine (Arg287) and one serine (Ser289) proposed to bind 2oxoglutarate. Crystallography of 2-ODDs revealed that the conserved motifs Hx(D/E)xnH and RxS are part of a characteristic double-stranded beta helix (Clifton et al., 2006) and two sets of four anti-parallel beta sheets forming a sandwich-like structure (jelly roll topology) which shield the ferrous iron in the active center for oxygen activation and catalysis (Koehntop et al., 2005). The active-site iron is coordinated to the histidine-rich motif which more often shows the Hx(D)xnH composition (Clifton et al., 2006). Four amino acids (Gly68, His75, Gly261, Pro207) are conserved with no obvious functionality but most likely required for proper folding of the polypeptide (Wellmann et al., 2002). Computational analysis (Chua et al., 2008) suggested further ve residues

(His132, Phe134, Lys202, Phe293 and Glu295), albeit less conserved, for substrate binding. Five 2-ODDs have been identied in the context of avonoid biosynthesis. FHT (hydroxylating), FLS (desaturating) and LDOX (hydroxylating/dehydrating) activities (Fig. 2) are widely distributed (Davies and Schwinn, 2007). FNS I (desaturating) appears to be conned to species of the Apiaceae (Gebhardt et al., 2005, 2007) with a sole nding in rice (Lee et al., 2008), while Cyt P450 take over the FNS I-equivalent function (Fig. 2) in other plants (Davies and Schwinn, 2007). It remains to be established, whether catalysis by these Cyt P450s follows the same mode, because a hydroxylated substrate intermediate was ruled out for the FNS I reaction (Britsch, 1990; Martens and Mithfer, 2005). It is noteworthy that an analogous scenario was reported for avonoid 6hydroxylation which is catalyzed by a 2-ODD in Chrysosplenium americanum Schwein. ex Hook. (American golden saxifrage, Saxifragaceae; Anzelotti and Ibrahim, 2004) but a Cyt P450 from soybean (Latunde-Dada et al., 2001). Among the avonoid-committed 2-ODDs most mechanistic and molecular investigations have been conducted on LDOX or FLS, and X-ray diffraction of LDOX-naringenin co-crystals already revealed the putative sites of substrate binding (Welford et al., 2001; Turnbull et al., 2004; Gebhardt et al., 2007). 3.3. Pivotal role of avonol synthases The full perception of factors and enzyme activities regulating the tissue-level of avonols is a prerequisie for breeding of avonol-enriched fruits and vegetables, but determines also the perspectives of metabolic engineering of plants or microorganisms (Ververidis et al., 2007b). FLS catalyzing the oxidation of dihydroavonols to avonols competes at a crucial branch point with dihydroavonol reductase (DFR) in the anthocyanin/PA pathway (Fig. 2). FLS was reported rst from irradiated parsley cells and characterized as a soluble 2-ODD requiring 2-oxoglutarate, FeII and ascorbate for full activity (Britsch et al., 1981). This enzyme was considered to be specic for natural (+)-(2R, 3R)-dihydroavonol substrates. Much later, a putative FLS cDNA was cloned from P. hybrida and functionally expressed in yeast and plants (Holton et al., 1993), which was followed by FLS sequences from various other plants, such as A. thaliana (Pelletier et al., 1997; Wisman et al., 1998), Citrus unshiu Marc. (satsuma mandarin, Rutaceae; Moriguchi et al., 2002), E. grandiorum (Nielsen et al., 2002), Solanum tuberosum L. (potato, Solanaceae; van Eldik et al., 1997), Malus domestica Borkh. (apple, Rosaceae; Lee et al. direct submission to Genbank), Matthiola incana (L.) R. Br. (stock, Brassicaceae; Henkel and Forkmann, direct submission to Genbank), G. max (Takahashi et al., 2007), Petroselinum crispum L. (parsley, Apiaceae; Martens et al., 2003a) or Fragaria ananassa Duch. (strawberry, Rosaceae; Almeida et al., 2007). The translated FLS sequences show a remarkable degree of conservation (about 85% similarity, 50% identity) and share partial similarity with LDOX (5060%), but much less with the related 2-ODD enzymes FNS I or FHT. It is noteworthy that FNS I is capable of oxidizing avanones, i.e. (2S)-naringenin (Fig. 3), to avones analogous to the 2,3-desaturation of dihydroavonols (Fig. 1B) by FLS. However, FNS I does not accept dihydroavonols as substrates, and provisional assays indicated that FLS does not form avones from avanones (Fig. 1B). The rst detailed FLS-study was carried out on the FLS from C. unshiu expressed in Escherichia coli (Wellmann et al., 2002). The recombinant enzyme surprisingly accepted (2S)-naringenin as substrate which was almost completely converted to kaempferol. Minute amounts of dihydrokaempferol recovered from these assays suggested that (2R, 3R)-dihydrokaempferol is an intermediate en route to kaempferol, and thus FHT/FLS bifunctionality (Fig. 3) must be assigned to FLS oxidizing both (+)-trans-dihydroavonols

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S. Martens et al. / Phytochemistry 71 (2010) 10401049

R1
3' 4'

OH

HO

O
2 3

OH

R1 = H; naringenin R1 = OH; eriodictyol

FHT

FLS1 LDOX
R1
3' 4'

FLS1/LDOX

R1 OH
3'

FLS1 & 3
HO O
2 3

OH
4'

HO

O
2 3

OH OH O

LDOX

OH OH O

DFR

R1 = H; dihydrokaempferol R1 = OH; dihydroquercetin

R1 = H; kaempferol R1 = OH; quercetin

R1
3' 4'

OH

LDOX
HO O
2 3

R1
3' 4'

OH

HO

O
2 3

OH OH OH OH

OH

R1 = H; leucopelargonidin R1 = OH; leucocyanidin

R1 = H; pelargonidin R1 = OH; cyanidin

Fig. 3. Postulated scheme of avonoid biosynthesis in A. thaliana. The side activities of LDOX replace partially the function of FLS1 and/or 3 in the formation of avonols and anthocyanidins/proanthocyanidins. Full arrows common pathway; dotted arrows side activities.

and (2S)-avanones to the respective avonols (Lukacin et al., 2003). Moreover, unnatural (2R)-naringenin was converted to ()-trans-dihydrokaempferol by the FLS from C. unshiu yielding only traces of kaempferol, and equivalent specicities at lower conversion rates were observed with FLSs from P. crispum, F. ananassa or A. thaliana (Prescott et al., 2002; Turnbull et al., 2004; Almeida et al., 2007; Preu et al., 2009). Nevertheless, all FLSs examined so far completely lacked FNS I activity. Thus, FLS belongs to a group of 2-ODDs with broad specicities, whereas FNS I and FHT show narrow substrate specicity and appear to form a separate group of dioxygenases in accordance with their high degree of sequence homology (Martens et al., 2003a; Gebhardt et al., 2005, 2007). 3.4. Formation of anthocyanidins Some information on the molecular regulation of anthocyanidin production is available from A. thaliana, where the PRODUCTION OF ANTHOCYANIN PIGMENTS1 (PAP1) gene, a conserved MYB regulator of phenylpropanoid biosynthesis, plays a key role in mediating the environmental regulation of anthocyanins but not avonols (Rowan et al., 2009). The reduction of dihydroavonols by DFR to colourless cis-(2R, 3S, 4S) leucoanthocyanidins (avan-3,4-diols) marks the beginning of the anthocyanidin-specic branch pathway (Fig. 2), and LDOX was shown to catalyze in planta the subsequent conversion to coloured, labile anthocyanidins (Fig. 2) (Saito et al., 1999; Welford et al., 2001). The latter step formally involves the dehydrogenation at C-2 and C-3/C-4 dehydration. Putative LDOX genes and cDNAs have been isolated from Antirrhinum majus L.

(snapdragon, Scrophulariaceae), Perilla hybrids, Torenia hybrids L. (torenia, Scrophulariaceae), Perilla frutescens L. (beefsteak plant, Lamiaceae), A. thaliana, Gerbera hybrida L. (gerbera, Asteraceae) and Z. mays (Saito et al., 1999; Nakajima et al., 2001; Turnbull et al., 2001; Wellmann et al., 2006). The essence of LDOX for leucoanthocyanidin-anthocyanidin conversion was demonstrated by restoration/complementation experiments in LDOX-minus lines of maize (A2 mutant) and Zantedeschia aethiopica L. (calla lily, Araceae; natural LDOX block in white spathe), where overexpression of LDOX cDNAs from maize, rice and Gerbera or transient expression by particle bombardment restored the anthocyanin formation (Menssen et al., 1990; Martens et al., 2003b and unpublished; Reddy et al., 2007). The alternative suppression of LDOX by antisense, sense or RNAi techniques clearly reduced the anthocyanin accumulation in owers of torenia (Nakamura et al., 2006). Nevertheless, the exact biochemical function of LDOX remained unknown till the recombinant enzyme from P. frustescens was assayed with leucoanthocyanidin as substrate (Saito et al., 1999). Translated LDOX sequences from different plants share 4878% similarity and less, albeit signicant, similarity with FLS (5060%) and FHT or FNS I (about 30%), respectively. The physiological role of LDOX has been put into question, because the recombinant enzyme from A. thaliana was classied as a non-specic 2-ODD catalyzing primarily the desaturation of trans-dihydroquercetin to quercetin, analogous to FLS, and the formation of 3,4-cis-dihydrokaempferol as a major product from (+/)-naringenin (Turnbull et al., 2001; Welford et al., 2001; Lukacin et al., 2003; Martens et al., 2003a; Wellmann et al., 2006). Furthermore, the proposed natural substrate cis-(2R,3S,4S)leucocy-

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anidin and trans-(2R,3S,4R)leucocyanidin were oxidized to quercetin as the predominant product (>85%) and cis-dihydroquercetin with only trace amounts of cyanidin (Turnbull et al., 2003). The in vivo signicance of the ndings has remained obscure, but the observed side activities of LDOX and also FLS raised the possibility that these enzymes provide an alternative route to avonols in situ in mutant lines lacking FHT or FLS activity. However, the proof of concept is still missing.

4. Flavonol and anthocyanidin biosynthesis in A. thaliana The avonoid pattern in A. thaliana is simple and consists solely of avonol glucosides derived from kaempferol, quercetin or isorhamnetin, beside anthocyanins and PAs (Pelletier et al., 1997, 1999; Veit and Pauli, 1999; Peer et al., 2001; Broun, 2005; Routaboul et al., 2006; Stracke et al., 2009). The avonols accumulate mainly in the vacuoles of epidermal cells (Feinbaum and Ausubel, 1988; Hartmann et al., 1998; Landry et al., 1995), whereas PAs are found in specialized tissues, i.e. the seed testa (Lepiniec et al., 2006), and the accumulation of anthocyanins might be induced by low temperature in correlation with the abundance of avonoid-committed transcripts (Leyva et al., 1995). The common substrates for all avonoids of A. thaliana are dihydroavonols which fuel the branch pathways to avonols, depending on FLS, and to anthocyanidins/PAs, catalyzed by DFR and LDOX (Fig. 2). While most of the biosynthetic enzymes appear to be encoded by single copy genes, e.g. LDOX, the A. thaliana genome contains a small family of up to six FLS isogenes (Pelletier et al., 1997; Stracke et al., 2007) as is also the case in S. tuberosum (van Eldik et al., 1997), E. grandiorum (Nielsen et al., 2002) and V. vinifera (Fujita et al., 2006). The FLS isogenes were tentatively identied by their homology to 2-ODDs and clustering with FLSs. All six genes are located on chromosome 5 with AtFLS2, -3, -4, and -5 arranged in a 7.5-kb tandem array. The four clustered genes are not more closely related to each other than to the other two genes (62 73% identity at the nucleotide level) with AtFLS2 being most distant (4851% identity), which suggests ancient duplications events (Owens et al., 2008a; Stracke et al., 2009). The putative A. thaliana FLS gene family and the LDOX gene were examined in detail only recently using genetic and biochemical approaches (Owens et al., 2008a; Stracke et al., 2009; Preu et al., 2009). Recombinant expression and enzyme assays revealed activities of only AtFLS1 and AtLDOX (Wisman et al., 1998; Saito et al., 1999; Winkel-Shirley, 2001; Prescott et al., 2002), whereas AtFLS2-6 were assigned inactive (Owens et al., 2008a; Stracke et al., 2009). Nevertheless, the contribution of AtFLS isoenzymes of variant substrate specicities or differential regulation had been suggested as a means of control of avonoid synthesis in A. thaliana, which might explain the variable composition of avonol glucosides in different tissues or upon a change in environmental conditions (Pelletier et al., 1997; Mehrtens et al., 2005; Owens et al., 2008a and references therein). The differential biosynthesis of avonols and anthocyanidins in A. thaliana, just like in maize and grape, is also controlled by specic transcription factors (TFs) (Mehrtens et al., 2005; Fujita et al., 2006). Regulation of anthocyanins and PA accumulation via combinatorial action of MYB and basic helix-loop-helix (bHLH)type TFs is a conserved feature in plants, and the R2R3-MYB factor MYB12 was identied recently as a avonol-specic regulator of avonoid biosynthesis in A. thaliana. MYB12 activates coordinately AtCHS, AtCHI, AtFHT and AtFLS1, the four genes required for the formation of a avonol. However, a preference for either one AtFLS isogene was not observed and the question whether MYB 12 is involved in any of the many stress signaling pathways that result in avonol accumulation is still unanswered (Mehrtens et al., 2005).

Interestingly, different avonols predominate in the various tissues with quercetin being the primary seed avonol and kaempferol the main avonol in whole owers, whereas similar levels of these two avonols are found in stamens (Pelletier et al., 1997). Only traces of isorhamnetin are found in seedlings and leaves (Stewart et al., 2001; Routaboul et al., 2006). Moreover, limitations in the supply of nitrogen or phosphorus induce higher concentrations of avonols in seedling and vegetative tissue of A. thaliana (Stewart et al., 2001). The nal composition of the avonol contents in different tissues of A. thaliana depends on the responsiveness of genes that encode avonol glucosyltransferases and other enzymes downstream (i.e. modifying kaempferol) in the biosynthetic path (Stracke et al., 2007). Experiments with fht mutant lines (transparent testa 6, tt6) or s1 knock-out lines of A. thaliana revealed some unforeseen accumulation of avonol, anthocyanin and PA, whereas chs mutants (tt4) literally lacked avonoids (Burbulis et al., 1996; Wisman et al., 1998; Pelletier et al., 1999; Routaboul et al., 2006; Stracke et al., 2007; Owens et al., 2008a,b; Ric de Vos, personal communication). The fht and s genotypes are generally characterized by pale-brown rather than yellow seeds, indicating the accumulation of anthocyanins and/or PAs. The Ats1 plants also exhibited a much more intense red colouration of the hyptocotyl and cotyledons during germination and at the base of the stalk of mature plants compared with the wild-type (Owens et al., 2008a; Stracke et al., 2009). A similar shift in metabolites of branch pathways has been reported for the avonoid mutant banyuls which is decient in anthocyanidin reductase (ANR; Fig. 2) (Devic et al., 1999). Furthermore, the synthesis of avonols was not eliminated entirely in Ats1 plants lacking a functional avonol-specic TF MYB12 factor (Mehrtens et al., 2005). The unexpected avonol accumulation in mutants together with the observed multifunctionality of FLS and ANS in vitro and the presence of FLS isogenes led to the following speculations: (a) AtFLS isozymes with different substrate specicities control the amount and type of avonols present in specic tissues, (b) avonols present in a given tissue or different AtFLS genes may be regulated independently of one another in a developmental- or tissue-specic manner, (c) an additional AtFLS isogene and/or AtLDOX side activity is responsible for avonol formation under in situ conditions, (d) AtFLSs and/or AtLDOX can complement the FHT or FLS step. First support for the hypothesis of contributing FLS/LDOX side activities in avonoid biosynthesis in planta was recently provided by overexpressing LDOX in rice plants (Reddy et al., 2007), by the biochemical characterization of AtFHT from A. thaliana mutant line tt6 (Owens et al., 2008b), and from metabolomic and genetic analyses in A. thaliana (Stracke et al., 2009). The accumulation of a mixture of avonols and anthocyanins increased concomitantly with a decrease of PAs in transgenic rice plants overexpressing homologous LDOX cDNA (Reddy et al., 2007). The authors suggested that LDOX may execute more than one dioxygenase activities on different avonoid substrates. The leaky phenotype of Atfht mutant alleles (tt6), producing pale-brown rather than yellow seeds, was biochemically and genetically characterized and provided evidence that FLS and LDOX compensate in vivo, at least partially, for the disrupted FHT enzyme. Similar leaky phenotypes apparently blocked in FHT were also reported from G. max and Dianthus caryophyllus L. (carnation, Asteraceae) suggesting an analogous compensation by related 2-ODDs in these plants as well (Owens et al., 2008b). The ndings of Fujita et al. (2006) may support thesis b for grape-vine, because each of the ve FLS genes was suggested to encode functional enzyme due to highly conserved amino acid residues at the relevant binding sites. Furthermore the transcrip-

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tion patterns of the ve FLSs varied with the organ and developmental stage. In a set of experiments employing A. thaliana tissue from seedlings and adult plants of wild-type (Col-0) and mutant lines (s1-2 mutant, ldox/s1-2 double mutant) the role of LDOX and/or FLSs for avonoid accumulation was investigated (Preu et al., 2009). A. thaliana ldox/s1-2 double mutant revealed a signicant reduction in avonol level as compared to the wild-type and the s1-2 in correlation with the genetic data (Stracke et al., 2009). Additionally, at the expense of avonols a signicant accumulation of dihydroavonol glycosides was observed (Stracke et al., 2009). Similar ndings were recorded from petunia and lisianthus owers expressing homologous antisense-FLS constructs (Holton et al., 1993; Nielsen et al., 2002). It must be emphasized again that extracts of all three genotypes contained avonols, albeit at greatly different levels, which assigns FLS-like enzyme activities to all genotypes (Stracke et al., 2009). Crude protein preparations from these plant tissues were prepared (Martens et al., 2003a), and standard enzyme assays with [14C]naringenin or [14C]dihydrokaempferol as substrate clearly revealed FHT and FLS activities in wild-type extracts, converting both substrates to kaempferol, whereas extracts of the s1-2 single mutant and the ldox/s1-2 double mutant lacked these activities (Preu et al., 2009). LDOX activity, however, has never been demonstrated in crude plant extracts of Arabidopsis or any other species, which can be explained by the instability of the enzyme during isolation and in vitro assay (Saito et al., 1999). Plant 2ODDs are commonly rather labile enzymes which might suffer rapid partial digestion after extraction prohibiting their functional identication (Lukacin et al., 2000a). Furthermore, low FLS activity of either one of the other putative FLS polypeptides or LDOX in crude extracts might have escaped the detection by in vitro assays which were commonly run for 30 min only (Martens et al., 2003a), while other groups used extended periods of incubation, i.e. up to 3 h with recombinant A. thaliana FLS1 (Prescott et al., 2002), for the detection of products on subsequent HPLC analysis. Arabidopsis AtFLS6 and AtFLS4 had been identied before as pseudogenes or non-functional copies (Owens et al., 2008a; Stracke et al., 2009). Therefore, only AtFLS1, AtFLS2, AtFLS3 and AtFLS5 as well as AtLDOX were expressed for further functional characterization in microbial systems. Standard assays of the recombinant polypeptides were conducted employing crude extracts or the puried enzyme fraction. As expected, AtFLS1 efciently converted dihydrokaempferol or dihydroquercetin to kaempferol and quercetin, respectively, with a clear bias towards dihydrokaempferol (kaempferol/quercetin ratio 1:0.7). These results are fully compatible with previous ndings (Prescott et al., 2002), although unnatural (2S,3S)-dihydrokaempferol does not appear to be an FLS substrate (Lukacin et al., 2003). AtLDOX also oxidized the substrates to the corresponding avonols, albeit to a lower extent (kaempferol/quercetin ratio 0.7:1). However, neither one of the other putative AtFLSs, tested in crude extracts or after purication, provided any detectable product under standard assay conditions. These results suggested that the FLS-like activity of AtLDOX significantly contributed to the formation of avonols in the A. thaliana s1-2 mutant. Additional support for this assumption was gained by the tightened reduction of avonol contents in the ldox/s1-2 double mutant (Stracke et al., 2009). Nevertheless, the small, but signicant, residual amount of avonols in the ldox/s1-2 mutant (Stracke et al., 2009) required further explanation. The minor FLS-like activity of this double mutant might thus be due to either one of the FLS isogenes which had been proposed from in vitro assays to encode catalytically inactive proteins or to other members of the 2-ODD family of enzymes with as yet unknown function (Owens et al., 2008a,b). Sequence alignments of AtFLSs and AtLDOX indicated that AtFLS2, analogous to truncated pseudogenes AtFLS4 and AtFLS6,

likely encodes a non-functional polypeptide because of a truncated C-terminus and lack of appropriate iron and 2-oxoglutarate binding sites (Owens et al., 2008a; Stracke et al., 2009; Preu et al., 2009). A limited loss of C-terminal amino acids may be tolerated, because, at least in Petunia FHT (Lukacin et al., 2000b), this reduced the activity only. However, marginal changes in the binding sites of substrate, ferrous iron and oxoglutarate cosubstrate strongly affect the substrate and/or product specicity of 2-ODD enzymes (Wilmouth et al., 2002). The crucial role of these residues for enzyme activity had been documented through knock down of AtFLS1 activity in studies mutating some of the residues proposed for ferrous iron or 2-oxoglutarate binding, and these mutations apparently affected the positioning of 2-oxoglutarate in the active site causing inefcient binding or total loss of function (Chua et al., 2008). Alignment of the AtFLS5 polypeptide with AtFLS1 also revealed substantial exchanges which likely affect the xation of substrate through hydrogen bonds and thus the catalytic activity (Owens et al., 2008a). In AtFLS3, however, the proposed substrate and cofactor binding sites appear fully conserved. Molecular details of substrate binding sites are not yet available for the FLSs, but LDOX may be used as a model enzyme. The high sequence similarity of LDOX and FLS implies a relationship much closer than to the other avonoid 2-ODDS, FHT and FNS I (Gebhardt et al., 2005, 2007). Furthermore, the mode of action of FLS must be very similar to that of LDOX, because the unnatural oxidation of dihydroquercetin to quercetin catalyzed in vitro by LDOX is identical to the reaction catalyzed by FLS. Since the crystal structure of LDOX was reported (Turnbull et al., 2001; Wilmouth et al., 2002), homology modeling might identify the amino acid residues essential for FLS activity. This approach had been used for FHT and FNS I from parsley as well as for AtFLS1 (Gebhardt et al., 2007; Chua et al., 2008) and should be amenable to describe the substrate and product selectivities of FLS2, 3 and 5. Model structures of AtLDOXnaringenin (2brt) (Welford et al., 2005) and AtLDOX-dihydroquercetin complexes (Wilmouth et al., 2002) were employed for homology modeling of AtFLS1, AtFLS3 and AtFLS5 (Prescott et al., 2002; Martens et al., 2003a). The conserved binding sites for 2-oxoglutarate and iron are distributed at an equivalent spacing in AtFLSs polypeptides. Furthermore, the models endorse the role of Glu327 in substrate binding and the lack of activity of AtFLS5 (Preu, 2009). Notably, AtFLS3 differs from AtFLS1 by replacement of Leu136 through Tyr136, and the AtFLS3-substrate model complex predicted that this phenolic residue changes the orientation of nearby His149 which is involved in substrate binding (Preu et al., 2009). Thus, a shift in location of substrate toward the active ferryl species is expected and the kinetic parameters of enzyme catalysis are likely affected. Point mutations in putative substrate binding sites commonly decrease considerably the activity of enzymes, which in turn conrms their relevance for catalytic turnover. However, replacing His132 by phenylalanine in the dihydroquercetin binding site of AtFLS generated a mutant of approximately 20% higher activity as compared to the wild-type (Chua et al., 2008). In this instance, kinetic analyses indicated an improved substrate binding afnity. Taking advantage of the in vivo bioconversion protocol yeast transformants expressing recombinant AtFLSs or AtLDOX were fed with various avanones and dihydroavonols (Preu et al., 2009). This method allows testing of a large number of potential substrates and may dene the in vivo substrate specicities with respect to the types of avonols accumulating in different tissues. Moreover, improved stability of enzymes is expected in situ and assays can be extended signicantly in time. Recent studies with recombinant E. coli overexpressing LDOX accordingly demonstrated the formation of plant-specic anthocyanins and avonols upon feeding relevant precursors, which conrms the reliability of the procedure (Yan et al., 2005). Bioconversions were performed by

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incubating cultures of AtFLS or AtLDOX yeast transformants in the presence of 5 mg of either one of the potential substrates (Witte et al., 2009) for 17 h, and similar experiments were run with E. coli transformants for 4 h. All dihydroavonols and avanones applied were converted to a variable degree and formation of the corresponding avonols was detected without any intermediates. AtLDOX transformants showed a considerably lower conversion rate than cells transformed with AtFLS1, whereas none of the transformants harbouring AtFLS2, 3 or 5 constructs was found to convert any of the dihydroavonol substrates under these conditions. Therefore, bioconversions were extended to two days, which nally revealed a signicant conversion of dihydrokaempferol or dihydroquercetin to kaempferol and quercetin, respectively, only in the incubations of AtFLS3-yeast transformants. AtFLS3 was therefore be annotated as a second functional gene in A. thaliana. Nevertheless, further site-directed mutagenesis is necessary to identify those amino acids which determine the turnover rate of FLSs in Arabidopsis. Furthermore, AtFLS3 is likely responsible for the formation of residual avonols in seedlings of the ldox/s1-2 mutant line, but nal proof requires the analysis of the respective triple mutant (ldox/s1-2/s3) (Preu et al., 2009).

The broad substrate specicity of multifunctional FLS and LDOX may be the result of incomplete evolution (Turnbull et al., 2001). Based on previous reports, the Arabidopsis FLS gene family was presumed to have originated from recent gene duplication events (Stracke et al., 2009; Owens et al., 2008a). Such duplications may lead to differentially regulated genes encoding enzymes of variable substrate specicities, which develop preferences for the synthesis of selected avonols and meet the dynamic physiological needs of the plants (Owens et al., 2008a). However, isogenes encoding inactive polypeptides and expressed independently of avonoid accumulation should no longer be assigned to the FLS gene family. The preservation of these non-functional pseudogenes (AtFLS4 and 6) over substantial evolutionary time rather suggests their former functional relevance, whereas functional AtFLS3 or AtFLS2 and 5, although coding for polypeptides lacking FLS activity, were still expressed in patterns overlapping with that of AtFLS1 and interact with other avonoid genes. Based on the recent results we postulate that a pseudogenisation/mutation process currently eliminates unnecessary genes and their protein functions. Alternatively, AtFLS2, 3 and 5 may have acquired another function while AtFLS3 at least preserved some FLS activity. Acknowledgments Funding by EU Project FLAVO (FOOD CT-2004-513960) and by Deutsche Forschungsgemeinschaft is grateful acknowledged. References
Almeida, J.R.M., DAmico, E., Preuss, A., Carbone, F., de Vos, C.H.R., Mourgues, F., Deiml, B., Perrotta, G., Fischer, T.C., Bovy, A.G., Martens, S., Rosati, C., 2007. Characterization of major enzymes and genes involved in avonoid and proanthocyanidin biosynthesis during fruit development in strawberry (Fragaria ananassa). Arch. Biochem. Biophys. 465, 6171. Anzelotti, D., Ibrahim, R., 2004. Molecular characterization and functional expression of avonol 6-hydroxylase. BMC Plant Biol. 4, 20. Bhm, H., Boeing, H., Hempel, J., Raab, B., Kroke, A., 1998. Flavonols, avone and anthocyanins as natural antioxidants of food and their possible role in the prevention of chronic diseases. Z. Ernhrungswiss. 37, 147163. Britsch, L., 1990. Purication and characterization of avone synthase I. Arch. Biochem. Biophys. 276, 348354. Britsch, L., Heller, W., Grisebach, H., 1981. Conversion of avanone to avone, dihydroavonol and avonol with an enzyme system from cell culture of parsley. Z. Naturforsch. 36, 742750. Broun, P., 2005. TrLDOXcriptional control of avonoid biosynthesis: a complex network of conserved regulators involved in multiple aspects of differentiation in Arabidopsis. Curr. Opin. Plant Biol. 8, 272279. Burbulis, I.E., Iacobuci, M., Shirley, B.W., 1996. A null mutation in the rst enzyme of avonoid biosynthesis does not affect male fertility in Arabidopsis. Plant Cell 8, 10131025. Chua, C.S., Biermann, D., Goo, K.S., Sim, T.-S., 2008. Elucidation of active site residues of Arabidospis thaliana avonol synthase provides a molecular platform for engineering avonols. Phytochemistry 69, 6675. Clifton, I.J., McDonough, M.A., Ehrismann, D., Kershaw, N.J., Granatino, N., Schoeld, C.J., 2006. Structural studies on 2-oxoglutarate oxygenases and related doublestranded -helix fold proteins. J. Inorg. Biochem. 100, 644669. Davies, K.M., Schwinn, K.E., 2007. Molecular biology and biotechnology of avonoid biosynthesis. In: Andersen, O.M., Markham, K.R. (Eds.), Flavonoids Chemistry, Biochemistry and Applications. Taylor and Francis Group, Boca Raton, pp. 143 218. Davies, K.M., Schwinn, K.E., Deroles, S.C., Manson, D.G., Lewis, D.H., Bloor, S.J., Bradley, J.M., 2003. Enhancing anthocyanin production by altering competition for substrate between avonol synthase and dihydroavonol 4-reductase. Euphytica 131, 259268. DeCarolis, E., DeLuca, V., 1994. 2-Oxoglutarate-dependent dioxygenase and related enzymes: biochemical characterization. Phytochemistry 36, 10941107. Degenhardt, J., Koellner, T.G., Gershenzon, J., 2009. Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70, 16211637. Devic, M., Guilleminot, J., Debeaujon, I., Bechtold, N., Bensaude, E., Koornneef, M., Pelletier, G., Delseny, M., 1999. The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development. Plant J. 19, 387398. Feinbaum, R.L., Ausubel, F.M., 1988. TrLDOXcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol. Cell. Biol. 8, 19851992. Feld, H., Zapp, J., Becker, H., 2003. Secondary metabolites from liverwort Tylimanthus renifolius phytochemistry 64, 13351340.

5. Conclusion A. thalana has served as a model plant for various biochemical and genetic studies of secondary metabolism including the avonoid pathway (Winkel-Shirley, 2001). Flavonols (quercetin, kaempferol, isorhamnetin glucosides) and the corresponding avan-3-ols or dihydroavonols (Fig. 1) as well as PAs and anthocyanins were reported from wild-type plants. At least thirty-ve genes committed to avonoid biosynthesis have been identied: ten structural genes (e.g. FLS, LDOX), ten genes coding for modifying enzymes and 12 regulatory genes (transcription factors; e.g. TT2, TT8), besides three genes involved in avonoid compartmentation (Yonekura-Sakakibara et al., 2008). A single LDOX gene and a family of six FLS genes were identied in the Arabidopsis genome, but only AtFLS1 was shown to yield a functional FLS (Wisman et al., 1998). LDOX and FLS were attributed to different branch pathways (Fig. 2), and FLS should correlate with the accumulation of avonols. Investigations of the expression levels of AtFLS1, 2, 3 and 5 revealed a wide tissue-distribution of AtFLS1 with highest transcript amounts during the reproductive stages. In contrast, the inactive AtFLS2 appears to be expressed at high levels in the shoot apex and lower stem, where AtFLS1 transcript was absent, and only to a low extent in owers and siliques. AtFLS5 and 3 were expressed at much lower intensities or extremely low and even falling to undetectable levels. Moreover, all AtFLS isogenes have tissueand cell-type specic promoter activities that overlap with those of AtFLS1 and interact with other avonoid proteins in yeast two hybrid assays (Owens et al., 2008a). Recent reports identied AtFLS3 as another active enzyme (Preu et al., 2009) and assigned a novel FLS-activity in situ to AtLDOX (Stracke et al., 2009). Both these enzymes and their expression patterns provide an ample explanation for the signicant quantities of avonols produced in s1-2 and s1-2/ldox mutant lines of A. thaliana (Stracke et al., 2009; Preu et al., 2009) and must be accounted for in the scheme of avonoid biosynthesis (Figs. 2 and 3). Nevertheless, the activities of FLS3 and LDOX are insufcient to fully substitute for FLS1 in s1-mutant Arabidopsis plants. This may reect structural differences that preclude efcient interaction with other avonoid-specic enzymes in the form of larger protein complexes. The data derived from Arabidopsis on FLS and on the FLS-like activity of LDOX are likely relevant also for other plant species and need to be considered, when metabolic engineering studies are conducted in plants or microorganisms.

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S. Martens et al. / Phytochemistry 71 (2010) 10401049 Nakajima, J., Tanaka, Y., Yamazaki, M., Saito, K., 2001. Reaction mechanism from leucoanthocyanidin to anthocyanidin 3-glucoside, a key reaction for coloring in anthocyanin biosynthesis. J. Biol. Chem. 276, 2579725803. Nakamura, N., Fukuchi-Mizutani, M., Miyazaki, K., Suzuki, K., Tanaka, Y., 2006. RNAi suppression of anthocyanidin synthase gene in Torenia hybrida yields white owers with higher frequency and better stability than antisense and sense suppression. Plant Biotechnol. 23, 1317. Nielsen, K., Deroles, S.C., Markham, K.R., Bradley, M.J., Podivinsky, E., Manson, D., 2002. Antisense avonol synthase alters copigmentation and ower color in lisianthus. Mol. Breeding 9, 217229. Oh, H., Kim, D.H., Cho, J.H., Kim, Y.C., 2004. Hepatoprotective and free radical scavenging activities of phenolic petrosins, avonoids isolated from Equisetum arvense. J. Ethnopharmacol. 95, 421424. Okamoto, T., 2005. 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Stefan Martens has earned a Ph.D. in plant breeding at the Technical University of Munich Freising-Weihenstephan. After two years Post Doc. in the Institute of Prof. Dr. Gert Forkamnn in 2002 he joined the Institute of Pharmaceutical Biology at the Philipps University of Marburg (AG Prof. Ulrich Matern) where he established an independent research group. In 2009 he was appointed Group Leader for Biochemistry and Molecular Biology of Soft Fruits at the Edmund Mach Foundation in San Michele all Adige (TN, Italy). His scientic interest is focused on the biosynthesis of secondary metabolites not only in soft fruits but also in apple, grapes, herbs and other plant species including breeding, metabolomics, enzymology and genomics.

Anja Preu studied Biochemistry (19992004) at ErnstMoritz-Arndt University of Greifswald. From 2005 2008 she was Ph.D. student at Institute for Pharmaceutical Biology of Philipps-University Marburg. Since 2008 she is Post Doc at Biomolecular Food Technology TU-Munich in Freising.

Ulrich Matern, Prof. (em.) holds a pharmacy license and received his Ph.D. in biochemistry (1972) from Freiburg University, Germany (supervisor Hans Grisebach). After postdoctoral training at the Dept. of Plant Pathology, Montana State University (1976/77), and the Dept. of Chemistry, University of Minnesota (1979), he returned to Freiburg University to continue studies in plant biochemistry and, after a second thesis (Habilitation, 1983), was entitled Professor of biochemistry. In 1995, he joined the faculty at Philipps-University Marburg, Germany, as Professor and Director of the Institute of Pharmaceutical Biology till retirement in 2008. His research focused on the biochemistry of secondary metabolites, ranging from antibiotics over phytotoxins to avonoids, coumarins and other phenylpropanoids, which has been published in numerous reports and was acknowledged by the Phytochemistry Pioneer Award (2008).