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The Strawberry Pathogenesis-related 10 (PR-10) Fra a Proteins Control Flavonoid Biosynthesis by Binding to Metabolic Intermediates*

  • Ana Casañal
    Footnotes
    Affiliations
    Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-Consejo Superior de Investigaciones Científicas), Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain
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  • Ulrich Zander
    Footnotes
    Affiliations
    European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France

    Unit of Virus Host-Cell Interactions, Université Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France
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  • Cristina Muñoz
    Affiliations
    Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-Consejo Superior de Investigaciones Científicas), Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain
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  • Florine Dupeux
    Affiliations
    European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France

    Unit of Virus Host-Cell Interactions, Université Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France
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  • Irene Luque
    Affiliations
    Department of Physical Chemistry and Institute of Biotechnology, University of Granada, Campus Fuentenueva s/n, 18071 Granada, Spain
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  • Miguel Angel Botella
    Affiliations
    Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-Consejo Superior de Investigaciones Científicas), Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain
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  • Wilfried Schwab
    Affiliations
    Biotechnology of Natural Products, Technische Universität München, 85354 Freising, Germany
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  • Victoriano Valpuesta
    Correspondence
    To whom correspondence may be addressed
    Affiliations
    Instituto de Hortofruticultura Subtropical y Mediterránea (IHSM-UMA-Consejo Superior de Investigaciones Científicas), Departamento de Biología Molecular y Bioquímica, Universidad de Málaga, 29071 Málaga, Spain
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  • José A. Marquez
    Correspondence
    To whom correspondence may be addressed
    Affiliations
    European Molecular Biology Laboratory, Grenoble Outstation, 6 rue Jules Horowitz, 38042 Grenoble, France

    Unit of Virus Host-Cell Interactions, Université Grenoble Alpes-EMBL-CNRS, 6 rue Jules Horowitz, 38042 Grenoble, France
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  • Author Footnotes
    * This work was supported in part by the P-CUBE project of the European Commission (FP7/2007-2013; Grant 227764), Ministerio de Ciencia e Innovacion Grant BIO2010-15630 (Spain), and Deutsche Forschungsgemeinschaft SFB 924 (Germany).
    1 Both authors contributed equally to this work.
    2 Recipient of a Formacion de Personal Investigador (FPI) fellowship from the Spanish government.
Open AccessPublished:October 16, 2013DOI:https://doi.org/10.1074/jbc.M113.501528
      Pathogenesis-related 10 (PR-10) proteins are involved in many aspects of plant biology but their molecular function is still unclear. They are related by sequence and structural homology to mammalian lipid transport and plant abscisic acid receptor proteins and are predicted to have cavities for ligand binding. Recently, three new members of the PR-10 family, the Fra a proteins, have been identified in strawberry, where they are required for the activity of the flavonoid biosynthesis pathway, which is essential for the development of color and flavor in fruits. Here, we show that Fra a proteins bind natural flavonoids with different selectivity and affinities in the low μm range. The structural analysis of Fra a 1 E and a Fra a 3-catechin complex indicates that loops L3, L5, and L7 surrounding the ligand-binding cavity show significant flexibility in the apo forms but close over the ligand in the Fra a 3-catechin complex. Our findings provide mechanistic insight on the function of Fra a proteins and suggest that PR-10 proteins, which are widespread in plants, may play a role in the control of secondary metabolic pathways by binding to metabolic intermediates.

      Introduction

      The family pathogenesis-related 10 (PR-10)
      The abbreviations used are: PR-10
      pathogenesis-related 10
      PAL
      phenylalanine ammonia-lyase
      ITC
      isothermal titration calorimetry
      START
      StAR-related lipid transfer
      PYR
      pyrabactin resistance
      PYL
      pyrabactin resistance-like
      RCAR
      regulatory components of ABA receptors
      TEV
      tobaco etch virus.
      proteins comprises a large number of sequences widely distributed among seed plants (
      • Liu J.-J.
      • Ekramoddoullah A.K.
      The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses.
      ). However, their function is still poorly understood. PR-10 proteins were initially characterized by their increased expression levels in response to infection by plant pathogens and under abiotic stress conditions. Today, a large number of PR-10 genes have been identified in different species, showing a diversity of expression patterns under both normal growth and stress conditions (
      • Liu J.-J.
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      The family 10 of plant pathogenesis-related proteins: their structure, regulation, and function in response to biotic and abiotic stresses.
      ,
      • Radauer C.
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      The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands.
      ). Some PR-10 proteins such as the white birch Bet v 1 and the apple Mal d 1 are highly abundant in pollen and fruits, respectively, and are responsible for allergic reactions, including seasonal and food allergies (
      • Gajhede M.
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      ,
      • Mirza O.
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      • Gajhede M.
      Dominant epitopes and allergic cross-reactivity: complex formation between a Fab fragment of a monoclonal murine IgG antibody and the major allergen from birch pollen Bet v 1.
      ). PR-10 proteins belong to the START superfamily. These proteins adopt a helix-grip fold with an internal cavity capable of binding hydrophobic ligands (
      • Radauer C.
      • Lackner P.
      • Breiteneder H.
      The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands.
      ,
      • Iyer L.M.
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      • Aravind L.
      Adaptations of the helix-grip fold for ligand binding and catalysis in the START domain superfamily.
      ,
      • Ponting C.P.
      • Aravind L.
      START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins.
      ,
      • Soccio R.E.
      • Breslow J.L.
      StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism.
      ). Only two protein families within the START superfamily have been extensively characterized at a functional and structural level: the lipid transport proteins, which are involved in non vesicular transport of lipids in eukaryotic cells (
      • Ponting C.P.
      • Aravind L.
      START: a lipid-binding domain in StAR, HD-ZIP and signalling proteins.
      ,
      • Lev S.
      Non-vesicular lipid transport by lipid-transfer proteins and beyond.
      ) and the plant PYR/PYL/RCAR proteins that function as intracellular receptors for the plant hormone abscisic acid (
      • Ma Y.
      • Szostkiewicz I.
      • Korte A.
      • Moes D.
      • Yang Y.
      • Christmann A.
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      Regulators of PP2C phosphatase activity function as abscisic acid sensors.
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      • Melcher K.
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      A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.
      ,
      • Miyazono K.
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      • Zhi Y.
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      • Yoshida T.
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      Structural basis of abscisic acid signalling.
      ,
      • Nishimura N.
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      • Rambo R.P.
      • Hitomi C.
      • Cutler S.R.
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      Structural mechanism of abscisic acid binding and signaling by dimeric PYR1.
      ,
      • Park S.Y.
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      • Schroeder J.I.
      • Volkman B.F.
      • Cutler S.R.
      Abscisic acid inhibits type 2C protein phosphatases via the PYR/PYL family of START proteins.
      ,
      • Santiago J.
      • Dupeux F.
      • Round A.
      • Antoni R.
      • Park S.Y.
      • Jamin M.
      • Cutler S.R.
      • Rodriguez P.L.
      • Márquez J.A.
      The abscisic acid receptor PYR1 in complex with abscisic acid.
      ). Some PR-10 proteins, including Bet v 1, the mung bean cytokinin-specific binding protein and the Prunus LPR10 protein, have been found to bind to a series of artificial and natural hydrophobic molecules, including cytokinins and phytosteroids (
      • Radauer C.
      • Lackner P.
      • Breiteneder H.
      The Bet v 1 fold: an ancient, versatile scaffold for binding of large, hydrophobic ligands.
      ,
      • Fernandes H.
      • Bujacz A.
      • Bujacz G.
      • Jelen F.
      • Jasinski M.
      • Kachlicki P.
      • Otlewski J.
      • Sikorski M.M.
      • Jaskolski M.
      Cytokinin-induced structural adaptability of a Lupinus luteus PR-10 protein.
      ,
      • Fernandes H.
      • Pasternak O.
      • Bujacz G.
      • Bujacz A.
      • Sikorski M.M.
      • Jaskolski M.
      Lupinus luteus pathogenesis-related protein as a reservoir for cytokinin.
      ,
      • Kofler S.
      • Asam C.
      • Eckhard U.
      • Wallner M.
      • Ferreira F.
      • Brandstetter H.
      Crystallographically mapped ligand binding differs in high and low IgE binding isoforms of birch pollen allergen bet v 1.
      ,
      • Marković-Housley Z.
      • Degano M.
      • Lamba D.
      • von Roepenack-Lahaye E.
      • Clemens S.
      • Susani M.
      • Ferreira F.
      • Scheiner O.
      • Breiteneder H.
      Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier.
      ,
      • Mogensen J.E.
      • Wimmer R.
      • Larsen J.N.
      • Spangfort M.D.
      • Otzen D.E.
      The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands.
      ,
      • Pasternak O.
      • Bujacz G.D.
      • Fujimoto Y.
      • Hashimoto Y.
      • Jelen F.
      • Otlewski J.
      • Sikorski M.M.
      • Jaskolski M.
      Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin.
      ,
      • Fernandes H.
      • Michalska K.
      • Sikorski M.
      • Jaskolski M.
      Structural and functional aspects of PR-10 proteins.
      ). However, the functional relevance of these interactions remains unclear. Recently, three new members of the PR-10 family, Fra a 1E, Fra a 2, and Fra a 3, have been identified and shown to play an important role in the control of phenylpropanoids and flavonoids biosynthesis in strawberry fruits (
      • Karlsson A.L.
      • Alm R.
      • Ekstrand B.
      • Fjelkner-Modig S.
      • Schiött A.
      • Bengtsson U.
      • Björk L.
      • Hjernø K.
      • Roepstorff P.
      • Emanuelsson C.S.
      Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens.
      ,
      • Hjernø K.
      • Alm R.
      • Canbäck B.
      • Matthiesen R.
      • Trajkovski K.
      • Björk L.
      • Roepstorff P.
      • Emanuelsson C.
      Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant.
      ,
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ).
      Flavonoids and phenolic compounds are among the most important secondary metabolites in plants. In addition to color and flavor development, they participate in many aspects of plant biology, including UV protection, as antioxidants, auxin transport regulators, and defense compounds against pathogens (
      • Fait A.
      • Hanhineva K.
      • Beleggia R.
      • Dai N.
      • Rogachev I.
      • Nikiforova V.J.
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      Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development.
      ,
      • Fraser C.M.
      • Chapple C.
      The phenylpropanoid pathway in Arabidopsis.
      ,
      • Halbwirth H.
      • Puhl I.
      • Haas U.
      • Jezik K.
      • Treutter D.
      • Stich K.
      Two-phase flavonoid formation in developing strawberry (Fragaria x ananassa) fruit.
      ,
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ,
      • Hassan S.
      • Mathesius U.
      The role of flavonoids in root-rhizosphere signalling: opportunities and challenges for improving plant-microbe interactions.
      ). Thus, injury by pathogens or pests induces the accumulation of flavonoids and other phenolic compounds with antimicrobial activity (
      • Ahuja I.
      • Kissen R.
      • Bones A.M.
      Phytoalexins in defense against pathogens.
      ). Flavonoids are also exuded by plant roots and act as signals that modify the transcriptional activity of nodulation genes in nitrogen-fixing bacteria, thereby promoting symbiotic association (
      • Kobayashi H.
      • Naciri-Graven Y.
      • Broughton W.J.
      • Perret X.
      Flavonoids induce temporal shifts in gene-expression of nod-box controlled loci in Rhizobium sp. NGR234.
      ,
      • Treutter D.
      Significance of flavonoids in plant resistance and enhancement of their biosynthesis.
      ). Other flavonoids have been implicated in pollen germination, seed resistance to pests and numerous other processes (
      • Treutter D.
      Significance of flavonoids in plant resistance and enhancement of their biosynthesis.
      ,
      • Mahajan M.
      • Ahuja P.S.
      • Yadav S.K.
      Post-transcriptional silencing of flavonol synthase mRNA in tobacco leads to fruits with arrested seed set.
      ). The effect of dietary flavonoids in human health is also a subject of study due to their antioxidative and anticarcinogenic activities (
      • Ghasemzadeh A.
      • Ghasemzadeh N.
      Flavonoids and phenolic acids: role and biochemical activity in plants and human.
      ). Flavonoids are synthesized via the phenylpropanoid and flavonoid pathways (see Fig. 1) (
      • Fraser C.M.
      • Chapple C.
      The phenylpropanoid pathway in Arabidopsis.
      ,
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ). The first step in the phenylpropanoid pathway is catalyzed by the enzyme phenylalanine ammonia-lyase (PAL) and leads to the production of cinnamic acid from l-phenylalanine. PAL is the gateway enzyme to the synthesis of phenolic and flavonoid compounds as well as many other secondary metabolites (
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ). In Arabidopsis and other species, PAL gene expression is responsive to developmental and environmental clues such as wounding, pathogen infection, or UV radiation, among others (
      • Huang J.
      • Gu M.
      • Lai Z.
      • Fan B.
      • Shi K.
      • Zhou Y.H.
      • Yu J.Q.
      • Chen Z.
      Functional analysis of the Arabidopsis PAL gene family in plant growth, development, and response to environmental stress.
      ,
      • Blount J.W.
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      • Lamb C.
      • Dixon R.A.
      Altering expression of cinnamic acid 4-hydroxylase in transgenic plants provides evidence for a feedback loop at the entry point into the phenylpropanoid pathway.
      ,
      • Moura J.C.
      • Bonine C.A.
      • de Oliveira Fernandes Viana J.
      • Dornelas M.C.
      • Mazzafera P.
      Abiotic and biotic stresses and changes in the lignin content and composition in plants.
      ,
      • Naoumkina M.A.
      • Zhao Q.
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      • Dai X.
      • Zhao P.X.
      • Dixon R.A.
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      ,
      • Olsen K.M.
      • Lea U.S.
      • Slimestad R.
      • Verheul M.
      • Lillo C.
      Differential expression of four Arabidopsis PAL genes; PAL1 and PAL2 have functional specialization in abiotic environmental-triggered flavonoid synthesis.
      ). Another important step in the synthesis of flavonoids is the production of naringenin, which is the first product in the pathway with a flavan structure and from which many other flavonoids are derived (see Fig. 1). This step is catalyzed by the enzyme chalcone synthase. Many of the final products of the flavonoid biosynthesis pathway accumulate as O-glycosyl derivatives at the position 3 of the C ring of the flavan nucleus and are accumulated in the vacuole or secreted through the plasma membrane into the apoplastic space (
      • Zhao J.
      • Dixon R.A.
      The “ins” and “outs” of flavonoid transport.
      ). A number of flavonoids that account for color of the fruit and contribute significantly to its taste are produced in the strawberry fruit in a developmental-specific pattern (
      • Fait A.
      • Hanhineva K.
      • Beleggia R.
      • Dai N.
      • Rogachev I.
      • Nikiforova V.J.
      • Fernie A.R.
      • Aharoni A.
      Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development.
      ,
      • Muñoz C.
      • Sánchez-Sevilla J.F.
      • Botella M.A.
      • Hoffmann T.
      • Schwab W.
      • Valpuesta V.
      Polyphenol composition in the ripe fruits of Fragaria species and transcriptional analyses of key genes in the pathway.
      ). Proanthocyanidins (condensed tannins) are mostly produced in the young fruits that make them bitter, whereas anthocyanins, mostly pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside (see Fig. 1), which confer color, are abundant in the later stages of fruit maturation.
      Figure thumbnail gr1
      FIGURE 1The phenolic compound biosynthesis pathway. A schematic representation of the phenylpropanoid and flavonoid biosynthesis pathway is shown. Major families of flavonoid compounds are highlighted. Flavonoids are characterized by the presence of the flavan nucleus with A, B, and C rings as indicated (inset). Final products of the flavonoid pathway such as pelargonidin 3-O-glucoside, are often glycosylated at the position 3 of the C ring of the flavan nucleus. Suppression of Fra a protein expression affects the expression of phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) genes (red inverted triangles) and alters phenolic compound accumulation with an increase in the levels of catechin and a decreased accumulation of anthocyanins (as indicated by arrows) (
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ).
      The strawberry Fra a transcripts are present in most plant organs; however, they show maximal expression levels in open flowers, fruits, and roots, depending on the member of the family (
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ). The first evidence of the involvement of Fra a proteins in the control of the synthesis of flavonoids was provided by Emanuelsson and co-workers (
      • Hjernø K.
      • Alm R.
      • Canbäck B.
      • Matthiesen R.
      • Trajkovski K.
      • Björk L.
      • Roepstorff P.
      • Emanuelsson C.
      Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant.
      ), who reported that fruits of colorless cultivars showed very low levels of Fra a 1 protein expression in contrast to red-colored fruits. Later, Fra a RNAi silencing experiments showed that suppression of the expression of Fra a proteins in strawberry fruits led to decreased accumulation of the main flavonoids responsible for the red color of fruits, including cyanidin 3-O-glucoside and pelargonidin 3-O-glucoside (see Fig. 1), whereas other aspects of fruit maturation were unaffected (
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ). Other flavonoids such as kaempferol 3-O-glucoside and pelargonidin 3-malonyl-glucoside also showed decreased levels in silenced fruits, whereas other intermediate metabolites of the flavonoid pathway such as catechin and proanthocyanidins accumulated at higher levels (see Fig. 1). Interestingly, silencing of Fra a proteins in strawberry also produced a decrease in the expression levels of genes encoding for PAL and chalcone synthase in the fruits, showing that Fra a proteins are required for the expression of structural genes in the flavonoid biosynthesis pathway and suggesting that their function may be regulatory (
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ). These studies indicated that the Fra a proteins participate in the control of flavonoid biosynthesis in strawberry fruits. Recently, a solution structure of the Fra a1 protein has been described (
      • Seutter von Loetzen C.
      • Schweimer K.
      • Schwab W.
      • Rösch P.
      • Hartl-Spiegelhauer O.
      Solution structure of the strawberry allergen Fra a 1.
      ). However, the molecular basis for the function of the Fra proteins remains unknown.
      In this work, we show that strawberry Fra a proteins bind natural flavonoids, providing a basis for their function in the control of flavonoid metabolisms. Moreover, we present crystallographic structures of Fra a 1E and Fra a 3 in complex with catechin. The analysis of these structures shows that flavonoid binding is associated with conformational changes in critical loop regions providing for the first time a molecular basis for the function of Fra a proteins in the control of flavonoid biosynthesis.

      DISCUSSION

      Fra a proteins are members of the pathogenesis-related 10 family and are required for the normal accumulation of flavonoids and the development of color in strawberry fruits (
      • Karlsson A.L.
      • Alm R.
      • Ekstrand B.
      • Fjelkner-Modig S.
      • Schiött A.
      • Bengtsson U.
      • Björk L.
      • Hjernø K.
      • Roepstorff P.
      • Emanuelsson C.S.
      Bet v 1 homologues in strawberry identified as IgE-binding proteins and presumptive allergens.
      ,
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ). However, their function, as that of PR-10 proteins in general, is still not clearly understood. The data presented here demonstrates that Fra a proteins bind natural flavonoids, providing for the first time mechanistic insight on the function of these proteins in the control of flavonoid biosynthesis.
      ITC experiments show that Fra a proteins can bind metabolites of the flavonoid pathway with affinities in the low μm range and with different selectivity. The three ligands identified in this study have been shown to be present in fruits as well as other parts of the strawberry plant (
      • Muñoz C.
      • Sánchez-Sevilla J.F.
      • Botella M.A.
      • Hoffmann T.
      • Schwab W.
      • Valpuesta V.
      Polyphenol composition in the ripe fruits of Fragaria species and transcriptional analyses of key genes in the pathway.
      ,
      • Aaby K.
      • Ekeberg D.
      • Skrede G.
      Characterization of phenolic compounds in strawberry (Fragaria x ananassa) fruits by different HPLC detectors and contribution of individual compounds to total antioxidant capacity.
      ,
      • Kosar M.
      • Kafkas E.
      • Paydas S.
      • Baser K.H.
      Phenolic composition of strawberry genotypes at different maturation stages.
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      • Kärenlampi S.
      • Aharoni A.
      Non-targeted analysis of spatial metabolite composition in strawberry (Fragaria x ananassa) flowers.
      ) and accumulate at the same organ and developmental stage where the highest expression levels of the Fra a proteins occur (
      • Hjernø K.
      • Alm R.
      • Canbäck B.
      • Matthiesen R.
      • Trajkovski K.
      • Björk L.
      • Roepstorff P.
      • Emanuelsson C.
      Down-regulation of the strawberry Bet v 1-homologous allergen in concert with the flavonoid biosynthesis pathway in colorless strawberry mutant.
      ). The structure of the Fra a 3-catechin complex provides details on the mechanism of ligand binding and stabilization. The catechin molecule adopts a linear disposition with its long axis approximately parallel to the axis of the long C-terminal α-helix of Fra a 3. It is stabilized by polar interactions with the side chains of Asp-28, Ser-63, His-70, Tyr-84, and Arg-139 and backbone atoms of residues Gln-37, Ala-38, and Gly-60, whereas the non-polar groups of catechin are surrounded by hydrophobic side chains including Ile-31, Val-39, Leu-59, and Leu-143. The amino acids facing the cavity in the three Fra a proteins are generally conserved. However, variability is observed between the three proteins at certain positions (Fig. 5), which could explain their different selectivity toward ligands. For example key amino acids Leu-59 and Arg-139, involved in Fra a 3 (+)-catechin interaction are replaced by Phe and Lys in both Fra a 1E and Fra a 2.
      Figure thumbnail gr5
      FIGURE 5Multiple sequence alignment of the strawberry Fra a 1E, Fra a 2, and Fra a 3 proteins and other related Bet v 1/START proteins. The secondary structural elements correspond to the Fra a 1E (black) and LIPR-10.2B (blue) structures. The L5 loop has been highlighted in yellow. Residues oriented toward the cavity and residues involved in catechin binding (for Fra a 3) are indicated by dark green boxes and light green triangles, respectively. Positions showing sequence variations, which are either important for catechin binding (cyan) or facing the cavity (black stars), are also indicated. The sequence alignment was performed using ClustalW (EBI server), and the figure was generated by ESPript (
      • Gouet P.
      • Robert X.
      • Courcelle E.
      ESPript/ENDscript: Extracting and rendering sequence and 3D information from atomic structures of proteins.
      ).
      Comparison of the structures of the apo forms of Fra a 1E and the Fra a 3-catechin complex indicates that Fra a proteins show considerably flexibility in the loop regions surrounding the cavity (loops L3, L5, and L7) and that ligand-binding induces important conformational changes. Fra a 3 adopts a more compact structure with a closed conformation of loop L5 that traps the catechin molecule inside the cavity. Interestingly, loop L5 in Fra a proteins is structurally equivalent to the Ω1 loop of the mammalian START proteins (
      • Tsujishita Y.
      • Hurley J.H.
      Structure and lipid transport mechanism of a StAR-related domain.
      ) and the β3-β4 loop of the plant PYRL/PYL/RCAR hormone receptors (
      • Santiago J.
      • Dupeux F.
      • Round A.
      • Antoni R.
      • Park S.Y.
      • Jamin M.
      • Cutler S.R.
      • Rodriguez P.L.
      • Márquez J.A.
      The abscisic acid receptor PYR1 in complex with abscisic acid.
      ), which also adopt closed conformations upon ligand binding (see Fig. 6). In the case of the mammalian START proteins, these conformational changes are thought to play a role in lipid extraction and solubilization (
      • Tsujishita Y.
      • Hurley J.H.
      Structure and lipid transport mechanism of a StAR-related domain.
      ,
      • Kudo N.
      • Kumagai K.
      • Tomishige N.
      • Yamaji T.
      • Wakatsuki S.
      • Nishijima M.
      • Hanada K.
      • Kato R.
      Structural basis for specific lipid recognition by CERT responsible for nonvesicular trafficking of ceramide.
      ,
      • Roderick S.L.
      • Chan W.W.
      • Agate D.S.
      • Olsen L.R.
      • Vetting M.W.
      • Rajashankar K.R.
      • Cohen D.E.
      Structure of human phosphatidylcholine transfer protein in complex with its ligand.
      ), whereas in the plant abscisic acid receptors the closed conformation stabilizes the hormone inside the cavity and promotes interaction between the receptor and protein phosphatases of the class 2C, leading to the activation of the ABA signaling pathway (
      • Melcher K.
      • Ng L.M.
      • Zhou X.E.
      • Soon F.F.
      • Xu Y.
      • Suino-Powell K.M.
      • Park S.Y.
      • Weiner J.J.
      • Fujii H.
      • Chinnusamy V.
      • Kovach A.
      • Li J.
      • Wang Y.
      • Li J.
      • Peterson F.C.
      • Jensen D.R.
      • Yong E.L.
      • Volkman B.F.
      • Cutler S.R.
      • Zhu J.K.
      • Xu H.E.
      A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors.
      ,
      • Miyazono K.
      • Miyakawa T.
      • Sawano Y.
      • Kubota K.
      • Kang H.J.
      • Asano A.
      • Miyauchi Y.
      • Takahashi M.
      • Zhi Y.
      • Fujita Y.
      • Yoshida T.
      • Kodaira K.S.
      • Yamaguchi-Shinozaki K.
      • Tanokura M.
      Structural basis of abscisic acid signalling.
      ,
      • Nishimura N.
      • Hitomi K.
      • Arvai A.S.
      • Rambo R.P.
      • Hitomi C.
      • Cutler S.R.
      • Schroeder J.I.
      • Getzoff E.D.
      Structural mechanism of abscisic acid binding and signaling by dimeric PYR1.
      ,
      • Santiago J.
      • Dupeux F.
      • Round A.
      • Antoni R.
      • Park S.Y.
      • Jamin M.
      • Cutler S.R.
      • Rodriguez P.L.
      • Márquez J.A.
      The abscisic acid receptor PYR1 in complex with abscisic acid.
      ). This suggests that ligand-induced conformational changes are a conserved feature in the START protein superfamily and might also play an important role in the function of other members of the PR-10 family.
      Figure thumbnail gr6
      FIGURE 6Function of PR-10, PYR/PYL/RCAR, and START proteins involve conformational changes in loop regions. a, Fra a 3 in ribbon representation with bound (+)-catechin. The flexible loops L3, L5, and L7 are shown in red. b, the abscisic acid receptor PYR1 (Protein Data Bank code 3K90) in ribbon representation. The gating loops undergoing conformational changes hormone binding and receptor activation are depicted in red. c, the START domain of the CERT protein (Protein Data Bank code 3H3S) in ribbon representation. The Ω1 loop is shown in red.
      The structural analysis presented here together with the molecular mechanisms previously described for LPTs and PYR/PYL/RCAR proteins suggest two possible functions for the Fra a proteins at molecular level. Fra a proteins could act as transporters or “chemical chaperones” binding to flavonoid intermediates and making them available to processing enzymes. The key enzymes of the phenylpropanoid and flavonoid biosynthesis pathways, including PAL, chalcone synthase, and C4H have been shown to co-localize to the endoplasmic reticulum in different plant species. These enzymes form multi-protein complexes at the cytosolic side of the membrane where synthesis of many flavonoid and phenylpropanoid compounds occurs (
      • Zhao J.
      • Dixon R.A.
      The “ins” and “outs” of flavonoid transport.
      ,
      • Lepiniec L.
      • Debeaujon I.
      • Routaboul J.M.
      • Baudry A.
      • Pourcel L.
      • Nesi N.
      • Caboche M.
      Genetics and biochemistry of seed flavonoids.
      ,
      • Winkel B.S.
      Metabolic channeling in plants.
      ). This association in multiprotein complexes has been proposed to help sequester unstable or toxic intermediates and to control the metabolic flux among the multiple branches of the pathway, thereby determining which compounds are synthesized preferentially (
      • Winkel B.S.
      Metabolic channeling in plants.
      ). Fra a proteins might form part of these complexes, contributing to limit diffusion of intermediates and making them available to downstream processing enzymes. Fra a proteins could also be involved in the transport of flavonoids from the ER to other cellular membranes, such as the tonoplast or the plasma membrane. Indeed, anthocyanins and other conjugated flavonoids such as glycosylated catechin and epicatechins are translocated to and accumulated into the vacuole through the action of specific membrane transport proteins, whereas other flavonoid compounds are secreted to the apoplast through the plasma membrane, especially in roots (
      • Zhao J.
      • Dixon R.A.
      The “ins” and “outs” of flavonoid transport.
      ,
      • Zhao J.
      • Huhman D.
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      • He X.Z.
      • Sumner L.W.
      • Tang Y.
      • Dixon R.A.
      MATE2 mediates vacuolar sequestration of flavonoid glycosides and glycoside malonates in Medicago truncatula.
      ,
      • Zhao J.
      • Dixon R.A.
      MATE transporters facilitate vacuolar uptake of epicatechin 3′-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatulaArabidopsis.
      ,
      • Marinova K.
      • Pourcel L.
      • Weder B.
      • Schwarz M.
      • Barron D.
      • Routaboul J.M.
      • Debeaujon I.
      • Klein M.
      The Arabidopsis MATE transporter TT12 acts as a vacuolar flavonoid/H+ -antiporter active in proanthocyanidin-accumulating cells of the seed coat.
      ). In this case, Fra a proteins might have a function analogous to that of the mammalian START proteins that act as cytosolic transporters of lipids shuttling between different cellular membranes (
      • Soccio R.E.
      • Breslow J.L.
      StAR-related lipid transfer (START) proteins: mediators of intracellular lipid metabolism.
      ,
      • Lev S.
      Non-vesicular lipid transport by lipid-transfer proteins and beyond.
      ).
      Another possibility is that Fra a proteins might play a role as regulatory components involved in intracellular signaling. In maize, Arabidopsis and other species, the genes coding for enzymes involved in phenylpropanoid and flavonoid biosynthesis are regulated at a transcriptional level through the activity of MYB and bHLH type transcription factors (
      • Vogt T.
      Phenylpropanoid biosynthesis.
      ,
      • Lepiniec L.
      • Debeaujon I.
      • Routaboul J.M.
      • Baudry A.
      • Pourcel L.
      • Nesi N.
      • Caboche M.
      Genetics and biochemistry of seed flavonoids.
      ,
      • Verdier J.
      • Zhao J.
      • Torres-Jerez I.
      • Ge S.
      • Liu C.
      • He X.
      • Mysore K.S.
      • Dixon R.A.
      • Udvardi M.K.
      MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula.
      ). Moreover, it has been recently shown that a blockage in downstream flavonoid processing enzymes results in transcriptional inhibition of PAL and that this inhibition is dependent on the accumulation of flavonoids, demonstrating that the expression of structural genes is mediated by a metabolic intermediate downstream of naringenin (
      • Yin R.
      • Messner B.
      • Faus-Kessler T.
      • Hoffmann T.
      • Schwab W.
      • Hajirezaei M.R.
      • von Saint Paul V.
      • Heller W.
      • Schäffner A.R.
      Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation.
      ). The capacity of Fra a proteins to bind specific flavonoids suggests that they could play a role as signaling components, monitoring the metabolic flux through different branches of the pathway and influencing the expression level of specific regulatory genes. This would be consistent with the effect of Fra a silencing on the transcriptional activity of PAL and chalcone synthase genes and the altered accumulation of certain flavonoids (
      • Muñoz C.
      • Hoffmann T.
      • Escobar N.M.
      • Ludemann F.
      • Botella M.A.
      • Valpuesta V.
      • Schwab W.
      The strawberry fruit Fra a allergen functions in flavonoid biosynthesis.
      ).
      Close homologues of the Fra a proteins have been found in apple, peach, and tomato, some of which are also expressed to high levels during fruit ripening (
      • Beuning L.
      • Bowen J.
      • Persson H.
      • Barraclough D.
      • Bulley S.
      • Macrae E.
      Characterisation of Mal d 1-related genes in Malus.
      ,
      • Botton A.
      • Andreotti C.
      • Costa G.
      • Ramina A.
      Peach (Prunus persica L. Batsch) allergen-encoding genes are developmentally regulated and affected by fruit load and light radiation.
      ). The amino acids involved in the Fra a 3-catechin interaction are also highly conserved in these proteins (see Fig. 5), which suggests that these proteins might also have the capacity to bind structurally close flavonoids. However, other PR-10 proteins show more divergent amino acids sequences in the cavity region (see Fig. 5) and might bind other ligands. The phenylpropanoid and flavonoid biosynthesis pathway is responsible for the production of a large proportion of secondary metabolites in plants and shows a high degree of variability among species. It is not only involved in development of color in fruits and flowers, but it is also important for many other biological functions in plants, including defense against pathogens, insect attraction, and pollination and UV protection, among others. The structural analysis of the Fra a proteins suggests that PR-10 proteins, which are widespread in plants, might function in the control of flavonoid or other secondary metabolic pathways in plants.

      Acknowledgments

      We thank the European Synchrotron Radiation Facility and the Grenoble Outstation of the European Molecular Biology Laboratory for access to Macromolecular Crystallography beam lines.

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