Planta DOI 10.1007/s00425-016-2475-9 ORIGINAL ARTICLE Transgenic apple plants overexpressing the chalcone 3-hydroxylase gene of Cosmos sulphureus show increased levels of 3-hydroxyphloridzin and reduced susceptibility to apple scab and fire blight 1 2 3 1 Olly Sanny Hutabarat • Henryk Flachowsky • Ionela Regos • Silvija Miosic • 4 1 1 Christine Kaufmann • Shadab Faramarzi • Mohammed Zobayer Alam • 1 2 5 2 Christian Gosch • Andreas Peil • Klaus Richter • Magda-Viola Hanke • 3 1 1 Dieter Treutter • Karl Stich • Heidi Halbwirth Received: 25 November 2015 / Accepted: 26 January 2016 Ó The Author(s) 2016. This article is published with open access at Springerlink.com Abstract be involved. Hydroxylation of phloretin in position 3 has Main conclusion Overexpression of chalcone-3-hy- high similarity to the B-ring hydroxylation of flavonoids droxylase provokes increased accumulation of 3-hy- catalysed by the well-known flavonoid 30-hydroxylase droxyphloridzin in Malus. Decreased flavonoid (F30H). Using recombinant F30H and chalcone 3-hydrox- concentrations but unchanged flavonoid class composi- ylase (CH3H) from Cosmos sulphureus we show that F30H tion were observed. The increased 3-hydroxyphlorizin and CH3H accept phloretin to some extent but higher contents correlate well with reduced susceptibility to conversion rates are obtained with CH3H. To test whether fire blight and scab. CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we The involvement of dihydrochalcones in the apple defence created transgenic apple trees harbouring CH3H from C. mechanism against pathogens is discussed but unknown sulphureus. The three transgenic lines obtained showed biosynthetic steps in their formation hamper studies on lower polyphenol concentrations but no shift between the their physiological relevance. The formation of 3-hydrox- main polyphenol classes dihydrochalcones, flavonols, yphloretin is one of the gaps in the pathway. Polyphenol hydroxycinnamic acids and flavan 3-ols. Increase of oxidases and cytochrome P450 dependent enzymes could 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were Electronic supplementary material The online version of this observed. Decreased activity of dihydroflavonol 4-reduc- article (doi:10.1007/s00425-016-2475-9) contains supplementary tase and chalcone synthase/chalcone isomerase could par- material, which is available to authorized users. tially explain the lower polyphenol concentrations. In & Heidi Halbwirth comparison to the parent line, the transgenic CH3H-lines [email protected] showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydrox- 1 Institute of Chemical Engineering, Technical University of yphlorizin contents. Vienna, Getreidemarkt 9, 1060 Vienna, Austria 2 Federal Research Centre for Cultivated Plants, Institute of Keywords Chalcone 3-hydroxylase (CH3H) Á Breeding Research on Horticultural and Fruit Crops, Julius Ku¨hn-Institut, Pillnitzer Platz 3a, 01326 Dresden, Germany Dihydrochalcones Á Erwinia amylovora Á 3-Hydroxyphloretin Á 3-Hydroxyphloridzin Á 3 Unit of Fruit Science, Technical University of Munich, Du¨rnast 2, 85350 Freising, Germany Malus 9 domestica Á Venturia inaequalis 4 Chair of Urban Water Systems Engineering, Technical University of Munich, Am Coulombwall, 85748 Garching, Abbreviations Germany CH3H Chalcone 3-hydroxylase 0 0 5 Federal Research Centre for Cultivated Plants, Institute for F3 H Flavonoid 3 -hydroxylase Resistance Research and Stress Tolerance, Julius Ku¨hn- PPO Polyphenol oxidase Institut, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany 123 Planta Introduction intermediates in the phloretin oxidation (Goodenough et al. 1983; Haruta et al. 1998; Ridgway and Tucker 1999), their Apple (Malus 9 domestica Borkh.) belongs to the most physiological relevance for the 3-hydroxyphloridzin popular fruits world-wide and their consumption is sug- biosynthesis in apple leaves has not yet been demonstrated. gested to be health-beneficial (Boyer and Liu 2004; It seems however unlikely that such an unspecific enzyme Ehrenkranz et al. 2005; Hyson 2011). Apple is unique which produces a spectrum of cell-toxic compounds should among plant species because it produces large amounts of be involved in the biosynthesis of the 3-hydroxyphloridzin dihydrochalcones, predominantly phloridzin (phloretin 20- that is constitutively present in Malus sp. O-glucoside) (Fig. 1), which are not or only found in low Hydroxylation in position 3 of dihydrochalcones shows levels in other plants. Some Malus species accumulate the high similarity to the introduction of a second hydroxyl 40-O-glucosides trilobatin (M. trilobata C. K. Schneid.) and group in the B-ring of flavonoids and chalcones (Fig. 2) sieboldin (M. sieboldii Rehder) besides the 20-O-glucosides which are catalysed by the cytochrome P450 dependent 0 0 (Fig. 1) (Williams 1982; Gaucher et al. 2013b). The monooxygenases flavonoid 3 -hydroxylase (F3 H) and physiological function of dihydrochalcones in apple chalcone 3-hydroxylase (CH3H) (Schlangen et al. 2010b). remains, however, unclear. A presumed involvement in The chemical structure of dihydrochalcones is closely pathogen defence has been divergently discussed, but no related to that of chalcones as both lack the heterocyclic final proof was presented so far (Gosch et al. 2010a, b). ring that is typically found in flavonoid structures (ring C) Although dihydrochalcones have antimicrobial activity (Fig. 2). Whereas CH3H depends on specific motifs in the (MacDonald and Bishop 1952; Barreca et al. 2014), their protein structure and was so far only described in Aster- 0 constitutive presence in apple tissues alone does not seem aceae species, F3 H can be found in many plant species to be decisive for disease resistance (Picinelli et al. 1995). (Schlangen et al. 2009, 2010a). In Malus sp., at least 3 0 Hydroxylation in position 3 is the first step in the oxidation genes encoding F3 Hs have been identified (Han et al. of phloretin by polyphenol oxidases (PPO), which results 2010) and it cannot be excluded that they are involved in in the formation of highly reactive quinoid structures that the 3-hydroxyphloridzin biosynthesis besides their essen- can interfere with cell invading pathogens (Le Guerneve´ tial role in the flavonoid pathway. In the present study we et al. 2004; Guyot et al. 2007; Overeem 1976). The rele- tested whether CH3H of the ornamental plant C. sul- vance of PPO and peroxidase reaction products such as phureus (CsCH3H) accepts phloretin as substrate and we 3-hydroxyphloretin, dihydrochalcone dimers and quinoid show that constitutive overexpression of CsCH3H in structures and of their formation speed was therefore dis- apple leaves results in plants with increased 3-hydrox- cussed as well (de Bernonville et al. 2010, 2011; Gaucher yphloridzin formation which seems to be correlated to et al. 2013a, b). In the case of the fire blight resistant reduced susceptibility to the biotic diseases scab and fire crabapple cv. Evereste, however, a correlation between blight which are caused by Venturia inaequalis (Cooke) pathogen resistance and the constitutive presence of sie- G. Winter and Erwinia amylovora (Burrill) Winslow boldin was not observed (de Bernonville et al. 2011). et al., respectively. Whereas dihydrochalcone biosynthesis has been studied in the past few years (Jugde´ et al. 2008; Gosch et al. 2009, 2010a; Dare et al. 2013; Ibdah et al. 2014), the introduction Materials and methods of a hydroxyl group in position 3 of dihydrochalcones was not elucidated so far. Two types of enzymes could catalyse Chemicals the reaction, PPOs and/or cytochrome P450 dependent monooxygenases. Although PPOs from apple have [14C ]-Phloretin was synthesized as described previously repeatedly been reported to produce 3-hydroxyphloretin as (Halbwirth et al. 2006). Fig. 1 Main phloretin 5 5 derivatives found in Malus 3 3 species. R=H: phloretin. R=OH: 2+ 2+ 0 3-hydroxyphloretin. R =H: 4’ 4 4’ 4 trilobatin. R0=OH: sieboldin +2 2+ *OX2 2+ 2’ 2’ 2*OX 2 2+ 2 123 Planta Fig. 2 Hydroxylation of a 2+ dihydrochalcones (a) and chalcones (b) in position 3 and 2+ 2+ flavonoids in position 30 (c) +2 2+ +2 2+ 2 2 phloretin 3-hydroxyphloretin b 2+ 2+ 2+ +2 2+ CH3H +2 2+ 2 2 isoliquiritigenin butein c 2+ 2+ 2+ F3'H +2 2 +2 2 2+ 2 2+ 2 naringenin eriodictyol 3-Hydroxyphloretin was purchased from Apin Chemi- promotor from A. thaliana. The cDNA clone was ligated cals (http://www.apinchemicals.com). 2,2-Diphenyl-1- between the SpeI and MluI restriction sites using the pri- picrylhydrazyl (DPPH) and 6-hydroxy-2,5,7,8-tetram- mers SpeCH3H and CH3HMlu (Suppl. Table S1). ethylchroman-2-carboxylic acid (trolox) were obtained from Sigma-Aldrich (www.sigmaaldrich.com/austria. Plant material html). Proliferating axillary shoot cultures of PinS (JKI, Dresden- Production and in vitro testing of recombinant CsCH3H Pillnitz, Germany), a seedling of the apple (Malus 9 do- mestica BORKH.) cv. ‘Pinova’ were used for plant trans- Heterologous expression of CsCH3H and F30H in yeast and formation. Transformation was performed using the testing of substrate acceptance was performed as described Agrobacterium tumefaciens strain EHA105 (Hood et al. previously (Schlangen et al. 2010b). 1993) containing the binary plasmid vector p9u10- 35S::CH3H. Regeneration, selection of transgenic plants, Vector construction for plant transformation rooting and acclimatization were carried out as