Abstract
Main conclusionOverexpression of chalcone-3-hydroxylase provokes increased accumulation of 3-hydroxyphloridzin inMalus. Decreased flavonoid concentrations but unchanged flavonoid class composition were observed. The increased 3-hydroxyphlorizin contents correlate well with reduced susceptibility to fire blight and scab.The involvement of dihydrochalcones in the apple defence mechanism against pathogens is discussed but unknown biosynthetic steps in their formation hamper studies on their physiological relevance. The formation of 3-hydroxyphloretin is one of the gaps in the pathway. Polyphenol oxidases and cytochrome P450 dependent enzymes could be involved. Hydroxylation of phloretin in position 3 has high similarity to the B-ring hydroxylation of flavonoids catalysed by the well-known flavonoid 3′-hydroxylase (F3′H). Using recombinant F3′H and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F3′H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H. To test whether CH3H catalyzes the hydroxylation of dihydrochalcones in planta and if this could be of physiological relevance, we created transgenic apple trees harbouring CH3H from C. sulphureus. The three transgenic lines obtained showed lower polyphenol concentrations but no shift between the main polyphenol classes dihydrochalcones, flavonols, hydroxycinnamic acids and flavan 3-ols. Increase of 3-hydroxyphloridzin within the dihydrochalcones and of epicatechin/catechin within soluble flavan 3-ols were observed. Decreased activity of dihydroflavonol 4-reductase and chalcone synthase/chalcone isomerase could partially explain the lower polyphenol concentrations. In comparison to the parent line, the transgenic CH3H-lines showed a lower disease susceptibility to fire blight and apple scab that correlated with the increased 3-hydroxyphlorizin contents.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-016-2475-9) contains supplementary material, which is available to authorized users.
Highlights
Apple (Malus 9 domestica Borkh.) belongs to the most popular fruits world-wide and their consumption is suggested to be health-beneficial (Boyer and Liu 2004; Ehrenkranz et al 2005; Hyson 2011)
Using recombinant flavonoid 30-hydroxylase (F30H) and chalcone 3-hydroxylase (CH3H) from Cosmos sulphureus we show that F30H and CH3H accept phloretin to some extent but higher conversion rates are obtained with CH3H
In the present study we tested whether CH3H of the ornamental plant C. sulphureus (CsCH3H) accepts phloretin as substrate and we show that constitutive overexpression of CsCH3H in apple leaves results in plants with increased 3-hydroxyphloridzin formation which seems to be correlated to reduced susceptibility to the biotic diseases scab and fire blight which are caused by Venturia inaequalis (Cooke) G
Summary
Apple (Malus 9 domestica Borkh.) belongs to the most popular fruits world-wide and their consumption is suggested to be health-beneficial (Boyer and Liu 2004; Ehrenkranz et al 2005; Hyson 2011). It seems unlikely that such an unspecific enzyme which produces a spectrum of cell-toxic compounds should be involved in the biosynthesis of the 3-hydroxyphloridzin that is constitutively present in Malus sp. Hydroxylation in position 3 of dihydrochalcones shows high similarity to the introduction of a second hydroxyl group in the B-ring of flavonoids and chalcones (Fig. 2) which are catalysed by the cytochrome P450 dependent monooxygenases flavonoid 30-hydroxylase (F30H) and chalcone 3-hydroxylase (CH3H) (Schlangen et al 2010b). In the present study we tested whether CH3H of the ornamental plant C. sulphureus (CsCH3H) accepts phloretin as substrate and we show that constitutive overexpression of CsCH3H in apple leaves results in plants with increased 3-hydroxyphloridzin formation which seems to be correlated to reduced susceptibility to the biotic diseases scab and fire blight which are caused by Venturia inaequalis (Cooke) G.
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