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

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.Austrian Sci-ence Fund (FWF

Measuring Flavonoid Enzyme Activities in Tissues of Fruit Species

Flavonoids are important secondary metabolites, which are ubiquitously present in plant-derived food. Since flavonoids may show beneficial effects on human health, there is increasing interest in the availability of plants with a tailor-made flavonoid spectrum. Determination of flavonoid enzyme activities and investigations into their substrate specificity are an important precondition for both classical and molecular approaches. We tested two different protocols for enzyme preparation from eight fruit species. In many cases, a protocol adapted for polyphenol-rich tissues was superior. Using a suitable protocol for investigations of kiwi fruits, we show that flavanone 3-hydroxylase is absent in the green-fleshed cultivar Hayward. As flavonoid enzyme activities could be detected in harvested kiwi fruits over a storage period of five months, postharvest modification of the flavonoid spectrum has to be expected

4-Deoxyaurone Formation in <i>Bidens ferulifolia</i> (Jacq.) DC

<div><p>The formation of 4-deoxyaurones, which serve as UV nectar guides in <i>Bidens ferulifolia</i> (Jacq.) DC., was established by combination of UV photography, mass spectrometry, and biochemical assays and the key step in aurone formation was studied. The yellow flowering ornamental plant accumulates deoxy type anthochlor pigments (6′-deoxychalcones and the corresponding 4-deoxyaurones) in the basal part of the flower surface whilst the apex contains only yellow carotenoids. For UV sensitive pollinating insects, this appears as a bicoloured floral pattern which can be visualized in situ by specific ammonia staining of the anthochlor pigments. The petal back side, in contrast, shows a faintly UV absorbing centre and UV absorbing rays along the otherwise UV reflecting petal apex. Matrix-free UV laser desorption/ionisation mass spectrometric imaging (LDI-MSI) indicated the presence of 9 anthochlors in the UV absorbing areas. The prevalent pigments were derivatives of okanin and maritimetin. Enzyme preparations from flowers, leaves, stems and roots of <i>B. ferulifolia</i> and from plants, which do not accumulate aurones e.g. <i>Arabidopsis thaliana</i>, were able to convert chalcones to aurones. Thus, aurone formation could be catalyzed by a widespread enzyme and seems to depend mainly on a specific biochemical background, which favours the formation of aurones at the expense of flavonoids. In contrast to 4-hydroxyaurone formation, hydroxylation and oxidative cyclization to the 4-deoxyaurones does not occur in one single step but is catalyzed by two separate enzymes, chalcone 3-hydroxylase and aurone synthase (catechol oxidase reaction). Aurone formation shows an optimum at pH 7.5 or above, which is another striking contrast to 4-hydroxyaurone formation in <i>Antirrhinum majus</i> L. This is the first example of a plant catechol oxidase type enzyme being involved in the flavonoid pathway and in an anabolic reaction in general.</p></div

Spatial distribution of okanin (<i>m/z</i> 287) along a <i>Bidens ferulifolia</i> petal fixed by adhesive tape on a Indium Tin Oxide glass slide.

<p>first row: front side, second row: back side; from left to right: daylight photos, UV photos, negative ion mode LDI-MSI images (green area indicates the presence of the target compound), negative ion mode LDI-MSI images overlapping the daylight photos. All mass peaks related to anthochlors showed a similar distribution.</p

HPLC chromatograms from incubation of enzyme preparations.

<p><i>Bidens ferulifolia</i> petals (a) and leaves (b), <i>Antirrhinum majus</i> petals (c) and leaves (d), <i>Arabidopsis thaliana</i> col-0 plants (e), <i>Tagetes erecta</i> petals (f), <i>Dianthus caryophyllus</i> petals (g), and <i>Petunia hybrida</i> petals (h) with butein and of enzyme preparations from <i>B. ferulifolia</i> petals (i) and <i>Antirrhinum majus</i> petals (j) with isoliquiritigenin.</p

UV nectar guides.

<p><i>Bidens ferulifolia</i> flower UV photography of front (a) and back (e) side, daylight photographies (b, f) and after (c, g) ammonia staining. Cross sections of <i>B. ferulifolia</i> petals base native (i) and stained (j) and petal apex native (k) and stained (l). Epi-illumination mode microscopic view of stained epidermis of petal front side base (d) and apex (h). <i>Coreopsis grandiflora</i> flower before (m) and after (n) ammonia staining. <i>Cosmos sulphureus</i> flower before (o) and after (p) ammonia staining.</p

LDI-MS of a <i>Bidens ferulifolia</i> petal.

<p>a: a representative spectrum of the petal base containing nine <i>m/z</i> signals of anthochlors. b: no anthochlors were detected in representative spectra of the petal apex. For the allocation of <i>m/z</i> signals to compounds refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061766#pone-0061766-g001" target="_blank">Figure 1</a>.</p

Overview on flavonoid and anthochlor formation from <i>p</i>-coumaroyl-CoA.

<p>abbrev.: ANS: anthocyanidin synthase, AUS: aurone synthase, CH3H: chalcone 3-hydroxylase, CHI: chalcone isomerase, CHR: chalcone reductase, CHS: chalcone synthase, DFR: dihydroflavonol 4-reductase, FHT: flavanone 3-hydroxylase, F3′H: flavonoid 3′-hydroxylase, FNSII: flavone synthase II.</p