Heidrun Halbwirth

Molecular cloning, substrate specificity of the functionally expressed dihydroflavonol 4-reductases from Malus domestica and Pyrus communis cultivars and the consequences for flavonoid metabolism

Treatment with the dioxygenase inhibitor prohexadione-Ca leads to major changes in the flavonoid metabolism of apple (Malus domestica) and pear (Pyrus communis) leaves. Accumulation of unusual 3-deoxyflavonoids is observed, which have been linked to an enhanced resistance toward fire blight. The committed step in this pathway is the reduction of flavanones. Crude extracts from leaves are able to perform this reaction. There was previous evidence that DFR enzymes of certain plants possess additional flavanone 4-reductase (FNR) activity. Such an FNR activity of DFR enzymes is proved here by heterologous expression of the enzymes. The heterologously expressed DFR/FNR enzymes of Malus and Pyrus possess distinct differences in substrate specificities despite only minor differences of the amino acid sequences. Kinetic studies showed that dihydroflavonols generally are the preferred substrates. However, with the observed substrate specificities the occurrence of 3-deoxyflavonoids in vivo after application of prohexadione-Ca can be explained.

Shift in polyphenol profile and sublethal phenotype caused by silencing of anthocyanidin synthase in apple (Malus sp.).

We have investigated the consequences of blocking anthocyanin biosynthesis by silencing a key enzyme, anthocyanidin synthase, in transgenic plants of a red-leaved apple cultivar. This is complementary to a previous study of induction of anthocyanin biosynthesis by overexpressing a heterologous transcription factor. Analysis of these opposite phenotypes allows one to study anthocyanin functions in apple and to test the influence of the genetic manipulation on other, related metabolites. As expected, anthocyanin biosynthesis was almost completely blocked and this was accompanied by a shift in the profile of flavonoids and related polyphenols. Most interestingly, a rise in epicatechin was found. A severe reduction of viability by necrotic leaf lesions was also observed, suggesting an essential function of anthocyanins in apple.

Transgenic apple plants overexpressing the Lc gene of maize show an altered growth habit and increased resistance to apple scab and fire blight

Transgenic apple plants (Malusxa0×xa0domestica cv. ‘Holsteiner Cox’) overexpressing the Leaf Colour (Lc) gene from maize (Zea mays) exhibit strongly increased production of anthocyanins and flavan-3-ols (catechins, proanthocyanidins). Greenhouse plants investigated in this study exhibit altered phenotypes with regard to growth habit and resistance traits. Lc-transgenic plants show reduced size, transversal gravitropism of lateral shoots, reduced trichome development, and frequently reduced shoot diameter and abnormal leaf development with fused leaves. Such phenotypes seem to be in accordance with a direct or an indirect effect on polar-auxin-transport in the transgenic plants. Furthermore, leaves often develop necrotic lesions resembling hypersensitive response lesions. In tests, higher resistance against fire blight (caused by the bacterium Erwinia amylovora) and against scab (caused by the fungus Venturia inaequalis) is observed. These phenotypes are discussed with respect to the underlying altered physiology of the Lc-transgenic plants. The results are expected to be considered in apple breeding strategies.

Formation of 6'-Deoxychalcone 4'-Glucosides by Enzyme Extracts from Petals of Dahlia variabilis.

Yellow colouration of Dahlia variabilis is mainly provided by isoliquiritigenin 4′-glucoside and butein 4′-glucoside. Incubation of petal extracts with uridin 5′-diphosphoglucose and isoliquiritigenin or butein led to the formation of one product which was identified as the respective 6′-deoxychalcone 4′-glucoside. Glucosylation of hydroxyl groups in other positions was not observed. Naringenin chalcone and eriodictyol chalcone were not accepted as substrates. The 4′-glucosylation of isoliquiritigenin and butein showed a broad pH optimum ranging from pH 7 to 8 and was stimulated by Mg2+, Ca2+ and Mn2+. N-Ethylmaleimide, p-hydroxymercuribenzoate, Cu2+, Zn2+, and Fe2+ clearly reduced the activity of the enzyme. The apparent Km values for UDP-glucose, isoliquiritigenin and butein were 90, 26 and 276 [μᴍ respectively.