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Flavonoids are important secondary metabolites in strawberry as they fulfill a wide variety of physiological functions. In addition, they are beneficial for human health. Previous studies have shown for selected enzymes from the flavonoid pathway that flavonoid biosynthesis shows two peaks during fruit development. We provide optimized protocols for the determination of the activities of the key flavonoid enzymes: phenylalanine ammonia lyase, chalcone synthase/chalcone isomerase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, flavonol synthase, flavonoid 3-O-glucosyltransferase, and flavonoid 7-O-glucosyltransferase. Using these protocols we were able to demonstrate two distinct activity peaks during fruit ripening at early and late developmental stages for all enzymes with the exception of flavonol synthase. The first activity peak corresponds to the formation of flavanols, while the second peak is clearly related to anthocyanin and flavonol accumulation. The results indicate that flavonoid 3-O-glucosyltransferase activity is not essential for redirection from flavanol to anthocyanin formation in strawberry.

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Pear (Pyrus sp.) is a major fruit crop of temperate regions with increasing extent of cultivation. Pear flavonoids contribute to its fruit color, pathogen defense, and are health beneficial ingredients of the fruits. Comparative Southern analyses with apple (Malus x domestica) cDNAs showed comparable genomic organization of flavonoid genes of both related genera. A homology-based cloning approach was used to obtain the cDNAs of most enzymes of the main flavonoid pathway of Pyrus: phenylalanine ammonia lyase, chalcone synthase, chalcone isomerase, flavanone 3b-hydroxylase, flavonol synthase, dihydroflavonol 4-reductase, leucoanthocyanidin reductase 1 and 2, anthocyanidin synthase, anthocyanidin reductase, and UDP-glucose : flavonoid 7-O-glucosyltransferase. The substrate specificities of the recombinant enzymes expressed in yeast were determined for physiological and non-physiological substrates and found to be in general agreement with the characteristic pear flavonoid metabolite pattern of mainly B-ring dihydroxylated anthocyanins, flavonols, catechins, and flavanones. Furthermore, significant differences in substrate specificities and gene copy numbers in comparison to Malus were identified. Cloning of the cDNAs and studying the enzymes of the Pyrus flavonoid pathway is an essential task toward a comprehensive knowledge of Pyrus polyphenol metabolism. It also elucidates evolutionary patterns of flavonoid/polyphenol pathways in the Rosaceae, which allocate several important crop plants.

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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.