The production of anthocyanins in fruit tissues is highly controlled at the developmental level. We have studied the expression of flavonoid biosynthesis genes during the development of bilberry (Vaccinium myrtillus) fruit in relation to the accumulation of anthocyanins, proanthocyanidins, and flavonols in wild berries and in color mutants of bilberry. The cDNA fragments of five genes from the flavonoid pathway, phenylalanine ammonia-lyase, chalcone synthase, flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase, were isolated from bilberry using the polymerase chain reaction technique, sequenced, and labeled with a digoxigenin-dUTP label. These homologous probes were used for determining the expression of the flavonoid pathway genes in bilberries. The contents of anthocyanins, proanthocyanidins, and flavonols in ripening bilberries were analyzed with high-performance liquid chromatography-diode array detector and were identified using a mass spectrometry interface. Our results demonstrate a correlation between anthocyanin accumulation and expression of the flavonoid pathway genes during the ripening of berries. At the early stages of berry development, procyanidins and quercetin were the major flavonoids, but the levels decreased dramatically during the progress of ripening. During the later stages of ripening, the content of anthocyanins increased strongly and they were the major flavonoids in the ripe berry. The expression of flavonoid pathway genes in the color mutants of bilberry was reduced. A connection between flavonol and anthocyanin synthesis in bilberry was detected in this study and also in previous data collected from flavonol and anthocyanin analyses from other fruits. In accordance with this, models for the connection between flavonol and anthocyanin syntheses in fruit tissues are presented.Fruit development from flower to ripe fruit is a complex process that involves modification of cellular compartments, loss of cell wall structure causing softening, and accumulation of carbohydrates (Brady, 1987). The production of secondary metabolites during the ripening process is an essential phenomenon for the contribution of seed dispersal of the plant in the form of accumulation of pigments and flavor compounds. The significance of secondary products in defense against diseases in developing fruits should also be remembered (Harborne, 1997;Mercier, 1997).Flavonoids are a large group of phenolic secondary metabolites that are widespread among plants and are involved in many plant functions. Anthocyanins, a flavonoid subclass, are the main pigments in flowers and fruits, acting as insect and animal attractants (Bohm, 1998;Harborne and Williams, 2000). Anthocyanins are synthesized via the phenylpropanoid pathway (Fig. 1). Anthocyanin biosynthesis has been extensively studied in several plant species, and, therefore, detailed information of the course of reactions is available. Two classes of genes are required for anthocyanin biosynthesis, the structural genes encoding the enzymes...

The growth conditions in different latitudes vary markedly with season, day length, light quality and temperature. Many plant species have adapted well to the distinct environments through different strategies, one of which is the production of additional secondary metabolites. Flavonoids are a widely spread group of plant secondary metabolites that are involved in many crucial functions of plants. Our understanding of the biosynthesis, occurrence and function of flavonoids has increased rapidly in recent decades. Numerous studies have been published on the influence of environmental factors on the biosynthesis of flavonoids. However, extensive long-term studies that examine the effect of the characteristics of northern climates on flavonoid biosynthesis are still scarce. This review focuses on the current knowledge about the effect of light intensity, photoperiod and temperature on the gene-environment interaction related to flavonoid biosynthesis in plants.

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.

Anthocyanins are important health-promoting phytochemicals that are abundant in many fleshy fruits. Bilberry (Vaccinium myrtillus) is one of the best sources of these compounds. Here, we report on the expression pattern and functional analysis of a SQUAMOSA-class MADS box transcription factor, VmTDR4, associated with anthocyanin biosynthesis in bilberry. Levels of VmTDR4 expression were spatially and temporally linked with color development and anthocyanin-related gene expression. Virus-induced gene silencing was used to suppress VmTDR4 expression in bilberry, resulting in substantial reduction in anthocyanin levels in fully ripe fruits. Chalcone synthase was used as a positive control in the virus-induced gene silencing experiments. Additionally, in sectors of fruit tissue in which the expression of the VmTDR4 gene was silenced, the expression of R2R3 MYB family transcription factors related to the biosynthesis of flavonoids was also altered. We conclude that VmTDR4 plays an important role in the accumulation of anthocyanins during normal ripening in bilberry, probably through direct or indirect control of transcription factors belonging to the R2R3 MYB family.