Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of flowers and fruits. In apples (Malus domestica Borkh.), several steps of the anthocyanin pathway are coordinately regulated, suggesting control by common transcription factors. A gene encoding an R2R3 MYB transcription factor was isolated from apple (cv Cripps' Pink) and designated MdMYB1. Analysis of the deduced amino acid sequence suggests that this gene encodes an ortholog of anthocyanin regulators in other plants. The expression of MdMYB1 in both Arabidopsis (Arabidopsis thaliana) plants and cultured grape cells induced the ectopic synthesis of anthocyanin. In the grape (Vitis vinifera) cells MdMYB1 stimulated transcription from the promoters of two apple genes encoding anthocyanin biosynthetic enzymes. In ripening apple fruit the transcription of MdMYB1 was correlated with anthocyanin synthesis in red skin sectors of fruit. When dark-grown fruit were exposed to sunlight, MdMYB1 transcript levels increased over several days, correlating with anthocyanin synthesis in the skin. MdMYB1 gene transcripts were more abundant in red skin apple cultivars compared to non-red skin cultivars. Several polymorphisms were identified in the promoter of MdMYB1. A derived cleaved amplified polymorphic sequence marker designed to one of these polymorphisms segregated with the inheritance of skin color in progeny from a cross of an unnamed red skin selection (a sibling of Cripps' Pink) and the non-red skin cultivar Golden Delicious. We conclude that MdMYB1 coordinately regulates genes in the anthocyanin pathway and the expression level of this regulator is the genetic basis for apple skin color.

The plant hormone auxin regulates various developmental processes including root formation, vascular development, and gravitropism. Mutations within the AUX1 gene confer an auxin-resistant root growth phenotype and abolish root gravitropic curvature. Polypeptide sequence similarity to amino acid permeases suggests that AUX1 mediates the transport of an amino acid-like signaling molecule. Indole-3-acetic acid, the major form of auxin in higher plants, is structurally similar to tryptophan and is a likely substrate for the AUX1 gene product. The cloned AUX1 gene can restore the auxin-responsiveness of transgenic aux1 roots. Spatially, AUX1 is expressed in root apical tissues that regulate root gravitropic curvature.

SummaryMost of the thousands of grapevine cultivars (Vitis vinifera L.) can be divided into two groups, red and white, based on the presence or absence of anthocyanin in the berry skin, which has been found from genetic experiments to be controlled by a single locus. A regulatory gene, VvMYBA1, which could activate anthocyanin biosynthesis in a transient assay, was recently shown not to be transcribed in white berries due to the presence of a retrotransposon in the promoter. We have found that the berry colour locus comprises two very similar genes, VvMYBA1 and VvMYBA2, located on a single bacterial artificial chromosome. Either gene can regulate colour in the grape berry. The white berry allele of VvMYBA2 is inactivated by two non-conservative mutations, one leads to an amino acid substitution and the other to a frame shift resulting in a smaller protein. Transient assays showed that either mutation removed the ability of the regulator to switch on anthocyanin biosynthesis. VvMYBA2 sequence analyses, together with marker information, confirmed that 55 white cultivars all contain the white berry allele, but not red berry alleles. These results suggest that all extant white cultivars of grape vines have a common origin. We conclude that rare mutational events occurring in two adjacent genes were essential for the genesis of the white grapes used to produce the white wines and white table grapes we enjoy today.

The TRANSPARENT TESTA GLABRA1 ( TTG1 ) locus regulates several developmental and biochemical pathways in Arabidopsis, including the formation of hairs on leaves, stems, and roots, and the production of seed mucilage and anthocyanin pigments. The TTG1 locus has been isolated by positional cloning, and its identity was confirmed by complementation of a ttg1 mutant. The locus encodes a protein of 341 amino acid residues with four WD40 repeats. The protein is similar to AN11, a regulator of anthocyanin biosynthesis in petunia, and more distantly related to those of the ␤ subunits of heterotrimeric G proteins, which suggests a role for TTG1 in signal transduction to downstream transcription factors. The 1.5-kb TTG1 transcript is present in all major organs of Arabidopsis. Sequence analysis of six mutant alleles has identified base changes producing truncations or single amino acid changes in the TTG1 protein. INTRODUCTIONThe TRANSPARENT TESTA GLABRA1 ( TTG1 ) locus controls many apparently unrelated characters of Arabidopsis (catalogued by Koornneef, 1981), several of which appear to be confined to the epidermal cell layer of different tissues. ttg1 mutants have a glabrous phenotype, possessing none of the leaf or stem hairs (trichomes) that normally are derived from the meristematic L1 cell layer. Purple anthocyanin pigments are absent from the ttg1 seed coat, causing the transparent testa phenotype in which the yellow cotyledons are visible through the testa. In wild-type plants, anthocyanins are present in the hypocotyl of seedlings and in the stem and leaves of plants as they age, and they are inducible by many forms of stress, including high light, poor nutrients, or water stress. ttg1 mutants completely lack anthocyanins in the epidermis and in subepidermal layers of leaves and stems. Mucilage normally found in the cell wall of the seed coat is absent in ttg1 mutants. Seeds of ttg1 plants do not require drying and cold treatments to germinate and therefore exhibit an altered seed dormancy when compared with ecotypes, such as Landsberg erecta (L er ;Koornneef, 1981;Léon-Kloosterziel et al., 1994). This characteristic of ttg1 mutants may be linked to an altered seed coat structure. The TTG1 gene appears to have the opposite effect on root hair formation when compared with its effect on leaf hair initiation. In Arabidopsis, root hairs extend from root epidermal cells only in files of cells that contact two underlying cortical cells, whereas in ttg1 mutants, extra root hairs occur in the atrichoblast cell files (Galway et al., 1994). Under laboratory growth conditions, mutations at the ttg1 locus do not greatly affect the viability of the plants.In ttg1 mutants, the anthocyanin biosynthetic pathway is blocked at the dihydroflavonol-4-reductase (DFR) step, because DFR-encoding transcripts have not been detected in these mutants (Shirley et al., 1995). By contrast, transcripts of the chalcone synthase and chalcone isomerase genes are unaffected. The point of regulation of the pathway by TTG1 was confirmed by the clon...

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Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and healthpromoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera 'Shiraz') R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.

Among the dramatic changes occurring during grape berry (Vitis vinifera) development, those affecting the flavonoid pathway have provoked a number of investigations in the last 10 years. In addition to producing several compounds involved in the protection of the berry and the dissemination of the seeds, final products of this pathway also play a critical role in berry and wine quality. In this article, we describe the cloning and functional characterization of VvMYB5b, a cDNA isolated from a grape berry (V. vinifera 'Cabernet Sauvignon') library. VvMYB5b encodes a protein belonging to the R2R3-MYB family of transcription factors and displays significant similarity with VvMYB5a, another MYB factor recently shown to regulate flavonoid synthesis in grapevine. The ability of VvMYB5a and VvMYB5b to activate the grapevine promoters of several structural genes of the flavonoid pathway was confirmed by transient expression of the corresponding cDNAs in grape cells. Overexpression of VvMYB5b in tobacco (Nicotiana tabacum) leads to an up-regulation of genes encoding enzymes of the flavonoid pathway and results in the accumulation of anthocyanin-and proanthocyanidin-derived compounds. The ability of VvMYB5b to regulate particularly the anthocyanin and the proanthocyanidin pathways is discussed in relation to other recently characterized MYB transcription factors in grapevine. Taken together, data presented in this article give insight into the transcriptional mechanisms associated with the regulation of the flavonoid pathway throughout grape berry development.

Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB-type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.