The TRANSPARENT TESTA8 ( TT8 ) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loophelix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE ( DFR ) and BANYULS ( BAN ), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 ( TTG1 ) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat. INTRODUCTIONFlavonoids are derived from phenylalanine and malonyl-CoA and constitute one of the largest groups of secondary metabolites in plants. They are based on a 15-carbon skeleton, which can be modified to yield different subclasses, including flavonols, anthocyanins, and proanthocyanidins ( Figure 1). In plants, flavonoid derivatives are responsible for the pigmentation pattern of vegetative parts and seeds and are involved in a wide range of biological functions. For example, they protect against UV radiation, serve as signal molecules in plant-microbe interactions, and participate in plant defense responses (reviewed in Dooner et al., 1991;Koes et al., 1994;Dixon and Paiva, 1995;Shirley, 1996). Recent studies have also stressed the involvement of flavonoids in seed coat-imposed dormancy as well as in seed storability (Winkel-Shirley, 1998;Debeaujon et al., 2000). Moreover, flavonoids are receiving increasing interest as health-promoting components of animal and human diets (Lairon and Amiot, 1999). These diverse roles can be correlated, at least in part, with the well-documented antioxidant properties of phenylpropanoid derivatives, especially flavonoids (RiceEvans et al., 1997), and with their inhibitory effect on enzymatic activities (Castelluccio et al., 1995).Investigating the structure and regulation of the flavonoid biosynthetic pathway in plants may thus help us to better understand and monitor flavonoid metabolism with regard to properties of the end products (Weisshaar and Jenkins, 1998). Flavonoid biosynthesis has been studied extensively by several methods, from protein purification to screening libraries with heterologous probes (reviewed in Holton and Cornish, 1995). The ubiquitous and nonessential nature of pigments for plant viability has made it possible to identify many flavonoid mutants, which has facilitated the genetic and molecular dissection of the pathway. To date, most of the enzymes involved in flavonoid biosynthesis have been c...

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In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2 , TT8 , and TTG1 , are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WDrepeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.

The TRANSPARENT TESTA8 (TT8) locus is involved in the regulation of flavonoid biosynthesis in Arabidopsis. The tt8-3 allele was isolated from a T-DNA-mutagenized Arabidopsis collection and found to be tagged by an integrative molecule, thus permitting the cloning and sequencing of the TT8 gene. TT8 identity was confirmed by complementation of tt8-3 and sequence analysis of an additional allele. The TT8 gene encodes a protein that displays a basic helix-loop-helix at its C terminus and represents an Arabidopsis ortholog of the maize R transcription factors. The TT8 transcript is present in developing siliques and in young seedlings. The TT8 protein is required for normal expression of two flavonoid late biosynthetic genes, namely, DIHYDROFLAVONOL 4-REDUCTASE (DFR) and BANYULS (BAN), in Arabidopsis siliques. Interestingly, TRANSPARENT TESTA GLABRA1 (TTG1) and TT2 genes also control the expression of DFR and BAN genes. Our results suggest that the TT8, TTG1, and TT2 proteins may interact to control flavonoid metabolism in the Arabidopsis seed coat.

In Arabidopsis, proanthocyanidins specifically accumulate in the endothelium during early seed development. At least three TRANSPARENT TESTA (TT) genes, TT2, TT8, and TTG1, are necessary for the normal expression of several flavonoid structural genes in immature seed, such as DIHYDROFLAVONOL-4-REDUCTASE and BANYULS (BAN). TT8 and TTG1 were characterized recently and found to code for a basic helix-loop-helix domain transcription factor and a WD-repeat-containing protein, respectively. Here the molecular cloning of the TT2 gene was achieved by T-DNA tagging. TT2 encoded an R2R3 MYB domain protein with high similarity to the rice OsMYB3 protein and the maize COLORLESS1 factor. A TT2-green fluorescent protein fusion protein was located mostly in the nucleus, in agreement with the regulatory function of the native TT2 protein. TT2 expression was restricted to the seed during early embryogenesis, consistent with BAN expression and the proanthocyanidin deposition profile. Finally, in gain-of-function experiments, TT2 was able to induce ectopic expression of BAN in young seedlings and roots in the presence of a functional TT8 protein. Therefore, our results strongly suggest that stringent spatial and temporal BAN expression, and thus proanthocyanidin accumulation, are determined at least partially by TT2.