anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

Abstract

The petunia loci anthocyanin1 ( an1 ), an2 , an4 , and an11 are required for the transcription of anthocyanin biosynthetic genes in floral organs. The an2 and an11 loci were recently cloned and shown to encode a MYB-domain transcriptional activator and a cytosolic WD40 protein, respectively. Here, we report the isolation of an1 by transposon tagging. an1 encodes a new member of the basic helix-loop-helix family of transcription factors that is functionally and evolutionarily distinct from JAF13, the apparent petunia ortholog of maize RED1 and snapdragon DELILA. We provide genetic evidence that the transcription factors encoded by an1 , an2 , and an4 operate in an unexpectedly complex regulatory hierarchy. In leaves, ectopic expression of AN2 induces an1 expression, whereas in anthers, an1 expression depends on an4 , encoding (or controlling) a MYB protein that is paralogous to AN2. Experiments with transgenic plants expressing a post-translationally controlled AN1-GLUCOCORTICOID RECEPTOR fusion protein indicated that independent of protein synthesis, AN1 directly activates the expression of the dfrA gene encoding the enzyme dihydroflavonol 4-reductase and of Pmyb27 encoding a MYB-domain protein of unknown function.

The Arabidopsis TT2 Gene Encodes an R2R3 MYB Domain Protein That Acts as a Key Determinant for Proanthocyanidin Accumulation in Developing Seed

“…The fact that a null allele displayed wild-type amounts of TT8 mRNA in siliques suggests that the loss of TT2 function may be compensated for by the presence of other protein(s) that have at least partially redundant functions during seed development. Similar results were obtained with petunia, in which the MYB protein AN2 did not appear to regulate AN1 mRNA levels in petals; but when ectopically expressed, AN2 apparently activated AN1 expression in other tissues such as the leaf (Spelt et al, 2000). In maize, there is no evidence that MYB and bHLH proteins control each others expression (Goff et al, 1990;Sainz et al, 1997).…”

“…AN1 and AN11 also are required for anthocyanin accumulation in almost all pigmented tissues, whereas AN2 expression apparently is restricted to the corolla limb. Paralogs of AN2 have been identified and apparently control pigmentation in different organs (Spelt et al, 2000).In Arabidopsis, we demonstrated previously that functional TTG1 (TRANSPARENT TESTA GLABROUS1), TT8, and TT2 proteins are required for the normal expression of at least two flavonoid LBGs, DFR and BAN , during seed formation (Nesi et al, 2000) (Figure 1). TTG1 was shown to encode a WD-repeat-containing protein and probably represents an ortholog of the petunia AN11 product (Walker et Enzymes are indicated in uppercase letters, with the corresponding genetic loci given in lowercase italic letters.…”

“…AN1 and AN11 also are required for anthocyanin accumulation in almost all pigmented tissues, whereas AN2 expression apparently is restricted to the corolla limb. Paralogs of AN2 have been identified and apparently control pigmentation in different organs (Spelt et al, 2000).…”

“…In petunia, the ANTHOCYANIN1 ( AN1 ), AN2 , and AN11 genes regulate, at least partially, the expression of flavonoid LBGs in flower and encode a bHLH protein, an R2R3 MYB factor, and a WD-repeat regulator, respectively (Quattrocchio et al, 1993(Quattrocchio et al, , 1999 de Vetten et al., 1997;Spelt et al, 2000). AN1 and AN11 also are required for anthocyanin accumulation in almost all pigmented tissues, whereas AN2 expression apparently is restricted to the corolla limb.…”

Reconstructing Historical Events that Occurred in the Petunia Hf1 Gene, which Governs Anthocyanin Biosynthesis, and Effects of Artificial Selection by Breeding

“…Petunia is a model system for studying floral anthocyanin biosynthesis, and most of the structural and regulatory genes controlling the biosynthetic pathway have been isolated (Holton et al 1993, Holton and Cornish 1995, Vetten et al 1997, Quattrocchio et al 1998, Spelt et al 2000, Winkel-Shirley 2001. Among the structural genes, the cytochrome P450 gene Hf1 is particularly important for breeding petunias with various floral colors because it encodes a key enzyme, flavonoid 3′, 5′-hydroxylase (F3′5′H).…”