ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms

Abstract

ANTHOCYANIN1 (AN1) of petunia is a transcription factor of the basic helix-loop-helix (bHLH) family that is required for the synthesis of anthocyanin pigments. Here, we show that AN1 controls additional aspects of cell differentiation: the acidification of vacuoles in petal cells, and the size and morphology of cells in the seed coat epidermis. We identified an1 alleles, formerly known as ph6, that sustain anthocyanin synthesis but not vacuolar acidification and seed coat morphogenesis. These alleles express truncated proteins lacking the C-terminal half of AN1, including the bHLH domain, at an approximately 30-fold higher level than wild-type AN1. An allelic series in which one, two, or three amino acids were inserted into the bHLH domain indicated that this domain is required for both anthocyanin synthesis and vacuolar acidification. These findings show that AN1 controls more aspects of epidermal cell differentiation than previously thought through partially separable domains.

Figure 1.

Mutations in Regulatory Anthocyanin Genes Increase the pH of Petal Homogenates. pH values (means ± sd; n ≥ 4) were measured in petal homogenates of various genotypes, as indicated on the horizontal axis: +, wild-type allele(s); m, mutable (unstable) alleles; r, derived full-revertant alleles.

Figure 2.

Phenotypes of Flowers Harboring Different an1 Alleles. Each image shows part of a flower and the pH value of its petal extracts at right (means ± sd; n ≥ 3). The an1 alleles present in each flower are indicated in italic type at top. For flowers that are homozygous for an an1 allele, only a single allele number is given. For flowers that are heterozygous, the numbers of both an1 alleles are given separated by a slash.

Figure 3.

Molecular Analysis of an1 Alleles. (A) Structure of the AN1 gene and mutant alleles. Exons are indicated by rectangles, and introns are indicated by a horizontal line. Exonic regions that encode the conserved N-terminal domain of AN1 and the bHLH region are shaded black; regions encoding less conserved parts of AN1 are indicated by hatching. Exon regions that are not translated are indicated by rectangles of half the height. Triangles (not drawn to scale) denote transposon insertions in the indicated alleles; their orientations are indicated with arrows (arrows pointing right indicate that the orientation of the transposon relative to AN1 is the same as the sequences in the corresponding GenBank accessions). (B) Analysis of RNAs expressed in AN1 and an1 flowers. RNAs of several genes (indicated at right) were detected by RNA gel blot analysis (AN1 mRNA) or RT-PCR (DFR, PAT, and GAPDH mRNA). For all alleles (indicated above the lanes) homozygous flowers were used, except for X2200 and W138R, which were heterozygous over the parental W138 allele. (C) Structure of mRNAs and proteins expressed by an1 alleles. The numbers of the alleles are indicated at left, the structure of their mRNAs are indicated in the middle and the encoded proteins are indicated at right. Exons and transposon sequences are drawn as in (A); protein-coding sequences that are not translated in the mutant mRNA are shaded white. The mRNA splicing patterns are indicated by thin lines. The lines connecting the allele numbers indicate how distinct excision alleles derived from the original transposon insertion alleles. (D) Gel blot analysis of AN1 proteins expressed in an1 mutants. The flowers analyzed were homozygous for the alleles indicated above the lanes, except for W138R, which was heterozygous over the parental mutable allele W138.

Figure 4.

Transient Expression Assays of Mutant an1 Alleles. The bars denote luciferase (LUC) activity expressed from a DFR:LUC reporter gene, normalized for β-glucuronidase (GUS) activity expressed from a codelivered 35S:GUS gene (means ± se; n = 5) in particle-bombarded leaves. Both reporters were codelivered with effector genes expressing AN2 and different mutant AN1 proteins from the 35S promoter, as indicated on the horizontal axis. A minus sign indicates that the corresponding effector was omitted.

Figure 5.

Molecular Analysis of bHLH Domain Mutants of AN1. (A) Sequences of the bHLH domains encoded by the wild-type (wt) allele R27, the transposon insertion allele W211, and derived excision alleles (W211R1 to W221W). Regions in which the protein differs from the wild type are underlined. The asterisk denotes the end of the protein. (B) RT-PCR analysis of mRNAs expressed from the AN1-controlled genes DFR and PAT1 and a housekeeping gene (GAPDH) in the corolla limbs of an1 mutants. The analyzed corolla limbs were homozygous for alleles R27 and W211. Alleles W211W to W211R3 were heterozygous over the parental mutable allele W211, whereas the full revertant allele, W138R, was heterozygous over the parental allele W138. (C) Analysis of the an1 transcripts expressed in the corolla limbs described in (B). The arrowhead indicates the wild-type 2.45-kb AN1 mRNA. (D) Gel blot analysis of AN1 proteins expressed in corolla limbs of the mutants described in (B).

Figure 6.

Effect of an1 Mutations on the Morphology of Seed Coat Cells. (A) Seed coats of the wild-type (WT) line R27. (B) Ovules of the wild-type (WT) line R27. (C) Seed coats of line W225, which is homozygous for the stable null allele an1-W225. (D) Ovules of line W225 (an1-W225). (E) Seed coats of line W138, which is homozygous for the mutable allele an1-W138. Note the large revertant sector of large brown seed coat cells (marked R) at left. Revertant cells in the scanning electron micrographs (middle and right) are marked R. (F) Seed coats of line W234, which is homozygous for a wild-type AN1 allele and a stable recessive an3 allele (an3-W1006) containing a large deletion (van Houwelingen et al., 1998). (G) Seed coats of a plant heterozygous for the intermediate allele X2200 and the mutable allele W138. Revertant cells are indicated with R. (H) Seed coats of a plant homozygous for an1-B2262. The color photographs show untreated mature seeds, and the black-and-white images are scanning electron micrographs. Bars = 50 μm.