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First published online August 23, 2002; 10.1105/tpc.003772 American Society of Plant Biologists ANTHOCYANIN1 of Petunia Controls Pigment Synthesis, Vacuolar pH, and Seed Coat Development by Genetically Distinct MechanismsDepartment of Developmental Genetics, Institute for Molecular Biological Sciences, Vrije Universiteit, de Boelelaan 1087, 1081 HV Amsterdam, The Netherlands 1 To whom correspondence should be addressed. E-mail koes{at}bio.vu.nl; fax 31-20-4447155
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  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.
The synthesis of anthocyanin pigments in floral tissue provides an excellent marker to study the differentiation of cells. Most of the structural genes that encode the 15 enzymes required for anthocyanin synthesis and modification have been isolated (Holton and Cornish, 1995 ; Winkel-Shirley, 2001). By mutational analyses in Antirrhinum, Arabidopsis, maize, and petunia, several regulatory genes have been identified that are required for the tissue-specific transcription of the structural anthocyanin genes (for reviews, see Mol et al., 1998; Winkel-Shirley, 2001).
ANTHOCYANIN1 (AN1) of petunia encodes a basic helix-loop-helix (bHLH) protein that activates the transcription of the structural anthocyanin gene DIHYDROFLAVONOL REDUCTASE (DFR) and a putative regulatory gene (MYB27) whose function is unknown (Spelt et al., 2000
AN2 is expressed in the petal limb only and encodes a MYB domain protein (Quattrocchio et al., 1999
In Arabidopsis, two other transcription factors, the homeodomain protein ANTHOCYANINLESS2 (ANL2) and the zinc finger protein TT1, have been implicated in the accumulation of proanthocyanidin polymers in the seed coat (Kubo et al., 1999
Although the regulatory genes mentioned above were identified initially by their role in anthocyanin synthesis, an increasing amount of evidence indicates that they control additional aspects of cell differentiation. Expression of a C1-R fusion protein in maize suspension cells causes the activation or repression of hundreds of genes (Bruce et al., 2000
Ectopic expression of R in Arabidopsis has the opposite effect: the formation of extra trichomes in stems and leaves (Lloyd et al., 1992
The vacuole of plant cells plays a central role in pH homeostasis, osmoregulation, ion transport, and the sequestration of (toxic) metabolites (Taiz, 1992
Because anthocyanins accumulate in vacuoles and because their absorption spectrum depends on pH, they provide a natural indicator of vacuolar pH (Mol et al., 1998
Petunia flowers normally do not turn blue upon opening, and their vacuolar pH stays on the reddish (low-pH) side of the anthocyanin color spectrum. By genetic analyses, seven loci, designated PH1 to PH7, were identified that mutate to flowers with a more bluish color (de Vlaming et al., 1983 In this article, we show that AN1, AN2, and AN11 control, in addition to anthocyanin synthesis, the vacuolar pH in petal cells and the morphology of the seed coat epidermis. Our finding that ph6 mutants do not define a separate locus, as was thought previously, but represent specific alleles of AN1 indicates that AN1 controls anthocyanin synthesis and vacuolar pH by distinct mechanisms.
Mutations at AN1, AN2, and AN11 Increase the pH of Flower Homogenates The petunia line W138 arose from the parental line R27 by the insertion of a dTph1 transposon in the AN1 gene and bears white flowers with red and pink revertant spots. During the phenotypic analysis of some new ph mutants that were identified among W138 progeny, we noticed that the an1-W138 mutation increased the pH of petal limb homogenates to a similar extent as did mutations in PH3 and PH4 (Quattrocchio, 1994 ). To establish the significance of this observation, we measured the pH of petal limb homogenates for a range of genotypes.
Figure 1 shows that extracts from an1-W138 homozygous flowers have a pH value that is
AN3 encodes the enzyme flavanone 3  -hydroxylase (F3H), which catalyzes the conversion of flavanones to the corresponding dihydroflavonols, and an3 mutations block anthocyanin synthesis well before the an1 block (van Houwelingen et al., 1998). Extracts of an3-R134 and AN3 revertant flowers had pH values comparable to that of the wild-type progenitor (R27). Similar results were obtained for mutants containing two other an3 alleles (an3-S205 and an3-S206; data not shown) and for transgenic plants in which CHS genes, which encode chalcone synthase, the first enzyme of flavonoid metabolism, were cosuppressed. Because the an3 mutation blocks the anthocyanin pathway at an earlier step than an1 mutations, an3 and an1 an3 double mutants accumulate the same flavonoids (eriodictyol in the R27 background). Even in an an3 background, the effect of the an1-W138 mutation on flower extract pH was observed (Figure 1; compare an1 an3 and AN1revan3). Thus, the pH shift observed in an1-W138 flowers cannot be explained by the absence of anthocyanins.
To determine whether AN2 and AN11 also are involved in pH control, we analyzed unstable mutants and revertants for these loci. Flowers harboring the unstable an11-W137 allele (de Vetten et al., 1997
Similar results were obtained when we compared pH values in a stable recessive an2 mutant and isogenic AN2 plants in which the mutation was complemented by a 35S:AN2 transgene (Quattrocchio et al., 1998
Analysis of Functional AN1 Domains by in Vivo Mutagenesis We analyzed these unstable an1 alleles for: (1) molecular alterations in the structure and expression of an1, (2) their capacity to drive anthocyanin synthesis and the expression of the structural anthocyanin gene DFR, and (3) their capacity to regulate intracellular pH and drive the expression of PAT1, a gene of unknown function that is downregulated in ph4 and ph3 mutants (F. Quattrocchio, I. Roobeek, W. Verweij, C. Spelt, J. Mol, and R. Koes, unpublished data). The results for the most informative alleles are presented below.
The Wild-Type AN1 Allele Expresses Distinct mRNA Species That Result from Alternative Polyadenylation
Rapid amplification of cDNA ends (Frohman et al., 1988 ) showed that the 1.4-kb an1 transcript resulted from premature polyadenylation in intron 6 and encodes a truncated AN1 protein (Figure 3C). Given that the removal of intron 7 is relatively slow (Spelt et al., 2000), we assume that the 2.9-kb an1 RNAs represent incompletely spliced transcripts.
an1 Alleles That Disrupt Both Short and Long AN1 mRNAs The an1-W17 allele, which is present in the R27 background, contains an insertion of a novel 3800-bp petunia transposon of the CACTA family, designated Tph6 (a more detailed characterization of Tph6 will be published elsewhere), that blocks expression of the wild-type 1.4- and 2.45-kb mRNAs and results in the expression of a 1.3-kb AN1 mRNA (Figures 3A and 3C). RT-PCR and 3' rapid amplification of cDNA ends analyses indicated that this RNA arose by premature polyadenylation within Tph6, followed by the splicing of exon 2 to a cryptic splice site in Tph6; it encodes a truncated AN1 protein of 114 amino acids (Figure 3C). Flowers homozygous for an1-W17 are white (and contain few revertant spots), have a high petal pH (Figure 2C), and express little or no DFR and PAT mRNA (Figure 3B), indicating that the W17 mutation is similar in effect to Z2309 and is essentially a null allele.
The Long AN1 mRNA Is Essential to Direct Anthocyanin Synthesis and Vacuolar Acidification
The dTph1 insertion in an1-W138 duplicated the acceptor splice of exon 7, and the derived excision alleles contain footprints that contain zero, one, two, or three potential acceptor splice sites at this position (Spelt et al., 2000 an1-W225 specifies white flowers with an increased petal pH and strongly reduced DFR and PAT1 mRNA levels (Figures 2E and 3B), similar to the null alleles Z2309 and W17. Because an1-W138 and an1-W225 still express the 1.4-kb AN1 mRNA but nevertheless have a null phenotype, we infer that the 1.4-kb AN1 mRNA has, at the levels at which it is expressed normally, little or no effect on the synthesis of anthocyanins and vacuolar acidification.
To examine how a reduction of the 2.45-kb an1 mRNA influenced anthocyanin synthesis and vacuolar acidification, we analyzed the partial revertants of an1-W138. Partial revertant alleles of an1-W138, such as an1-X2200, contain both the original and a potential alternative acceptor splice site (Spelt et al., 2000 Interestingly, an1-X2200 encodes a pink (i.e., light red) flower color, indicating that anthocyanin synthesis is diminished, whereas the pH value is similar to that of wild-type R27 (Figure 2F). Thus, anthocyanin synthesis is affected more strongly by reduced AN1 expression than is vacuolar pH, even though the amount of DFR and PAT mRNA is reduced equally (approximately twofold).
Mutant an1 Alleles That Partially Separate the Anthocyanin and pH Function
More than a decade later, Chuck et al. (1993) To determine whether ph6 mutants represent specific alleles of AN1, we analyzed the structure of the AN1 gene in progeny of the original G621 plants. The progeny of two of these mutants consisted of 34 plants that all bore purplish flowers with red spots (Figure 2G). DNA analysis indicated that they harbored a mutant an1 allele (an1-G621) that contained near the end of exon 6 an insertion of a new 873-bp transposon (Figure 3A) that we designated dTph5 (details on dTph5 will be published elsewhere). Molecular analysis of the stable an1-W160 allele, which arose in G621 progeny, showed that the dTph5 element had excised and produced an 8-bp footprint that disrupts the AN1 coding sequence (data not shown). Because these results suggested that the G621 mutant represents a dTph5 insertion allele of AN1, rather than an allele of a separate PH6 locus, we performed complementation experiments. Crosses between G621 mutants and null an1 mutants (an1-W225 and an1-W138) all yielded progeny with a (purplish) ph mutant flower color rather than a (red) wild-type color, indicating that the G621 mutation was allelic to an1 (Figure 2H). Therefore, we concluded that ph6 mutants do not define a separate locus; rather, they represent particular alleles of AN1 that lost the control of vacuolar pH but retained the capacity to direct the nearly normal synthesis of anthocyanins. Consistent with this conclusion, DFR mRNA expression was reduced approximately twofold in an1-G621 petals, whereas PAT1 mRNA was abolished almost completely (Figure 3B). In derived an1-W160 mutants (Figure 2I), both DFR and PAT1 were downregulated strongly (Figure 3B).
RNA analysis showed that an1-G621 expresses a 1.4-kb an1 transcript that is polyadenylated prematurely in the dTph5 sequence (Figure 3C). This truncated transcript appears to be relatively stable, because it accumulates at threefold higher levels than the wild-type 2.45-kb an1 mRNA (Figure 3B). By contrast, the excision allele W160 expresses a reduced amount (
From the transposon mutagenesis screens, we isolated two additional an1 alleles, B3196 and B2262, with a strong effect on pH control and a mild effect on anthocyanin synthesis (Figure 2J and data not shown), which is reflected by a mild reduction of DFR mRNA and an almost complete reduction of PAT1 mRNA (Figure 3B). B2262 and B3196 contain a dTph1 insertion in exon 6 in the inverted orientation (Figure 3A and data not shown). This results in premature polyadenylation of the an1 mRNA in dTph1 sequences and the formation of a small amount of mRNAs encoding a truncated AN1 protein (Figures 3B and 3C). The AC-induced ph6-m1 allele also expresses a truncated transcript, but its structure and the sequence of the encoded protein are not known (Chuck et al., 1993
Mutations That Truncate an1 mRNA and Reduce Both Anthocyanin Synthesis and Vacuolar Acidification Among the progeny of line M87 (Figure 2L), we found the unstable allele an1-X2366 containing a dTph1 insertion in exon 6 (Figure 3A). an1-X2366 clearly is not a null allele, because the flowers have a "blush" of variable intensity, dotted with full-colored revertant spots (Figure 2M). This incomplete block in anthocyanin synthesis is reflected by the relatively large amount of residual DFR mRNAs in X2366 homozygous flowers.
Strikingly, the X2366 mutation has a much stronger effect on PAT1 expression, because PAT1 mRNA is abolished almost completely (Figure 3B). X2366 petals express a small amount (
Excision of dTph1 from X2366 produced a stable recessive allele, an1-W242, that is stronger than the parental X2366 allele, because it encodes completely white flowers with a slightly higher petal pH (Figure 2N) and a complete reduction of both DFR and PAT1 expression (Figure 3B). an1-W242 expresses a small amount of full-size an1 transcript (
Analysis of Mutant AN1 Proteins
On protein blots, the anti-AN1 serum detected several bands (Figure 3D). The
In an1-G621 petals, we detected a new 32-kD protein, a size expected on the basis of the an1-G621 mRNA sequence (Figure 3D). Apparently, this mutant AN1 protein is very stable, because it is accumulated at
Transient Expression of an1 Alleles To reduce the contribution of the high protein stability and the consequent high protein levels to the activities that are measured, we expressed mutant an1 alleles from the strong and nearly constitutive 35S promoter of Cauliflower mosaic virus and assayed the activation of the DFR promoter in transient expression assays. The rationale behind these experiments is that the 35S promoter causes strong expression of all alleles, whereas the short time span of the experiment (24 h, compared with several days for the development of a mature flower on the plant) reduces the large differences in protein levels that result from differences in protein stability.
To generate 35S-driven gene constructs for distinct an1 alleles, we placed cDNA fragments amplified from mutant petals between the 35S promoter and the 3' end of the NOPALINE SYNTHASE gene (see Methods). Each construct was introduced into leaf cells, in combination with a 35S-driven AN2 gene (35S:AN2), a LUCIFERASE reporter gene driven by the DFR promoter (DFR:LUC), and a constitutively expressed control gene (35S:
Figure 4 shows that coexpression of AN1 and AN2 strongly induced DFR:LUC expression in leaf cells, whereas either factor alone did not, consistent with previous results (Quattrocchio et al., 1998
These results indicate that AN1-G621 has a reduced capacity to induce the DFR promoter, which in vivo apparently is compensated for by the 30-fold overexpression of the protein, resulting in nearly normal DFR mRNA expression levels.
An Allelic Series Resulting from Alterations in the bHLH Domain PCR and sequence analyses of one of these alleles, an1-W211, showed that the dTph1 element had excised from the original position at the border of intron 6 and exon 7 and created a footprint that would have restored an1 function (Figure 2T) if a dTph1 insertion had not disrupted the region encoding the first helix of the bHLH domain. This bHLH insertion apparently creates a null allele, because an1-W211 homozygous petal limbs lack anthocyanins, have increased pH (Figure 2O), and do not express DFR and PAT1 mRNA (Figure 5).
Somatic and germinal excisions of dTph1 from an1-W211 produced a series of four new stable an1 alleles with activities ranging from null to nearly wild type (Figures 2P to 2S). The an1-W211W allele specifies white flowers that completely lack DFR and PAT mRNA and a petal homogenate pH that is increased by 0.6 (Figure 2P), indicating that this is a null allele. PCR and sequence analysis showed that this allele contains a 7-bp footprint that results in a shift of the AN1 reading frame. The an1-W211R3 allele has some remaining activity and specifies light pink flowers in which the amount of DFR and PAT mRNA is reduced strongly (to 10% of the wild-type level); it also produces homogenates in which the pH is increased by 0.4 (Figures 2Q and 5B).
DNA analysis showed that an1-W211R3 arose from an1-W211 by excision of the dTph1 element, which resulted in the formation of a 9-bp footprint and the insertion of three amino acids in the bHLH domain (Figure 5A). The allele an1-W211R2 specifies pink flowers that contain 20% DFR mRNA (relative to the wild type) and produce extracts in which the pH is increased by 0.2 (Figure 2R). PCR and sequence analysis showed that this allele contained a 6-bp footprint resulting in the insertion of two amino acids in AN1. The an1-W211R1 allele, which specifies dark pink flowers with wild-type pH (Figure 2S) and nearly wild-type levels of DFR mRNA ( RNA gel blot analyses (Figure 5C), RT-PCR experiments (data not shown), and protein gel blot experiments (Figure 5D) showed that the three in-frame excision alleles (W211R1, W211R2, and W211R3) all produce wild-type amounts of an1 mRNA and AN1 protein, indicating that the different phenotypes result from a differences in the activity, not the amount, of the encoded AN1 protein. The dTph1 insertion in an1-W211 and the derived frameshift in an1-W211W result in strongly reduced amounts of an1 mRNA and AN1 protein, which explains why these are null alleles.
Regulatory Anthocyanin Genes Control Cell Shape in the Seed Coat
Figure 6A shows that the seed coat epidermis of mature wild-type seeds has a brown color and consists of relatively large cells with a rough, "bubbly" surface and thick, serrated crests where they are attached to neighboring cells. These cells derive from the outer layer of the single petunia integument, which is formed during ovule development, by an extensive differentiation process that involves cell enlargement and thickening of tangential walls (Colombo et al., 1997
The seed coat epidermis of an1 seeds has a very different morphology: it is yellow and consists of relatively small cells that have a smooth surface and thinner, less serrated crests (Figure 6C). Because the reduced cell size is compensated for by an increase in cell number, an1 seeds are enclosed within a complete epidermis. Analysis of an1 ovules showed that the epidermis of the integument contained a similar number of cells than that of wild-type ovules (Figure 6D). This finding is consistent with expression data showing that AN1 is expressed in the integument endothelium but not in the epidermis (C. Spelt, M. Bliek, and R. Koes, unpublished data). Thus, the supernumerary cells in an1 seed coats arise from cell divisions that occur after fertilization, during development of the seed. To determine whether AN1 acts cell autonomously during seed coat development, we analyzed seeds from unstable mutants. The epidermis of W138 seeds (and other unstable an1 mutants) mostly consists of small yellow cells, but some seeds contain patches of revertant cells with a wild-type phenotype (Figure 6E). At the borders of revertant sectors, the relatively large AN1 cells contact a larger than usual number of the much smaller neighboring mutant cells (Figure 6C). The crest between AN1 and an1 cells seems to consist of two halves with a wild-type and a mutant appearance, respectively, and crests between an1 cells have a mutant phenotype even when they contact an AN1 revertant cell on one end. Thus, the role of AN1 during the morphogenesis of seed coat cells is highly cell autonomous. Seed coats of an an11 line had the same morphology as an1 seeds, whereas seed coats of an2, an4, and an2 an4 double mutants had a wild-type morphology (data not shown). Given the restricted expression domains of AN2 and AN4, it is possible that seed coat morphogenesis requires a distinct member of this gene family. To determine whether the altered morphology of an1 and an11 seeds is caused by the block in flavonoid synthesis, we analyzed seeds in which the expression of flavonoid biosynthetic enzymes is blocked by either mutation or cosuppression. The epidermis of an3 seeds has a yellow color that apparently is caused by the absence of flavonoid pigments, but the size and the structure of the cells are similar to those of the wild type (Figure 6F). Similar results were obtained for seed coats of petunia lines in which the expression of CHS was blocked by cosuppression (data not shown). Thus, the altered morphology of an1 and an11 seed coat cells cannot be explained by the absence of flavonoid compounds. To determine whether the AN1 domains involved in the specification of seed coat morphology were separable from those regulating vacuolar pH or anthocyanin synthesis, we analyzed the seed coats from various an1 mutants. Plants heterozygous for the an1 alleles X2200 and W138 express, in petals, an approximately fourfold reduced amount of wild-type an1 mRNA. The seed coats of such plants, however, appear fully mutant, because they consist of small yellow cells with occasional patches of revertant cells, which presumably result from reversion of the W138 allele (Figure 6G). Also, seeds that are homozygous for an1-B2262 or an1-G621, alleles that strongly affect PAT expression and pH control but that have little or no effect on DFR expression and anthocyanin synthesis, appear to have a full mutant phenotype (Figure 6H).
In this article, we show that AN1 of petunia acts on at least two and possibly more pathways: the well-defined anthocyanin biosynthesis pathway, and one or more poorly defined pathways involved in the acidification of the vacuole and the morphogenesis of the seed coat epidermis.
Control of Intracellular pH
AN1 is required for the expression of genes that encode at least eight different enzymes of the anthocyanin pathway (reviewed by Mol et al., 1998
High anthocyanin pumping activity in the revertant cells would result in rapid vacuolar sequestration of anthocyanins, including those that arrive by diffusion from neighboring mutant cells with weak AN1 and pump activity, resulting in depletion of anthocyanins in those cells. Consistent with this idea, the halos are several cells wide (large revertant sectors have wider halos than small revertant spots) and have a sharp inner edge with white cells neighboring the full-colored revertant cells, whereas the outer edge consists of a gradient of cells with colors ranging from white to the normal mutant color. If this idea is true, it suggests that the unknown transporter also is downregulated in an1-G621 and ph6-m1 mutants, because An1+ revertant spots on such flowers also are surrounded by a white halo (Figures 2G and 2H) (Chuck et al., 1993
To date, two different transporters have been implicated in the transport of flavonoids: (1) the MATE-type (for multidrug and toxic compound extrusion) protein TT12, which is required for vacuolar localization of proanthocyanidins in the Arabidopsis seed coat (Debeaujon et al., 2001
Two genes have been identified that are coregulated by AN1 and one or more PH genes. One of them, MYB27, encodes a MYB domain protein of unknown function that is regulated by AN1 and PH4 (Mur, 1995
Control of Seed Coat Cell Morphology
After fertilization, the seed increases severalfold in size, which requires that the epidermis extends proportionally. This extension seems primarily the result of cell expansion, not cell division, because the epidermis of the ovule integument and the mature seed coat consist of a similar number of cells (Figure 6). Cell expansion is accompanied by other morphological changes, such as extensive thickening of the cell wall and formation of starch granules (Colombo et al., 1997 At least two explanations can account for this phenotype, and they are not mutually exclusive. First, the primary role of AN1 may be to prevent the growing epidermal cells from undergoing cell division. Alternatively, the primary role of AN1 may be in cell enlargement, and the induction of cell division in an1 seeds may be a secondary effect that is induced indirectly, for instance, by the more rapid expansion of underlying cell layers. Although the mechanism by which AN1 controls seed coat development is unclear, some possibilities can be excluded. First, alterations in the flavonoid metabolism of an1 seeds cannot account for the observed morphological changes, because these are not seen in seeds in which the expression of the enzyme F3H (an3 mutants) or CHS (cosuppressed lines) is blocked. The synthesis of flavonoid (polymers) apparently is not required for the enlargement of these epidermal cells or for the thickening of their walls, because these processes show little or no alterations in an3 mutants or chs cosuppressed lines. Because the mutations an1-G621 and an1-B2262, which in the flower primarily affect vacuolar acidification, have strong effects on the size and shape of seed coat cells, one may argue that this is an indirect result of the misregulation of vacuolar pH. However, we consider this possibility unlikely, because the seed coats of the known ph mutants consist of cells of nearly normal size and shape, even though some of the mutations affect seed color. Therefore, we favor the hypothesis that AN1 may control a third set of genes, in addition to those involved in anthocyanin synthesis and pH control, that is required for the normal morphology of seed coat cells.
Role of the bHLH Domain
Transient expression experiments showed that deletion of the bHLH domain of B or R diminished the transcriptional activation of the BRONZE1 target promoter by only 50%, suggesting that the bHLH domain has a marginal role (Goff et al., 1992 Mutational analyses of AN1, which is functionally and evolutionarily distinct from but structurally related to R, shed more light on this issue. Insertions of one, two, or three amino acids into the AN1 bHLH domain (as in the mutants W211R1 to W211R3) result in a progressive reduction of the capacity (but not in the amount) of AN1 to induce the expression of PAT1 and DFR and to drive anthocyanin synthesis and vacuolar acidification. This indicates that the bHLH domain is involved in the activation of anthocyanin synthesis and vacuolar acidification. The removal of the C-terminal half of AN1, including the bHLH domain, while the amount of AN1 is kept approximately normal (as in an1-X2366 mutants) reduces anthocyanin synthesis and DFR expression strongly (to approximately the same extent as the insertion of two or three amino acids in the HLH region), but not completely. Although this result seems to indicate that AN1 is more sensitive to C-terminal deletions than is R or B, this is not certain. First, we cannot exclude the possibility that the transcription activation capacity of AN1-X2366 is underestimated as a result of difficulties with nuclear import. Second, because the bHLH deletion derivatives of R and B were analyzed in transient assays only, the stability and the specific activity of mutant R/B proteins are unknown.
Strikingly, removal of the C-terminal part of AN1 affects vacuolar acidification, seed coat morphogenesis, and PAT1 expression much stronger than anthocyanin synthesis or DFR expression. When overexpressed at levels This fact indicates that the C-terminal region of AN1, including the bHLH domain, plays a more prominent role in vacuolar acidification and seed coat morphogenesis than it does in anthocyanin synthesis, which may be the reason for the evolutionary conservation of this domain. Whether this is because AN1 regulates these different processes by interactions with different protein partners or via different cis-acting elements in the target gene promoters is the subject of further research.
Evolutionary Aspects
In Arabidopsis, TTG controls hair cell fate by interaction with MYB proteins (GLABROUS1 [GL1] and WEREWOLF [WER]; Lee and Schiefelbein, 1999
However, the multicellular trichomes of Solanaceae appear to be specified by regulators that are distinct from those that specify the fate of the unicellular Arabidopsis hairs. First, neither mutations nor ectopic overexpression of AN1, AN2, and AN11 results in an obvious trichome phenotype (Quattrocchio et al., 1998
Similarly the function of AN1 and AN11 in seed coat morphogenesis appears to be less widespread than their function in anthocyanin synthesis, because mutations in the homologous Arabidopsis genes seem to have little effect on the size and shape of seed coat epidermal cells (Debeaujon et al., 2000
Petunia Lines and Mutants Unless stated otherwise, all mutant lines used in this study were generated in the genetic background of the wild-type Petunia hybrida line R27. The alleles an1-W17 (formerly an1s/-+) and an1-W138 (formerly an1s/p-+) were isolated in progeny of R27 (Bianchi et al., 1978 ; Doodeman et al., 1984). an1-W219 was isolated in progeny of an1-W17 (T. Gerats and P. de Vlaming, unpublished data). The insertion alleles Z2083, B2261, and W2175 were identified among progeny of an1-W138 (van Houwelingen et al., 1998; this work). The excision allele X2200, derived from an1-W138, has been described previously (Spelt et al., 2000). The insertion alleles G621 (P. de Vlaming, unpublished data) and X2366 (van Houwelingen et al., 1998) arose in genetic backgrounds that were not related directly to R27.
Scanning Electron Microscopy
pH of Petal Extracts
Molecular Analysis of Mutant an1 Alleles
Transient Expression Assays
Details regarding the fusion genes 35S:AN2, DFR:LUCIFERASE, and 35S:
RNA Analyses
Expression of AN1 in Escherichia coli and Immunological Methods
Distribution of Biomaterials
Accession Numbers
We thank Saskia Kars for help with scanning electron microscopy, Daisy Kloos for help with the genetic experiments, and Pieter Hoogeveen and Martina Meesters for their care of the plants.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003772. Received April 9, 2002; accepted June 4, 2002.
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Abstract
INTRODUCTION
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.
