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Petunia embryos carrying the no apical meristem (nam) mutation fail to develop a shoot apical meristem. Occasional shoots on nam- seedlings bear flowers that develop ten instead of five primordia in the second whorl. Double mutants with the homeotic gene green petals show that nam acts independently of organ identify in whorl 2 and now also affects primordium number in whorl 3. The nam gene was isolated by transposon tagging. The encoded protein shares a conserved N-terminal domain with several other proteins of unknown function and thus represents a novel class of proteins. Strikingly, nam mRNA accumulates in cells at the boundaries of meristems and primordia. These data indicate a role for nam in determining positions of meristems and primordia.

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Flavonoids are a class of low molecular weight phenolic compounds that is widely distributed in the plant kingdom. They exhibit a diverse spectrum of biological functions and play an important role in the interaction between plants and their environment. Flavonoids not only protect the plant from the harmful effects of UV irradiation but also play a crucial role in the sexual reproduction process. A special class of flavonoid polymers, the tannins, plays a structural role in the plant. Yet other classes of flavonoids, flavonols and anthocyanins, have been implicated in the attraction of pollinators. Certain flavonoids participate in the interaction between plants and other organisms such as symbiotic bacteria and parasites. This raises the intriguing question as to how these different compounds arose and evolved. Based on taxonomy and molecular analysis of gene expression patterns it is possible to deduce a putative sequence of acquisition of the different branches of the biosynthetic pathway and their regulators.

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

In petunia flowers, the loci anl, an2, and anll control the pigmentation of the flower by stimulating the transcription of anthocyanin biosynthetic genes. The anl and an2 locus were recently cloned and encode a basic helix-loop-helix (bHLH) and MYB-domain transcriptional activator, respectively. Here, we report the isolation of the anll locus by transposon tagging. RNA gel blot experiments show that anll is expressed independently from anl and an2 throughout plant development, as well as in tissues that do not express the anthocyanin pathway. It encodes a novel WD-repeat protein that is highly conserved even in species that do not produce anthocyanins such as yeast, nematodes, and mammals. The observation that the human anll homolog partially complements the anll petunia mutant in transient assays shows that sequence similarity reflects functional conservation. Overexpression of an2 in anll-petals restored the activity of a structural anthocyanin gene in transient assays, indicating that AN11 acts upstream of AN2. Cell fractionation experiments show that the bulk of the ANll protein is localized in the cytoplasm. Taken together, this indicates that ANll is a cytoplasmic component of a conserved signal transduction cascade that modulates AN2 function in petunia, thereby linking cellular signals with transcriptional activation.

The regulatory anthocyanin loci, an1, an2, an4 and an11 of Petunia hybrida, and r and c1 from Zea mays, control transcription of different sets of target genes. Both an2 and c1 encode a MYB-type protein. This study reports the isolation of a P. hybrida gene, jaf13, encoding a basic helix-loop-helix protein that, on the basis of sequence homology and intron/exon structure, represents the P. hybrida orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 is sufficient for activation of the dihydroflavonol 4-reductase-A (dfrA) promoter and enhanced pigment accumulation in P. hybrida. This indicates that an2 and jaf13 play a key role in determining the tissue-specific expression pattern of structural genes. However, because chalcone synthase (chs) and flavanone-3-hydroxylase (f3h) are not activated, the pattern of pigmentation is not fundamentally altered. Expression of an2 in Z. mays complements a mutation in pl, a c1 paralogue, indicating that an2 activates a wider set of target genes in this host. Transient expression assays in Z. mays and P. hybrida tissues showed that C1 and R or AN2 and JAF13 can activate the promoter of the c2 gene, encoding Z. mays CHS, but not the chsA promoter from P. hybrida. These results indicate that regulatory anthocyanin genes are conserved between species and that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks.

The shape and color of flowers are important for plant reproduction because they attract pollinators such as insects and birds. Therefore, it is thought that alterations in these traits may result in the attraction of different pollinators, genetic isolation, and ultimately, (sympatric) speciation. Petunia integrifolia and P. axillaris bear flowers with different shapes and colors that appear to be visited by different insects. The anthocyanin2 ( an2 ) locus, a regulator of the anthocyanin biosynthetic pathway, is the main determinant of color differences. Here, we report an analysis of molecular events at the an2 locus that occur during Petunia spp evolution. We isolated an2 by transposon tagging and found that it encodes a MYB domain protein, indicating that it is a transcription factor. Analysis of P. axillaris subspecies with white flowers showed that they contain an2 Ϫ alleles with two alternative frameshifts at one site, apparently caused by the insertion and subsequent excision of a transposon. A third an2 ؊ allele has a nonsense mutation elsewhere, indicating that it arose independently. The distribution of polymorphisms in an2 ؊ alleles suggests that the loss of an2 function and the consequent changes in floral color were not the primary cause for genetic separation of P. integrifolia and P. axillaris. Rather, they were events that occurred late in the speciation process, possibly to reinforce genetic isolation and complete speciation. INTRODUCTIONFlowers are the structures containing the male and female sex organs of angiosperms. Flowers of diverse species display a wide range of different morphologies and pollination strategies. For instance, flowers of wind-pollinated species usually possess small and inconspicuous petals or no petals at all, whereas flowers of insect-pollinated plants usually possess large, brightly colored, and patterned petals that serve as visual signals and a landing site for visiting insects.Recent experiments suggest that the wide variety of plant and flower morphologies may have depended on the evolution of a relatively small number of genes. First, mutations at single loci, usually isolated by breeders or researchers, are sufficient to cause fundamental alterations in inflorescence architecture (Doebley et al., 1997;Souer et al., 1998). Similarly, the different shapes, colors, and color patterns of naturally occurring Mimulus (monkeyflower) spp are due to alterations at only a few (major) loci (Bradshaw et al., 1995).Second, even very different inflorescence and flower architectures appear to be determined by genes that often encode conserved proteins but that differ in their expression patterns (reviewed in Doebley and Lukens, 1998).The isolation of key regulatory loci and analysis of the molecular alterations that have taken place in them will provide new insights into the evolution and diversification of flower morphology. The biosynthesis of anthocyanin flower pigments is particularly suited for such studies, because it is a well-defined biochemical pathway that is being...