Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

Abstract

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...

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

“…pigmentation in the petal limb. These results suggest that an2 function is redundant with and taken over by other loci in distinct tissues (see Quattrocchio et al, 1998Quattrocchio et al, , 1999. In contrast, an1 is expressed in all tissues that express dfr and jaf13 (Figure 4).…”

“…gus and dfr mRNA amounts, expressed in arbitrary units, were determined by hybridization of RT-PCR products, quantification of the radioactivity by PhosphorImaging, and normalization to the amount of gapdh mRNA. (Quattrocchio et al, 1999), and those from derived An2 ϩ germinal revertants contained similar amounts of an1 transcripts (data not shown). However, because an2-W82 is not a null allele, this result is difficult to interpret.…”

“…However, because an2-W82 is not a null allele, this result is difficult to interpret. Therefore, we analyzed an1 transcripts in an F 1 hybrid of the lines W115 and W59, both of which contain frameshift an2 alleles that have completely lost the ability to activate dfrA (Quattrocchio et al, 1999), and compared them with an1 mRNA in complemented W115/W59 plants harboring a 35S-an2 transgene. Figure 7 shows that the 35S-an2 complementants express high amounts of an2 mRNA, which restores dfr expression (lanes 4 to 6) and pigmentation (see Quattrocchio et al, 1998).…”

“…The an2 locus encodes a MYB-domain protein that is functionally interchangeable with C1 from maize (Quattrocchio et al, 1998(Quattrocchio et al, , 1999. jaf13 of petunia and delila from snapdragon encode highly similar bHLH proteins thought to be orthologs of R1 from maize (Goodrich et al, 1992;Quattrocchio et al, 1998).…”

A chimeric repressor of petunia PH4 R2R3-MYB family transcription factor generates margined flowers in torenia

“…PhAN1, PhAN2 and PhAN11 are genes controlling both pigmentation and vacuolar pH in petunia petals. 8,9,10 Petunia also has a series of PhPH genes regulating vacuolar pH in the petals. 11,12,13 Because anthocyanins are accumulated in the vacuole and their colors change with pH (reddish at lower pH and bluish at higher pH), the PhPH genes also regulate petal color.…”