Petunia

By: Merrilee G. Anderson

Anthocyanins are responsible for many of the vivid colors we see in higher plants. The variety of flower color attributable to flavonoid compounds ranges from creamy yellow to pale pink to flaming red and from pale blue to bright violet. Petunia hybrida has been extensively studied out of an interest to develop new and different flower colors for commercial operations. In petunia, all anthocyanins found in flowers are glucosylated at the 3' position and may also possess a glucose group at the 5' position. Anthocyanins can be found in various petunia flower organs including the pollen wall (Jonsson et al. 1984). In seeds, chs, chi, and dfr genes are activated in the pericarp, resulting in the synthesis of an as yet undefined class of flavonoids, likely proanthocyanididins (Quattrocchio et al. 1993) Current applications to modulating flower color include changing the amounts and/or specificity of the pigment produced. (Courtney-Gutterson 1994) Because the action of genes controlling anthocyanin pigment biosynthesis can be detected visually, numerous mutants have been created in which pigmentation is blocked or spatially altered.

In petunia, genes have been identified for enzyme regulation of the anthocyanin biosynthetic pathway. In 1991, (Dooner et al. 1991) reported three loci, po, an3, and an6 had been identified for effects on enzymes CHI, F3H, and DFR respectively. More recently (Kroon et al. 1994) distinguish at least 30 genes controlling pigmentation of specific floral organs. Their focus on an1, an2, and an11 as the primary sites of regulation in petunia flower color is devoted to analysis of locus Rt. The Rt locus controls the rhamnosylation of reddish anthocyanin-3-O-glucosides which are the first in a series of modifications leading to magenta or blue/purple anthocyanins. The an1, an2, and an11 loci comprise a class that controls the activity of multiple enzymes of the pathway including DFR, UDP-glucose, UF3GT, UF5GT, and four anthocyanin methyl transferases. They conclude that the Rt locus encodes an anthocyanin rhamnosyl transferase.

The regulation of the biosynthetic pathway has also been well characterized. In petunia, regulation was identified at DFR and UF3GT by mutations at loci of four regulatory genes: an1, an2, an10, and an11. Mutant plants accumulate dihydroflavonols suggesting that CHS, CHI and F3H are not regulated, indicating that regulation occurs from DFR onwards. This is one step further than in Antirrhinum . This has led researchers to speculate that petunia and snapdragon select for synthesis of flavones and flavonols, products of the flavonoid pathway that contribute to copigmentation these species but not in maize. (Dooner et al. 1991) This has been supported by Quattrocchio et al. (1993) with findings that separate units of structural anthocyanin genes regulate anthocyanin production in petal limbs and anthers of petunia. These two separately regulated pathways are conserved in snapdragon, with division of the two before F3H, and not after F3H as in petunia.

Regulation by the five genes an1, an2, an6, an10, and an11 results as control of the steady state level of dfr mRNA in the petal. The an6 gene appears to be identical to dfrA genes, while the four other genes an1, an2, an10, and an11 likely act in trans on dfr expression. Mutations in petunia an1, an2,and an11 genes can be complemented by maize Lc and C1 factors, suggesting that regulation is functionally similar. (Quattrocchio et al. 1993).

Expression of maize and petunia flavonoid biosynthetic genes is also subject to control by phytohormones. For example, transcriptional activation of chs genes in the flower corolla of petunia is dependent on gibberellic acid, while abscisic acid is involved in pigmentation of the maize aleurone layer (Quattrocchio et al. 1993).

More recent studies of the regulatory genes have explained differences in regulation through divergent evolution of target anthocyanin genes (Quattrocchio et al. 1998). Four petunia loci, an1, an2, an4, and an11 are compared with two maize loci r and c1. Both an2 and c1 were found to encode MYB-type proteins. Isolation of a P. hybrida gene, jaf13, was found to encode a helix-loop-helix protein representing an orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 activated the dfrA promoter to enhance pigment accumulation, and indicated tissue-specific expression. In Z. mays, expression of an2 complements a mutation in pl, a c1 paralogue, indicating activation of a wider set of target genes by an2 in corn. Results indicate conserved regulatory anthocyanin genes between species and suggest that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks. Analysis of distribution of flavonoids among plant species and conserved sequence homology has allowed proposed models in the evolution of flavonoid biosynthesis (Quattrocchio et al. 1993, Quattrocchio et al. 1998). In this regard, petunia has served as a model for anthocyanin experimentation.

[LINK TO PETUNIA BIOSYNTHETIC PATHWAY]

[CONCLUSIONS]

REFERENCES

Courtney-Gutterson, N. 1994. The biologist's palette: genetic engineering of anthocyanin biosynthesis and flower color. In, Genetic Engineering of Plant Secondary Metabolism, Ellis, B.E., G.W. Kuroki, and H.A. Stafford, eds. Plenum Press NY. 93:124.

Dooner, H.K., T.P. Robbins, and R.A. Jorgensen. 1991. Annu. Rev. Genet. 25:173-199.

Gutterson, N. 1995. Anthocyanin biosynthetic genes and their application to flower color modification through sense suppression. HortSci. 30(5):964966.

Kroon, J, E. Souer, A. de Graaff, UY. Xue, J. Mol and R. Koes. 1994. Cloning and structural analysis of the anthocyanin pigmentation locus Rt of Petunia hybrida: characterization of insertion sequences in two mutant alleles. Plant Journal. 5(1):69-80.

Jonsson, L.M.V., M.E.G. Aarsman, J. van Diepen, P. de Vlaming, N. Smit, and A.W. Schram. 1984. Properties and genetic control of anthocyanin 5-O-glucosyltransferase in flowers of Petunia hybrida. Planta. 160:341-347.

Quattrocchio, F., J.F. Wing, H.T.C. Leppen, J. N.M. Mol, and R.E. Koes. 1993. Regulatory genes controlling anthocyanin pigmentation are functionally conserved among plant species and have distinct sets of target genes. Plant Cell. 5:1497-1512.

Quattrocchio, F., J.F. Wing, K. van der Woude, J.N.M. Mold, and R. Koes. 1998. Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes. Plant J. 13(4):475-488.