Colour-enhancing protein in blue petals

Spectacular morning glory blooms rely on a behind-the-scenes proton exchanger.

Abstract

The coloration of blue flowers depends on the production of the appropriate anthocyanin pigments, the presence of metal ions and co-pigments, and the vacuolar pH¹. An increase in vacuolar pH enhances blue coloration^1,2, but little is known about the proteins responsible for raising the vacuolar pH. Here we show that a mutant purple-flowering Japanese morning glory (Ipomoea nil) plant that carries a recessive mutation in the gene encoding a vacuolar Na⁺/H⁺-exchanger is unable to increase its vacuolar pH to create the normal bright blue petals.

Main

In the morning glory Ipomoea tricolor, reddish-purple buds unfurl into blue flowers. As the bloom opens, the vacuolar pH in the flower epidermis increases from 6.6 to 7.7 (ref. 2). The colour is very similar to that of I. nil, and the flowers of both plants contain the same anthocyanin as their principal pigment^2,3.

Ipomoea nil plants bearing the purple-mutable (pr-m; Fig. 1a) allele in the Purple (Pr) locus have purple flowers with blue sectors⁴ (Fig. 1a) owing to recurrent somatic mutation from the recessive pr-m to the blue Purple-revertant (Pr-r; Fig. 1b) allele. We have produced germinal revertants (heterogenotes, Pr-r/pr-m ) that have blue flowers (Fig. 1b) and, after self-pollination, we chose pairs of siblings carrying either the pr-m or Pr-r allele homozygously. We found no difference in the pigment composition of the pr-m and Pr-r lines, but the pH of the sap from the Pr-r flower epidermis was higher than that in pr-m by about 0.7, indicating that the pr mutant fails to increase the vacuolar pH.

Figure 1: Ipomoea nil flower phenotypes and the structure of the Pr gene.

a, Mutable line (pr-m). b, Germinal revertant ( Pr-r). c, The Pr gene. White and blue boxes represent the Pr exons and open reading frame, respectively. d, Sequences at the Tpn4 insertion site in the Pr-r (wild type), pr-m and Pr-r lines.

As Tpn1-related transposons are a common spontaneous mutagen in I. nil⁵, we used a transposon-display procedure⁶ to identify the Pr gene. Sequencing of Pr complementary DNA (accession no. AB033989) revealed that it contains an open reading frame that is highly homologous to the Arabidopsis and rice vacuolar Na⁺/H⁺-exchangers^7,8 and that it complements the yeast nhx1 mutation⁷ (our unpublished results), confirming that the Pr gene encodes a vacuolar Na⁺/H⁺-exchanger. We named this transporter InNhx1 (for I. nil Nhx1).

The Pr gene itself (accession no. AB033990) comprises 15 exons and a Tpn1-related element, Tpn4, which is integrated into its first exon in the pr-m lines (Fig. 1c). This integration of Tpn4 generates a 3-base-pair target duplication, and the germinal revertants studied here contain footprints generated by Tpn4 excision (Fig. 1d). Moreover, blue sectors of the variegated flowers contain footprints that result from somatic excision of Tpn4, whereas we found no evidence of such excision in the purple regions of these flowers in six different pr-m lines. To our knowledge, the Pr gene encoding InNhx1 is the first gene to be identified that increases the vacuolar pH in petals in order to confer blue coloration.