Identification of AN1, PH4 and PH3 target genes through an mRNA profiling approach

Proton gradients are the endocellular forces required to drive the transport of ions and other microand macromolecules across membranes. In petunia, mutations in ANTHOCYANIN 1, (AN1), AN2, and AN11 and in PH1-PH7 affect the pH of the vacuolar lumen. Because AN1, AN2, AN11 and PH2, PH3, PH4 and PH6 encode regulatory proteins, they are likely to control transcription of genes involved in an unknown vacuolar pH-regulating pathway. In an mRNA-profiling assay (micro-array and cDNA-AFLP) we isolated of a set of transcripts, which expression is decreased in an1, ph3 and ph4 mutants. All identified target genes are unaffected in ph2 mutants, suggesting that PH2 operates in a different pHregulating pathway or that PH2 modifies proteins at post-translational level. Transgenic plants in which the expression of two of these target genes was reduced by RNA interference showed blue flowers and increased vacuolar pH in the petal epidermis, demonstrating the role of these genes in acidification of the vacuolar lumen. MAC9F1 shows similarity to proteins with unknown function whereas MACF55 encodes a proton pump. As none of the already described regulators of vacuolar pH (vacuolar-ATPases, pyrophosphatases and enzymes for synthesis of organic acids) are present in this collection of target genes, it is possible that a novel acidification pathway involving not yet described structural genes is responsible for pH regulation of the lumen of anthocyanin accumulating vacuoles in flower petals.