Inactivation of a tonoplast P-type ATPase reduces vacuolar acidification and results in a blue flower color

The pH control in different cell compartments is essential for the trafficking of vesicles, proteins and small molecules. Vacuolar type H + -ATPases (v-ATPases) and pyrophosphatases (PPases) in plants control the pH of endomembranes compartments whereas P-type H + -ATPases control the pH of the cytosol by pumping protons across the plasma membrane. In this way an electrochemical gradient is created, which is important for transport of ions across membranes. The regulatory proteins ANTHOCYANIN 1 (AN1), AN2 and AN11 control transcription of genes of the anthocyanin biosynthesis pathway. These same regulators, in combination with PH4 and PH3, induce genes of another pathway, which leads to the acidification of the vacuole. Here we report the isolation of one AN1, PH4 and PH3 target gene, MACF55, which encodes a P-type ATPase. Silencing of MACF55 results in blue flowers with a high pH of the crude petal homogenate indicating that MACF55 is directly involved in vacuolar acidification. Isolation of the PH5 locus from a transposon tagged mutant showed that it encodes MACF55. Complementation experiments showed that 35S:PH5 could fully complement ph5 mutants but not an1, ph3 and ph4, indicating that PH5 activity depends on other genes controlled by AN1, PH4 and PH3. PH5-GFP fusion protein localizes the tonoplast of cowpea protoplast, suggesting that PH5 is responsible for proton pumping from the cytosol into the vacuolar lumen. Thus, PH5 plays a key role in a trait (flower color), which is important for plant reproduction.