A new small subunit of the mitochondrial F-ATPase, PhDC, determines flower color by acidifying vacuoles

The pH within the plant vacuole determines the anthocyanin absorption spectrum and affects flower color. V-ATPase, P-ATPase, and pyrophosphatase proton pumps involved in vacuolar acidification have been reported. However, whether other proteins regulate vacuolar pH in Petunia hybrida remains unclear. Here, by using VIGS technology to silence numerous genes annotated as 'unknown protein', we identified a gene whose silencing darkened petal color and named it PhDC (DEEPENING COLOR). PhDC silencing mediated by RNAi also darkened petal color. PhDC has low similarity to yeast mitochondrial F-ATPase subunit and Arabidopsis ATP17 (a predicted mitochondrial F-ATPase subunit). PhATPd and PhATPO, encoding the mitochondrial F-ATPase subunit d and OSCP, respectively, shared similar spatiotemporal expression with PhDC. PhDC localized to mitochondria and interacted with four subunits of mitochondrial F-ATPase. PhDC, PhATPd, and PhATPO silencing led to a similar phenotype in petunia. PhDC silencing reduced mitochondrial F-ATPase activity and impaired mitochondria. The anthocyanin content in PhDC-silenced petals did not significantly change, while the vacuoles of petal cells were alkalized, and the P-ATPase activity and ATP content were reduced. These results suggest that PhDC is a new subunit of the mitochondrial F-ATPase. Furthermore, PhDC silencing affects petal color by altering the vacuole pH and P-ATPase activity.