The plasma membrane in plant cells is energized with an electrical potential and proton gradient generated through the action of H
+
pumps belonging to the P-type ATPase superfamily. The
Arabidopsis
genome encodes 11 plasma membrane H
+
pumps. Auto-inhibited H
+
-ATPase isoform 10 (AHA10) is expressed primarily in developing seeds. Here we show that four independent gene disruptions of
AHA10
result in seed coats with a
transparent testa
(
tt
) phenotype (light-colored seeds). A quantitative analysis of extractable flavonoids in
aha10
seeds revealed an ≈100-fold reduction of proanthocyanidin (PA), one of the two major end-product pigments in the flavonoid biosynthetic pathway. In wild-type seed coat endothelial cells, PA accumulates in a large central vacuole. In
aha10
mutants, the formation of this vacuole is impaired, as indicated by the predominance of multiple small vacuoles observed by fluorescence microscopy using a vacuole-specific dye, 5-(and -6)-carboxy 2′,7′-dichlorofluorescein diacetate. A similar vacuolar defect was also observed for another
tt
mutant,
tt12
, a proton-coupled multidrug and toxic compound extrusion transporter potentially involved in loading provacuoles with a flavonoid intermediate required for PA production. The endothelial cells in
aha10
mutants are otherwise healthy, as indicated by the lack of a significant decrease in (
i
) the accumulation of other flavonoid pathway end products, such as anthocyanins, and (
ii
) mRNA levels for two endothelium-specific transcripts (
TT12
and
BAN
). Thus, the specific effect of
aha10
on vacuolar and PA biogenesis provides genetic evidence to support an unexpected endomembrane function for a member of the plasma membrane H
+
-ATPase family.
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