TRANSPARENT TESTA 13 is a tonoplast P_3A‐ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

Abstract

SUMMARYIntracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H + -ATPases in the plasma membrane, and multimeric vacuolar-type H + -ATPases (V-ATPases) and vacuolar H + -pyrophosphatases (H + -PPases) in endomembranes. Here, we show that reduced accumulation of proanthocyanidins (PAs) and hence the diminished brown seed coloration found in the Arabidopsis thaliana mutant transparent testa 13 (tt13) is caused by disruption of the gene encoding the P 3A -ATPase AHA10. Identification of the gene encoded by the tt13 locus completes the molecular characterization of the classical set of transparent testa mutants. Cells of the tt13 seed coat endothelium do not contain PA-filled central vacuoles as observed in the wild-type. tt13 phenocopies tt12, a mutant that is defective in vacuolar import of the PA precursor epicatechin. Our data show that vacuolar loading with PA precursors depends on TT13. Consistent with the tt13 phenotype, but in contrast to other isoforms of P-type H + -ATPases, TT13 localizes to the tonoplast. PA accumulation in tt13 is partially restored by expression of the tonoplast localized H + -PPase VHP1. Our findings indicate that the P 3A -ATPase TT13 functions as a proton pump in the tonoplast of seed coat endothelium cells, and generates the driving force for TT12-mediated transport of PA precursors to the vacuole.

Job Sharing in the Endomembrane System: Vacuolar Acidification Requires the Combined Activity of V-ATPase and V-PPase

“…In Arabidopsis, AHA10 is required for the vacuolar accumulation of proanthocyanidins (Baxter et al, 2005) and has recently been shown to be localized at the tonoplast of seed coat endothelial cells (Appelhagen et al, 2015). In agreement with its preferential expression in the integument layers of developing seeds, no other phenotypes than the altered seed color have been reported for aha10 mutants.…”

“…Although a slight increase of vacuolar pH was detected in the roots of wild-type, vha-a2 vha-a3, and fugu5-1 plants, vacuolar pH in the triple mutant fugu5-1 vha-a2 vha-a3 was not affected by ortho-vanadate ( Figure 7A). Moreover, although transcripts of AHA10, the only tonoplast-localized P-type H + -ATPase of Arabidopsis identified to date (Appelhagen et al, 2015), are detectable in roots, transcript levels are not increased in any of the V-ATPase and/or V-PPase mutants (Supplemental Figure 5B). In contrast, the V-ATPase inhibitor concanamycin A (ConcA) abolished vacuolar acidification in all genotypes tested, including vha-a2 vha-a3 and fugu5-1 vha-a2 vha-a3 ( Figure 7B), leading to the accumulation of autophagic bodies without affecting BCECF accumulation or vacuolar morphology ( Figure 7C).…”

“…In recent years, it has become clear that P-type H + -ATPases can be found at the tonoplast in some cell types (Verweij et al, 2008;Appelhagen et al, 2015), and we thus assessed the effect of ortho-vanadate, an inhibitor of P-type ATPases on media acidification (Supplemental Figure 5A), and vacuolar pH in roots using BCECF. Although a slight increase of vacuolar pH was detected in the roots of wild-type, vha-a2 vha-a3, and fugu5-1 plants, vacuolar pH in the triple mutant fugu5-1 vha-a2 vha-a3 was not affected by ortho-vanadate ( Figure 7A).…”

Functionally Similar WRKY Proteins Regulate Vacuolar Acidification in Petunia and Hair Development in Arabidopsis

“…The finding that blue flowering soybean (Glycine max) mutants have lesions in the apparent PH4 homolog (Takahashi et al, 2013) and that AHA10 is homologous and functionally interchangeable with PH5 (Appelhagen et al, 2015) suggests that the role of MBW complexes in vacuolar acidification is also widespread. The role of the MBW complex in specification of trichome or atrichoblast fate, by contrast, was established later in evolution as it is seen in some Rosid species only (Arabidopsis) and not in dicot species belonging to the Asterids or in monocots (Serna and Martin, 2006).…”

“…PH3 promotes vacuolar acidification via activation of PH5, which encodes a tonoplast H + pump that is also required for tannin accumulation in the seed (Verweij et al, 2008). Because a mutation in the Arabidopsis PH5 homolog AHA10 also abolishes tannin accumulation in the seed (Baxter et al, 2005;Appelhagen et al, 2015), just like ttg2, it is likely that TTG2 enables tannin accumulation, at least in part, by activating AHA10.…”