A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana

doi:10.1073/pnas.0406377102

A plasma membrane H⁺-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana

^*Center for Phytoremediation Research and Development, ^¶Purdue–UT-Pan American Phytoremediation Program, and ^∥Department of Horticulture, Purdue University, West Lafayette, IN 47907; ^§Department of Biology, Western Washington University, Bellingham, WA 98225; ^**Biochemistry Department, University of Nevada, MS200, Reno, NV 89557; and ^†The Scripps Research Institute, La Jolla, CA 92037

Edited by Maarten Koornneef, Max Planck Institute for Plant Breeding Research, Cologne, Germany (received for review August 31, 2004)

Abstract

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.

Footnotes

↵ †† To whom correspondence should be addressed. E-mail: jfharper{at}unr.edu.
↵ ‡ I.R.B. and J.C.Y. contributed equally to this work.
Author contributions: J.C.Y., W.A.P., A.S.M., and J.F.H. designed research; J.C.Y., G.A., N.F., N.B., T.C., W.A.P., S.P.H., and A.S.M. performed research; J.C.Y., W.A.P., S.P.H., A.S.M., and J.F.H. analyzed data; and J.F.H. wrote the paper.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: AHA10, autoinhibited H⁺-ATPase isoform 10; PA, proanthocyanidin; MATE, multidrug and toxic compound extrusion; Ws, Wassilewskija; DMACA, dimethylaminocinnamaldehyde; tt, transparent testa; T-DNA, transfer DNA; LDOX, leucoanthocyanidin dioxygenase; BAN, banyuls.