title: A plasma membrane H+-ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana.
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 approximately 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.
authors.affiliation: Center for Phytoremediation Research and Development, Purdue University, West Lafayette, IN 47907, USA.
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Baxter
authors.forename: Jeffery C
authors.initials: JC
authors.lastname: Young
authors.forename: Gordon
authors.initials: G
authors.lastname: Armstrong
authors.forename: Nathan
authors.initials: N
authors.lastname: Foster
authors.forename: Naomi
authors.initials: N
authors.lastname: Bogenschutz
authors.forename: Tatiana
authors.initials: T
authors.lastname: Cordova
authors.forename: Wendy Ann
authors.initials: WA
authors.lastname: Peer
authors.forename: Samuel P
authors.initials: SP
authors.lastname: Hazen
authors.forename: Angus S
authors.initials: AS
authors.lastname: Murphy
authors.forename: Jeffrey F
authors.initials: JF
authors.lastname: Harper
mesh_terms: D000872:Anthocyanins / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002392:Catechin / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D018744:DNA, Plant / Q000235:genetics; D004727:Endothelium / Q000378:metabolism; D017343:Genes, Plant; D005816:Genetic Complementation Test; D009154:Mutation; D010641:Phenotype; D030821:Plants, Genetically Modified; D044945:Proanthocyanidins / Q000096:biosynthesis*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018749:RNA, Plant / Q000235:genetics / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
chemical_list: D000872:Anthocyanins; D018744:DNA, Plant; D044945:Proanthocyanidins; D012333:RNA, Messenger; D018749:RNA, Plant; D002392:Catechin; D006180:Proton-Translocating ATPases