Molecular Characterization of Mutant Arabidopsis Plants with Reduced Plasma Membrane Proton Pump Activity

Abstract

Arabidopsis mutants containing gene disruptions in AHA1 and AHA2, the two most highly expressed isoforms of the In animals, the sodium pump is the primary active transport system and creates a membrane potential and sodium gradient that are used by all ion channels and cotransporters (1, 2). In higher plants and fungi, however, the transport of all solutes across the plasma membrane is coupled to a proton gradient rather than a sodium gradient. Thus, in these organisms, a plasma membrane proton pump creates a protonmotive force at the plasma membrane that drives all channels and cotransporters. Given the known importance of transport at the plasma membrane for life functions, it is not surprising that genetic studies of the sodium pump in nematodes, fruit flies, zebrafish, and mice, as well as with the proton pump of yeast, all conclusively demonstrate the lethal effects of loss-of-function mutations for a gene encoding the primary active transporter (Table 1) (3-11). In contrast, although there have been several reports of altered growth of mutant plants containing genetic alterations in the plasma membrane proton pump (12-17), none of these studies have provided evidence indicating that this enzyme performs an essential function for plant life. In this study, we present evidence clearly demonstrating that the plasma membrane proton pump is essential for plant growth. We show that AHA1 and AHA2 (for Arabidopsis H ϩ -ATPase isoforms 1 and 2), the two most highly expressed members of the AHA gene family, perform overlapping functions that mask the lethality in single gene loss-of-function mutants. We also describe phenotypic screening that supports the in planta role of the proton pump in generating a protonmotive force and mass spectrometric methods that allow a more detailed and quantitative analysis of the in vivo regulation of these proteins at the post-translational level. (aha1-6, SALK016325; aha1-7, SALK065288; and aha1-8, SALK118350) and AHA2 (aha2-4, SALK082786, and aha2-5, SALK022010) were obtained from the Arabidopsis Biological Resource Center (Ohio State University) (18). Seeds were germinated on plates containing half-strength M&S 3 salts, 1% (w/v) sucrose, and 0.7% (w/v) agar. Plants that were transferred to soil/perlite mixture (Jiffy-Mix, Jiffy Products of America, Lorrain, OH; horticultural perlite, The Schundler Co., Metuchen, NJ) were grown at 21°C under constant light or 22°C with a regime of 16 h of light/8 h of dark. EXPERIMENTAL PROCEDURES Plant Materials and Growth Conditions-Mutants (ecotype Columbia) carrying T-DNA insertions in AHA1T-DNA Mutant Identification and Plant Genotyping-Plant genomic DNA was extracted using the method of Krysan et al. (19), with the elimination of the phenol/chloroform extraction step. The location of the T-DNA insertion in AHA1 or AHA2 was determined by sequencing PCR fragments containing the * This work was supported by grants from the Department of Energy and the National Science Foundation (to M. R. S.) and by National Science Foundation Grant MCB-0619...

An Arabidopsis Plasma Membrane Proton ATPase Modulates JA Signaling and Is Exploited by the Pseudomonas syringae Effector Protein AvrB for Stomatal Invasion

“…In addition, the gain-of-function mutant ost2-2D exhibits constitutive COI1-JAZ9 interaction in the absence of exogenous JA. Although the functional redundancy of AHA1 and AHA2 and lethality of the loss-of-function aha1 aha2 double mutant (Arango et al, 2003;Haruta et al, 2010) prevented us from testing if the PM H + -ATPases are required for JA signaling and AvrB-induced stomatal opening, it is highly likely that RIN4 and AHA1 act upstream of the COI1-JAZ interaction to regulate JA signaling and stomatal opening. Consistent with this possibility, the constitutive expression of JA response genes and enhanced disease susceptibility in ost2-2D is largely abolished by the coi1 mutation.…”

Phytosulfokine Regulates Growth in Arabidopsis through a Response Module at the Plasma Membrane That Includes CYCLIC NUCLEOTIDE-GATED CHANNEL17, H+-ATPase, and BAK1

“…To explore the possibility that PSK signaling of protoplast expansion may involve PM H + -ATPase, we tested for interactions of PSKR1 and CNGC17 with AHA1 and AHA2, the two most highly expressed and crucial isoforms of PM H + -ATPase (Haruta et al, 2010). Strong interactions of PSKR1-Nub and CNGC17-Nub with AHA1-Cub and AHA2-Cub were observed (Figures 3A and 3B).…”

SORTING NEXIN1 Is Required for Modulating the Trafficking and Stability of the Arabidopsis IRON-REGULATED TRANSPORTER1

“…This was found to be only a small part of the extensive posttranscriptional regulation of the iron uptake system (Brumbarova and Bauer, 2005;Brumbarova et al, 2008;Donnini et al, 2010;Rellán-Alvarez et al, 2010;Lan et al, 2011). AHA1 and AHA2 activity is regulated by phosphorylation of a single amino acid (Fuglsang et al, 2007;Haruta et al, 2010). FRO2 activity is upregulated only under iron deficiency, even if the gene is artificially expressed under sufficient iron supply (Connolly et al, 2003).…”