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. 2010 Jun 4;285(23):17918-29.
doi: 10.1074/jbc.M110.101733. Epub 2010 Mar 26.

Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity

Miyoshi Haruta  1 , Heather L Burch, Rachel B Nelson, Greg Barrett-Wilt, Kelli G Kline, Sheher B Mohsin, Jeffery C Young, Marisa S Otegui, Michael R Sussman
Affiliations

Molecular characterization of mutant Arabidopsis plants with reduced plasma membrane proton pump activity

Miyoshi Haruta et al. J Biol Chem. .

Abstract

Arabidopsis mutants containing gene disruptions in AHA1 and AHA2, the two most highly expressed isoforms of the Arabidopsis plasma membrane H(+)-ATPase family, have been isolated and characterized. Plants containing homozygous loss-of-function mutations in either gene grew normally under laboratory conditions. Transcriptome and mass spectrometric measurements demonstrate that lack of lethality in the single gene mutations is not associated with compensation by increases in RNA or protein levels. Selected reaction monitoring using synthetic heavy isotope-labeled C-terminal tryptic peptides as spiked standards with a triple quadrupole mass spectrometer revealed increased levels of phosphorylation of a regulatory threonine residue in both isoforms in the mutants. Using an extracellular pH assay as a measure of in vivo ATPase activity in roots, less proton secreting activity was found in the aha2 mutant. Among 100 different growth conditions, those that decrease the membrane potential (high external potassium) or pH gradient (high external pH) caused a reduction in growth of the aha2 mutant compared with wild type. Despite the normal appearance of single mutants under ideal laboratory growth conditions, embryos containing homozygous double mutations are lethal, demonstrating that, as expected, this protein is absolutely essential for plant cell function. In conclusion, our results demonstrate that the two genes together perform an essential function and that the effects of their single mutations are mostly masked by overlapping patterns of expression and redundant function as well as by compensation at the post-translational level.

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Figures

FIGURE 1.
FIGURE 1.
Phylogenetic and expression analyses of 11 AHA members. A, phylogenetic tree and expression level of AHA members based on microarray and expression sequence tag analyses. Protein sequences were aligned by an algorithm, ClustalV (PAM250), provided by Megalign (Lasergene, DNASTAR, Inc., Madison, WI). Bootstrap values (%) obtained by resampling 1000 times were indicated at the branches of the tree. n.a., not available. a, data were obtained on line. B, developmental expression study of AHA members with Genevestigator. Data were obtained on line.
FIGURE 2.
FIGURE 2.
Molecular characterization of loss-of-function mutants of AHA1 and AHA2. A, description of the T-DNA insertional alleles of AHA1 and AHA2. Black boxes represent exons. Vertical lines indicate the location of T-DNAs. aha1–6, SALK016325; aha1–7, SALK065288; aha1–8, SALK118350; aha2–4, SALK082786; and aha2–5, SALK022010. B, quantitative RT-PCR of AHA1 and AHA2 in wild type (WT), aha1–6, and aha2–4 mutants. C, transcript signal intensities of the 11 AHA members in WT, aha1–6, and aha2–4 mutants determined by microarray analyses. Graph shows mean values with S.D. D and E, AHA1 and AHA2 protein abundances in WT, aha1–6, and aha2–4 plants. Isoform-specific polypeptides (the amino acid position 407–420 and 471–490) were determined by quantifying the peak areas of the peptide ions found in the total ion chromatogram of mass spectrometric analyses. Data are shown as means ± S.E. of three separate measurements. F, selective reaction monitoring mass spectrometry analysis for AHA1 and -2 C-terminal peptides of nonphosphorylated and phosphorylated forms. Proportion of AHA1 phosphopeptide Thr(P)-948 and AHA2 phosphopeptide Thr(P)-947 was quantified and shown ± S.D. Col WT, wild type plant of A. thaliana, ecotype Columbia.
FIGURE 3.
FIGURE 3.
Seed developments in the double mutants and AHA1 and AHA2 promoter activity. A, phenotype of seed development in WT viewed under a stereomicroscope. B, immature siliques of aha1–6/aha1–6 AHA2/aha2–4 plants containing empty spaces. C, immature siliques of AHA1/aha1–6 aha2–4/aha2–4 plants containing empty spaces. D, magnified view of an aborted seed in a silique of aha1–6/aha1–6 AHA2/aha2–4 plant. E, percentage of developing seeds from the double mutants compared with WT or the AHA single mutant plants. Bars indicate S.E. F, normal growing embryo from self-pollinated aha1–6/aha1–6 AHA2/aha2–4 plants. G, arrested embryos from the same silique as in F. H, normal growing embryo from self-pollinated AHA1/aha1–6 aha2–4/aha2–4 plants. I, arrested embryos from the same silique as in H. Scale bar, 20 μm. J–N, GUS staining in embryos for AHA1 promoter activity. J, developing seed; K, 8-cell stage; L, early globular stage; M, late globular stage; N, heart stage. Scale bar, 20 μm. O, GUS staining in developing seed for AHA2 promoter activity. Scale bar, 10 μm. E; embryo.
FIGURE 4.
FIGURE 4.
Root growth phenotypes of aha1 and aha2 mutants in response to treatments that perturb the protonmotive force. A, high pH sensitivity of root growth in aha2 mutant. B, high potassium sensitivity of root growth in aha2 mutant. C, dose-response effects of potassium on aha1 and aha2 root growth. D, images of root growth on control, KCl, and hygromycin media. E, effects of aminoglucoside antibiotics on aha2 root growth. Hygro, 5 μg/ml hygromycin; Gent, 10 μg/ml gentamicin; Strept, 100 μg/ml streptomycin. F, dose-response effects of hygromycin B on aha1 and aha2 root growth. G, cesium and lithium tolerance in aha2 mutant. H, suppression of hygromycin inhibitory effect by potassium. Col WT, wild type plant of A. thaliana, ecotype Columbia.
FIGURE 5.
FIGURE 5.
Proton-secreting activity in roots of wild type and aha1 and aha2 mutants. Representative extracellular pH assay reflecting plasma membrane proton pump activity. pH of bathing media was quantified with fluorescein dye. n = 7; error bars indicate S.E. Col WT, wild type plant of A. thaliana, ecotype Columbia.
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