A Transgene Encoding a Plasma Membrane H⁺-ATPase That Confers Acid Resistance in Arabidopsis thaliana Seedlings

Corresponding author: Michael R. Sussman, Biotechnology Center, 425 Henry Mall, University of Wisconsin, Madison, WI 53706, msussman{at}facstaff.wisc.edu (E-mail).

Communicating editor: D. PREUSS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Proton pumps (H⁺-ATPases) are the primary active transport systems in the plasma membrane of higher plant cells. These enzymes are encoded by a large gene family expressed throughout the plant, with specific isoforms directed to various specialized cells. While their involvement in membrane energetics has been suggested by a large body of biochemical and physiological studies, a genetic analysis of their role in plants has not yet been performed. We report here that mutant Arabidopsis thaliana plants containing a phloem-specific transgene encoding a plasma membrane H⁺-ATPase with an altered carboxy terminus show improved growth at low pH during seedling development. These observations provide the first genetic evidence for a role of the plasma membrane H⁺-ATPase in cytoplasmic pH homeostasis in plants.

IN fungi and higher plants, proton pumps provide the primary active transport system at the plasma membrane (reviewed in GOFFEAU and SLAYMAN 1981 ; SERRANO 1989 ; SUSSMAN 1994 ; MICHELET and BOUTRY 1995 ). These enzymes belong to a large family of cation pumps that generate ion gradients across the plasma membrane (MOLLER et al. 1996 ). By coupling ATP hydrolysis with the translocation of a proton, the H⁺-ATPase enzyme generates electrical and pH gradients that mediate the transport of nutrients and metabolites at the cellular, tissue, and organ levels. In addition to a role in transport, the plant proton pump is thought to be involved in signal transduction and responses to the environment. For example, the plasma membrane H⁺-ATPase has been implicated as playing a regulatory role in processes such as cell division, cell wall extension, and cell elongation (CLELAND 1987 ; SENN and GOLDSMITH 1988 ; SERRANO 1989 ; HAGER et al. 1991 ; KIM and KAUFMAN 1995 ; BARBIER-BRYGOO et al. 1996 ).

The biological role of the plasma membrane H⁺-ATPase in higher plants has been inferred from genetic studies performed with a similar enzyme in yeast (CID et al. 1987 ; MCCUSKER et al. 1987 ; NAKAMOTO et al. 1991 ), and from heterologous expression studies in which plant H⁺-ATPases are expressed in yeast (VILLALBA et al. 1992 ; PALMGREN and CHRISTENSEN 1993 ; PALMGREN and CHRISTENSEN 1994 ; MORSOMME et al. 1996 ). In Saccharomyces cerevisiae, two genes code for the plasma membrane H⁺-ATPase (SERRANO et al. 1986 ; SCHLESSER et al. 1988 ), but only one, PMA1, is constitutively expressed and required for growth (SERRANO et al. 1986 ). This relatively simple circumstance has facilitated many physiological and cloning studies of the plant ATPase in the fungi, but the models derived from these approaches have been difficult to test in planta.

Gene cloning and sequencing studies in Arabidopsis and other higher plants has demonstrated that the plant plasma membrane H⁺-ATPase is encoded by a large gene family containing nine or more members (EWING and BENNET 1994 ; HARPER et al. 1994 ; SUSSMAN 1994 ). Promoter studies using reporter genes and direct measurements of mRNA levels indicate that individual gene isoforms are differentially expressed in a tissue-specific manner (HARPER et al. 1990, HARPER et al. 1994 ; DEWITT et al. 1991 ; HOULNE and BOUTRY 1994 ). For example, one isoform has been found to be predominantly expressed within the plasma membrane of phloem companion cells, a specialized tissue involved in the long distance transport of sugars, nutrients and hormones (DEWITT et al. 1991 ; DEWITT and SUSSMAN 1995 ). The energy to drive this active process has long been thought to be derived from the proton pump (H⁺-ATPase), and not surprisingly, immunocytochemical localization studies demonstrate that the companion cell contains one of the highest concentrations of the pump in the plant (VILLALBA et al. 1991; DEWITT and SUSSMAN 1995 ).

In yeast, the plasma membrane H⁺-ATPase is activated in vivo by glucose metabolism, and deletion analysis has shown the C terminus of the enzyme to be involved in this regulation (PALMGREN et al. 1990 ; PORTILLO et al. 1991 ). Moreover, single point mutations in the C terminus of the H⁺-ATPase result in increased proton pumping that permits yeast to grow at low pH (MORSOMME et al. 1996 ). Using a yeast expression system for structure-function studies of the Arabidopsis H⁺-ATPase, PALMGREN and coworkers have shown that deletion of amino acid residues in the C terminus of the plant pump results in a constitutively activated H⁺-ATPase (PALMGREN and CHRISTENSEN 1993 ; REGENBERG et al. 1995 ).

To study how the C-terminal region of the plasma membrane H⁺-ATPase is involved in signal transduction and membrane transport processes in plants, we have generated mutant Arabidopsis that express an H⁺-ATPase (AHA3) with a disrupted C terminus. This ATPase isoform was previously shown to be localized to the plasma membrane of companion cells (DEWITT et al. 1995, DEWITT et al. 1996 ). We show here that Arabidopsis seedlings expressing AHA3 with an altered C terminus, have improved growth at low pH.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant materials and growth conditions:
Arabidopsis mutant and wild-type seeds were surface sterilized in 70% (v/v) ETOH/0.1% (v/v) Triton X-100 for 5 min, rinsed 2x with 95% (v/v) ETOH and dried on sterile Whatmann paper. Sterile seeds were plated on 0.5x Murishige and Skoog media (Gibco BRL, Grand Island NY), no sucrose, with either 5 mM MES (pH 5.7 ± 0.1) or 3 mM 3,3-dimethylglutaric (DMG; Sigma, St. Louis). The pH of the medium was titrated, as indicated in Figure 1 Figure 2 Figure 3, with 1 N KOH prior to the addition of Agar to 1% (Sigma).

View larger version (76K): [in this window] [in a new window] [Download PPT slide]   Figure 1. —Wild-type (Bensheim) and 944/GUS seedlings grown on 3 mM DMG at different pHs. (a) Dark-grown plants, and (b) light-grown plants, 7 days after germination.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Figure 2. —pH dependence of dark-grown hypocotyl elongation. Wild-type (Bensheim) and 944/GUS transgenic seedlings were germinated and grown on 3 mM DMG at different pHs. Hypocotyl lengths of treated seedlings were compared with seedlings grown on 5 mM MES (pH 5.7). The data shown represent at least 100 seedlings per treatment ±SE.

View larger version (46K): [in this window] [in a new window] [Download PPT slide]   Figure 3. —Inhibition of hypocotyl elongation on low pH media. The inhibition of wild-type (a) ecotype Bensheim and (b) ecotype LER, and transgenic seedlings grown on 3 mM DMG (pH 4.7). Hypocotyl lengths of treated seedlings were compared with seedlings grown on 5 mM MES (pH 5.7). The (c) control represents the average from twelve independent lines for plants containing the 12-kb AHA3 genomic insert with no modification of the C terminus. The 5' control (d) contains the AHA3 promoter alone fused to GUS, in the LER background (5 independent lines). The 3' (e) control contains 3 kb of the 12-kb insert downstream of AHA3 (6 independent lines). Bars labeled 904 and 944 denote the C-terminal epitopes c-myc (), HA () and GUS () at amino acid position 904 or 944 of the AHA3 gene. The data shown represent at least 100 seedlings per treatment ±SE.

To synchronize germination, plated seeds were kept in the dark at 4° for 3–4 days, then treated with red light (30 µmol m^-2 s^-1). Seeds were then transferred to either dark or light (45 µmol m^-2 s^-1) conditions for 7 days. Dark-grown seedlings were measured to the nearest millimeter with a ruler. To control for differences in germination and elongation rates in the various plant lines, each experiment included growth on 5 mM MES plates (pH 5.7). The inhibition of hypocotyl elongation for each treatment is expressed as the percentage of hypocotyl length of treated seedlings compared to control seedlings.

Genetic techniques:
The epitope-tagged plants and other controls were constructed as described in (DEWITT and SUSSMAN 1995 ). Plants containing the transgenes were crossed with wild-type plants of the same ecotype. The F₁ progeny were scored for hypocotyl elongation in the various media, then transferred to soil and allowed to self. The F₂ progeny were similarly scored. The measurement of beta-glucuronidase (GUS) was done as described in BLEECKER and PATTERSON 1997 .

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Previously, we described the use of epitope tagging as a means of identifying the cellular and subcellular localization of individual members within the AHA gene family (DEWITT and SUSSMAN 1995 ; DEWITT et al. 1996 ). The coding regions for two well-characterized 21 amino-acid epitopes from the c-myc mammalian oncogene and the influenza hemagglutinin (HA) proteins (KOLODZIEJ and YOUNG 1991 ) were separately fused to a 12-kb genomic clone of AHA3. The AHA3 clone contains 4 kb upstream of the AHA3 gene, including the native promoter, and 2.5 kb of 3' flanking sequence. The epitope tags were inserted so that they would be expressed at amino acid residues 904 and 944 in the 150-amino-acid-long carboxy terminus of AHA3. This portion of AHA3 is hydrophilic and predicted to be cytoplasmically located, based on studies of the yeast H⁺-ATPase (DAVIS and HAMMES 1989 ; MONK et al. 1991 ). Additionally, in separate experiments, the 550-amino-acid-long bacterial protein, beta-glucuronidase (GUS) was also inserted into the 904 and 944 locations of AHA3. After transformation, all lines were selected and maintained by resistance to kanamycin (DEWITT et al. 1991 ).

Indirect immunofluorescence microscopy and Western blot analyses using antibodies directed against c-myc, HA or GUS epitopes demonstrated that transgenic plants containing these epitope-tagged transgenes produced fusion proteins that were targeted to the plasma membrane (DEWITT et al. 1996 ), and were expressed predominantly within companion cells of the phloem (DEWITT and SUSSMAN 1995 ).

To determine whether the expression of these transgenes altered plant growth or development, we grew the transformants under a variety of physiological conditions, including assays comparing wild-type and mutant responses to phytohormones, light and gravity, and osmotic and salt stresses. In these, and other assays, the only significant difference from wild-type growth was noted when the pH of the medium was changed. The "normal" pH of our growth medium (1x Murashige-Skoog) is 5.7. To assay seedling growth at low pH, the media were buffered with a nonmetabolizable, weak DMG (GREENBERG and GOLDSCHMIDT 1989 ). The difference in pH susceptibility as observed in both darkness and in light is shown in Figure 1. In this experiment, wild-type and mutant seeds were germinated and grown on petri plates containing 3 mM DMG with pH adjusted to 4.0, 4.5, 5.0, 5.5 or 6.0 using KOH. We observed that seeds from the transgenic lines were better able to tolerate the decreased pH, compared to wild type. To quantify this observation, we measured the hypocotyl lengths of seedlings germinated in the dark, in media containing a variety of pHs ranging from 3.7 to 5.7 (Figure 2). Wild-type growth was significantly reduced below pH 5, whereas with the transgenic line 944/GUS, a significant reduction in hypocotyl elongation was noted only at pHs below 4.0. The shape of these curves implies that the mutant is able to tolerate a tenfold higher concentration of external protons.

In order to determine whether the difference in growth at low pH is due to an altered H⁺-ATPase transgene, rather than an additional copy of the gene, we generated transgenic lines containing the AHA3 transgene with no modifications. Additionally, recombinant plants with only the 5' (4-kb), or the 3' (3-kb) portions of the AHA3 clone were generated to test whether the regions upstream or downstream of the AHA3 gene contained coding sequences that improved growth at low pH. The acid-resistant phenotype was scored for all of these genotypes using the hypocotyl length assay with 3 mM DMG medium buffered to pH 4.7. The results for the lines assayed are presented in Table 1, and are summarized in Figure 3. As shown in Table 1 and Figure 3, the hypocotyl length of the Bensheim ecotype (used as the starting material for all transgenic lines) was inhibited by ca. 50% at pH 4.7. The transgenic plants with disruptions in the C terminus of the AHA3 transgene show a range of effects, from 25% growth reduction (line 471-208; 904/c-myc) to none (line 457A; 904/HA). The average of at least two lines per construct is shown in Figure 3, except for 904/HA and 944/GUS for which only one line was assayed. As observed for wild-type control plants, all of the transgenic seedlings containing an unmodified H⁺-ATPase transgene showed ca. 50% growth reductions. In addition to the controls listed in Table 1, eleven nonrelated transgenic lines were assayed for growth at low pH, including a line in which the GUS gene is driven by the CaMV 35S promoter. All of these additional lines responded similarly to wild-type seedlings at low pH (data not shown). These results suggest that the acid resistant phenotype is conferred by the modification of the C terminus, rather than just an extra copy of the AHA3 gene or a transformation artifact.

If the acid resistant phenotype is caused by expression of the transgene containing a disruption of the AHA3 C terminus, it should segregate as an inherited single gene. To test this, we crossed six lines (expressing a variety of epitopes, at both 904 and 944 positions) with wild type, and then tested them for acid resistance in both the F₁ and F₂ generations (Table 2). In each case, the F₁ seeds were all resistant, but in the F₂ generation, three-fourths were resistant, consistent with a dominant effect of the transgene. The cosegregation of the transgene with acid resistance was tested by assaying for GUS expression in F₂ families of lines with GUS inserted in the C terminus of AHA3. Two independent lines, 904/GUS and 944/GUS, were assayed, and both showed the segregation of GUS expression with improved hypocotyl elongation at low pH (Table 2). These results are consistent with the conclusion that any plant containing a single copy of the transgene is significantly more resistant to acid conditions than wild type.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Earlier studies comparing the structure and function of genetically altered H⁺-ATPase proteins in yeast established that the carboxy-terminal domain of the fungal proton pump is an autoinhibitory domain (PORTILLO et al. 1991 ; SEKLER et al. 1994 ). Similarly, when plant H⁺-ATPase genes were expressed in yeast and tested biochemically, it was observed that changes in the carboxy terminus were associated with increases in catalytic activity (VILLALBA et al. 1992 ; PALMGREN and CHRISTENSEN 1993 , PALMGREN and CHRISTENSEN 1994 ; MORSOMME et al. 1996 ). These changes were not due to differences in expression, biogenesis or protein stability. Rather, they reflected true catalytic differences in affinity for substrates (K_m) or in the rate at which the enzyme turns over during the catalytic cycle (V_max). In vitro biochemical studies with the plant enzyme, in which the carboxy terminus was proteolytically cleaved, further confirmed that the carboxy terminus down-regulates the catalytic activity of the H⁺-ATPase (RASI-CALDOGNOV et al. 1993 ).

Despite the numerous in vitro experiments with purified plasma membranes, or with the enzyme expressed in yeast, direct in situ evidence for this phenomenon within a higher plant has not been reported. In this article we have presented the first in planta evidence that the carboxy terminus is indeed acting as an autoinhibitory domain within the intact plant. This conclusion is based on our observation that plants containing a variety of transgenes with a carboxy-terminal disruption, all show increased resistance to growth inhibition at low pH.

It is striking to note that this transgene, which behaves genetically as a single dominant gene, encodes an enzyme which is predominantly expressed only in companion cells of the phloem. This raises the interesting question of why such an expression pattern should cause resistance of the overall plant to growth at acid pH. A likely answer is that in these young seedlings lacking a cuticle, the phloem may be a primary target of low pH. It is known that the phloem sap is alkaline at pH 8.0 or higher (VREUGDENHILL and KOOT-GRONSVELD 1989 ). Thus, the phloem may be more sensitive to the steeper difference in pH across the plasma membrane than other cells within the stem, leaves and roots. Furthermore, it is likely that a deleterious change in the phloem pH could result in an overall change in growth rates, since the phloem mediates the long distance transport of essential sugars, nutrients and hormones. In the transgenic plants, a modified proton pump expressed in the phloem appears to allow these cells to maintain proper function at low pHs. It is likely that the modified AHA3 gene produces an unregulated pump, and, as in yeast with point mutations in the carboxy terminus (MORSOMME et al. 1996 ), growth is maintained at low pH. However, further studies will be needed to test our hypothesis that the phenotype is caused by post-translational changes in catalytic activity, rather than changes in expression, stability, or targeting of the AHA3-tagged mRNA or protein.

A final word should be made about expression of plasma membrane transporters as transgenes. Although we have attempted to overexpress and underexpress H⁺-ATPase genes in Arabidopsis and tobacco using the 35S promoter with a variety of H⁺-ATPase sense and antisense constructs, we have never been able to generate plants in which the H⁺-ATPase concentration is noticeably altered or which show an altered phenotype. It is possible that we were successful in the experiments reported here because a natural promoter, which directed expression to a single cell type, was used. For enzymes that perform essential functions, it may be important to direct expression of transgenes to precise locations and developmental stages that are not deleterious to growth and development.

	ACKNOWLEDGMENTS

We gratefully acknowledge LESLIE DICKMANN and SARA PATTERSON for technical assistance. This work was supported by grants from the U.S. Department of Energy/National Science Foundation/U.S. Department of Agriculture Collaborative Program in Plant Biology (BIR 92-20331), and the U.S. Department of Energy (DE-F602-88ER13938).

Manuscript received January 5, 1998; Accepted for publication March 6, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BARBIER-BRYGOO, H., S. ZIMMERMANN, S. THOMINE, I. R. WHITE, and P. MILLNER et al., 1996  Elementary auxin response chains at the plasma membrane involve external abp1 and multiple electrogenic ion transport proteins. Plant Growth Regul. 18:23-28.

BLEECKER, A. B. and S. E. PATTERSON, 1997  Last exit: senescence, abscission, and meristem arrest in Arabidopsis.. Plant Cell 9:1169-1179[Medline].

CID, A., R. PERONA, and R. SERRANO, 1987  Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences. Curr. Genet. 12:105-110[Medline].

CLELAND, R. E., 1987 Auxin and cell elongation, pp. 132–148 in Plant Hormones and Their Role in Plant Growth and Development, edited by P. J. DAVIES. Martinus Nijhoff, Boston.

DAVIS, C. and G. G. HAMMES, 1989  Topology of the yeast plasma membrane proton-translocating ATPase. J. Biol. Chem. 264:370-374[Abstract/Free Full Text].

DEWITT, N. D., J. F. HARPER, and M. R. SUSSMAN, 1991  Evidence for a plasma membrane proton pump in the phloem cells of higher plants. Plant J. 1:121-128[Medline].

DEWITT, N. D. and M. R. SUSSMAN, 1995  Immunocytological localization of an epitope-tagged plasma membrane proton pump (H⁺-ATPase) in phloem companion cells. Plant Cell 7:2053-2067[Abstract].

DEWITT, N. D., B. HONG, M. R. SUSSMAN, and J. F. HARPER, 1996  Targeting of two Arabidopsis H⁺-ATPase isoforms to the plasma membrane. Plant Physiol. 112:833-844[Abstract].

EWING, N. N. and A. B. BENNET, 1994  Assessment of the number and expression of P-type H⁺-ATPase genes in tomato. Plant Physiol. 106:547-557[Abstract].

GOFFEAU, A. and C. W. SLAYMAN, 1981  The proton-translocating ATPase of the fungal plasma membrane. Biochim. Biophys. Acta 639:197-223[Medline].

GREENBERG, J. and E. E. GOLDSCHMIDT, 1989  Acidifying agents, uptake, and physiological activity of gibberellin A-3 in Citrus. Hortscience 24:791-793.

HAGER, A., G. DEBUS, H. G. EDEL, H. STRANSKY, and R. SERRANO, 1991  Auxin induces exocytosis and the rapid synthesis of a high-turnover pool of plasma-membrane proton-ATPase. Planta 185:527-537.

HARPER, J. F., L. MANNEY, and M. R. SUSSMAN, 1994  The plasma membrane H⁺-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds. Mol. Gen. Genet. 244:572-587[Medline].

HOULNE, G. and M. BOUTRY, 1994  Identification of an Arabidopsis thaliana gene encoding a plasma membrane H⁺-ATPase whose expression is restricted to anther tissues. Plant J. 5:311-317[Medline].

KIM, D. and P. B. KAUFMAN, 1995  Basis for changes in the auxin-sensitivity of Avena sativa (oat) leaf-sheath pulvini during the gravitropic response. J. Plant Physiol. 145:113-120[Medline].

KOLODZIEJ, P. and R. A. YOUNG, 1991  Epitope tagging and protein surveillance. Methods Enzymol. 194:508-519[Medline].

MCCUSKER, J. H., D. S. PERLIN, and J. E. HABER, 1987  Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae.. Mol. Cell. Biol. 7:4082-4088[Abstract/Free Full Text].

MICHELET, N. and M. BOUTRY, 1995  The plasma membrane H⁺-ATPase. Plant Physiol. 108:1-6[Medline].

MØLLER, J. V., B. JUUL, and M. LE MARIE, 1996  Structural organization, ion transport, and energy transduction of P-type ATPases. Biochim. Biophys. Acta 1286:1-51[Medline].

MONK, B. C., C. MONTESINOS, C. FERGUSON, K. LEONARD, and R. SERRANO, 1991  Immunological approaches to the transmembrane topology and conformational changes of the carboxyl-terminal regulatory domain of yeast plasma membrane proton-ATPase. J. Biol. Chem. 266:18097-18103[Abstract/Free Full Text].

MORSOMME, P., A. D. K. D'EXAERDE, S. DE-MEESTER, D. THINES, and A. GOFFEAU et al., 1996  Single point mutations in various domains of a plant plasma membrane H⁺-ATPase expressed in Saccharomyces cerevisiae increase H⁺-pumping and permit yeast growth at low pH. EMBO J. 15:5513-5526[Medline].

NAKAMOTO, R., R. RAO, and C. W. SLAYMAN, 1991  Expression of the yeast plasma membrane proton ATPase in secretory vesicles: a new strategy for directed mutagenesis. J. Biol. Chem. 266:7940-7949[Abstract/Free Full Text].

PALMGREN, M. G., C. LARSSON, and M. SOMMARIN, 1990  Proteolytic activation of the plant plasma membrane H⁺-ATPase by removal of a terminal segment. J. Biol. Chem. 265:13423-13426[Abstract/Free Full Text].

PALMGREN, M. G. and G. CHRISTENSEN, 1993  Complementation in situ of the yeast plasma membrane proton-ATPase gene pmal by proton-ATPase gene from a heterologous species. FEBS Lett. 317:216-222[Medline].

PALMGREN, M. G. and G. CHRISTENSEN, 1994  Functional comparisons between plant plasma membrane H⁺-ATPase isoforms expressed in yeast. J. Biol. Chem. 269:3027-3033[Abstract/Free Full Text].

PORTILLO, F., P. ERASO, and R. SERRANO, 1991  Analysis of the regulatory domain of yeast plasma membrane proton-ATPase by directed mutagenesis and intragenic suppression. FEBS Lett. 287:71-74[Medline].

RASI-CALDOGNOV, F., M. C. PUGLIARELLO, C. OLIVARI, and M. I. DEMICHELIS, 1993  Controlled proteolysis mimics the effect of fusicoccin on the plasma membrane H⁺-ATPase. Plant Physiol. 103:391-398[Abstract].

REGENBERG, B., J. M. VILLALBA, F. C. LANFERMEIJER, and M. G. PALMGREN, 1995  C-terminal deletion analysis of plant plasma membrane H⁺-ATPase: yeast as a model system for solute transport across the plant plasma membrane. Plant Cell 7:1655-1666[Abstract].

SCHLESSER, A., S. ULASZEWSKI, M. GHISLAIN, and A. GOFFEAU, 1988  A second transport ATPase gene in Saccharomyces cerevisiae.. J. Biol. Chem. 263:19480-19487[Abstract/Free Full Text].

SEKLER, I., M. WEISS, and U. PICK, 1994  Activation of the Dunaliella acidophila plasma membrane H⁺-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site. Plant Physiol. 105:1125-1132[Abstract].

SENN, A. P. and M. H. M. GOLDSMITH, 1988  Regulation of electrogenic proton pumping by auxin and fusicoccin as related to the growth of Avena coleoptiles. Plant Physiol. 88:131-138[Abstract/Free Full Text].

SERRANO, R., 1989  Structure and function of plasma membrane ATPase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40:61-91.

SERRANO, R., M. C. KIELLAND-BRANDT, and G. R. FINK, 1986  Yeast plasma membrane ATPase is essential for growth and has homology with (Na⁺ + K⁺), K⁺- and Ca²⁺-ATPases. Nature 319:689-693[Medline].

SOKAL, R. R., and F. J. ROHLF, 1969 Biometry. W. H. Freeman and Co., San Franscisco.

SUSSMAN, M. R., 1994  Molecular analyses of proteins in the plasma membrane. Annu. Rev. Plant Physiol. Plant Mol. Biol. 45:211-234.

VILLALBA, J. M., M. G. PALMGREN, G. E. BERBERIAN, C. FERGUSON, and R. SERRANO, 1992  Functional expression of plant plasma membrane proton ATPase in yeast endoplasmic reticulum. J. Biol. Chem. 267:12341-12349[Abstract/Free Full Text].

VREUGDENHILL, D. and E. A. M. KOOT-GRONSVELD, 1989  Measurements of pH, sucrose and potassium ions in the phloem sap of castor bean (Ricinus communis) plants. Physiol. Plant. 77:385-388.

This article has been cited by other articles:

				K. A. Duncan and S. C. Huber Sucrose Synthase Oligomerization and F-actin Association are Regulated by Sucrose Concentration and Phosphorylation Plant Cell Physiol., November 1, 2007; 48(11): 1612 - 1623. [Abstract] [Full Text] [PDF]

				F. Gevaudant, G. Duby, E. von Stedingk, R. Zhao, P. Morsomme, and M. Boutry Expression of a Constitutively Activated Plasma Membrane H+-ATPase Alters Plant Development and Increases Salt Tolerance Plant Physiology, August 1, 2007; 144(4): 1763 - 1776. [Abstract] [Full Text] [PDF]

				W. R. Robertson, K. Clark, J. C. Young, and M. R. Sussman An Arabidopsis thaliana Plasma Membrane Proton Pump Is Essential for Pollen Development Genetics, November 1, 2004; 168(3): 1677 - 1687. [Abstract] [Full Text] [PDF]

				T. P. Jahn, A. Schulz, J. Taipalensuu, and M. G. Palmgren Post-translational Modification of Plant Plasma Membrane H+-ATPase as a Requirement for Functional Complementation of a Yeast Transport Mutant J. Biol. Chem., February 15, 2002; 277(8): 6353 - 6358. [Abstract] [Full Text] [PDF]

				S. J. Ahn, M. Sivaguru, H. Osawa, G. C. Chung, and H. Matsumoto Aluminum Inhibits the H+-ATPase Activity by Permanently Altering the Plasma Membrane Surface Potentials in Squash Roots Plant Physiology, August 1, 2001; 126(4): 1381 - 1390. [Abstract] [Full Text] [PDF]

				R. Zhao, V. Dielen, J.-M. Kinet, and M. Boutry Cosuppression of a Plasma Membrane H+-ATPase Isoform Impairs Sucrose Translocation, Stomatal Opening, Plant Growth, and Male Fertility PLANT CELL, April 1, 2000; 12(4): 535 - 546. [Abstract] [Full Text]

				H. Sze, X. Li, and M. G. Palmgren Energization of Plant Cell Membranes by H+-Pumping ATPases: Regulation and Biosynthesis PLANT CELL, April 1, 1999; 11(4): 677 - 690. [Full Text]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Young, J. C. Articles by Sussman, M. R. Search for Related Content PubMed PubMed Citation Articles by Young, J. C. Articles by Sussman, M. R.