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Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H+–adenosine triphosphatase 2 at Ser899, mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion.

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...

In plants and fungi, energetics at the plasma membrane is provided by a large protonmotive force (PMF) generated by the family of P-type ATPases specialized for proton transport (commonly called PM H+-ATPases or, in Arabidopsis, AHAs for Arabidopsis H+-ATPases). Studies have demonstrated that this 100-kDa protein is essential for plant growth and development. Posttranslational modifications of the H+-ATPase play critical roles in its regulation. Phosphorylation of several Thr and Ser residues within the carboxy terminal regulatory domain composed of ~ 100 amino acids change in response to environmental stimuli, endogenous hormones, and nutrient conditions. Recently developed mass spectrometric technologies provide a means to carefully quantify these changes in H+-ATPase phosphorylation at the different sites. These chemical modifications can then be genetically tested in planta by complementing the loss-of-function aha mutants with phosphomimetic mutations. Interestingly, recent data suggest that phosphatase-mediated changes in PM H+-ATPase phosphorylation are important in mediating auxin-regulated growth. Thus, as with another hormone (abscisic acid), dephosphorylation by phosphatases, rather than kinase mediated phosphorylation, may be an important focal point for regulation during plant signal transduction. Although interactions with other proteins have also been implicated in ATPase regulation, the very hydrophobic nature and high concentration of this polytopic protein presents special challenges in evaluating the biological significance of these interactions. Only by combining biochemical and genetic experiments can we attempt to meet these challenges to understand the essential molecular details by which this protein functions in planta.

The plasma membrane proton gradient is an essential feature of plant cells. In Arabidopsis (Arabidopsis thaliana), this gradient is generated by the plasma membrane proton pump encoded by a family of 11 genes (abbreviated as AHA, for Arabidopsis H + -ATPase), of which AHA1 and AHA2 are the two most predominantly expressed in seedlings and adult plants. Although double knockdown mutant plants containing T-DNA insertions in both genes are embryonic lethal, under ideal laboratory growth conditions, single knockdown mutant plants with a 50% reduction in proton pump concentration complete their life cycle without any observable growth alteration. However, when grown under conditions that induce stress on the plasma membrane protonmotive force (PMF), such as high external potassium to reduce the electrical gradient or high external pH to reduce the proton chemical gradient, aha2 mutant plants show a growth retardation compared with wild-type plants. In this report, we describe the results of studies that examine in greater detail AHA2's specific role in maintaining the PMF during seedling growth. By comparing the wild type and aha2 mutants, we have measured the effects of a reduced PMF on root and hypocotyl growth, ATP-induced skewed root growth, and rapid cytoplasmic calcium spiking. In addition, genome-wide gene expression profiling revealed the up-regulation of potassium transporters in aha2 mutants, indicating, as predicted, a close link between the PMF and potassium uptake at the plasma membrane. Overall, this characterization of aha2 mutants provides an experimental and theoretical framework for investigating growth and signaling processes that are mediated by PMF-coupled energetics at the cell membrane.