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...

Extracellular matrix (ECM) molecules are known to play a pivotal role in the morphogenesis of the secondary palate. The maintenance and degradation of the ECM is mediated in part by the matrix metalloproteinases (MMPs) and their endogenous inhibitors TIMPs. MMPs and TIMPs have previously been shown to be developmentally regulated within the palatal shelf during secondary palate morphogenesis. This study was conducted to examine the temporospatial distribution of these enzymes and their inhibitors within the palatal shelves using immunofluorescent localization to determine if specific changes occur in their distribution concomitant with events in palatal shelf formation and reorientation. Frontal sections through the posterior palatal shelves at gestational day (gd) 12, 13 and 14 were immunofluorescently stained for MMPs 2, 3, 9, and 13 and TIMPs 1, 2, and 3 using standard protocols and commercially available antibodies. The results demonstrated that MMPs and TIMPs were already present within the palatal shelf mesenchyme 30 h prior to reorientation and closure and that their expression within the shelf mesenchyme increased as the shelves remodeled, then decreased with closure and fusion. Increased distribution of MMPs and TIMPs within specific regions of the palatal mesenchyme and palatal epithelial basement membrane preceded decreases previously observed within these areas for their substrates, fibronectin, collagen III and collagen I. In addition, MMP-3 and TIMP-3 were immunolocalized to regions of the palatal epithelium that undergo reorganization concomitant with reorientation. The results of this study indicate that MMPs and TIMPs are developmentally regulated during palatal shelf morphogenesis and that their distribution correlates with the distribution of the ECM components of the palatal shelf they regulate. These results provide support for the idea that temporospatially controlled interactions between MMPs and their substrates may be pivotal in modulating events in palatal morphogenesis.

A plasma membrane receptor protein kinase, FERONIA (FER), regulates various aspects of plant reproductive and vegetative growth. In roots, binding of a peptide ligand to FER causes rapid suppression of cell elongation whereas in ovules, FER is involved in gametophyte interactions. Here, we examined the effect of a mutation that eliminates kinase activity, on both ovule fertilization and root growth, using the same batch of seeds containing a kinase-dead mutation. The kinase-dead mutation of FER reduced the ability to complement fer-4 knockout phenotypes, compared with wild-type sequence in root, but not in ovules. Our results support a model in which cell type-specific regulatory mechanisms, such as different interacting partners and/or downstream signaling events, lead to cell type-specific functions of FER.

Within the past two decades, the biological application of mass spectrometric technology has seen great advances in terms of innovations in hardware, software, and reagents. Concurrently, the burgeoning field of proteomics has followed closely (Yates et al., Annu Rev Biomed Eng 11:49-79, 2009)-and with it, importantly, the ability to globally assay altered levels of posttranslational modifications in response to a variety of stimuli. Though many posttranslational modifications have been described, a major focus of these efforts has been protein-level phosphorylation of serine, threonine, and tyrosine residues (Schreiber et al., Proteomics 8:4416-4432, 2008). The desire to examine changes across signal transduction cascades and networks in their entirety using a single mass spectrometric analysis accounts for this push-namely, preservation and enrichment of the transient yet informative phosphoryl side group. Analyzing global changes in phosphorylation allows inferences surrounding cascades/networks as a whole to be made. Towards this same end, much work has explored ways to permit quantitation and combine experimental samples such that more than one replicate or experimental condition can be identically processed and analyzed, cutting down on experimental and instrument variability, in addition to instrument run time. One such technique that has emerged is metabolic labeling (Gouw et al., Mol Cell Proteomics 9:11-24, 2010), wherein biological samples are labeled in living cells with nonradioactive heavy isotopes such as (15)N or (13)C. Since metabolic labeling in living organisms allows one to combine the material to be processed at the earliest possible step, before the tissue is homogenized, it provides a unique and excellent method for comparing experimental samples in a high-throughput, reproducible fashion with minimal technical variability. This chapter describes a pipeline used for labeling living Arabidopsis thaliana plants with nitrogen-15 ((15)N) and how this can be used, in conjunction with a technique for enrichment of phosphorylated peptides (phosphopeptides), to determine changes in A. thaliana's phosphoproteome on an untargeted, global scale.

The Arabidopsis thaliana plasma membrane proton ATPase genes, AHA1 and AHA2, are the two most highly expressed isoforms of an 11 gene family and are collectively essential for embryo development. We report the translational fusion of a tandem affinity-purification tag to the 5′ end of the AHA1 open reading frame in a genomic clone. Stable expression of TAP-tagged AHA1 in Arabidopsis rescues the embryonic lethal phenotype of endogenous double aha1/aha2 knockdowns. Western blots of SDS-PAGE and Blue Native gels show enrichment of AHA1 in plasma membrane fractions and indicate a hexameric quaternary structure. TAP-tagged AHA1 rescue lines exhibited reduced vertical root growth. Analysis of the plasma membrane and soluble proteomes identified several plasma membrane-localized proteins with alterred abundance in TAP-tagged AHA1 rescue lines compared to wild type. Using affinity-purification mass spectrometry, we uniquely identified two additional AHA isoforms, AHA9 and AHA11, which copurified with TAP-tagged AHA1. In conclusion, we have generated transgenic Arabidopsis lines in which a TAP-tagged AHA1 transgene has complemented all essential endogenous AHA1 and AHA2 functions and have shown that these plants can be used to purify AHA1 protein and to identify in planta interacting proteins by mass spectrometry.