Abscisic acid (ABA) is a hormone that controls seed dormancy and germination as well as the overall plant response to important environmental stresses such as drought. Recent studies have demonstrated that the ABA-bound receptor binds to and inhibits a class of protein phosphatases. To identify more broadly the phosphoproteins affected by this hormone in vivo, we used 14 N/ 15 N metabolic labeling to perform a quantitative untargeted mass spectrometric analysis of the Arabidopsis thaliana phosphoproteome following ABA treatment. We found that 50 different phosphopeptides had their phosphorylation state significantly altered by ABA over a treatment period lasting 5-30 min. Among these changes were increases in phosphorylation of subfamily 2 SNF1-related kinases and ABA-responsive basic leucine zipper transcription factors implicated in ABA signaling by previous in vitro studies. Furthermore, four members of the aquaporin family showed decreased phosphorylation at a carboxy-terminal serine which is predicted to cause closure of the water-transporting aquaporin gate, consistent with ABA's role in ameliorating the effect of drought. Finally, more than 20 proteins not previously known to be involved with ABA were found to have significantly altered phosphorylation levels. Many of these changes are phosphorylation decreases, indicating that an expanded model of ABA signaling, beyond simple phosphatase inhibition, may be necessary. This quantitative proteomics dataset provides a more comprehensive, albeit incomplete, view both of the protein targets whose biochemical activities are likely to be controlled by ABA and of the nature of the emerging phosphorylation and dephosphorylation cascades triggered by this hormone.mass spectrometry | proteomics | quantitation | metabolic labeling | Arabidopsis thalianaA bscisic acid (ABA) is a phytohormone that initiates the water removal and overall dormancy program that plant embryos undergo during seed formation and germination and also mediates the cold and drought stress responses that occur in vegetative tissues (1, 2). The receptor for this hormone recently has been identified as a family of small soluble proteins known as PYR/ PYLs that are encoded by 14 genes in the Arabidopsis genome (3, 4). Conformational studies using crystallography and NMR, as well as in vitro biochemical experiments, indicate that upon ABA binding these proteins undergo a conformational change that results in an increased affinity for protein phosphatase 2C (PP2C), and this interaction results in inhibition of phosphatase activity (5-8). These results are consistent with genetic data using stable transgenic lines containing knockout and overexpression mutations and with transient expression studies of the receptors and phosphatase (9, 10).The resulting model predicts that downstream effects of ABA are mediated by early changes in the phosphoproteome induced by changes in PP2C phosphatase activity, which then can activate downstream sucrose non-fermenting 1-related subfamily 2 (SnRK2) kinases (11). To ...
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...
Proteomics research is beginning to expand beyond the more traditional shotgun analysis of protein mixtures to include targeted analyses of specific proteins using mass spectrometry. Integral to the development of a robust assay based on targeted mass spectrometry is prior knowledge of which peptides provide an accurate and sensitive proxy of the originating gene product (i.e., proteotypic peptides). To develop a catalog of "proteotypic peptides" in human heart, TRIzol extracts of leftventricular tissue from nonfailing and failing human heart explants were optimized for shotgun proteomic analysis using Multidimensional Protein Identification Technology (MudPIT). Ten replicate MudPIT analyses were performed on each tissue sample and resulted in the identification of 30 605 unique peptides with a q-value ≤ 0.01, corresponding to 7138 unique human heart proteins. Experimental observation frequencies were assessed and used to select over 4476 proteotypic peptides for 2558 heart proteins. This human cardiac data set can serve as a public reference to guide the selection of proteotypic peptides for future targeted mass spectrometry experiments monitoring potential protein biomarkers of human heart diseases.
Genetic, chemical, and environmental perturbations can all induce large changes in cellular proteomes, and research aimed at quantifying these changes are an important part of modern biology. Although improvements in the hardware and software of mass spectrometers have produced increased throughput and accuracy of such measurements, new uses of heavy isotope internal standards that assist in this process have emerged. Surprisingly, even complex life forms such as mammals can be grown to near-complete replacement with heavy isotopes of common biological elements such as (15)N, and these isotopically labeled organisms provide excellent controls for isolating and identifying experimental variables such as extraction or fractionation efficiencies. We discuss here the theory and practice of these technologies, as well as provide a review of significant recent biological applications.
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