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Protein footprinting is a useful method for studying protein higher order structure and conformational changes induced by interactions with various ligands via addition of covalent modifications onto the protein. Compared to other methods that provide single amino acid-level structural resolution, such as cryo-EM, X-ray diffraction, and NMR, mass spectrometry (MS)-based methods can be advantageous as they require lower protein amounts and purity. As with other MS-based proteomic methods, such as post-translational modification analysis, enrichment techniques have proven necessary for both optimal sensitivity and sequence coverage when analyzing highly complex proteomes. Currently used reagents for footprinting via covalent labeling, such as hydroxyl radicals and carbodiimide-based methods, do not yet have a suitable enrichment method, limiting their applicability to whole proteome analysis. Here, we report a method for enrichable covalent labeling built upon the GEE/EDC system commonly used to covalently label aspartic acid and glutamic acid residues. Novel labeling reagents containing alkynyl functionality can be "clicked" to any azido-containing molecule with copper-catalyzed azide-alkyne cycloaddition (CuAAC), allowing for enrichment or further labeling. Multiple azide- and alkyne-containing GEE-like molecules were tested, and the most efficient method was determined to be the EDC-facilitated coupling of glycine propargyl amide (GPA) to proteins. The pipeline we report includes clicking via CuAAC to a commercially available biotin-azide containing a photocleavable linker, followed by enrichment using a streptavidin resin and subsequent cleavage under ultraviolet light. The enrichment process was optimized through the screening of clickable amines, coupling reagents, and enrichment scaffolds and methods with pure model proteins and has also been applied to complex mixtures of proteins isolated from the model plant, Arabidopsis thaliana , suggesting that our method may ultimately be used to measure protein conformation on a proteomic scale.

The plasma membrane proton pump is the primary energy transducing, electrogenic ion pump of the plasma membrane in plants and fungi. Compared to its fungal counterpart, the plant plasma membrane proton pump's regulatory C-terminal domain (CTD) contains an additional regulatory segment that links multiple sensory pathways regulating plant cell length through phosphorylation and recruitment of regulatory 14-3-3 proteins. However, a complete structural model of a plant proton pump is lacking. Here, we performed covalent labeling with mass spectrometric analysis (CL-MS) on the Arabidopsis pump AHA2 to identify potential interactions between the CTD and the catalytic domains. Our results suggest that autoinhibition in the plant enzyme is much more structurally complex than in the fungal enzyme., (© 2024 Federation of European Biochemical Societies.)

Premise: A multi-omic approach was used to explore proteins and networks hypothetically important for establishing filament dimorphisms in heterostylous Turnera subulata (Sm.) as an exploratory method to identify genes for future empirical research., Methods: Mass spectrometry (MS) was used to identify differentially expressed proteins and differentially phosphorylated peptides in the developing filaments between the L- and S-morphs. RNAseq was used to generate a co-expression network of the developing filaments, MS data were mapped to the co-expression network to identify hypothetical relationships between the S-gene responsible for filament dimorphisms and differentially expressed proteins., Results: Mapping all MS identified proteins to a co-expression network of the S-morph's developing filaments identified several clusters containing SPH1 and other differentially expressed or phosphorylated proteins. Co-expression analysis clustered CDKG2, a protein that induces endoreduplication, and SPH1-suggesting a shared biological function. MS analysis suggests that the protein is present and phosphorylated only in the S-morph, and thus active only in the S-morph. A series of CDKG2 regulators, including ATM1, and cell cycle regulators also correlated with the presence of reciprocal herkogamy, supporting our interest in the protein., Conclusions: This work has built a foundation for future empirical work, specifically supporting the role of CDKG2 and ATM1 in promoting filament elongation in response to SPH1 perception., (© 2024 The Author(s). American Journal of Botany published by Wiley Periodicals LLC on behalf of Botanical Society of America.)

Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green alga Chlamydomonas reinhardtii to establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway., (© 2024. The Author(s).)

Hydrophobic microdomains, also known as hydrophobic patches, are essential for many important biological functions of water-soluble proteins. These include ligand or substrate binding, protein-protein interactions, proper folding after translation, and aggregation during denaturation. Unlike transmembrane domains, which are easily recognized from stretches of contiguous hydrophobic sidechains in amino acids via primary protein sequence, these three-dimensional hydrophobic patches cannot be easily predicted. The lack of experimental strategies for directly determining their locations hinders further understanding of their structure and function. Here, we posit that the small triatomic anion N 3 - (azide) is attracted to these patches and, in the presence of an oxidant, forms a radical that covalently modifies C-H bonds of nearby amino acids. Using two model proteins (BSA and lysozyme) and a cell-free lysate from the model higher plant Arabidopsis thaliana , we find that radical-mediated covalent azidylation occurs within buried catalytic active sites and ligand binding sites and exhibits similar behavior to established hydrophobic probes. The results herein suggest a model in which the azido radical is acting as an "affinity reagent" for nonaqueous three-dimensional protein microenvironments and is consistent with both the nonlocalized electron density of the azide moiety and the known high reactivity of azido radicals widely used in organic chemistry syntheses. We propose that the azido radical is a facile means of identifying hydrophobic microenvironments in soluble proteins and, in addition, provides a simple new method for attaching chemical handles to proteins without the need for genetic manipulation or specialized reagents.

We have developed a method for complete dissolution of whole eggs in formic acid that provides a new approach to analyzing egg biomolecules. As expected from prior work with extracted lipids, phosphatidylcholine represents the most abundant 31 P NMR signal. A simplified methanol/chloroform partitioning method for separating the dissolved egg solution into metabolites, lipids and protein was performed and after ultra-high mass resolution and tandem MS fragmentation analyses several phosphatidylcholine molecules containing different fatty acid chain lengths as well as number and position of double bonds was detected. The MS based proteomic analysis further revealed 6 Gallus sequences annotated as 'uncharacterized' because they show no sequence homology with any other protein found in nature and thus, may represent proteins uniquely evolved to perform functions specific to chickens. Overall, this procedure is a rapid and facile means of characterizing in a high throughput and comprehensive manner, the molecular components of whole eggs., Competing Interests: Declaration of Competing Interest The Wisconsin Alumni Research Foundation, which is responsible for the intellectual property arising from UW-Madison research has applied for a patent entitled “Complete dissolution of dried whole egg in formic acid and/or nitric acid and use of the resulting solution in the food industry and for research in human nutrition”., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions-which are more difficult to study-dynamically regulate the proteome's 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based "structural" proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Oxidative proteome damage has been implicated as a major contributor to cell death and aging. Protein damage and aging has been a particular theme of the recent research of Miroslav Radman. However, the study of how cellular proteins are damaged by oxidative processes is still in its infancy. Here we examine oxidative changes in the proteomes of four bacterial populations-wild type E. coli , two isolates from E. coli populations evolved for high levels of ionizing radiation (IR) resistance, and D. radiodurans -immediately following exposure to 3000 Gy of ionizing radiation. By a substantial margin, the most prominent intracellular oxidation events involve hydroxylation of methionine residues. Significant but much less frequent are carbonylation events on tyrosine and dioxidation events on tryptophan. A few proteins are exquisitely sensitive to targeted oxidation events, notably the active site of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in E. coli . Extensive experimental evolution of E. coli for IR resistance has decreased overall proteome sensitivity to oxidation but not to the level seen in D. radiodurans . Many observed oxidation events may reflect aspects of protein structure and/or exposure of protein surfaces to water. Proteins such as GAPDH and possibly Ef-Tu may have an evolved sensitivity to oxidation by H 2 O 2 .

Coronaviruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), encode a nucleotidyl transferase in the N-terminal (NiRAN) domain of the nonstructural protein (nsp) 12 protein within the RNA dependent RNA polymerase. Here we show the detection of guanosine monophosphate (GMP) and uridine monophosphate-modified amino acids in nidovirus proteins using heavy isotope-assisted mass spectrometry (MS) and MS/MS peptide sequencing. We identified lysine-143 in the equine arteritis virus (EAV) protein, nsp7, as a primary site of in vitro GMP attachment via a phosphoramide bond. In SARS-CoV-2 replicase proteins, we demonstrate nsp12-mediated nucleotidylation of nsp7 lysine-2. Our results demonstrate new strategies for detecting GMP-peptide linkages that can be adapted for higher throughput screening using mass spectrometric technologies. These data are expected to be important for a rapid and timely characterization of a new enzymatic activity in SARS-CoV-2 that may be an attractive drug target aimed at limiting viral replication in infected patients., (© 2021. The Author(s).)

Ionizing radiation (IR) is lethal to most organisms at high doses, damaging every cellular macromolecule via induction of reactive oxygen species (ROS). Utilizing experimental evolution and continuing previous work, we have generated the most IR-resistant Escherichia coli populations developed to date. After 100 cycles of selection, the dose required to kill 99% the four replicate populations (IR9-100, IR10-100, IR11-100, and IR12-100) has increased from 750 Gy to approximately 3,000 Gy. Fitness trade-offs, specialization, and clonal interference are evident. Long-lived competing sub-populations are present in three of the four lineages. In IR9, one lineage accumulates the heme precursor, porphyrin, leading to generation of yellow-brown colonies. Major genomic alterations are present. IR9 and IR10 exhibit major deletions and/or duplications proximal to the chromosome replication terminus. Contributions to IR resistance have expanded beyond the alterations in DNA repair systems documented previously. Variants of proteins involved in ATP synthesis (AtpA), iron-sulfur cluster biogenesis (SufD) and cadaverine synthesis (CadA) each contribute to IR resistance in IR9-100. Major genomic and physiological changes are emerging. An isolate from IR10 exhibits protein protection from ROS similar to the extremely radiation resistant bacterium Deinococcus radiodurans , without evident changes in cellular metal homeostasis. Selection is continuing with no limit to IR resistance in evidence as our E. coli populations approach levels of IR resistance typical of D. radiodurans ., (Copyright © 2020 Bruckbauer, Martin, Minkoff, Veling, Lancaster, Liu, Trimarco, Bushnell, Lipzen, Wood, Sussman, Pennacchio and Cox.)

Lipo-chitooligosaccharides (LCOs) are signaling molecules produced by rhizobial bacteria that trigger the nodulation process in legumes, and by some fungi that also establish symbiotic relationships with plants, notably the arbuscular and ecto mycorrhizal fungi. Here, we show that many other fungi also produce LCOs. We tested 59 species representing most fungal phyla, and found that 53 species produce LCOs that can be detected by functional assays and/or by mass spectroscopy. LCO treatment affects spore germination, branching of hyphae, pseudohyphal growth, and transcription in non-symbiotic fungi from the Ascomycete and Basidiomycete phyla. Our findings suggest that LCO production is common among fungi, and LCOs may function as signals regulating fungal growth and development.

Recent work has begun to investigate the role of protein damage in cell death because of ionizing radiation (IR) exposure, but none have been performed on a proteome-wide basis, nor have they utilized MS (MS) to determine chemical identity of the amino acid side chain alteration. Here, we use Escherichia coli to perform the first MS analysis of IR-treated intact cells on a proteome scale. From quintuplicate IR-treated (1000 Gy) and untreated replicates, we successfully quantified 13,262 peptides mapping to 1938 unique proteins. Statistically significant, but low in magnitude (<2-fold), IR-induced changes in peptide abundance were observed in 12% of all peptides detected, although oxidative alterations were rare. Hydroxylation (+15.99 Da) was the most prevalent covalent adduct detected. In parallel with these studies on E. coli , identical experiments with the IR-resistant bacterium, Deinococcus radiodurans, revealed orders of magnitude less effect of IR on the proteome. In E. coli , the most significant target of IR by a wide margin was glyceraldehyde 3'-phosphate dehydrogenase (GAPDH), in which the thiol side chain of the catalytic Cys residue was oxidized to sulfonic acid. The same modification was detected in IR-treated human breast carcinoma cells. Sensitivity of GAPDH to reactive oxygen species (ROS) has been described previously in microbes and here, we present GAPDH as an immediate, primary target of IR-induced oxidation across all domains of life., Competing Interests: Conflict of interest—Authors declare no competing interests.., (© 2020 Bruckbauer et al.)

In plants and fungi, the plasma membrane proton pump (H + -ATPase) establishes an electrochemical gradient across the plasma membrane, which serves as the driving force for the secondary transport of ions and nutrients across the cell membrane. This is an essential enzyme that functions in many important processes including stomatal movement, cell elongation, and cellular responses to stimuli from hormones, light, and other environmental conditions. Therefore, understanding how the activity of the H + -ATPase is regulated is important to understand how plants adapt to different growth conditions. The autoinhibitory effect of the C-terminal regulatory domain of H + -ATPase is well-established and is thought to be mediated by interactions with the catalytic domains. Here, using the lysine reactive mass spectrometry cleavable cross-linker DSSO, we found that the C-terminal domain of the Arabidopsis H + -ATPase 2 (AHA2) cross-linked extensively with the actuator, nucleotide-binding, and phosphorylation domains, suggesting that the C-terminal domain regulates the catalytic cycle by modulating the relative positions of these domains. Interestingly, several C-terminal cross-links occurred near a predicted proton binding site (Asp-684 in TM6), suggesting that the C-terminal domain may regulate proton efflux. Additionally, cross-links between the C-terminal domain and other domains of AHA2 were detected in a monomeric protein resolved on SDS-PAGE, suggesting that intramolecular interactions may also be involved in the regulation of enzyme activity. Finally, we observed mixed-isotope cross-linking between the C-terminal domain and other domains of 14 N-AHA2 (unlabeled) and 15 N-AHA2 (labeled), supporting our model that oligomeric H + -ATPase may autoinhibit the neighboring monomer in a "head-to-tail" configuration.

Secondary metabolites (SMs) of medicinal plants are the material basis of their clinically curative effects. They are also important indicators for evaluating the quality of medicinal materials. However, the synthesis and accumulation of SMs are very complex, which are affected by many factors including internal developmental genetic circuits (regulated gene, enzyme) and by external environment factors (light, temperature, water, salinity, etc.). Currently, lots of literatures focused on the effect of environmental factors on the synthesis and accumulation of SMs of medicinal plants, the effect of the developmental growth and genetic factors on the synthesis and accumulation of SMs still lack systematic classification and summary. Here, we have given the review base on our previous works on the morphological development of medicinal plants and their secondary metabolites, and systematically outlined the literature reports how different environmental factors affected the synthesis and accumulation of SMs. The results of our reviews can know how developmental and environmental factors qualitatively and quantitatively influence SMs of medicinal plants and how these can be integrated as tools to quality control, as well as on the improvement of clinical curative effects by altering their genomes, and/or growth conditions., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Background: A major roadblock to reducing the mortality of colorectal cancer (CRC) is prompt detection and treatment, and a simple blood test is likely to have higher compliance than all of the current methods. The purpose of this report is to examine the utility of a mass spectrometry-based blood serum protein biomarker test for detection of CRC., Materials and Methods: Blood was drawn from individuals (n = 213) before colonoscopy or from patients with nonmetastatic CRC (n = 50) before surgery. Proteins were isolated from the serum of patients using targeted liquid chromatography-tandem mass spectrometry. We designed a machine-learning statistical model to assess these proteins., Results: When considered individually, over 70% of the selected biomarkers showed significance by Mann-Whitney testing for distinguishing cancer-bearing cases from cancer-free cases. Using machine-learning methods, peptides derived from epidermal growth factor receptor and leucine-rich alpha-2-glycoprotein 1 were consistently identified as highly predictive for detecting CRC from cancer-free cases. A five-marker panel consisting of leucine-rich alpha-2-glycoprotein 1, epidermal growth factor receptor, inter-alpha-trypsin inhibitor heavy-chain family member 4, hemopexin, and superoxide dismutase 3 performed the best with 70% specificity at over 89% sensitivity (area under the curve = 0.86) in the validation set. For distinguishing regional from localized cancers, cross-validation within the training set showed that a panel of four proteins consisting of CD44 molecule, GC-vitamin D-binding protein, C-reactive protein, and inter-alpha-trypsin inhibitor heavy-chain family member 3 yielded the highest performance (area under the curve = 0.75)., Conclusions: The minimally invasive blood biomarker panels identified here could serve as screening/detection alternatives for CRC in a human population and potentially useful for staging of existing cancer., (Copyright © 2019 Elsevier Inc. All rights reserved.)

We report the recombinant preparation from Escherichia coli cells of samples of two closely related, small, secreted cysteine-rich plant peptides: rapid alkalinization factor 1 (RALF1) and rapid alkalinization factor 8 (RALF8). Purified samples of the native sequence of RALF8 exhibited well-resolved nuclear magnetic resonance (NMR) spectra and also biological activity through interaction with a plant receptor kinase, cytoplasmic calcium mobilization, and in vivo root growth suppression. By contrast, RALF1 could only be isolated from inclusion bodies as a construct containing an N-terminal His-tag; its poorly resolved NMR spectrum was indicative of aggregation. We prepared samples of the RALF8 peptide labeled with 15 N and 13 C for NMR analysis and obtained near complete 1 H, 13 C, and 15 N NMR assignments; determined the disulfide pairing of its four cysteine residues; and examined its solution structure. RALF8 is mostly disordered except for the two loops spanned by each of its two disulfide bridges., (© 2019 The Authors. Protein Science published by Wiley Periodicals, Inc. on behalf of The Protein Society.)

To identify modifications to amino acids that are directly induced by ionizing radiation, free amino acids and 3-residue peptides were irradiated using a linear accelerator (Linac) radiotherapy device. Mass spectrometry was performed to detail the relative sensitivity to radiation as well as identify covalent, radiation-dependent adducts. The order of reactivity of the 20 common amino acids was generally in agreement with published literature except for His (most reactive of the 20) and Cys (less reactive). Novel and previously identified modifications on the free amino acids were detected. Amino acids were far less reactive when flanked by glycine residues in a tripeptide. Order of reactivity, with GVG most and GEG least, was substantially altered, as were patterns of modification. Radiation reactivity of amino acids is clearly and strongly affected by conversion of the α-amino and α-carboxyl groups to peptide bonds, and the presence of neighboring amino acid residues.

A major challenge for the reduction of colon cancer is to detect patients carrying high-risk premalignant adenomas with minimally invasive testing. As one step, we have addressed the feasibility of detecting protein signals in the serum of patients carrying an adenoma as small as 6-9 mm in maximum linear dimension. Serum protein biomarkers, discovered in two animal models of early colonic adenomagenesis, were studied in patients using quantitative mass-spectrometric assays. One cohort included patients bearing adenomas known to be growing on the basis of longitudinal computed tomographic colonography. The other cohort, screened by optical colonoscopy, included both patients free of adenomas and patients bearing adenomas whose risk status was judged by histopathology. The markers F5, ITIH4, LRG1, and VTN were each elevated both in this patient study and in the studies of the Pirc rat model. The quantitative study in the Pirc rat model had demonstrated that the elevated level of each of these markers is correlated with the number of colonic adenomas. However, the levels of these markers in patients were not significantly correlated with the total adenoma volume. Postpolypectomy blood samples demonstrated that the elevated levels of these four conserved markers persisted after polypectomy. Two additional serum markers rapidly renormalized after polypectomy: growth-associated CRP levels were enhanced only with high-risk adenomas, while PI16 levels, not associated with growth, were reduced regardless of risk status. We discuss biological hypotheses to account for these observations, and ways for these signals to contribute to the prevention of colon cancer., Competing Interests: Conflict of interest statement: P.J.P. is cofounder of VirtuoCTC, consultant for Bracco and Check-Cap, and shareholder of SHINE, Elucent, and Cellectar. M.M.I., P.J.P., M.R., M.R.S., and W.F.D. are inventors on patent application PCTUS2015065049 submitted by The Wisconsin Alumni Research Foundation that covers the quantitative proteomic analysis, animal model, and patient resource design described in this report. The other authors declare no potential conflicts of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Signals and signaling pathways underlying the symbiosis between legumes and rhizobia have been studied extensively over the past decades. In a previous phosphoproteomic study on the Medicago truncatula-Sinorhizobium meliloti symbiosis, we identified plant proteins that are differentially phosphorylated upon the perception of rhizobial signals, called Nod factors. In this study, we provide experimental evidence that one of these proteins, Early Phosphorylated Protein 1 (EPP1), is required for the initiation of this symbiosis. Upon inoculation with rhizobia, MtEPP1 expression was induced in curled root hairs. Down-regulation of MtEPP1 in M. truncatula roots almost abolished calcium spiking, reduced the expression of essential symbiosis-related genes (MtNIN, MtNF-YB1, MtERN1 and MtENOD40) and strongly decreased nodule development. Phylogenetic analyses revealed that orthologs of MtEPP1 are present in legumes and specifically in plant species able to host arbuscular mycorrhizal fungi, suggesting a possible role in this association too. Short chitin oligomers induced the phosphorylation of MtEPP1 like Nod factors. However, the down-regulation of MtEPP1 affected the colonization of M. truncatula roots by arbuscular mycorrhizal fungi only moderately. Altogether, these findings indicate that MtEPP1 is essential for the establishment of the legume-rhizobia symbiosis but might plays a limited role in the arbuscular mycorrhizal symbiosis., (© The Author(s) 2018. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Modern tandem MS-based sequencing technologies allow for the parallel measurement of concentration and covalent modifications for proteins within a complex sample. Recently, this capability has been extended to probe a proteome's three-dimensional structure and conformational state by determining the thermal denaturation profile of thousands of proteins simultaneously. Although many animals and their resident microbes exist under a relatively narrow, regulated physiological temperature range, plants take on the often widely ranging temperature of their surroundings, possibly influencing the evolution of protein thermal stability. In this report we present the first in-depth look at the thermal proteome of a plant species, the model organism Arabidopsis thaliana By profiling the melting curves of over 1700 Arabidopsis proteins using six biological replicates, we have observed significant correlation between protein thermostability and several known protein characteristics, including molecular weight and the composition ratio of charged to polar amino acids. We also report on a divergence of the thermostability of the core and regulatory domains of the plant 26S proteasome that may reflect a unique property of the way protein turnover is regulated during temperature stress. Lastly, the highly replicated database of Arabidopsis melting temperatures reported herein provides baseline data on the variability of protein behavior in the assay. Unfolding behavior and experiment-to-experiment variability were observed to be protein-specific traits, and thus this data can serve to inform the design and interpretation of future targeted assays to probe the conformational status of proteins from plants exposed to different chemical, environmental and genetic challenges., (© 2019 Volkening et al.)

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p -benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a "head-to-tail" organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers., (© 2018 Nguyen et al.)

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is routinely used to determine the spatial distributions of various biomolecules in tissues. Recently, there has been an increased interest in creating higher resolution images using sources with more focused beams. One such source, an atmospheric pressure (AP) MALDI source from MassTech, has a laser capable of reaching spatial resolutions of 10 μm. Here, the AP-MALDI source coupled with a Q Exactive HF Orbitrap platform is compared to the commercial MALDI LTQ Orbitrap XL system using Medicago truncatula root nodules. AP-MALDI parameters, such as the S-lens value, capillary temperature, and spray voltage, were optimized on the Q Exactive-HF platform for optimal detection of plant metabolites. The performance of the two systems was evaluated for sensitivity, spatial resolution, and overall ability to detect plant metabolites. The commercial MALDI LTQ Orbitrap XL was superior regarding the number of compounds detected, as at least two times more m/z were detected compared to the AP-MALDI system. However, although the AP-MALDI source requires a spatial resolution higher than 10 μm to get the best signal, the spatial resolution at 30 μm is still superior compared to the 75 μm spatial resolution achieved on the MALDI platform. The AP-MALDI system was also used to investigate the metabolites present in M. truncatula roots and root nodules under high salt and low salt conditions. A discriminative analysis with SCiLS software revealed m/z ions specific to the control and salt conditions. This analysis revealed 44 m/z ions present at relatively higher abundances in the control samples, and 77 m/z enriched in the salt samples. Liquid chromatography-tandem MS was performed to determine the putative molecular identities of some of the mass ions enriched in each sample, including, asparagine, adenosine, and nicotianamine in the control samples, and arginine and soyasaponin I in the salt treated samples.

FERONIA (FER), one of the 17 malectin-like receptor-like kinases encoded in the Arabidopsis genome, acts as a receptor for a 5 kDa growth-inhibiting secreted protein hormone, rapid alkalinization factor 1 (RALF1). Upon binding the peptide ligand, FER is involved in a variety of signaling pathways eliciting ovule fertilization and vegetative root cell expansion. Here, we report the use of mass spectrometry-based, carbodiimide-mediated protein carboxyl group (aspartic and glutamic acid) footprinting to map solvent accessible amino acids of the ectodomain of FER (ectoFER), including those involved in RALF1 binding and/or allosteric changes. Aspartate and glutamate residues labeled in this procedure were located in various regions, including the N-terminus, malectin-like domains, and juxtamembrane region, and these correlated well with a three-dimensional structural model of ectoFER predicted from the crystal structure of a related receptor. Covalent cross-linking experiments also revealed the N-terminus of ectoFER linked to the highly conserved C-terminus of RALF1. RALF1 binding assays performed with truncation mutants of ectoFER further implicated the receptor N-terminal and juxtamembrane regions in the binding of RALF1. In conclusion, our results of mass spectrometry-based footprinting methods provide a framework for understanding ligand-induced changes in solvent accessibility and their positions within the three-dimensional structure of a plant receptor kinase.

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., (© 2018 Federation of European Biochemical Societies.)

A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Protein three-dimensional structure dynamically changes in solution depending on the presence of ligands and interacting proteins. Methods for detecting these changes in protein conformation include 'protein footprinting,' using mass spectrometry. We describe herein a new technique, PLIMB (Plasma Induced Modification of Biomolecules), that generates µs bursts of hydroxyl radicals from water, to measure changes in protein structure via altered solvent accessibility of amino acid side chains. PLIMB was first benchmarked with model compounds, and then applied to a biological problem, i.e., ligand (EGF) induced changes in the conformation of the external (ecto) domain of Epidermal Growth Factor Receptor (EGFR). Regions in which oxidation decreased upon adding EGF fall along the dimerization interface, consistent with models derived from crystal structures. These results demonstrate that plasma-generated hydroxyl radicals from water can be used to map protein conformational changes, and provide a readily accessible means of studying protein structure in solution.

The electric eel ( Electrophorus electricus ) is unusual among electric fishes because it has three pairs of electric organs that serve multiple biological functions: For navigation and communication, it emits continuous pulses of weak electric discharge (<1 V), but for predation and defense, it intermittently emits lethal strong electric discharges (10 to 600 V). We hypothesized that these two electrogenic outputs have different energetic demands reflected by differences in their proteome and phosphoproteome. We report the use of isotope-assisted quantitative mass spectrometry to test this hypothesis. We observed novel phosphorylation sites in sodium transporters and identified a potassium channel with unique differences in protein concentration among the electric organs. In addition, we found transcription factors and protein kinases that show differential abundance in the strong versus weak electric organs. Our findings support the hypothesis that proteomic differences among electric organs underlie differences in energetic needs, reflecting a trade-off between generating weak voltages continuously and strong voltages intermittently.

There are more than 600 receptor-like kinases (RLKs) in Arabidopsis , but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Mutations in the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP) lead to the rare and fatal disorder, Alexander disease (AxD). A prominent feature of the disease is aberrant accumulation of GFAP. It has been proposed that this accumulation occurs because of an increase in gene transcription coupled with impaired proteasomal degradation, yet this hypothesis remains untested. We therefore sought to directly investigate GFAP turnover in a mouse model of AxD that is heterozygous for a disease-causing point mutation ( Gfap R236H /+ ) (and thus expresses both wild-type and mutant protein). Stable isotope labeling by amino acids in cell culture, using primary cortical astrocytes, indicated that the in vitro half-lives of total GFAP in astrocytes from wild-type and mutant mice were similar at ∼3-4 days. Surprisingly, results obtained with stable isotope labeling of mammals revealed that, in vivo , the half-life of GFAP in mutant mice (15.4 ± 0.5 days) was much shorter than that in wild-type mice (27.5 ± 1.6 days). These unexpected in vivo data are most consistent with a model in which synthesis and degradation are both increased. Our work reveals that an AxD-causing mutation alters GFAP turnover kinetics in vivo and provides an essential foundation for future studies aimed at preventing or reducing the accumulation of GFAP. In particular, these data suggest that elimination of GFAP might be possible and occurs more quickly than previously surmised., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase family (approximately 5% of the protein coding genes), although the specific function for only a few dozen of these kinases is clearly established. Recent comparative genomics studies have revealed that parasitic nematodes and pathogenic microbes produce plant peptide hormone mimics that target specific plant plasma membrane receptor-like protein kinases, thus usurping endogenous signaling pathways for their own pathogenic purposes. With biochemical, genetic, and physiological analyses of the regulation of hormone receptor signal pathways, we are thus just now beginning to understand how plants optimize the development of their body shape and cope with constantly changing environmental conditions., (© 2017 Elsevier Inc. All rights reserved.)

Plant science is an important, rapidly developing area of study. Within plant science, one area of study that has grown tremendously with recent technological advances, such as mass spectrometry, is the field of plant-omics; however, plant peptidomics is relatively underdeveloped in comparison with proteomics and metabolomics. Endogenous plant peptides can act as signaling molecules and have been shown to affect cell division, development, nodulation, reproduction, symbiotic associations, and defense reactions. There is a growing need to uncover the role of endogenous peptides on a molecular level. Mass spectrometric imaging (MSI) is a valuable tool for biological analyses as it allows for the detection of thousands of analytes in a single experiment and also displays spatial information for the detected analytes. Despite the prediction of a large number of plant peptides, their detection and imaging with spatial localization and chemical specificity is currently lacking. Here we analyzed the endogenous peptides and proteins in Medicago truncatula using matrix-assisted laser desorption/ionization (MALDI)-MSI. Hundreds of endogenous peptides and protein fragments were imaged, with interesting peptide spatial distribution changes observed between plants in different developmental stages.

Legumes are essential components of agricultural systems because they enrich the soil in nitrogen and require little environmentally deleterious fertilizers. A complex symbiotic association between legumes and nitrogen-fixing soil bacteria called rhizobia culminates in the development of root nodules, where rhizobia fix atmospheric nitrogen and transfer it to their plant host. Here we describe a quantitative proteomic atlas of the model legume Medicago truncatula and its rhizobial symbiont Sinorhizobium meliloti, which includes more than 23,000 proteins, 20,000 phosphorylation sites, and 700 lysine acetylation sites. Our analysis provides insight into mechanisms regulating symbiosis. We identify a calmodulin-binding protein as a key regulator in the host and assign putative roles and targets to host factors (bioactive peptides) that control gene expression in the symbiont. Further mining of this proteomic resource may enable engineering of crops and their microbial partners to increase agricultural productivity and sustainability.

Unlike the major cereal crops corn, rice, and wheat, leguminous plants such as soybean and alfalfa can meet their nitrogen requirement via endosymbiotic associations with soil bacteria. The establishment of this symbiosis is a complex process playing out over several weeks and is facilitated by the exchange of chemical signals between these partners from different kingdoms. Several plant components that are involved in this signaling pathway have been identified, but there is still a great deal of uncertainty regarding the early events in symbiotic signaling, i.e., within the first minutes and hours after the rhizobial signals (Nod factors) are perceived at the plant plasma membrane. The presence of several protein kinases in this pathway suggests a mechanism of signal transduction via posttranslational modification of proteins in which phosphate is added to the hydroxyl groups of serine, threonine and tyrosine amino acid side chains. To monitor the phosphorylation dynamics and complement our previous untargeted 'discovery' approach, we report here the results of experiments using a targeted mass spectrometric technique, Selected Reaction Monitoring (SRM) that enables the quantification of phosphorylation targets with great sensitivity and precision. Using this approach, we confirm a rapid change in the level of phosphorylation in 4 phosphosites of at least 4 plant phosphoproteins that have not been previously characterized. This detailed analysis reveals aspects of the symbiotic signaling mechanism in legumes that, in the long term, will inform efforts to engineer this nitrogen-fixing symbiosis in important non-legume crops such as rice, wheat and corn.

The wall-associated kinases (WAKs)(1)are receptor protein kinases that bind to long polymers of cross-linked pectin in the cell wall. These plasma-membrane-associated protein kinases also bind soluble pectin fragments called oligo-galacturonides (OGs) released from the wall after pathogen attack and damage. WAKs are required for cell expansion during development but bind water soluble OGs generated from walls with a higher affinity than the wall-associated polysaccharides. OGs activate a WAK-dependent, distinct stress-like response pathway to help plants resist pathogen attack. In this report, a quantitative mass-spectrometric-based phosphoproteomic analysis was used to identify Arabidopsis cellular events rapidly induced by OGsin planta Using N(14/)N(15)isotopicin vivometabolic labeling, we screened 1,000 phosphoproteins for rapid OG-induced changes and found 50 proteins with increased phosphorylation, while there were none that decreased significantly. Seven of the phosphosites within these proteins overlap with those altered by another signaling molecule plants use to indicate the presence of pathogens (the bacterial "elicitor" peptide Flg22), indicating distinct but overlapping pathways activated by these two types of chemicals. Genetic analysis of genes encoding 10 OG-specific and two Flg22/OG-induced phosphoproteins reveals that null mutations in eight proteins compromise the OG response. These phosphorylated proteins with genetic evidence supporting their role in the OG response include two cytoplasmic kinases, two membrane-associated scaffold proteins, a phospholipase C, a CDPK, an unknown cadmium response protein, and a motor protein. Null mutants in two proteins, the putative scaffold protein REM1.3, and a cytoplasmic receptor like kinase ROG2, enhance and suppress, respectively, a dominantWAKallele. Altogether, the results of these chemical and genetic experiments reveal the identity of several phosphorylated proteins involved in the kinase/phosphatase-mediated signaling pathway initiated by cell wall changes., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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 crucial 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., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

In plants and fungi the plasma membrane proton pump generates a large proton-motive force that performs essential functions in many processes, including solute transport and the control of cell elongation. Previous studies in yeast and higher plants have indicated that phosphorylation of an auto-inhibitory domain is involved in regulating pump activity. In this report we examine the Medicago truncatula plasma membrane proton pump gene family, and in particular MtAHA5. Yeast complementation assays with phosphomimetic mutations at six candidate sites support a phosphoregulatory role for two residues, suggesting a molecular model to explain early Nod factor-induced changes in the plasma membrane proton-motive force of legume root cells., (Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Abscisic acid (ABA)¹ is a plant hormone that controls many aspects of plant growth, including seed germination, stomatal aperture size, and cellular drought response. ABA interacts with a unique family of 14 receptor proteins. This interaction leads to the activation of a family of protein kinases, SnRK2s, which in turn phosphorylate substrates involved in many cellular processes. The family of receptors appears functionally redundant. To observe a measurable phenotype, four of the fourteen receptors have to be mutated to create a multilocus loss-of-function quadruple receptor (QR) mutant, which is much less sensitive to ABA than wild-type (WT) plants. Given these phenotypes, we asked whether or not a difference in ABA response between the WT and QR backgrounds would manifest on a phosphorylation level as well. We tested WT and QR mutant ABA response using isotope-assisted quantitative phosphoproteomics to determine what ABA-induced phosphorylation changes occur in WT plants within 5 min of ABA treatment and how that phosphorylation pattern is altered in the QR mutant. We found multiple ABA-induced phosphorylation changes that occur within 5 min of treatment, including three SnRK2 autophosphorylation events and phosphorylation on SnRK2 substrates. The majority of robust ABA-dependent phosphorylation changes observed were partially diminished in the QR mutant, whereas many smaller ABA-dependent phosphorylation changes observed in the WT were not responsive to ABA in the mutant. A single phosphorylation event was increased in response to ABA treatment in both the WT and QR mutant. A portion of the discovery data was validated using selected reaction monitoring-based targeted measurements on a triple quadrupole mass spectrometer. These data suggest that different subsets of phosphorylation events depend upon different subsets of the ABA receptor family to occur. Altogether, these data expand our understanding of the model by which the family of ABA receptors directs rapid phosphoproteomic changes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: With its unique ability to produce high-voltage electric discharges in excess of 600 volts, the South American strong voltage electric eel (Electrophorus electricus) has played an important role in the history of science. Remarkably little is understood about the molecular nature of its electric organs., Results: We present an in-depth analysis of the genome of E. electricus, including the transcriptomes of eight mature tissues: brain, spinal cord, kidney, heart, skeletal muscle, Sachs' electric organ, main electric organ, and Hunter's electric organ. A gene set enrichment analysis based on gene ontology reveals enriched functions in all three electric organs related to transmembrane transport, androgen binding, and signaling. This study also represents the first analysis of miRNA in electric fish. It identified a number of miRNAs displaying electric organ-specific expression patterns, including one novel miRNA highly over-expressed in all three electric organs of E. electricus. All three electric organ tissues also express three conserved miRNAs that have been reported to inhibit muscle development in mammals, suggesting that miRNA-dependent regulation of gene expression might play an important role in specifying an electric organ identity from its muscle precursor. These miRNA data were supported using another complete miRNA profile from muscle and electric organ tissues of a second gymnotiform species., Conclusions: Our work on the E. electricus genome and eight tissue-specific gene expression profiles will greatly facilitate future research on determining the coding and regulatory sequences that specify the function, development, and evolution of electric organs. Moreover, these data and future studies will be informed by the first comprehensive analysis of miRNA expression in an electric fish presented here.

The development of noninvasive methods for early detection of colon cancer is critical for the successful management of this disease. Using a targeted quantitative proteomics technique, we assessed the ability of 12 serum proteins to detect the presence of colonic polyps in the Apc(Pirc) (/+) rat model of familial colon cancer. Serum protein candidates were selected from gene transcripts upregulated in colonic tumors of Apc(Pirc) (/+) rats and from a prior study of serum proteins differentially expressed in mice carrying intestinal adenomas. Proteins were quantified at early stages of polyp formation in a rat cohort monitored longitudinally by colonoscopy over a period of 75 days. Of the 12 proteins monitored at three distinct time points, seven showed differential expression in at least one time point in the serum from Apc(Pirc) (/+) rats compared with wild-type rats. Tumor multiplicity correlated with protein expression changes, and most tumors grew during the study. EGFR, LRG1, ITIH4, and F5 displayed the most robust tumor-associated protein expression changes over time. Receiver operator characteristic analysis using these four proteins resulted in a sensitivity of 100%, a specificity of 80%, and an area under the curve of 0.93 at 135 days of age, when the Pirc rats bore an average of 19 tumors in the colon and seven in the small intestine. The results of this study demonstrate that the quantitative analysis of a panel of serum proteins can detect the presence of early intestinal tumors in a rat model, and provides support for future measurements in humans., (©2014 American Association for Cancer Research.)

Elucidating how plants sense and respond to water loss is important for identifying genetic and chemical interventions that may help sustain crop yields in water-limiting environments. Currently, the molecular mechanisms involved in the initial perception and response to dehydration are not well understood. Modern mass spectrometric methods for quantifying changes in the phosphoproteome provide an opportunity to identify key phosphorylation events involved in this process. Here, we have used both untargeted and targeted isotope-assisted mass spectrometric methods of phosphopeptide quantitation to characterize proteins in Arabidopsis (Arabidopsis thaliana) whose degree of phosphorylation is rapidly altered by hyperosmotic treatment. Thus, protein phosphorylation events responsive to 5 min of 0.3 m mannitol treatment were first identified using 15 N metabolic labeling and untargeted mass spectrometry with a high-resolution ion-trap instrument. The results from these discovery experiments were then validated using targeted Selected Reaction Monitoring mass spectrometry with a triple quadrupole. Targeted Selected Reaction Monitoring experiments were conducted with plants treated under nine different environmental perturbations to determine whether the phosphorylation changes were specific for osmosignaling or involved cross talk with other signaling pathways. The results indicate that regulatory proteins such as members of the mitogen-activated protein kinase family are specifically phosphorylated in response to osmotic stress. Proteins involved in 5' messenger RNA decapping and phosphatidylinositol 3,5-bisphosphate synthesis were also identified as targets of dehydration-induced phosphoregulation. The results of these experiments demonstrate the utility of targeted phosphoproteomic analysis in understanding protein regulation networks and provide new insight into cellular processes involved in the osmotic stress response., (© 2014 American Society of Plant Biologists. All Rights Reserved.)

Little is known about the genetic basis of convergent traits that originate repeatedly over broad taxonomic scales. The myogenic electric organ has evolved six times in fishes to produce electric fields used in communication, navigation, predation, or defense. We have examined the genomic basis of the convergent anatomical and physiological origins of these organs by assembling the genome of the electric eel (Electrophorus electricus) and sequencing electric organ and skeletal muscle transcriptomes from three lineages that have independently evolved electric organs. Our results indicate that, despite millions of years of evolution and large differences in the morphology of electric organ cells, independent lineages have leveraged similar transcription factors and developmental and cellular pathways in the evolution of electric organs., (Copyright © 2014, American Association for the Advancement of Science.)

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H + -ATPases (PM H + -ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H + -ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H + -ATPase function. We find that SAUR19 stimulates PM H + -ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H + -ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H + -ATPases, and negatively regulate PM H + -ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion., (© 2014 American Society of Plant Biologists. All rights reserved.)

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.

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 Ser(899), 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.