Mass spectrometry-based technologies for probing the 3D world of plant proteins.

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.

Ionizing Radiation-induced Proteomic Oxidation in Escherichia coli.

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.

Conserved serum protein biomarkers associated with growing early colorectal adenomas.

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.

Proteome-wide Analysis of Protein Thermal Stability in the Model Higher Plant Arabidopsis thaliana.

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.

Intermolecular and Intramolecular Interactions of the Arabidopsis Plasma Membrane Proton Pump Revealed Using a Mass Spectrometry Cleavable Cross-Linker.

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 14N-AHA2 (unlabeled) and 15N-AHA2 (labeled), supporting our model that oligomeric H+-ATPase may autoinhibit the neighboring monomer in a "head-to-tail" configuration.

Noninvasive Detection of Colorectal Carcinomas Using Serum Protein Biomarkers.

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.

In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase.

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.

A Novel Positive Regulator of the Early Stages of Root Nodule Symbiosis Identified by Phosphoproteomics.

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.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

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.

Probing a Plant Plasma Membrane Receptor Kinase's Three-Dimensional Structure Using Mass Spectrometry-Based Protein Footprinting.

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.

Glial fibrillary acidic protein exhibits altered turnover kinetics in a mouse model of Alexander disease.

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 (GfapR236H/+) (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.

A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

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.

Rapid Oligo-Galacturonide Induced Changes in Protein Phosphorylation in Arabidopsis.

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.

Algal ancestor of land plants was preadapted for symbiosis.

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.

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis.

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.

Rapid Phosphoproteomic Effects of Abscisic Acid (ABA) on Wild-Type and ABA Receptor-Deficient A. thaliana Mutants.

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.

Examination of Endogenous Peptides in Medicago truncatula Using Mass Spectrometry Imaging.

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.

Deletion of a tandem gene family in Arabidopsis: increased MEKK2 abundance triggers autoimmunity when the MEKK1-MKK1/2-MPK4 signaling cascade is disrupted.

An Arabidopsis thaliana mitogen-activated protein (MAP) kinase cascade composed of MEKK1, MKK1/MKK2, and MPK4 was previously described as a negative regulator of defense response. MEKK1 encodes a MAP kinase kinase kinase and is a member of a tandemly duplicated gene family with MEKK2 and MEKK3. Using T-DNA insertion lines, we isolated a novel deletion mutant disrupting this gene family and found it to be phenotypically wild-type, in contrast with the mekk1 dwarf phenotype. Follow-up genetic analyses indicated that MEKK2 is required for the mekk1, mkk1 mkk2, and mpk4 autoimmune phenotypes. We next analyzed a T-DNA insertion in the MEKK2 promoter region and found that although it does not reduce the basal expression of MEKK2, it does prevent the upregulation of MEKK2 that is observed in mpk4 plants. This mekk2 allele can rescue the mpk4 autoimmune phenotype in a dosage-dependent manner. We also found that expression of constitutively active MPK4 restored MEKK2 abundance to wild-type levels in mekk1 mutant plants. Finally, using mass spectrometry, we showed that MEKK2 protein levels mirror MEKK2 mRNA levels. Taken together, our results indicate that activated MPK4 is responsible for regulating MEKK2 RNA abundance. In turn, the abundance of MEKK2 appears to be under cellular surveillance such that a modest increase can trigger defense response activation.

Unique patterns of transcript and miRNA expression in the South American strong voltage electric eel (Electrophorus electricus).

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.

Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

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.