Benjamin B. Minkoff

A Peptide Hormone and Its Receptor Protein Kinase Regulate Plant Cell Expansion

Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H+–adenosine triphosphatase 2 at Ser899, mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion. A signaling system important in the regulation of plant cell size during development is identified. A Peptide Finds Its Receptor Because plant cells cannot move around within the plant, developmental changes are orchestrated by changes in cell size and shape. Using quantitative phosphoproteomics, genetics, and chemical analyses, Haruta et al. (p. 408) identified a signaling chain that links a secreted peptide, RALF (rapid alkalinization factor), with its receptor kinase, FERONIA, at the cell surface. FERONIA is involved in reproductive and vegetative development–processes that require the changes in cell size initiated by RALF signaling.

Phosphoproteomic Analyses Reveal Early Signaling Events in the Osmotic Stress Response

Rapid changes in protein phosphorylation across abiotic stress, hormone, and pathogen treatments reveal proteins undergoing phosphoregulation in response to dehydration. 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 15N 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.

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.

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 OGs in planta. Using N14/N15 isotopic in vivo metabolic 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 dominant WAK allele. 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.

A Pipeline for 15 N Metabolic Labeling and Phosphoproteome Analysis in Arabidopsis thaliana

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

Expression of a translationally fused TAP-tagged plasma membrane proton pump in Arabidopsis thaliana.

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.

Plasma-Generated OH Radical Production for Analyzing Three-Dimensional Structure in Protein Therapeutics

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.

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.

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.

Transmission of oxygen radicals through free-standing single-layer and multilayer silicon-nitride and silicon-dioxide films

Free radicals from processing plasmas are known to cause damage to dielectric films used in semiconductor devices. Many radicals are highly reactive and can readily interact with the material exposed to the plasma. This can modify the chemical structure of the material causing deterioration of electrical and mechanical properties of the films. This work detects the transmission of oxygen radicals through single- and double-layer silicon-nitride and silicon-dioxide freestanding films. The films were exposed to oxygen plasma. A fluorophore dye was used to detect the oxygen radicals traversing through the films. By measuring the fluorescence of the dye before and after multiple timed-plasma exposures, the transmission properties of oxygen radicals through the material were found. The results indicate that the absorption length of oxygen radicals increases with increasing plasma exposure times for Si3N4 films because the oxygen plasma oxidizes the top layer of the film and forms a less dense silicon oxynitrid...