Sylwia Kierszniowska

PAMP (Pathogen-associated Molecular Pattern)-induced Changes in Plasma Membrane Compartmentalization Reveal Novel Components of Plant Immunity

Plasma membrane compartmentalization spatiotemporally regulates cell-autonomous immune signaling in animal cells. To elucidate immediate early protein dynamics at the plant plasma membrane in response to the bacterial pathogen-associated molecular pattern (PAMP) flagellin (flg22) we employed quantitative mass spectrometric analysis on detergent-resistant membranes (DRMs) of Arabidopsis thaliana suspension cells. This approach revealed rapid and profound changes in DRM protein composition following PAMP treatment, prominently affecting proton ATPases and receptor-like kinases, including the flagellin receptor FLS2. We employed reverse genetics to address a potential contribution of a subset of these proteins in flg22-triggered cellular responses. Mutants of three candidates (DET3, AHA1, FER) exhibited a conspicuous defect in the PAMP-triggered accumulation of reactive oxygen species. In addition, these mutants showed altered mitogen-activated protein kinase (MAPK) activation, a defect in PAMP-triggered stomatal closure as well as altered bacterial infection phenotypes, which revealed three novel players in elicitor-dependent oxidative burst control and innate immunity. Our data provide evidence for dynamic elicitor-induced changes in the membrane compartmentalization of PAMP signaling components.

Definition of Arabidopsis sterol-rich membrane microdomains by differential treatment with methyl-beta-cyclodextrin and quantitative proteomics.

Plasma membranes are dynamic compartments with key functions in solute transport, cell shape, and communication between cells and the environment. In mammalian cells and yeast, the plasma membrane has been shown to be compartmented into so-called lipid rafts, which are defined by their resistance to treatment with non-ionic detergents. In plants, the existence of lipid rafts has been postulated, but the precise composition of this membrane compartment is still under debate. Here we were able to experimentally clearly distinguish (i) true sterol-dependent “raft proteins” and (ii) sterol-independent “non-raft” proteins and co-purifying “contaminants” in plant detergent-resistant membranes. We used quantitative proteomics techniques involving 15N metabolic labeling and specific disruption of sterol-rich membrane domains by methyl-β-cyclodextrin. Among the sterol-dependent proteins we found an over-representation of glycosylphosphatidylinositol-anchored proteins. A large fraction of these proteins has functions in cell wall anchoring. We were able to distinguish constant and variable components of plant sterol-rich membrane microdomains based on their responsiveness to the drug methyl-β-cyclodextrin. Predominantly proteins with signaling functions, such as receptor kinases, G-proteins, and calcium signaling proteins, were identified as variable members in plant lipid rafts, whereas cell wall-related proteins and specific proteins with unknown functions make up a core set of sterol-dependent plant plasma membrane proteins. This allows the plant to maintain a balance between static anchoring of cell shape forming elements and variable adjustment to changing external conditions.

Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3

The phytohormone abscisic acid (ABA) plays a key role in the plant response to drought stress. Hence, ABA-dependent gene transcription and ion transport is regulated by a variety of protein kinases and phosphatases. However, the nature of the membrane-delimited ABA signal transduction steps remains largely unknown. To gain insight into plasma membrane-bound ABA signaling, we identified sterol-dependent proteins associated with detergent resistant membranes from Arabidopsis thaliana mesophyll cells. Among those, we detected the central ABA signaling phosphatase ABI1 (abscisic-acid insensitive 1) and the calcium-dependent protein kinase 21 (CPK21). Using fluorescence microscopy, we found these proteins to localize in membrane nanodomains, as observed by colocalization with the nanodomain marker remorin Arabidopsis thaliana remorin 1.3 (AtRem 1.3). After transient coexpression, CPK21 interacted with SLAH3 [slow anion channel 1 (SLAC1) homolog 3] and activated this anion channel. Upon CPK21 stimulation, SLAH3 exhibited the hallmark properties of S-type anion channels. Coexpression of SLAH3/CPK21 with ABI1, however, prevented proper nanodomain localization of the SLAH3/CPK21 protein complex, and as a result anion channel activation failed. FRET studies revealed enhanced interaction of SLAH3 and CPK21 within the plasma membrane in response to ABA and thus confirmed our initial observations. Interestingly, the ABA-induced SLAH3/CPK21 interaction was modulated by ABI1 and the ABA receptor RCAR1/PYL9 [regulatory components of ABA receptor 1/PYR1 (pyrabactin resistance 1)-like protein 9]. We therefore propose that ABA signaling via inhibition of ABI1 modulates the apparent association of a signaling and transport complex within membrane domains that is necessary for phosphorylation and activation of the S-type anion channel SLAH3 by CPK21.

Ratio-dependent significance thresholds in reciprocal 15N-labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Metabolic labeling of plant tissues with 15N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on 15N/14N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio‐dependent SD is used to define p‐values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.

The use of heavy nitrogen in quantitative proteomics experiments in plants

In the growing field of plant systems biology, there is an undisputed need for methods allowing accurate quantitation of proteins and metabolites. As autotrophic organisms, plants can easily metabolize different nitrogen isotopes, resulting in proteins and metabolites with distinct molecular mass that can be separated on a mass spectrometer. In comparative quantitative experiments, treated and untreated samples are differentially labeled by nitrogen isotopes and jointly processed, thereby minimizing sample-to-sample variation. In recent years, heavy nitrogen labeling has become a widely used strategy in quantitative proteomics and novel approaches have been developed for metabolite identification. Here, we present an overview of currently used experimental strategies in heavy nitrogen labeling in plants and provide background on the history and function of this quantitation technique.

Remodeling of Leaf Cellular Glycerolipid Composition under Drought and Re-hydration Conditions in Grasses from the Lolium-Festuca Complex.

Drought tolerant plant genotypes are able to maintain stability and integrity of cellular membranes in unfavorable conditions, and to regenerate damaged membranes after stress cessation. The profiling of cellular glycerolipids during drought stress performed on model species such as Arabidopsis thaliana does not fully cover the picture of lipidome in monocots, including grasses. Herein, two closely related introgression genotypes of Lolium multiflorum (Italian ryegrass) × Festuca arundinacea (tall fescue) were used as a model for other grass species to describe lipid rearrangements during drought and re-hydration. The genotypes differed in their level of photosynthetic capacity during drought, and in their capacity for membrane regeneration after stress cessation. A total of 120 lipids, comprising the classes of monogalactosyldiacyloglycerol, digalactosyldiacyloglycerol, sulfoquinovosyldiacylglycerol, phosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, diacylglicerol, and triacylglicerol, were analyzed. The results clearly showed that water deficit had a significant impact on lipid metabolism in studied forage grasses. It was revealed that structural and metabolic lipid species changed their abundance during drought and re-watering periods and some crucial genotype-dependent differences were also observed. The introgression genotype characterized by an ability to regenerate membranes after re-hydration demonstrated a higher accumulation level of most chloroplast and numerous extra-chloroplast membrane lipid species at the beginning of drought. Furthermore, this genotype also revealed a significant reduction in the accumulation of most chloroplast lipids after re-hydration, compared with the other introgression genotype without the capacity for membrane regeneration. The potential influence of observed lipidomic alterations on a cellular membrane stability and photosynthetic capacity, are discussed. HIGHLIGHTS A higher drought tolerance of grasses could be associated with an earlier lipidome response to a stress signal and with a membrane regeneration after stress cessation accompanied by a turnover of chloroplast lipids

Medicinal Bioprospecting of the Amazon Rainforest: A Modern Eldorado?

Ignorant of the New World, Europeans believed in El Dorado, a hidden city of immense wealth in gold. Many consider the Amazonian forest to be a medicinal treasure chest and potentially the largest drug dispensary in the world. Yet, the quest to obtain drugs from indigenous tropical plants remains elusive. Here, we assess the potential of new technologies to tap into the metabolic diversity of tropical plants. We also consider how regulations affect access to plant resources. We conclude that, although the road to this medicinal El Dorado may be long and arduous, many other smaller but still valuable finds are hidden along the way.

System-wide detection of protein-small molecule complexes suggests extensive metabolite regulation in plants

Protein small molecule interactions are at the core of cell regulation controlling metabolism and development. We reasoned that due to the lack of system wide approaches only a minority of those regulatory molecules are known. In order to see whether or not this assumption is true we developed an effective approach for the identification of small molecules having potential regulatory role that obviates the need of protein or small molecule baits. At the core of this approach is a simple biochemical co-fractionation taking advantage of size differences between proteins and small molecules. Metabolomics based analysis of small molecules co-fractionating with proteins identified a multitude of small molecules in Arabidopsis suggesting the existence of numerous, small molecules/metabolites bound to proteins representing potential regulatory molecules. The approach presented here uses Arabidopsis cell cultures, but is generic and hence applicable to all biological systems.

Metabolic labeling and membrane fractionation for comparative proteomic analysis of Arabidopsis thaliana suspension cell cultures.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% (1). Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient (2). Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K(15)NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest (3). By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control (4).

PROMIS, global analysis of PROtein–metabolite interactions using size separation in Arabidopsis thaliana

Small molecules not only represent cellular building blocks and metabolic intermediates, but also regulatory ligands and signaling molecules that interact with proteins. Although these interactions affect cellular metabolism, growth, and development, they have been largely understudied. Herein, we describe a method, which we named PROtein–Metabolite Interactions using Size separation (PROMIS), that allows simultaneous, global analysis of endogenous protein–small molecule and of protein–protein complexes. To this end, a cell-free native lysate from Arabidopsis thaliana cell cultures was fractionated by size-exclusion chromatography, followed by quantitative metabolomic and proteomic analyses. Proteins and small molecules showing similar elution behavior, across protein-containing fractions, constituted putative interactors. Applying PROMIS to an A. thaliana extract, we ascertained known protein–protein (PPIs) and protein–metabolite (PMIs) interactions and reproduced binding between small-molecule protease inhibitors and their respective proteases. More importantly, we present examples of two experimental strategies that exploit the PROMIS dataset to identify novel PMIs. By looking for similar elution behavior of metabolites and enzymes belonging to the same biochemical pathways, we identified putative feedback and feed-forward regulations in pantothenate biosynthesis and the methionine salvage cycle, respectively. By combining PROMIS with an orthogonal affinity purification approach, we identified an interaction between the dipeptide Tyr–Asp and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. In summary, we present proof of concept for a powerful experimental tool that enables system-wide analysis of PMIs and PPIs across all biological systems. The dataset obtained here comprises nearly 140 metabolites and 5000 proteins, which can be mined for putative interactors.