Target of rapamycin signaling couples energy to oxygen sensing to modulate hypoxic gene expression in Arabidopsis.

Plants respond to oxygen deprivation by activating the expression of a set of hypoxia-responsive genes (HRGs). The master regulator of this process is a small group of transcription factors belonging to group VII of the ethylene response factors (ERF-VIIs). ERF-VIIs are highly unstable under aerobic conditions due to the continuous oxidation of their characteristic Cys residue at the N terminus by plant cysteine oxidases (PCOs). Under hypoxia, PCOs are inactive and the ERF-VIIs activate transcription of the HRGs required for surviving hypoxia. However, if the plant exposed to hypoxia has limited sugar reserves, the activity of ERF-VIIs is severely dampened. This suggests that oxygen sensing by PCO/ERF-VII is fine-tuned by another sensing pathway, related to sugar or energy availability. Here, we show that oxygen sensing by PCO/ERF-VII is controlled by the energy sensor target of rapamycin (TOR). Inhibition of TOR by genetic or pharmacological approaches leads to a much lower induction of HRGs. We show that two serine residues at the C terminus of RAP2.12, a major ERF-VII, are phosphorylated by TOR and are needed for TOR-dependent activation of transcriptional activity of RAP2.12. Our results demonstrate that oxygen and energy sensing converge in plants to ensure an appropriate transcription of genes, which is essential for surviving hypoxia. When carbohydrate metabolism is inefficient in producing ATP because of hypoxia, the lower ATP content reduces TOR activity, thus attenuating the efficiency of induction of HRGs by the ERF-VIIs. This homeostatic control of the hypoxia-response is required for the plant to survive submergence.

Purification of MAP-kinase protein complexes and identification of candidate components by XL-TAP-MS.

The purification of low-abundance protein complexes and detection of in vivo protein-protein interactions in complex biological samples remains a challenging task. Here, we devised crosslinking and tandem affinity purification coupled to mass spectrometry (XL-TAP-MS), a quantitative proteomics approach for analyzing tandem affinity-purified, crosslinked protein complexes from plant tissues. We exemplarily applied XL-TAP-MS to study the MKK2-Mitogen-activated protein kinase (MPK4) signaling module in Arabidopsis thaliana. A tandem affinity tag consisting of an in vivo-biotinylated protein domain flanked by two hexahistidine sequences was adopted to allow for the affinity-based isolation of formaldehyde-crosslinked protein complexes under fully denaturing conditions. Combined with 15N stable isotopic labeling and tandem MS we captured and identified a total of 107 MKK2-MPK4 module-interacting proteins. Consistent with the role of the MPK signaling module in plant immunity, many of the module-interacting proteins are involved in the biotic and abiotic stress response of Arabidopsis. Validation of binary protein-protein interactions by in planta split-luciferase assays and in vitro kinase assays disclosed several direct phosphorylation targets of MPK4. Together, the XL-TAP-MS approach purifies low abundance protein complexes from biological samples and discovers previously unknown protein-protein interactions.

Innate immune memory: An evolutionary perspective.

Over the last decades, there was increasing evidence for the presence of innate immune memory in living organisms. In this review, we compare the innate immune memory of various organisms with a focus on phylogenetics. We discuss the acquisition and molecular basis of immune memory and we describe the innate immune memory paradigm and its role in host defense during evolution. The molecular characterization of innate immunological memory in diverse organisms and host-parasite systems reconciles mechanisms with phenomena and paves the way to molecular comprehension of innate immune memory. We also revise the traditional classification of innate and adaptive immunity in jawed vertebrates. We emphasize that innate immune responses have the capacity to be "primed" or "trained", thereby exerting a yet unknown type of immunological memory upon re-infection.

ABA-Induced Stomatal Closure Involves ALMT4, a Phosphorylation-Dependent Vacuolar Anion Channel of Arabidopsis.

Stomatal pores are formed between a pair of guard cells and allow plant uptake of CO2 and water evaporation. Their aperture depends on changes in osmolyte concentration of guard cell vacuoles, specifically of K+ and Mal2- Efflux of Mal2- from the vacuole is required for stomatal closure; however, it is not clear how the anion is released. Here, we report the identification of ALMT4 (ALUMINUM ACTIVATED MALATE TRANSPORTER4) as an Arabidopsis thaliana ion channel that can mediate Mal2- release from the vacuole and is required for stomatal closure in response to abscisic acid (ABA). Knockout mutants showed impaired stomatal closure in response to the drought stress hormone ABA and increased whole-plant wilting in response to drought and ABA. Electrophysiological data show that ALMT4 can mediate Mal2- efflux and that the channel activity is dependent on a phosphorylatable C-terminal serine. Dephosphomimetic mutants of ALMT4 S382 showed increased channel activity and Mal2- efflux. Reconstituting the active channel in almt4 mutants impaired growth and stomatal opening. Phosphomimetic mutants were electrically inactive and phenocopied the almt4 mutants. Surprisingly, S382 can be phosphorylated by mitogen-activated protein kinases in vitro. In brief, ALMT4 likely mediates Mal2- efflux during ABA-induced stomatal closure and its activity depends on phosphorylation.

Substrate thiophosphorylation by Arabidopsis mitogen-activated protein kinases.

BACKGROUND: Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites. RESULTS: We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases. CONCLUSION: Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.

ABC transporter PEN3/PDR8/ABCG36 interacts with calmodulin that, like PEN3, is required for Arabidopsis nonhost resistance.

Nonhost resistance (NHR) is the most prevalent form of plant immunity. In Arabidopsis, NHR requires membrane-localized ATP-binding cassette (ABC) transporter PENETRATION (PEN) 3. Upon perception of pathogen-associated molecular patterns, PEN3 becomes phosphorylated, suggestive of PEN3 regulation by post-translational modification. Here, we investigated the PEN3 protein interaction network. We probed the Arabidopsis protein microarray AtPMA-5000 with the N-terminal cytoplasmic domain of PEN3. Several of the proteins identified to interact with PEN3 in vitro represent cellular Ca(2+) sensors, including calmodulin (CaM) 3, CaM7 and several CaM-like proteins, pointing to the importance of Ca(2+) sensing to PEN3-mediated NHR. We demonstrated co-localization of PEN3 and CaM7, and we confirmed PEN3-CaM interaction in vitro and in vivo by PEN3 pull-down with CaM Sepharose, CaM overlay assay and bimolecular fluorescence complementation. We also show that just like in pen3, NHR to the nonadapted fungal pathogens Phakopsora pachyrhizi and Blumeria graminis f.sp. hordei is compromised in the Arabidopsis cam7 and pen3 cam7 mutants. Our study discloses CaM7 as a PEN3-interacting protein crucial to Arabidopsis NHR and emphasizes the importance of Ca(2+) sensing to plant immunity.

Identification of novel in vivo MAP kinase substrates in Arabidopsis thaliana through use of tandem metal oxide affinity chromatography.

Mitogen-activated protein kinase (MPK) cascades are important for eukaryotic signal transduction. They convert extracellular stimuli (e.g. some hormones, growth factors, cytokines, microbe- or damage-associated molecular patterns) into intracellular responses while at the same time amplifying the transmitting signal. By doing so, they ensure proper performance, and eventually survival, of a given organism, for example in times of stress. MPK cascades function via reversible phosphorylation of cascade components MEKKs, MEKs, and MPKs. In plants the identity of most MPK substrates remained elusive until now. Here, we provide a robust and powerful approach to identify and quantify, with high selectivity, site-specific phosphorylation of MPK substrate candidates in the model plant Arabidopsis thaliana. Our approach represents a two-step chromatography combining phosphoprotein enrichment using Al(OH)(3)-based metal oxide affinity chromatography, tryptic digest of enriched phosphoproteins, and TiO(2)-based metal oxide affinity chromatography to enrich phosphopeptides from complex protein samples. When applied to transgenic conditional gain-of-function Arabidopsis plants supporting in planta activation of MPKs, the approach allows direct measurement and quantification ex vivo of site-specific phosphorylation of several reported and many yet unknown putative MPK substrates in just a single experiment.

Priming for stress resistance: from the lab to the field.

Upon treatment with necrotizing pathogens, many plants develop an enhanced capacity for activating defense responses to biotic and abiotic stress--a process called priming. The primed state can also be induced by colonization of plant roots with beneficial micro-organisms or by treatment of plants with various natural and synthetic compounds. Priming is thought to be the mechanism by which plants can show induced resistance against ostensibly virulent pathogens after a conditioning treatment. Although the phenomenon has been known for years, it has been appreciated just recently that priming for enhanced defense responses can result from plant-plant communication in nature and that priming can also boost the resistance of crops to biotic and abiotic stresses in the field.

Microarray data analysis made easy.

DNA microarrays are valuable tools for analyzing global gene expression. Because of the increasing popularity and the large volume of data produced, tools for facile microarray data analysis are essential. FiRe, a recently introduced computer program, has now solved the seemingly insuperable discrepancy between simplicity and evaluation of DNA microarray data. The program is available as a macro for the popular Microsoft Office Excel software and is user-friendly, interactive, versatile and platform-independent, paving the way for a further push in the evaluation of DNA microarrays.

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis.

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking.

Priming: getting ready for battle.

Infection of plants by necrotizing pathogens or colonization of plant roots with certain beneficial microbes causes the induction of a unique physiological state called "priming." The primed state can also be induced by treatment of plants with various natural and synthetic compounds. Primed plants display either faster, stronger, or both activation of the various cellular defense responses that are induced following attack by either pathogens or insects or in response to abiotic stress. Although the phenomenon has been known for decades, most progress in our understanding of priming has been made over the past few years. Here, we summarize the current knowledge of priming in various induced-resistance phenomena in plants.

Combining Metabolic ¹5N Labeling with Improved Tandem MOAC for Enhanced Probing of the Phosphoproteome.

In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool for studying protein phosphorylation because it enables unbiased localization, and site-specific quantification of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy for identifying phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. This is particularly true for plants in which the presence of secondary metabolites and endogenous compounds, the overabundance of ribulose-1,5-bisphosphate carboxylase and other components of the photosynthetic apparatus, and the concurrent difficulties in protein extraction necessitate two-step phosphoprotein/phosphopeptide enrichment strategies (Nakagami et al., Plant Cell Physiol 53:118-124, 2012).Approaches for label-free peptide quantification are advantageous due to their low cost and experimental simplicity, but they lack precision. These drawbacks can be overcome by metabolic labeling of whole plants with heavy nitrogen ((15)N) which allows combining two samples very early in the phosphoprotein enrichment workflow. This avoids sample-to-sample variation introduced by the analytical procedures and it results in robust relative quantification values that need no further standardization. The integration of (15)N metabolic labeling into tandem metal-oxide affinity chromatography (MOAC) (Hoehenwarter et al., Mol Cell Proteomics 12:369-380, 2013) presents an improved and highly selective approach for the identification and accurate site-specific quantification of low-abundance phosphoproteins that is based on the successive enrichment of light and heavy nitrogen-labeled phosphoproteins and peptides. This improved strategy combines metabolic labeling of whole plants with the stable heavy nitrogen isotope ((15)N), protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, and tryptic digest of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides and quantitative phosphopeptide measurement by liquid chromatography (LC) and high-resolution accurate mass (HR/AM) mass spectrometry (MS). Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and allows probing of the phosphoproteome to unprecedented depth, while (15)N metabolic labeling enables accurate relative quantification of measured peptides and direct comparison between samples.

Priming for enhanced defense.

When plants recognize potential opponents, invading pathogens, wound signals, or abiotic stress, they often switch to a primed state of enhanced defense. However, defense priming can also be induced by some natural or synthetic chemicals. In the primed state, plants respond to biotic and abiotic stress with faster and stronger activation of defense, and this is often linked to immunity and abiotic stress tolerance. This review covers recent advances in disclosing molecular mechanisms of priming. These include elevated levels of pattern-recognition receptors and dormant signaling enzymes, transcription factor HsfB1 activity, and alterations in chromatin state. They also comprise the identification of aspartyl-tRNA synthetase as a receptor of the priming activator ß-aminobutyric acid. The article also illustrates the inheritance of priming, exemplifies the role of recently identified priming activators azelaic and pipecolic acid, elaborates on the similarity to defense priming in mammals, and discusses the potential of defense priming in agriculture.

Tandem metal-oxide affinity chromatography for enhanced depth of phosphoproteome analysis.

In eukaryotic cells many diverse cellular functions are regulated by reversible protein phosphorylation. In recent years, phosphoproteomics has become a powerful tool to study protein phosphorylation because it allows unbiased localization, and site-specific quantification, of in vivo phosphorylation of hundreds of proteins in a single experiment. A common strategy to identify phosphoproteins and their phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. However, despite the high sensitivity of modern mass spectrometers the large dynamic range of protein abundance and the transient nature of protein phosphorylation remained major pitfalls in MS-based phosphoproteomics. Tandem metal-oxide affinity chromatography (MOAC) represents a robust and highly selective approach for the identification and site-specific quantification of low abundant phosphoproteins that is based on the successive enrichment of phosphoproteins and -peptides. This strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based MOAC, tryptic digestion of enriched phosphoproteins followed by TiO2-based MOAC of phosphopeptides. Thus, tandem MOAC effectively targets the phosphate moiety of phosphoproteins and phosphopeptides and, thus, allows probing of the phosphoproteome to unprecedented depth.

High subretinal fluid procoagulant activity in rhegmatogenous retinal detachment.

PURPOSE: An increased mRNA expression of genes related to blood coagulation has been demonstrated in an experimental retinal detachment model but has not yet been confirmed in human clinical specimens. Tissue factor (TF), the initiating factor of blood coagulation, may be a determinant of the extent of tissue injury after rhegmatogenous retinal detachment (RRD). This study was conducted to determine whether subretinal fluid and vitreous fluid collected from patients with RRD have a procoagulant effect. METHODS: Calibrated thrombin generation (CAT) was used to investigate the thrombogenic properties of 28 subretinal fluids collected during scleral buckling surgery for RRD. Further, the thrombogenic properties of vitreous fluids from RRD (n = 12), macular pucker (n = 5), macular hole (n = 6), and proliferative diabetic retinopathy (n = 5) were compared with the properties of eye bank eyes (n = 11), which served as control specimens. The procoagulant activity of TF was determined with Western blot analysis. RESULTS: The addition of subretinal fluid from all RRD patients (28/28, 100%) induced thrombin generation in normal and severely factor (F)XII-deficient plasma. Contrary to the subretinal fluid, the addition of vitreous fluids from various ocular disorders evoked very little thrombin generation in normal and severely FXII-deficient plasma (4/12, 33% RRD; 1/5, 20% macular pucker; 0/6, 0% macular hole; 0/5, 0% proliferative diabetic retinopathy; and 2/11, 18% eye bank eyes). The procoagulant activity in subretinal fluid was almost completely neutralized by antibodies against human TF. The presence of TF in subretinal fluid was confirmed by Western blot. CONCLUSIONS: Subretinal fluid of patients with RRD induces high procoagulant activity, determined by measuring the level of tissue factor.

Mitogen-activated protein kinases 3 and 6 are required for full priming of stress responses in Arabidopsis thaliana.

In plants and animals, induced resistance (IR) to biotic and abiotic stress is associated with priming of cells for faster and stronger activation of defense responses. It has been hypothesized that cell priming involves accumulation of latent signaling components that are not used until challenge exposure to stress. However, the identity of such signaling components has remained elusive. Here, we show that during development of chemically induced resistance in Arabidopsis thaliana, priming is associated with accumulation of mRNA and inactive proteins of mitogen-activated protein kinases (MPKs), MPK3 and MPK6. Upon challenge exposure to biotic or abiotic stress, these two enzymes were more strongly activated in primed plants than in nonprimed plants. This elevated activation was linked to enhanced defense gene expression and development of IR. Strong elicitation of stress-induced MPK3 and MPK6 activity is also seen in the constitutive priming mutant edr1, while activity was attenuated in the priming-deficient npr1 mutant. Moreover, priming of defense gene expression and IR were lost or reduced in mpk3 or mpk6 mutants. Our findings argue that prestress deposition of the signaling components MPK3 and MPK6 is a critical step in priming plants for full induction of defense responses during IR.