The cAMP-dependent phosphorylation footprint in response to heat stress.

KEY MESSAGE: cAMP modulates the phosphorylation status of highly conserved phosphosites in RNA-binding proteins crucial for mRNA metabolism and reprogramming in response to heat stress. In plants, 3',5'-cyclic adenosine monophosphate (3',5'-cAMP) is a second messenger that modulates multiple cellular targets, thereby participating in plant developmental and adaptive processes. Although its role in ameliorating heat-related damage has been demonstrated, mechanisms that govern cAMP-dependent responses to heat have remained elusive. Here we analyze the role cAMP-dependent phosphorylation during prolonged heat stress (HS) with a view to gain insight into processes that govern plant responses to HS. To do so, we performed quantitative phosphoproteomic analyses in Nicotiana tabacum Bright Yellow-2 cells grown at 27 °C or 35 °C for 3 days overexpressing a molecular "sponge" that reduces free intracellular cAMP levels. Our phosphorylation data and analyses reveal that the presence of cAMP is an essential factor that governs specific protein phosphorylation events that occur during prolonged HS in BY-2 cells. Notably, cAMP modulates HS-dependent phosphorylation of proteins that functions in mRNA processing, transcriptional control, vesicular trafficking, and cell cycle regulation and this is indicative for a systemic role of the messenger. In particular, changes of cAMP levels affect the phosphorylation status of highly conserved phosphosites in 19 RNA-binding proteins that are crucial during the reprogramming of the mRNA metabolism in response to HS. Furthermore, phosphorylation site motifs and molecular docking suggest that some proteins, including kinases and phosphatases, are conceivably able to directly interact with cAMP thus further supporting a regulatory role of cAMP in plant HS responses.

The discovery of hidden guanylate cyclases (GCs) in the Homo sapiens proteome.

Recent discoveries have established functional guanylate cyclase (GC) catalytic centers with low activity within kinase domains in plants. These crypto GCs generate guanosine 3',5'-cyclic monophosphate (cGMP) essential for both intramolecular and downstream signaling. Here, we have set out to search for such crypto GCs moonlighting in kinases in the H. sapiens proteome and identified 18 candidates, including the neurotropic receptor tyrosine kinase 1 (NTRK1). NTRK1 shows a domain architecture much like plant receptor kinases such as the phytosulfokine receptor, where a functional GC essential for downstream signaling is embedded within a kinase domain. In vitro characterization of the NTRK1 shows that the embedded NTRK1 GC is functional with a marked preference for Mn2+ over Mg2+. This therefore points to hitherto unsuspected roles of cGMP in intramolecular and downstream signaling of NTRK1 and the role of cGMP in NTRK1-dependent growth and neoplasia.

Plant adenylate cyclases have come full circle.

In bacteria, fungi and animals, 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylate cyclases (ACs), enzymes that catalyse the formation of 3',5'-cAMP from ATP, are recognized as key signalling components. In contrast, the presence of cAMP and its biological roles in higher plants have long been a matter of controversy due to the generally lower amounts in plant tissues compared with that in animal and bacterial cells, and a lack of clarity on the molecular nature of the generating and degrading enzymes, as well as downstream effectors. While treatment with 3',5'-cAMP elicited many plant responses, ACs were, however, somewhat elusive. This changed when systematic searches with amino acid motifs deduced from the conserved catalytic centres of annotated ACs from animals and bacteria identified candidate proteins in higher plants that were subsequently shown to have AC activities in vitro and in vivo. The identification of active ACs moonlighting within complex multifunctional proteins is consistent with their roles as molecular tuners and regulators of cellular and physiological functions. Furthermore, the increasing number of ACs identified as part of proteins with different domain architectures suggests that there are many more hidden ACs in plant proteomes and they may affect a multitude of mechanisms and processes at the molecular and systems levels.

Quantitative phosphoproteomics reveals novel roles of cAMP in plants.

3',5'-cyclic adenosine monophosphate (cAMP) is finally recognized as an essential signaling molecule in plants where cAMP-dependent processes include responses to hormones and environmental stimuli. To better understand the role of 3',5'-cAMP at the systems level, we have undertaken a phosphoproteomic analysis to elucidate the cAMP-dependent response of tobacco BY-2 cells. These cells overexpress a molecular "sponge" that buffers free intracellular cAMP level. The results show that, firstly, in vivo cAMP dampening profoundly affects the plant kinome and notably mitogen-activated protein kinases, receptor-like kinases, and calcium-dependent protein kinases, thereby modulating the cellular responses at the systems level. Secondly, buffering cAMP levels also affects mRNA processing through the modulation of the phosphorylation status of several RNA-binding proteins with roles in splicing, including many serine and arginine-rich proteins. Thirdly, cAMP-dependent phosphorylation targets appear to be conserved among plant species. Taken together, these findings are consistent with an ancient role of cAMP in mRNA processing and cellular programming and suggest that unperturbed cellular cAMP levels are essential for cellular homeostasis and signaling in plant cells.

Functional Crypto-Adenylate Cyclases Operate in Complex Plant Proteins.

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of many plant responses. Here, we show that an amino acid search motif based on annotated adenylate cyclases (ACs) identifies 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9-cis-epoxycarotenoid dioxygenase (NCED3 and At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally with two different methods. Given that the in vitro activity is low (fmoles cAMP pmol-1 protein min-1), but highly reproducible, we term the enzyme a crypto-AC. Our results are consistent with a role for ACs with low activities in multi-domain moonlighting proteins that have at least one other distinct molecular function, such as catalysis or ion channel activation. We propose that crypto-ACs be examined from the perspective that considers their low activities as an innate feature of regulatory ACs embedded within multi-domain moonlighting proteins. It is therefore conceivable that crypto-ACs form integral components of complex plant proteins participating in intra-molecular regulatory mechanisms, and in this case, potentially linking cAMP to ABA synthesis.

Mg2+ Is a Missing Link in Plant Cell Ca2+ Signalling and Homeostasis-A Study on Vicia faba Guard Cells.

Hyperpolarization-activated calcium channels (HACCs) are found in the plasma membrane and tonoplast of many plant cell types, where they have an important role in Ca2+-dependent signalling. The unusual gating properties of HACCs in plants, i.e., activation by membrane hyperpolarization rather than depolarization, dictates that HACCs are normally open in the physiological hyperpolarized resting membrane potential state (the so-called pump or P-state); thus, if not regulated, they would continuously leak Ca2+ into cells. HACCs are permeable to Ca2+, Ba2+, and Mg2+; activated by H2O2 and the plant hormone abscisic acid (ABA); and their activity in guard cells is greatly reduced by increasing amounts of free cytosolic Ca2+ ([Ca2+]Cyt), and hence closes during [Ca2+]Cyt surges. Here, we demonstrate that the presence of the commonly used Mg-ATP inside the guard cell greatly reduces HACC activity, especially at voltages ≤ -200 mV, and that Mg2+ causes this block. Therefore, we firstly conclude that physiological cytosolic Mg2+ levels affect HACC gating and that channel opening requires either high negative voltages (≥ -200 mV) or displacement of Mg2+ away from the immediate vicinity of the channel. Secondly, based on structural comparisons with a Mg2+-sensitive animal inward-rectifying K+ channel, we propose that the likely candidate HACCs described here are cyclic nucleotide gated channels (CNGCs), many of which also contain a conserved diacidic Mg2+ binding motif within their pores. This conclusion is consistent with the electrophysiological data. Finally, we propose that Mg2+, much like in animal cells, is an important component in Ca2+ signalling and homeostasis in plants.

An Arabidopsis thaliana leucine-rich repeat protein harbors an adenylyl cyclase catalytic center and affects responses to pathogens.

Adenylyl cyclases (ACs) catalyze the formation of the second messenger cAMP from ATP. Here we report the characterization of an Arabidopsis thaliana leucine-rich repeat (LRR) protein (At3g14460; AtLRRAC1) as an adenylyl cyclase. Using an AC-specific search motif supported by computational assessments of protein models we identify an AC catalytic center within the N-terminus and demonstrate that AtLRRAC1 can generate cAMP in vitro. Knock-out mutants of AtLRRAC1 have compromised immune responses to the biotrophic fungus Golovinomyces orontii and the hemibiotrophic bacteria Pseudomonas syringae, but not against the necrotrophic fungus Botrytis cinerea. These findings are consistent with a role of cAMP-dependent pathways in the defense against biotrophic and hemibiotrophic plant pathogens.

An Arabidopsis Clathrin Assembly Protein with a Predicted Role in Plant Defense Can Function as an Adenylate Cyclase.

Adenylate cyclases (ACs), much like guanylate cyclases (GCs), are increasingly recognized as essential parts of many plant processes including biotic and abiotic stress responses. In order to identify novel ACs, we have applied a search motif derived from experimentally tested GCs and identified a number of Arabidopsis thaliana candidates including a clathrin assembly protein (AT1G68110; AtClAP). AtClAP contains a catalytic centre that can complement the AC-deficient mutant cyaA in E. coli, and a recombinant AtClAP fragment (AtClAP261-379) can produce cyclic adenosine 3',5' monophosphate (cAMP) from adenosine triphosphate (ATP) in vitro. Furthermore, an integrated analysis of gene expression and expression correlation implicate cAMP in pathogen defense and in actin cytoskeletal remodeling during endocytic internalization.

Cyclic Nucleotide Monophosphates and Their Cyclases in Plant Signaling.

The cyclic nucleotide monophosphates (cNMPs), and notably 3',5'-cyclic guanosine monophosphate (cGMP) and 3',5'-cyclic adenosine monophosphate (cAMP) are now accepted as key signaling molecules in many processes in plants including growth and differentiation, photosynthesis, and biotic and abiotic defense. At the single molecule level, we are now beginning to understand how cNMPs modify specific target molecules such as cyclic nucleotide-gated channels, while at the systems level, a recent study of the Arabidopsis cNMP interactome has identified novel target molecules with specific cNMP-binding domains. A major advance came with the discovery and characterization of a steadily increasing number of guanylate cyclases (GCs) and adenylate cyclases (ACs). Several of the GCs are receptor kinases and include the brassinosteroid receptor, the phytosulfokine receptor, the Pep receptor, the plant natriuretic peptide receptor as well as a nitric oxide sensor. We foresee that in the near future many more molecular mechanisms and biological roles of GCs and ACs and their catalytic products will be discovered and further establish cNMPs as a key component of plant responses to the environment.

In Vitro Assessment of Guanylyl Cyclase Activity of Plant Receptor Kinases.

Cyclic nucleotides such as 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) are increasingly recognized as key signaling molecules in plants, and a growing number of plant mononucleotide cyclases, both adenylate cyclases (ACs) and guanylate cyclases (GCs), have been reported. Catalytically active cytosolic GC domains have been shown to be part of many plant receptor kinases and hence directly linked to plant signaling and downstream cellular responses. Here we detail, firstly, methods to identify and express essential functional GC domains of receptor kinases, and secondly, we describe mass spectrometric methods to quantify cGMP generated by recombinant GCs from receptor kinases in vitro.

The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling.

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per µg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.

Cyclic Nucleotide Monophosphates in Plants and Plant Signaling.

Cyclic nucleotide monophosphates (cNMPs) and the enzymes that can generate them are of increasing interest in the plant sciences. Arguably, the major recent advance came with the release of the complete Arabidopsis thaliana genome that has enabled the systematic search for adenylate (ACs) or guanylate cyclases (GCs) and did eventually lead to the discovery of a number of GCs in higher plants. Many of these proteins have complex domain architectures with AC or GC centers moonlighting within cytosolic kinase domains. Recent reports indicated the presence of not just the canonical cNMPs (i.e., cAMP and cGMP), but also the noncanonical cCMP, cUMP, cIMP, and cdTMP in plant tissues, and this raises several questions. Firstly, what are the functions of these cNMPs, and, secondly, which enzymes can convert the substrate triphosphates into the respective noncanonical cNMPs? The first question is addressed here by comparing the reactive oxygen species (ROS) response of cAMP and cGMP to that elicited by the noncanonical cCMP or cIMP. The results show that particularly cIMP can induce significant ROS production. To answer, at least in part, the second question, we have evaluated homology models of experimentally confirmed plant GCs probing the substrate specificity by molecular docking simulations to determine if they can conceivably catalytically convert substrates other than ATP or GTP. In summary, molecular modeling and substrate docking simulations can contribute to the evaluation of cyclases for noncanonical cyclic mononucleotides and thereby further our understanding of the molecular mechanism that underlie cNMP-dependent signaling in planta.

Changes in the Arabidopsis thaliana Proteome Implicate cAMP in Biotic and Abiotic Stress Responses and Changes in Energy Metabolism.

The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) is increasingly recognized as having many different roles in plant responses to environmental stimuli. To gain further insights into these roles, Arabidopsis thaliana cell suspension culture was treated with 100 nM of cell permeant 8-bromo-cAMP for 5 or 10 min. Here, applying mass spectrometry and comparative proteomics, 20 proteins were identified as differentially expressed and we noted a specific bias in proteins with a role in abiotic stress, particularly cold and salinity, biotic stress as well as proteins with a role in glycolysis. These findings suggest that cAMP is sufficient to elicit specific stress responses that may in turn induce complex changes to cellular energy homeostasis.

The Arabidopsis thaliana K(+)-uptake permease 7 (AtKUP7) contains a functional cytosolic adenylate cyclase catalytic centre.

Adenylate cyclases (ACs) catalyse the formation of the second messenger cyclic adenosine 3',5'-monophosphate (cAMP) from adenosine 5'-triphosphate (ATP). Although cAMP is increasingly recognised as an important signalling molecule in higher plants, ACs have remained somewhat elusive. Here we used a search motif derived from experimentally tested guanylyl cyclases (GCs), substituted the residues essential for substrate specificity and identified the Arabidopsis thaliana K(+)-uptake permease 7 (AtKUP7) as one of several candidate ACs. Firstly, we show that a recombinant N-terminal, cytosolic domain of AtKUP7(1-100) is able to complement the AC-deficient mutant cyaA in Escherichia coli and thus restoring the fermentation of lactose, and secondly, we demonstrate with both enzyme immunoassays and mass spectrometry that a recombinant AtKUP7(1-100) generates cAMP in vitro.

Cyclic mononucleotides modulate potassium and calcium flux responses to H2O2 in Arabidopsis roots.

Cyclic mononucleotides are messengers in plant stress responses. Here we show that hydrogen peroxide (H2O2) induces rapid net K(+)-efflux and Ca(2+)-influx in Arabidopsis roots. Pre-treatment with either 10µM cAMP or cGMP for 1 or 24h does significantly reduce net K(+)-leakage and Ca(2+)-influx, and in the case of the K(+)-fluxes, the cell permeant cyclic mononucleotides are more effective. We also examined the effect of 10µM of the cell permeant 8-Br-cGMP on the Arabidopsis microsomal proteome and noted a specific increase in proteins with a role in stress responses and ion transport, suggesting that cGMP is sufficient to directly and/or indirectly induce complex adaptive changes to cellular stresses induced by H2O2.

Phosphodiesterase activity is regulated by CC2D1A that is implicated in non-syndromic intellectual disability.

BACKGROUND: Cyclic adenosine 3',5'-monophosphate (cAMP) is a key regulator of many cellular processes, including in the neuronal system, and its activity is tuned by Phosphodiesterase (PDE) activation. Further, the CC2D1A protein, consisting of N-Terminal containing four DM14 domains and C-terminal containing C2 domain, was shown to regulate the cAMP-PKA pathway. A human deletion mutation lacking the fourth DM14 and the adjacent C2 domain results in Non Syndromic Intellectual Disability (NSID) also referred to as Non Syndromic Mental Retardation (NSMR). FINDINGS: Here we demonstrate that in Mouse Embryonic Fibroblasts (MEF) CC2D1A co-localizes with PDE4D in the cytosol before cAMP stimulation and on the periphery after stimulation, and that the movement to the periphery requires the full-length CC2D1A. In CC2D1A mouse mutant cells, the absence of three of the four DM14 domains abolishes migration of the complex to the periphery and causes constitutive phosphorylation of PDE4D Serine 126 (S126) via the cAMP-dependent protein kinase A (PKA) resulting in PDE4D hyperactivity. Suppressing PDE4D activity with Rolipram in turn restores the down-stream phosphorylation of the "cAMP response element-binding protein" (CREB) that is defective in mouse mutant cells. CONCLUSION: Our findings suggest that CC2D1A is a novel regulator of PDE4D. CC2D1A interacts directly with PDE4D regulating its activity and thereby fine-tuning cAMP-dependent downstream signaling. Based on our in vitro evidence we propose a model which links CC2D1A structure and function to cAMP homeostasis thereby affecting CREB phosphorylation. We speculate that CC2D1A and/or PDE4D may be promising targets for therapeutic interventions in many disorders with impaired PDE4D function such as NSID.

Proteomic signatures implicate cAMP in light and temperature responses in Arabidopsis thaliana.

The second messenger 3'-5'-cyclic adenosine monophosphate (cAMP) and adenylyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, are increasingly recognized as important signaling molecules in a number of physiological responses in higher plants. Here we used proteomics to identify cAMP-dependent protein signatures in Arabidopsis thaliana and identify a number of differentially expressed proteins with a role in light- and temperature-dependent responses, notably photosystem II subunit P-1, plasma membrane associated cation-binding protein and chaperonin 60 ß. Based on these proteomics results we conclude that, much like in cyanobacteria, algae and fungi, cAMP may have a role in light signaling and the regulation of photosynthesis as well as responses to temperature and we speculate that ACs could act as light and/or temperature sensors in higher plants. BIOLOGICAL SIGNIFICANCE: This current study is significant since it presents the first proteomic response to cAMP, a novel and key second messenger in plants. It will be relevant to researchers in plant physiology and in particular those with an interest in second messengers and their role in biotic and abiotic stress responses.

The Arabidopsis thaliana cysteine-rich receptor-like kinase CRK20 modulates host responses to Pseudomonas syringae pv. tomato DC3000 infection.

In plants, the cysteine-rich repeat kinases (CRKs) are a sub-family of receptor-like protein kinases that contain the DUF26 motif in their extracellular domains. It has been shown that in Arabidopsis thaliana, CRK20 is transcriptionally induced by pathogens, salicylic acid and ozone (O(3)). However, its role in responses to biotic and abiotic stress remains to be elucidated. To determine the function of CRK20 in such responses, two CRK20 loss-of-function mutants, crk20-1 and crk20-2, were isolated from public collections of Arabidopsis T-DNA tagged lines and examined for responses to O(3) and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O(3) sensitivities and no differences in the expression of defense genes when compared with the wild-type. However, pathogen growth was significantly reduced, while there were no differences in the induction of salicylic acid related defense genes or salicylic acid accumulation. Furthermore, correlation analysis of CRK20 gene expression suggests that it has a role in the control of H(2)O and/or nutrient transport. We therefore propose that CRK20 promotes conditions that are favorable for Pst DC3000 growth in Arabidopsis, possibly through the regulation of apoplastic homeostasis, and consequently, of the environment of this biotrophic pathogen.

Adenyl cyclases and cAMP in plant signaling - past and present.

In lower eukaryotes and animals 3'-5'-cyclic adenosine monophosphate (cAMP) and adenyl cyclases (ACs), enzymes that catalyse the formation of cAMP from ATP, have long been established as key components and second messengers in many signaling pathways. In contrast, in plants, both the presence and biological role of cAMP have been a matter of ongoing debate and some controversy. Here we shall focus firstly on the discovery of cellular cAMP in plants and evidence for a role of this second messenger in plant signal transduction. Secondly, we shall review current evidence of plant ACs, analyse aspects of their domain organisations and the biological roles of candidate molecules. In addition, we shall assess different approaches based on search motifs consisting of functionally assigned amino acids in the catalytic centre of annotated and/or experimentally tested nucleotide cyclases that can contribute to the identification of novel candidate molecules with AC activity such as F-box and TIR proteins.

Ozone and nitric oxide induce cGMP-dependent and -independent transcription of defence genes in tobacco.

Here, we analyse the temporal signatures of ozone (O3)-induced hydrogen peroxide(H2O2) and nitric oxide (NO) and the role of the second messenger guanosine3',5'-cyclic monophosphate (cGMP) in transcriptional changes of genes diagnostic for biotic and abiotic stress responses. Within 90 min O3 induced H2O2 and NO peaks and we demonstrate that NO donors cause rapid H2O2 accumulation in tobacco (Nicotiana tabacum) leaf. Ozone also causes highly significant, late (> 2 h) and sustained cGMP increases, suggesting that the second messenger may not be required in all early (< 2 h) responses to O3,but is essential and sufficient for the induction of some O3-dependent pathways.This hypothesis was tested resolving the time course of O3-induced transcript accumulation of alternative oxidase (AOX1a), glutathione peroxidase (GPX),aminocyclopropancarboxylic acid synthase (ACS2) that is critical for the synthesis of ethylene, phenylalanine ammonia lyase (PALa) and the pathogenesis-related protein PR1a.The data show that early O3 and NO caused transcriptional activation of the scavenger encoding proteins AOX1a, GPX and the induction of ethylene production through ACS2 are cGMP independent. By contrast, the early response of PALa and the late response of PR1a show critical dependence on cGMP.