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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. [ABSTRACT FROM AUTHOR]

Background: Second messengers link external cues to complex physiological responses. One such messenger, 3',5'-cyclic guanosine monophosphate (cGMP), has been shown to play a key role in many physiological responses in plants. However, in higher plants, guanylyl cyclases (GCs), enzymes that generate cGMP from guanosine-5'-triphosphate (GTP) have remained elusive until recently. GC search motifs constructed from the alignment of known GCs catalytic centers form vertebrates and lower eukaryotes have led to the identification of a number of plant GCs that have been characterized in vitro and in vivo. Presentation of the hypothesis Recently characterized GCs in Arabidopsis thaliana contributed to the development of search parameters that can identify novel candidate GCs in plants. We hypothesize that there are still a substantial number (> 40) of multi-domain molecules with potentially functional GC catalytic centers in plants that remain to be discovered and characterized. Testing the hypothesis: The hypothesis can be tested, firstly, by computational methods constructing 3D models of selected GC candidates using available crystal structures as templates. Homology modeling must include substrate docking that can provide support for the structural feasibility of the GC catalytic centers in those candidates. Secondly, recombinant peptides containing the GC domain need to be tested in in vitro GC assays such as the enzyme-linked immune-sorbent assay (ELISA) and/or in mass spectrometry based cGMP assays. In addition, quantification of in vivo cGMP transients with fluorescent cGMP-reporter assays in wild-type or selected mutants will help to elucidate the biological role of novel GCs. Implications of the hypothesis If it turns out that plants do harbor a large number of functional GC domains as part of multi-domain enzymes, then major new insights will be gained into the complex signal transduction pathways that link cGMP to fundamental processes such as ion transport and homeostasis, biotic and abiotic stress responses as well as cGMP-dependent responses to hormones. [ABSTRACT FROM AUTHOR]

Higher plants contain biologically active proteins that are recognized by antibodies against human atrial natriuretic peptide (ANP). We identified and isolated two Arabidopsis thaliana immunoreactive plant natriuretic peptide (PNP)-encoding genes, AtPNP-A and AtPNP-B, which are distantly related members of the expansin superfamily and have a role in the regulation of homeostasis in abiotic and biotic stresses, and have shown that AtPNP-A modulates the effects of ABA on stomata. Arabidopsis PNP (PNP-A) is mainly expressed in leaf mesophyll cells, and in protoplast assays we demonstrate that it is secreted using AtPNP-A:green fluorescent protein (GFP) reporter constructs and flow cytometry. Transient reporter assays provide evidence that AtPNP-A expression is enhanced by heat, osmotica and salt, and that AtPNP-A itself can enhance its own expression, thereby generating a response signature diagnostic for paracrine action and potentially also autocrine effects. Expression of native AtPNP-A is enhanced by osmotica and transiently by salt. Although AtPNP-A expression is induced by salt and osmotica, ABA does not significantly modulate AtPNP-A levels nor does recombinant AtPNP-A affect reporter expression of the ABA-responsive RD29A gene. Together, these results provide experimental evidence that AtPNP-A is stress responsive, secreted into the apoplastic space and can enhance its own expression. Furthermore, our findings support the idea that AtPNP-A, together with ABA, is an important component in complex plant stress responses and that, much like in animals, peptide signaling molecules can create diverse and modular signals essential for growth, development and defense under rapidly changing environmental conditions. [ABSTRACT FROM AUTHOR]

World food security is increasingly dependent on continuous crop improvement and, in particular, the development of crops with increased drought and salinity tolerance. The completed genomic sequence of the model plant Arabidopsis thaliana and the development of whole-genome microarrays, together with increasing repositories of publicly available data and data analysis tools, have opened new avenues to genome-wide systemic analysis of plant stress responses. Here we outline examples of how this full-genome expression profiling can contribute to our understanding of complex stress responses and the identification and evaluation of novel transgenes that could hold the key to the development of commercially viable and sustainable crop plants. [Copyright &y& Elsevier]

Why do highly expressed genes have small introns? This is an important issue, not least because it provides a testing ground to compare selectionist and neutralist models of genome evolution. Some argue that small introns are selectively favoured to reduce the costs of transcription. Alternatively, large introns might permit complex regulation, not needed for highly expressed genes. This "genome design" hypothesis evokes a regionalized model of control of expression and hence can explain why intron size covaries with intergene distance, a feature also consistent with the hypothesis that highly expressed genes cluster in genomic regions with high deletion rates. As some genes are expressed in the haploid stage and hence subject to especially strong purifying selection, the evolution of genes in Arabidopsis provides a novel testing ground to discriminate between these possibilities. Importantly, controlling for expression level, genes that are expressed in pollen have shorter introns than genes that are expressed in the sporophyte. That genes flanking pollen-expressed genes have average-sized introns and intergene distances argues against regional mutational biases and genomic design. These observations thus support the view that selection for efficiency contributes to the reduction in intron length and provide the first report of a molecular signature of strong gametophytic selection. [ABSTRACT FROM AUTHOR]

Multiple ancient genome duplications in Arabidopsis thaliana provide unique opportunities to assess factors that influence the fates of duplicated genes. We have found that genes retained in duplicate following one round of genome duplication are significantly more likely to be retained in duplicate again after a subsequent genome duplication. Genes retained in duplicate form a functionally biased set and include a significant over-representation of genes involved in the regulation of transcription. [Copyright &y& Elsevier]

Presents results of a research which identified a novel protein with guanylyl cyclase (GC) activity in Arabidopsis thaliana (At) Finding during catalytic activity in vitro that AtGC1 can function either as a monomer or homo-oligomer; Identification of AtGC1 as the first functional plant GC.

Plant natriuretic peptides (PNPs) are a group of systemically acting peptidic hormones affecting solute and solvent homeostasis and responses to biotrophic pathogens. Although an increasing body of evidence suggests PNPs modulate plant responses to biotic and abiotic stress, which could lead to their potential biotechnological application by conferring increased stress tolerance to plants, the exact mode of PNPs action is still elusive. In order to gain insight into PNP-dependent signalling, we set out to identify interactors of PNP present in the model plant Arabidopsis thaliana, termed AtPNP-A. Here, we report identification of rubisco activase (RCA), a central regulator of photosynthesis converting Rubisco catalytic sites from a closed to an open conformation, as an interactor of AtPNP-A through affinity isolation followed by mass spectrometric identification. Surface plasmon resonance (SPR) analyses reveals that the full-length recombinant AtPNP-A and the biologically active fragment of AtPNP-A bind specifically to RCA, whereas a biologically inactive scrambled peptide fails to bind. These results are considered in the light of known functions of PNPs, PNP-like proteins, and RCA in biotic and abiotic stress responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Metabolic processes in prokaryotic and eukaryotic organisms are often modulated by kinases which are in turn, dependent on Ca2+ and the cyclic mononucleotides cAMP and cGMP. It has been established that some proteins have both kinase and cyclase activities and that active cyclases can be embedded within the kinase domains. Here, we identified phosphodiesterase (PDE) sites, enzymes that hydrolyse cAMP and cGMP, to AMP and GMP, respectively, in some of these proteins in addition to their kinase/cyclase twin-architecture. As an example, we tested the Arabidopsis thaliana KINγ, a subunit of the SnRK2 kinase, to demonstrate that all three enzymatic centres, adenylate cyclase (AC), guanylate cyclase (GC) and PDE, are catalytically active, capable of generating and hydrolysing cAMP and cGMP. These data imply that the signal output of the KINγ subunit modulates SnRK2, consequently affecting the downstream kinome. Finally, we propose a model where a single protein subunit, KINγ, is capable of regulating cyclic mononucleotide homeostasis, thereby tuning stimulus specific signal output. [Display omitted] • Some regulatory subunits of kinases harbour catalytic centres for cNMP metabolism. • Plant proteomes contain proteins with triple AC, GC, and PDE activities. • Computational and in vitro analyses confirmed the triple activities of AtKINγ. • cNMPs enable fine-tuning of kinase complex activities and downstream signals. [ABSTRACT FROM AUTHOR]

Cellular and physiological evidence suggests the presence of a novel class of systemically mobile plant molecules that are recognized by antibodies against vertebrate atrial natriuretic peptides (ANPs). In order to characterize the function of these immunoanalogues we have expressed the full-length recombinant (AtPNP-A[1–126]) and demonstrate that this molecule induces osmoticum-dependent H2O uptake into protoplasts at nanomolar concentrations and thus affects cell volume. A similar response is also seen with a recombinant that does not contain the signal peptide (AtPNP-A[26–126]) as well as a short domain (AtPNP-A[33–66]) that shows homology to the vertebrate peptide. Taken together, these findings suggest that AtPNP-A has an important and systemic role in plant growth and homeostasis. [Copyright &y& Elsevier]

Background: RNA-binding proteins (RBPs) are increasingly recognized as regulatory component of post-transcriptional gene expression. RBPs interact with mRNAs via RNA-binding domains and these interactions affect RNA availability for translation, RNA stability and turn-over thus affecting both RNA and protein expression essential for developmental and stimulus specific responses. Here we investigate the effect of severe drought stress on the RNA-binding proteome to gain insights into the mechanisms that govern drought stress responses at the systems level. Results: Label-free mass spectrometry enabled the identification 567 proteins of which 150 significantly responded to the drought-induced treatment. A gene ontology analysis revealed enrichment in the "RNA binding" and "RNA processing" categories as well as biological processes such as "response to abscisic acid" and "response to water deprivation". Importantly, a large number of the stress responsive proteins have not previously been identified as RBPs and include proteins in carbohydrate metabolism and in the glycolytic and citric acid pathways in particular. This suggests that RBPs have hitherto unknown roles in processes that govern metabolic changes during stress responses. Furthermore, a comparative analysis of RBP domain architectures shows both, plant specific and common domain architectures between plants and animals. The latter could be an indication that RBPs are part of an ancient stress response. Conclusion: This study establishes mRNA interactome capture technique as an approach to study stress signal responses implicated in environmental changes. Our findings denote RBP changes in the proteome as critical components in plant adaptation to changing environments and in particular drought stress protein-dependent changes in RNA metabolism. [ABSTRACT FROM AUTHOR]

Abstract 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The majority of proteins in both prokaryote and eukaryote proteomes consist of two or more functional centers, which allows for intramolecular tuning of protein functions. Such architecture, as opposed to animal orthologs, applies to the plant cyclases (CNC) and phosphodiesterases (PDEs), the vast majority of which are part of larger multifunctional proteins. In plants, until recently, only two cases of combinations of CNC-PDE in one protein were reported. Here we propose that in plants, multifunctional proteins in which the PDE motif has been identified, the presence of the additional CNC center is common. Searching the Arabidopsis thaliana proteome with a combined PDE-CNC motif allowed the creation of a database of proteins with both activities. One such example is methylenetetrahydrofolate dehydrogenase, in which we determined the activities of adenylate cyclase (AC) and PDE. Based on biochemical and mutagenesis analyses we assessed the impact of the AC and PDE catalytic centers on the dehydrogenase activity. This allowed us to propose additional regulatory mechanism that govern folate metabolism by cAMP. It is therefore conceivable that the combined CNC-PDE architecture is a common regulatory configuration, where control of the level of cyclic nucleotides (cNMP) influences other catalytic activities of the protein. [Display omitted] • Increasing number of proteins with twin cyclase and PDE domain are being discovered by motifs. • Combined cyclase and PDE domain architecture is a common feature in plant proteins. • Such architecture allows for tuning of cNMP-dependent processes by single proteins. [ABSTRACT FROM AUTHOR]

Glyphosate is a widely applied broad-spectrum systemic herbicide that inhibits competitively the penultimate enzyme 5-enolpyruvylshikimate 3-phosphate synthase ( EPSPS) from the shikimate pathway, thereby causing deleterious effects. A glyphosate-resistant Arabidopsis mutant ( gre1) was isolated and genetic analyses indicated that a dysfunctional red ( R) and far-red ( FR) light receptor, phytochrome B (phy B), caused this phenotype. This finding is consistent with increased glyphosate sensitivity and glyphosate-induced shikimate accumulation in low R: FR light, and the induction of genes encoding enzymes of the shikimate pathway in high R: FR light. Expression of the shikimate pathway genes exhibited diurnal oscillation and this oscillation was altered in the phyB mutant. Furthermore, transcript analysis suggested that this diurnal oscillation was not only dependent on phyB but was also due to circadian regulatory mechanisms. Our data offer an explanation of the well documented observation that glyphosate treatment at various times throughout the day, with their specific composition of light quality and intensity, results in different efficiencies of the herbicide. [ABSTRACT FROM AUTHOR]

Highlights: [•] We show that H2O2 modulates potassium and calcium net fluxes in Arabidopsis roots. [•] We examine the effect of cyclic mononucleotide pre-treatment on these fluxes. [•] We also investigate the effect of cGMP on the microsomal proteome. [•] cGMP is sufficient to induce complex cellular changes to responses induced by H2O2. [Copyright &y& Elsevier]

Abstract: 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 β. 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. [Copyright &y& Elsevier]

Background: Increasing structural and biochemical evidence suggests that post-translational methionine oxidation of proteins is not just a result of cellular damage but may provide the cell with information on the cellular oxidative status. In addition, oxidation of methionine residues in key regulatory proteins, such as calmodulin, does influence cellular homeostasis. Previous findings also indicate that oxidation of methionine residues in signaling molecules may have a role in stress responses since these specific structural modifications can in turn change biological activities of proteins. Findings: Here we use tandem mass spectrometry-based proteomics to show that treatment of Arabidopsis thaliana cells with a non-oxidative signaling molecule, the cell-permeant second messenger analogue, 8-bromo-3,5-cyclic guanosine monophosphate (8-Br-cGMP), results in a time-dependent increase in the content of oxidised methionine residues. Interestingly, the group of proteins affected by cGMP-dependent methionine oxidation is functionally enriched for stress response proteins. Furthermore, we also noted distinct signatures in the frequency of amino acids flanking oxidised and un-oxidised methionine residues on both the C- and N-terminus. Conclusions: Given both a structural and functional bias in methionine oxidation events in response to a signaling molecule, we propose that these are indicative of a specific role of such post-translational modifications in the direct or indirect regulation of cellular responses. The mechanisms that determine the specificity of the modifications remain to be elucidated. [ABSTRACT FROM AUTHOR]

The quest for the discovery of mathematical principles that underlie biological phenomena is ancient and ongoing. We present a geometric analysis of the complex interdigitated pavement cells in the Arabidopsis thaliana (Col.) adaxial epidermis with a view to discovering some geometric characteristics that may govern the formation of this tissue. More than 2,400 pavement cells from 10, 17 and 24 day old leaves were analyzed. These interdigitated cells revealed a number of geometric properties that remained constant across the three age groups. In particular, the number of digits per cell rarely exceeded 15, irrespective of cell area. Digit numbers per 100 μm² cell area reduce with age and as cell area increases, suggesting early developmental programming of digits. Cell shape proportions as defined by length:width ratios were highly conserved over time independent of the size and, interestingly, both the mean and the medians were close to the golden ratio 1.618034. With maturity, the cell area:perimeter ratios increased from a mean of 2.0 to 2.4. Shape properties as defined by the medial axis transform (MAT) were calculated and revealed that branch points along the MAT typically comprise one large and two small angles. These showed consistency across the developmental stages considered here at 140° (± 5°) for the largest angles and 110° (μ 5°) for the smaller angles. Voronoi diagram analyses of stomatal center coordinates revealed that giant pavement cells (≥500 μm°) tend to be arranged along Voronoi boundaries suggesting that they could function as a scaffold of the epidermis. In addition, we propose that pavement cells have a role in spacing and positioning of the stomata in the growing leaf and that they do so by growing within the limits of a set of 'geometrical rules.' [ABSTRACT FROM AUTHOR]

Abstract: 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 (O3). 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 O3 and Pseudomonas syringae pv. tomato (Pst) DC3000. crk20-1 and crk20-2 showed similar O3 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 H2O 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. [Copyright &y& Elsevier]

Abstract: Plant natriuretic peptides (PNPs) belong to a novel class of peptidic signaling molecules that share some structural similarity to the N-terminal domain of expansins and affect physiological processes such as water and ion homeostasis at nano-molar concentrations. Here we show that a recombinant Arabidopsis thaliana PNP (AtPNP-A) rapidly increased the rate of dark respiration in treated leaves after 5min. In addition, we observed increases in lower leaves, and with a lag time of 10min, the effect spread to the upper leaves and subsequently (after 15min) to the opposite leaves. This response signature is indicative of phloem mobility of the signal, a hypothesis that was further strengthened by the fact that cold girdling, which affects phloem but not xylem or apoplastic processes, delayed the long distance AtPNP-A effect. We conclude that locally applied AtPNP-A can induce a phloem-mobile signal that rapidly modifies plant homeostasis in distal parts. [Copyright &y& Elsevier]

Abstract: While there is evidence of nitric oxide (NO)-dependent signalling via the second messenger cyclic guanosine 3′,5′-monophosphate (cGMP) in plants, guanylate cyclases (GCs), enzymes that catalyse the formation of cGMP from guanosine 5′-triphosphate (GTP) have until recently remained elusive and none of the candidates identified to-date are NO-dependent. Using both a GC and heme-binding domain specific (H-NOX) search motif, we have identified an Arabidopsis flavin monooxygenase (At1g62580) and shown electrochemically that it binds NO, has a higher affinity for NO than for O2 and that this molecule can generate cGMP from GTP in vitro in an NO-dependent manner. [Copyright &y& Elsevier]

Background: The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana. Results: A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of chlorophyll biosynthesis genes in a manner that is consistent with the increased synthesis of carotenoid precursors for ABA biosynthesis. In all tissues examined, induction of b-carotene hydroxylase transcript levels are linked to an increased demand for ABA. Conclusions: This analysis provides compelling evidence to suggest that coordinated transcriptional regulation of isoprenoid-related biosynthesis pathway genes plays a major role in coordinating the synthesis of functionally related chloroplast localized isoprenoid-derived compounds. [ABSTRACT FROM AUTHOR]

Background: Plant natriuretic peptides (PNPs) are a class of systemically mobile molecules distantly related to expansins. While several physiological responses to PNPs have been reported, their biological role has remained elusive. Here we use a combination of expression correlation analysis, meta-analysis of gene expression profiles in response to specific stimuli and in selected mutants, and promoter content analysis to infer the biological role of the Arabidopsis thaliana PNP, AtPNP-A. Results: A gene ontology analysis of AtPNP-A and the 25 most expression correlated genes revealed a significant over representation of genes annotated as part of the systemic acquired resistance (SAR) pathway. Transcription of these genes is strongly induced in response to salicylic acid (SA) and its functional synthetic analogue benzothiadiazole S-methylester (BTH), a number of biotic and abiotic stresses including many SA-mediated SAR-inducing conditions, as well as in the constitutive SAR expressing mutants cpr5 and mpk4 which have elevated SA levels. Furthermore, the expression of AtPNP-A was determined to be significantly correlated with the SAR annotated transcription factor, WRKY 70, and the promoters of AtPNP-A and the correlated genes contain an enrichment in the core WRKY binding W-box cis-elements. In constitutively expressing WRKY 70 lines the expression of AtPNP-A and the correlated genes, including the SAR marker genes, PR-2 and PR-5, were determined to be strongly induced. Conclusion: The co-expression analyses, both in wild type and mutants, provides compelling evidence that suggests AtPNP-A may function as a component of plant defence responses and SAR in particular. The presented evidence also suggests that the expression of AtPNP-A is controlled by WRKY transcription factors and WRKY 70 in particular. AtPNP-A shares many characteristics with PR proteins in that its transcription is strongly induced in response to pathogen challenges, it contains an N-terminal signalling peptide and is secreted into the extracellular space and along with PR-1, PR-2 and PR-5 proteins it has been isolated from the Arabidopsis apoplast. Based on these findings we suggest that AtPNP-A could be classified as a newly identified PR protein. [ABSTRACT FROM AUTHOR]

Plant natriuretic peptides (PNPs) belong to a novel class of systemically mobile molecules that are structurally similar to the N-terminal domain of expansins and affect physiological processes such as protoplast volume regulation at nano-molar concentrations. Here we demonstrate that AtPNP-A, a recombinant Arabidopsis thaliana PNP causes rapid H+ influx in the elongation zone of A. thaliana roots but not in the mature zone. AtPNP-A also induces significant K+ and Na+ efflux and this effect is seen in the mature root zone only. These observations suggest that responses to AtPNP-A are developmental stage and tissue specific and point to a complex role in plant growth and homeostasis. [ABSTRACT FROM AUTHOR]

A guanylyl cyclase has been recently identified in Arabidopsis but, despite the use of pharmacological inhibitors to infer roles of the second messenger 3′,5′-cyclic guanosine monophosphate (cGMP), very few measurements of actual cGMP levels in plants are available. Here, we demonstrate that cGMP levels in Arabidopsis seedlings increase rapidly (⩽5 s) and to different degrees after salt and osmotic stress, and that the increases are prevented by treatment with LY, an inhibitor of soluble guanylyl cyclases. In addition, we provide evidence to suggest that salt stress activates two cGMP signalling pathways – an osmotic, calcium-independent pathway and an ionic, calcium-dependent pathway. [Copyright &y& Elsevier]

In plants, much like in animals, nitric oxide (NO) has been established as an important gaseous signaling molecule. However, contrary to animal systems, NO-sensitive or NO-responsive proteins that bind NO in the form of a sensor or participating in redox reactions have remained elusive. Here, we applied a search term constructed based on conserved and functionally annotated amino acids at the centers of Heme Nitric Oxide/Oxygen (H-NOX) domains in annotated and experimentally-tested gas-binding proteins from lower and higher eukaryotes, in order to identify candidate NO-binding proteins in Arabidopsis thaliana. The selection of candidate NO-binding proteins identified from the motif search was supported by structural modeling. This approach identified AtLRB3 (At4g01160), a member of the Light Response Bric-a-Brac/Tramtrack/Broad Complex (BTB) family, as a candidate NO-binding protein. AtLRB3 was heterologously expressed and purified, and then tested for NO-response. Spectroscopic data confirmed that AtLRB3 contains a histidine-ligated heme cofactor and importantly, the addition of NO to AtLRB3 yielded absorption characteristics reminiscent of canonical H-NOX proteins. Furthermore, substitution of the heme iron-coordinating histidine at the H-NOX center with a leucine strongly impaired the NO-response. Our finding therefore established AtLRB3 as a NO-interacting protein and future characterizations will focus on resolving the nature of this response. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]