Based on the PDE2 crystal structure 11 residues were proposed to be involved in cyclic nucleotide binding 18, but comparison to the cAMP binding GAF domain of the Anabaena adenylate cyclase shows that these residues may only be strictly conserved in mammals. Further complicating our analysis is the fact that GAF domains are known to bind other small molecules such as 2-oxoglutarate, formate and bilins 242526. GAF domains form a structural scaffold which can be utilized to bind several possible small molecules depending on the functional groups on that scaffold. Therefore, their role in cyclic nucleotide signalling must be verified by biochemical means rather than strictly by sequence analysis. Our analysis indicated that in plants there are two types of proteins which contain GAF domains. These are the phytochrome proteins and the ethylene receptor proteins.

Phytochromes are light sensing signal transduction molecules which function to control several aspects of plant biology. Interestingly phytochromes were found to function upstream of cyclic nucleotides in their signal transduction pathways since their functions can be mimicked by cGMP and calcium in phytochrome knockout cells 272829. The light sensing portion of the phytochrome molecule is a covalently linked bilin molecule which is known to be bound by the GAF domain. Therefore it is unlikely that the GAF domain of the phytochrome is also able to bind cyclic nucleotides directly, although it is clear that cyclic nucleotides are somehow involved in this signalling pathway.

Ethylene responses have been documented for nearly a century in plants. This gaseous hormone is involved in many aspects of plant physiology, including fruit ripening, organ development, germination, seedling growth, flowering and response to challenges such as pathogens and stress 30. There are five putative ethylene receptor isoforms in both Arabidopsis and rice as determined by genome sequencing 31. All known ethylene receptors contain a GAF domain in a cytoplasmic region amino-terminal to the kinase domain. It has been speculated that this domain may be involved in cyclic nucleotide signalling but examination of heterologously expressed, functional ETR1 [Swiss-Prot: P49333] showed no detectable cyclic nucleotide binding (G. E. Schaller, personal communication). There are other ethylene receptors which have GAF domains and which to our knowledge have not been tested for cNMP binding, however, to date there is no evidence of cNMP regulation of ethylene receptors. Currently the function and ligands of the GAF domain in ethylene receptors is unknown.

From the alignment of CNB domains in animals, bacteria and plants, it was apparent that there are some strong similarities, as well as some significant differences (Figure 1A). In order to visualize whether plant CNB domains could fold in a similar manner to the other well characterized CNB domains, we generated an in silico model. We chose the plant protein which showed highest similarity to known crystal structures (Arabidopsis thaliana CNTE1) and based our model on the solved crystal structures of RIα, RIIβ, HCN2, CAP and Epac2 (Figure 1B and see additional file 5).

We then examined our model's overall topology as well as its cyclic nucleotide binding site. The basic fold of the domain is two anti-parallel beta sheets consisting of four strands forming a sandwich, ending with an alpha helix (the hinge region). Connecting these sheets are exposed loops, the most important of which is the phosphate binding cassette 32. It is important to note that our structure models very well against all CNB domains with excellent conservation of all secondary structure and most loops. We calculated the backbone root mean square deviation for our model versus each of the templates as: RIIβ domain 1: 0.76 Å; RIIβ domain 2: 0.83 Å; Epac1 domain 1: 1.08 Å; RIα domain 1: 0.85 Å; RIα domain 2: 0.94 Å; HCN2: 0.82 Å and CAP: 1.12 Å. Our model agrees in general with a previously reported model for the Arabidopsis CNGC2 33, although a detailed comparison between the two models was not performed. The use of a less distant target (atCNTE1) as well as several templates (seven compared to one) adds to the reliability of our structure. In all mammalian cAMP-binding structures solved, there is a key arginine residue (Arg 209 in RIα) which forms a salt bridge with the cNMP's phosphate group. This residue is absent in some CNB domains, despite evidence that at least some of these domains do in fact bind cyclic nucleotides. For example, this residue is absent in the Drosophila ether-a-go-go channel, which is known to be modulated by cyclic nucleotides 34. Examination of our model shows that in the region near the phosphate, there are two residues which may functionally replace the arginine, Tyr 91 and Ser 92 (Figure 1B). In bacterial CAP, hydrogen bonding is provided to the phosphate by the Arg 82 sidechain, Ser 83 amide nitrogen atom and sidechain hydroxyl group, as well as a water molecule. In some mammalian isoforms, the serine residue is changed to an alanine and therefore is only able to provide backbone hydrogen-bonding. In our plant atCNTE1 model, the serine residue is conserved, but the arginine residue is missing. Since there was no good template to model the phosphate binding cassette onto, our model only approximates the position of this loop, and will require verification by other structural studies.

The hydroxyl and amide groups of Ser 92, as well as the hydroxyl group of Tyr 91 are all within proximity of the cNMP phosphate and could play a role in stabilizing the cyclic nucleotide (see blue residues in Figure 1). Examination of the region which contacts the base, indicates that our model is most similar to the structure of CAP in this region, so it is likely that the base moeity of a cNMP is bound in a syn orientation as in CAP. This is in agreement with a previously reported atCNGC2 model 33. Further analysis of the binding site for the nucleotide indicates that it is likely cGMP which binds to atCNTE1. This conclusion is based upon the presence of three residues (Tyr 80, Ser 92 and Ser 109) which could potentially differentiate between cyclic nucleotides, each of which has a preference for cGMP (Figure 1 and 3536). Other conserved structural features of our model are the hydrophobic pocket forming residues Tyr 36, Val 42, Val 43, Tyr 53, Leu 55, Ala 60, Phe 82, Ala 93 and Val 95 and Leu 105 (see green residues in Figure 1) as well as several conserved glycine residues which are involved in turns between the beta strands (39, 58 and 83) and the helix capping Asp 109. This residue signals the end of the hinge region alpha helix and is present in most CNB domains examined.

Phylogenetic analysis indicates that the plant CNB domains segregate into three subfamilies (Figures 2 and 3). The phylogenetic distribution of the CNB domain matches their domain context in that CNGC, shaker-type and CNTE proteins form separate groups. Furthermore, for each of the three protein classes, the CNB phylogeny matches the phylogeny of the full-length protein, implying that these proteins obtained the CNB domain prior to isoform duplication (Figure 3, [Additional file 1], 37). Since all three branches have been detected in both Arabidopsis and Oryza, it is likely that the specific plant cyclic nucleotide responses developed prior to monocot-dicot divergence. We did not find CNB domains in any protein kinases, transcription factors or guanine nucleotide exchange factors in our analysis. Each of the three classes of CNB domain containing proteins will be discussed below.

Plant CNGC ion channels were first identified in a screen for calmodulin binding partners in barley 38. There are now known to be 20 CNGC proteins in Arabidopsis likely indicating a high level of channel redundancy 3739. We also detected 16 CNGC proteins in rice by examination of the TIGR Rice Genome Annotaion Resource 40.

Electrophysiological studies have shown the CNGC channels to be permeable to potassium, sodium and calcium 131415414243. Cyclic nucleotides have been shown to activate channel opening in all CNGC proteins examined thus far leading to an influx of cations into the cell 13141533. Mutagenic screens have shown that mutations in atCNGC2 and atCNGC4 create faulty pathogenic reactions 1344. When taken together with data showing that cyclic nucleotides are necessary for pathogen responses and that calcium and potassium influxes are characteristic of early phases of plant pathogen responses 45, this seems to imply that cyclic nucleotides may play a role in controlling plant immune responses.

Finally, work by Maathuis and Sanders 8 has shown that cyclic nucleotides can modulate sodium uptake in Arabidopsis plants, implying that there is a cyclic nucleotide controlled channel which plays a role in salinity tolerance. They showed that cyclic nucleotides are required for limiting sodium uptake in root protoplasts, but the exact molecule (cAMP or cGMP) responsible for this effect has not been pinpointed.

Plant potassium channels fall into two classes, the KCO channels and the shaker-type channels 37. In addition to the 9 shaker-type channels described in Arabidopsis thaliana, we have found 10 channels in Oryza sativa. A variety of mutational studies have implicated the shaker-type channels in several key processes involving the movement of potassium including: from the soil (AKT1, KAT3), long distance transport (AKT2), transport into growing pollen tube (AKT6), secretion into xylem sap (SKOR) and transport during guard cell opening either into the cell (KAT1, KAT2) or out of the cell (GORK) 37.

Shaker-type channels are voltage dependent outward (GORK, SKOR) or inward (KAT and AKT) rectifying channels. Analysis of heterologously expressed channels have shown that cyclic nucleotides function to adjust the activation potential of these channels 1112. Since cyclic nucleotides have already been implicated in some of the processes controlled by shaker-type channels 78946, it is reasonable to believe that cyclic nucleotides are physiological regulators of shaker-type potassium channels.

Initially we detected a short CNB containing protein which was only slightly larger than the domain itself. Sequencing of the EST provided by the Arabidopsis Biological Resource Center 47 showed the protein was actually mis-annotated by the automated gene-finding algorithm. Further analysis indicated that there are two isoforms of this protein in Arabidopsis and one in rice. Each protein contains an amino-terminal CNB domain and a carboxy-terminal acyl-CoA thioesterase domain. Searches of other partially sequenced plant genomes and EST databases indicated that these proteins are present in several plant species, but not in any other division of life and thus represents a novel plant-specific cyclic nucleotide target. Comparison of these protein sequences indicates a high level of conservation, including residues conserved for both catalysis and cyclic nucleotide binding domain structure. Arabidopsis CNTE1 had previously been partially characterized as a thioesterase and shown to have activity versus both 16:0-CoA and 18:1-CoA when over-expressed and partially purified from E. coli 48.

Fatty acid synthesis requires the use of acyl-CoA's as building blocks for incorporation into lipids. It is therefore possible, that these thioesterases function as scavengers which remove "irregular" fatty acids from the pool of available building blocks 48. Furthermore, the thioesterases could divert fatty acids away from biosynthetic pathways and β-oxidation during germination or during stressful conditions. In most cases when a small molecule binding domain is connected to a catalytic domain on the same polypeptide, the catalytic domain is regulated by the small molecule 49. The conservation of this protein across planta indicates that the CNB domain likely has a role in controlling the thioesterase activity of this enzyme, but it is unknown at this time exactly what role cyclic nucleotides play in this process. In order to address this we cloned and tried to express the atCNTE1 protein in E. coli, however after extensive trials we were unable to express and purify soluble protein.

We found no PKG or PKA regulatory subunit homologs in the Arabidopsis genome. There has been a long standing controversy in the plant field as to the existence of a plant cyclic nucleotide dependent kinase 505152. As PKA is the major cAMP target in mammalian cells we chose a biochemical approach to further explore the possibility that a PKA-like enzyme may be present in Arabidopsis.

We performed protein kinase assays with extracts of Arabidopsis thaliana using the PKA substrate Kemptide. Kemptide is a peptide which has a motif which was confirmed as the optimal substrate for PKA 5354 and is routinely used in mammalian PKA assays. As Figure 4A shows, there was no detectable increase in kinase activity in the plant cell extracts when cAMP or cGMP are added. Fractionation of extracts, as well as testing a range of cyclic nucleotide concentrations also did not allow us to detect any differences in kinase activity with addition of cyclic nucleotides (data not shown). For comparison, adipocyte extracts (a cAMP responsive mammalian tissue) were assayed as well, illustrating the large increase in protein kinase activity in these cells when cAMP is added. Furthermore, blotting of A. thaliana extracts with polyclonal antibodies raised against mammalian PKA subunits (both the catalytic and the RIIα subunit) reveals that no structurally similar proteins are present in this extract (Figure 4B). Blotting with a monoclonal antibody to the RIIβ subunit gave similar results (not shown). Although there is a weak band present in the Arabidopsis extract which cross-reacted with the catalytic subunit polyclonal antibody, it is likely unrelated to cyclic nucleotide signalling. Protein kinase catalytic domains are very highly conserved 5556 and therefore a minor amount of cross-reactivity is not unexpected. Further adding to the validity of the western blotting experiment is the observation that several studies have shown that true PKA-like enzymes in non-mammalian eukaryotes do cross react with antibodies raised against the regulatory subunits of mammalian PKA 5758, whereas this is not detected in our plant extracts.

The lack of evidence for kinase activity could be attributed to substrate specificity, differences in binding affinity or expression levels in plant extracts relative to mammalian extracts, so our experimental approach does not exhaustively rule out the possibility of a cNMP dependent kinase activity. However, we feel that these data in concert with the genomic and blotting data strongly suggest that there is no cNMP dependent kinase in plants. Finally, our data imply that if such a protein exists, it would bear little or no sequence, structural or biochemical similarity to the classically studied mammalian enzyme.

In order to identify the proteins which contain CNB or GAF domains, we initially used the Simple Modular Architecture Research Tool (SMART at smart.embl.heidelberg.de; 606162) to scan all predicted Arabidopsis proteins for CNB and GAF domains in the EMBL, TIGR or NCBI databases. Once redundancies were removed, a list of proteins was generated [see additional file 2]. In order to ensure broad coverage of possible variants, we also examined the Interpro collection of protein sequence analysis algorithms, all of which use slightly different methods 63. As an additional method, the predicted proteins of the Arabidopsis genome were searched using the BLAST algorithm 64. As search bait, we used several known cyclic nucleotide binding domains including those from GAF domains (human PDE2A [Swiss-Prot: O00408] and Anabaena cyaB1 [Trembl: P94181]) as well as CNB domains (human CGK2 [Swiss-Prot: Q13237], human RIIβ [Swiss-Prot: P31323], human Epac2 [Swiss-Prot: Q8WZA2], human rod CNGC [Swiss-Prot: P29973] and E. coli CAP [pir: E86000]). This yielded no new inclusions to our list of proteins, but did confirm each of our previous entries. For examination of the Oryza sativa spp. Japonica genome we performed BLAST searches using the aforementioned baits, as well as each of the Arabidopsis proteins. This search was performed using the Blast utility of the TIGR rice database 40. The criterion for inclusion was that the CNB or GAF domain had to match the consensus motif with an E-value of less than 0.5 over the entire domain as determined by SMART. For newly identified proteins from the Orzya sativa, we named them so that they agreed best with the nomenclature of Maser et al. 37 [see additional files 1, 2, 3]. Sequence alignments were performed using the ClustalX 65 or T-COFFEE algorithms 66and then inspected visually. Neighbor-joining trees were generated by ClustalX or PHYLIP 67, then were visualized with TreeView 68. Trees generated using a variety of analysis methods (parsimony, distance and maximum likelihood) yielded similar results to the neighbor-joining trees.

One protein, which appeared to contain only a cyclic nucleotide binding domain and no other motifs was found in the Arabidopsis database. We obtained the clone corresponding to this putative gene from the Arabidopsis Biological Resource Center and sequenced it. Sequencing was performed at the University of Calgary Core Sequencing Facility. We determined that the gene prediction algorithm which scanned the genome improperly predicted the intron/exon structure of this gene. The new gene, which we named cyclic nucleotide regulated thioesterase 1 (atCNTE1) was deposited in the NCBI database [GenBank: AY874170]. A subsequent BLAST search using this gene found another isoform of this gene in Arabidopsis (atCNTE2) and one isoform in Rice (osCNTE1) which we also included in our analysis.

We generated a model of the atCNTE1 cyclic nucleotide binding domain (residues 28–117) based on an alignment of atCNTE1 with the CNB domains of RIIβ [pdb: 1CX4] 69, RIα [pdb: 1RGS] 70, CAP [pdb: 1CGP] 71, HCN2 [pdb: 1Q3E] 72 and Epac1 [pdb: 1O7F] 32. This was submitted to the SWISS-MODEL server via the DeepView program 73. The alignment was iteratively refined to allow for best agreement of sequence and structural similarity.

Unless otherwise indicated all chemicals were purchased from Sigma-Aldrich. Assays were performed on extracts of Arabidopsis cells grown in suspension culture 74 or isolated male Wistar rat adipocytes from epididymal fat pads 75. Both cell types were homogenized in 50 mM Tris pH 7.5, 5% (v/v) glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine and 0.1% (v/v) 2-mercaptoethanol. Adipocytes were lysed by 10 strokes of a dounce homogenizer while plant cells were lysed by two passages through a french press cell at 15 000 psi. The extracts were clarified by centrifugation for 15 min at 4000 RPM in a SS34 rotor at 4°C. These extracts were assayed for kinase activity using ³²P-γ-ATP (Amersham-Pharmacia), 30 μM Kemptide substrate, 50 mM HEPES pH 7.4, 1 mM dithiothreitol and 10 μM cyclic nucleotide as specified. Reactions were allowed to occur for 10 minutes at 30°C and assays were terminated by spotting onto squares of P81 paper followed by extensive washing with 75 mM phosphoric acid 76. Assays were performed in duplicate on three separate preparations with error bars indicating the standard error between preparations (n = 3). Protein concentration was determined by the method of Bradford with bovine serum albumin (ICN Biomedicals) as a standard 77.

Bovine heart PKA catalytic subunit and RII were purified to homogeneity 7879. Purified protein was injected into rabbits and serum was obtained according to standard methods 80. Extracts of adipose and plant cells were prepared as described above. Samples were boiled into SDS-PAGE buffer and separated on a 10% denaturing gel 81. The proteins were then transferred to nitrocellulose for 2 h at 100V and blocked overnight with 5% (w/v) skim milk powder. Blots were probed with antibodies for 1 h and visualized by enhanced chemiluminesence. The PKA catalytic subunit antibody was affinity purified according to 82 and used at 0.5 μg/mL while RII was used as crude immune serum at a 5000X dilution. For the RII western blots, both a polyclonal and a monoclonal antibody (anti-RIIβ BD Transduction Laboratories) gave identical results.