The Arabidopsis thaliana Brassinosteroid Receptor (AtBRI1) Contains a Domain that Functions as a Guanylyl Cyclase In Vitro

Background Guanylyl cyclases (GCs) catalyze the formation of the second messenger guanosine 3′,5′-cyclic monophosphate (cGMP) from guanosine 5′-triphosphate (GTP). Cyclic GMP has been implicated in an increasing number of plant processes, including responses to abiotic stresses such as dehydration and salt, as well as hormones. Principle Findings Here we used a rational search strategy based on conserved and functionally assigned residues in the catalytic centre of annotated GCs to identify candidate GCs in Arabidopsis thaliana and show that one of the candidates is the brassinosteroid receptor AtBR1, a leucine rich repeat receptor like kinase. We have cloned and expressed a 114 amino acid recombinant protein (AtBR1-GC) that harbours the putative catalytic domain, and demonstrate that this molecule can convert GTP to cGMP in vitro. Conclusions Our results suggest that AtBR1 may belong to a novel class of GCs that contains both a cytosolic kinase and GC domain, and thus have a domain organisation that is not dissimilar to that of atrial natriuretic peptide receptors, NPR1 and NPR2. The findings also suggest that cGMP may have a role as a second messenger in brassinosteroid signalling. In addition, it is conceivable that other proteins containing the extended GC search motif may also have catalytic activity, thus implying that a significant number of GCs, both in plants and animals, remain to be discovered, and this is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains over 90 annotated putative CGs.


INTRODUCTION
Guanylyl cyclases (GCs) have been identified in many diverse prokaryotes and eukaryotes where they catalyse the formation of the second messenger guanosine 39,59-cyclic monophosphate (cGMP) from guanosine 59-triphosphate (GTP) [1]. In higher plants cGMP has been shown to act as a second messenger in a large number of physiological responses [2,3] that include cGMP-mediated changes of the transcriptome [4], NO-dependent signaling [5] as well as gravitropic responses [6] and plant hormone-dependent responses [7][8][9]. Furthermore, significant and transient increases in intracellular cGMP levels have also been reported in response to plant natriuretic peptides (PNPs) [8,10] as well as NaCl and drought stress [11]. The first functional GC in higher plants was identified with a search motif based on several functionally assigned amino acids in the catalytic domain of known GCs from lower eukaryotes and animals [12].
Here we show that a rationally designed search motif of the catalytic domain identifies several members of the family of leucine rich repeat receptor-like kinases (LRR RLKs) including an Arabidopsis thaliana brassinosteroid receptor (AtBRI1). A recombinant domain protein was made which tested positive for GC activity in vitro. The implications of this finding for both the projected number of different classes of GCs and the role of cGMP in brassinosteroid signaling are discussed.

Extending the search GC search motif and identification of AtBRI1
The original GC search motif [ [12] ( Figure 1A) yielded seven Arabidopsis candidate proteins including AtGC1 that has been demonstrated to have GC activity in vitro [12]. Two of seven proteins are annotated kinases and one of the two (At1g79680) belongs to the group of wall associated kinase-like proteins (WAKLs). In a quest to identify further candidate GCs in plants we mutated the position 7 in the original search motif. This position is assigned as having a role in dimerisation that may not be a critical requirement for GC functionality. When [D] in position 7 is substituted by [L] in a 100 amino acid recombinant AtGC1(1-100), a domain that is encoded by the first exon of AtGC1 ( Figure 1B), no significant loss in catalytic GC activity occurs (Fig. 1D). This implies firstly that a 100 amino acid domain is sufficient for activity and secondly, that [D] in position 7 is not essential for AtGC1 GC activity. These observation may hold for a number of GCs. Consequently we added [L] to make position 7 [DNAL] ( Fig. 2A . All three motifs are specific for GC rather than adenylyl cyclases (ACs) since they contain the residues [GCTH] in position 3 facing the purine and determining substrate specificity for GTP rather than ATP [13][14][15] and GenBank contains no annotated ACs that conform to either of the motifs.
The third and most relaxed motif appears in 171 Arabidopsis proteins of which 88 contain [EQCTDRVHY] at position 16 or 17 which is presumed responsible for metal ion binding [13]. Metal binding is normally associated with aspartic or glutamic acid ([DE]). Within the group of 88 proteins; 27 contain a PPi-binding [R] between 5 and 20 amino acids N-terminal of the core motif.
These 27 Arabidopsis proteins (Table 1) represent a highly significant (p,1e 25 ) enrichment for the gene ontology GO categories of phosphorus metabolic processes, protein metabolic process, cellular macromolecular metabolic process and biopolymer metabolic process. It is noteworthy that several are annotated as leucine rich repeat receptor like kinases (LRR RLKs) including AtBRI1 (Fig. 2). In AtBRI1, the putative catalytic core was identified within the cytosolic kinase domain.
The choice of for further testing AtBRI1 was informed by several factors including the fact that brassinosteroids are physiologically well characterised growth regulators that await further elucidation of their signal transduction networks as well as the availability of a number of AtBRI1 mutants that can support these investigations. Brassinosteroid receptors have been identified in several other species and these also contain the conserved GC motif (Fig. 2D).
The recombinant protein that we decided to synthesise and test for in vitro activity contains the predicted GC catalytic centre of AtBRI1 (At4g39400) and 50 additional amino acids on both the N-and the C-terminus (Fig. 2C). This peptide (AtBRI1-GC) is part of the cytoplasmic domain containing the N-terminal part aspartic acid ([D] at 233 from the catalytic centre) implicated in

Testing a recombinant GC domain (AtBRI1-GC) for activity
The capacity of the recombinant putative GC domain of AtBRI1 (AtBRI1-GC) to generate cGMP from GTP was assessed with two independent methods. Firstly, we used an enzyme immunoassay to check if a reconstituted recombinant AtBRI1-GC could function as a GC in vitro. The results indicate that the recombinant protein can cyclase GTP and does so preferably in the presence of Mg 2+ (Fig. 3A). In order to verify the result obtained with this anti-body based detection method we also used mass spectrometry. Firstly, we established that the Q-TOF mass chromatogram could detect cGMP at fmol concentrations (Fig. 3D, right inset) much like the enzyme immunoassay. We detected neither cGMP in the solution containing the recombinant protein only (Fig. 3B) nor in the reaction mix in the absence of the protein (Fig. 3C). Our sample generates cGMP in a time dependent way (Fig. 3D). We calculated that after 5 min. incubation in the presence of 1 mM GTP 100 fmoles cGMP/mg protein were generated and after 20 min. .3 pmoles cGMP/mg protein (Fig. 3D). We noted that values of the amount of cGMP generated obtained with the mass spectroscopy read higher than those obtained with the enzymatic assay and this observation has been made consistently in independent in vitro experiments with recombinant proteins (data not shown). In addition, it is noteworthy that plant GC activities are reportedly low and certainly not at the levels observed for some soluble animal GCs [17]. The main reason for this is that higher activities might require co-factors (e.g. Ca 2+ , chaperones or co-proteins) or post-translational modifications that are not present in the recombinant tested in vitro.
We also used mass spectrometry to test AtBRI1-GC for adenylyl cyclase activity in the presence of 1 mM ATP as the substrate and could not detect significant amounts of cAMP generated after 20 min. of incubation (result not shown) thus indicating that, at least in vitro, the recombinant protein has the predicted substrate preference for GTP.

DISCUSSION
Brassinosteroids (BRs) are polyhydroxylated plant steroid hormones with an essential role in co-regulating many processes including embryogenesis, cell elongation and vascular differentiation [25,26]. Brassinosteroid Insensitive-1 (BRI1) was first identified from mutant analysis and then cloned and found to be a leucine rich repeat receptor like kinase [27] located in the plasma membrane [28,29]. Based on the binding of the ligand BR to the leucine rich repeat extracellular domain, BRI1 has been identified as a BR receptor in Arabidopsis [29,30] and therefore a critical signal component. BRI1 is ubiquitously expressed in Arabidopsis and potential BRI1 kinase substrates have been identified such as transthyretin-like protein which is phosphorylated in vitro by the kinase domain of BRI1 [31]. Several models have been developed to describe the signaling events following perception of BR by BRI1 (see [32]) involving other membrane associated proteins and activation of transcription factors. The observation that AtBRI1 does harbor a functional GC domain within the cytosolic part of the molecule might suggest that cGMP is a second messenger in some BR dependent processes. However, this hypothesis remains to be tested. Several genes that regulate physiological functions are stimulated by BR as well as being influenced by cGMP. An example for this dual dependence is plant cell elongation [26]. Microarray studies revealed that genes involved in cell wall expansion such as expansins and pectinesterases are up-regulated by both BR [32] and membrane permeable cGMP treatments [4].
Both BR and gibberellin interact to regulate plant growth. Some of these interactions are antagonistic but in other cases, BR can potentiate gibberellin activity [33]. Gibberellin, itself, stimulates increases in cGMP [7]. It is conceivable that in some instances the GC domain of BRI1 could stimulate cGMP production and so potentiate gibberellin activity. On a speculative note, there may be key molecules within specific cells that specify decreased cytoplasmic kinase activity and enhance the GC activity of the AtBRI1.
There are several recessive alleles of AtBRI1 in the cytoplasmic kinase domain. Of these mutants, bri-101 is the only mutant in the GC catalytic region ([E] 1078 to [L]) and it is insensitive to BR and also has reduced kinase activity when tested in a heterologous system [27,28]. Interestingly, this mutation should not affect the GC activity as it occurs at position 8 which can be any amino acid. Three other mutants have been found in the region that we show confers GC activity in vitro, being:  [28]. The domain that we have identified (Fig. 2) occurs within the kinase domain [28]. We demonstrate that the isolated 114 amino acid recombinant peptide (AtBRI1-GC) has GC activity in vitro (Fig. 3). The relative importance of the two functions in the action of the receptor remains to be demonstrated bearing in mind that previously work has focused on the kinase domain as the GC domain had not been identified. Interestingly, a number of enzymes have recently been identified as ''moonlighting'' proteins with dual functions [34]; the kinase and GC activity of AtBRI1 could be yet another example.
On a more general level, the finding implies that functional GC domains may be part of a large variety of different multifunctional signaling molecules and receptors in particular. It is noteworthy that the atrial natriuretic peptide receptors NPR1 and NPR2 both signal through cGMP and have an AtBRI1-like domain organisation with an extracellular ligand-binding domain, a transmembrane domain and an intracellular kinase and GC domain [35,36].
Finally, the fact that two recombinant proteins (AtGC1(1-100) and AtBRI1 -GC ) of less than 120 amino acids have GC activity in vitro begs a reexamination of the minimal catalytic requirement for GCs and may suggest that the number of different GC domains is significantly higher than currently assumed. This is in keeping with the fact that the single cellular green alga Chlamydomonas reinhardtii contains a surprisingly large number (.90) of annotated putative GCs [1] and with the increasing number of biological processes discovered that are modulated by the second messenger cGMP [2,3].

Identification of GC catalytic domain
Annotated GCs were retrieved from NCBI and their catalytic domains [13] were used for Blast [37] queries of ''The Arabidopsis Information Resource'' (TAIR) database and GenBank. The catalytic domains were aligned using Clustal X [38] and the alignments at the catalytic centre of the catalytic domain was used to derive the search motifs [12]. Derived search motifs were tested

Site-directed mutagenesis
A non-methylated double strand was synthesized using 0.5 mM Forward (59 -ATACTGCCTATTCGATCTTCCCTTGGTGA-GTGATG-39) and 0.5 mM Reverse (59 -CATCACTCACCA-AGGGAAGATCGAATAGGCAGTAT-39) primers from a clone  Fig. 2A). The left inset represents the mass of the peak in the chromatogram, the right inset is the calibration curve with 1. 25 Leon-Rot, Germany). The thermal cycling parameters were: initial denaturation at 96uC for 3 min., followed by 30 sec. at 96uC, 50uC for 45 sec. and 72uC for 1 min. for 32 cycles, followed by a final extension at 72uC for 10 min. A fragment of 340 bp was excised from the gel and purified using the GFX purification kit as per manufacturer's instruction (Amersham Biosciences, Little Chalfont, UK). The fragment was cloned into the pCRHT7 TOPOH-NT vector (Invitrogen Ltd., Paisley, UK) and used to transform E. coli BL21 (DE3) pLysS cells (Invitrogen Ltd., Paisley, UK); colonies were selected and inserts verified by sequencing.
cGMP measurements GC activity in vitro was assessed by measuring cGMP generated from GTP in the presence of 10 mg purified protein, 50 mM Tris-HCl (pH 7.5), 2 mM isobutyl methylxanthine (IBMX), 5 mM Mg 2+ and/or 5 mM Mn 2+ and 1 mM GTP [39]. Product levels were measured by cGMP enzyme immunoassay Biotrak (EIA) System (Amersham Biosciences, Little Chalfont, UK) with the acetylation protocol as described in the supplier's manual. The anti-cGMP antibody is highly specific for cGMP and has approximately 10 6 times lower affinity for cAMP. Mass spectroscopic determinations of cGMP were done with a Waters API Q-TOF Ultima in the W-mode. The samples were introduced with a Waters Acquity UPLC (Waters Microsep, Johannesburg, South Africa) at a flow rate of 180 mL/min. and separation was achieved by a Phenomenex Synergi (Torrance, CA) 4 mm Fusion -RP (25062.0 mm) column. A gradient of solvent ''A'' (0.1% formic acid) and solvent ''B'' (100% acetonitrile) over 18 min was applied. During the first 7 min. the solvent composition was kept at 100% ''A'' followed by a linear gradient of 3 min. to 80% ''B'' and re-equilibration to the initial conditions. Electrospray ionisation in the negative mode was used at a cone voltage of 35 V. The running parameters were optimised for sensitivity and specificity.