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

Site-directed mutagenesis and functional testing of AtGC1(1–100).

<p>(A) Original 14 amino acid search motif for GCs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000449#pone.0000449-Ludidi1" target="_blank">[12]</a>; substitutions are in square brackets, X represents any amino acid and curly brackets define the number of amino acids. (B) AtGC1 (At5g05930): The position of the GC catalytic centre is marked in red; the underlined aspartic acid [D] is the amino acid that has been changed into a leucine [L] by site directed mutagenesis. The open arrow marks the conserved PPi-binding arginine [R] and the C-terminal putative metal binding site is highlighted in aquamarine. The green triangles point to exon borders and the solid arrow shows the border of the fragment that we have tested for GC activity. (C) SDS-PAGE of the 3 purification steps of the recombinant protein AtGC1(1–100); “M” is the molecular weight marker, “FT” is the protein in the flow-through, “W” is the wash and “E” is the eluted recombinant protein. (D) <i>In vitro</i> GC activity assay. The control (cont.; empty bar) was obtained by omitting protein in the reaction mix and the concentration of GTP was 1mM and that of Mn²⁺ or Mg²⁺ was 5 mM. The values for the wild-type protein (N-terminal fragment of 100 amino acids containing a [D] in position 7 of the catalytic centre) and the mutated protein (N-terminal fragment of 100 amino acids containing [L] in position 7 of the catalytic centre) are represented with solid bars. The bar values represent the mean cGMP (+/−SEM) generated in 15 minutes in three samples and the response pattern is representative of 3 independent experiments.</p

Preparation of recombinant AtBRI1-GC and functional testing <i>in vitro.</i>

<p>(A) Testing of GC activity with an enzyme immunoassay. The control contains the reaction mixture without the substrate (10 µg recombinant protein in 50 mM Tris-HCl (pH 7.5), 2 mM isobutyl methylxanthine (IBMX), 5 mM Mg²⁺ and 5 mM Mn²⁺), the other columns represent cGMP generated in the presence of 1 mM GTP and either Mn²⁺ or Mg²⁺ after 20 min. The bar values represent the mean (+/−SEM). (B) Extracted mass chromatogram of <i>m/z</i> 344 [M-1]⁻¹ ion of cGMP generated by 10 µg recombinant protein. The inset shows an SDS-PAGE of AtBRI1-GC expressed in <i>E. coli</i> BL21 (pLysS) (DE3) and purified with Ni-NTA agarose under denaturing conditions. Cleared lysate (lane 1), flow through (lane 2), first wash (lane 3), second wash (lane 4) and eluted recombinant protein (lane 5). ‘M’ is the molecular weight marker. (C) Extracted mass chromatogram of <i>m/z</i> 344 [M-1]⁻¹ ion of the reaction mix without AtBRI1-GC. (D) Two superimposed extracted mass chromatogram of <i>m/z</i> 344 [M-1]⁻¹ ion of cGMP generated by 10 µg recombinant protein after 5 and 20 min. respectively in the presence of 5 mM Mg²⁺. (Note that the sample was diluted 200 times as compared to the experiment presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000449#pone-0000449-g002" target="_blank">Fig. 2A</a>). The left inset represents the mass of the peak in the chromatogram, the right inset is the calibration curve with 1.25, 10 and 50 fmoles on the column.</p