Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling

Computational prediction and in vivo protein coupling experiments identify candidate plant G-protein coupled receptors in Arabidopsis, rice and poplar

Common and unique elements of the ABA-regulated transcriptome of Arabidopsis guard cells

<p>Abstract</p> <p>Background</p> <p>In the presence of drought and other desiccating stresses, plants synthesize and redistribute the phytohormone abscisic acid (ABA). ABA promotes plant water conservation by acting on specialized cells in the leaf epidermis, guard cells, which border and regulate the apertures of stomatal pores through which transpirational water loss occurs. Following ABA exposure, solute uptake into guard cells is rapidly inhibited and solute loss is promoted, resulting in inhibition of stomatal opening and promotion of stomatal closure, with consequent plant water conservation. There is a wealth of information on the guard cell signaling mechanisms underlying these rapid ABA responses. To investigate ABA regulation of gene expression in guard cells in a systematic genome-wide manner, we analyzed data from global transcriptomes of guard cells generated with Affymetrix ATH1 microarrays, and compared these results to ABA regulation of gene expression in leaves and other tissues.</p> <p>Results</p> <p>The 1173 ABA-regulated genes of guard cells identified by our study share significant overlap with ABA-regulated genes of other tissues, and are associated with well-defined ABA-related promoter motifs such as ABREs and DREs. However, we also computationally identified a unique <it>cis</it>-acting motif, GTCGG, associated with ABA-induction of gene expression specifically in guard cells. In addition, approximately 300 genes showing ABA-regulation unique to this cell type were newly uncovered by our study. Within the ABA-regulated gene set of guard cells, we found that many of the genes known to encode ion transporters associated with stomatal opening are down-regulated by ABA, providing one mechanism for long-term maintenance of stomatal closure during drought. We also found examples of both negative and positive feedback in the transcriptional regulation by ABA of known ABA-signaling genes, particularly with regard to the PYR/PYL/RCAR class of soluble ABA receptors and their downstream targets, the type 2C protein phosphatases. Our data also provide evidence for cross-talk at the transcriptional level between ABA and another hormonal inhibitor of stomatal opening, methyl jasmonate.</p> <p>Conclusions</p> <p>Our results engender new insights into the basic cell biology of guard cells, reveal common and unique elements of ABA-regulation of gene expression in guard cells, and set the stage for targeted biotechnological manipulations to improve plant water use efficiency.</p

Boolean modeling of transcriptome data reveals novel modes of heterotrimeric G-protein action

Classical mechanisms of heterotrimeric G-protein signaling are observed to function in regulation of the transcriptome. Conversely, many theoretical regulatory modes of the G-protein are not manifested in the transcriptomes we investigate.A new mechanism of G-protein signaling is revealed, in which the β subunit regulates gene expression identically in the presence or absence of the α subunit.We find evidence of cross-talk between G-protein-mediated and hormone-mediated transcriptional regulation.We find evidence of system specificity in G-protein signaling

Proportional Venn diagrams detailing the number of predicted and co-predicted 7TM protein sequences in the non-redundant , , and proteomes

Signal peptides were removed prior to topology analyses.<p><b>Copyright information:</b></p><p>Taken from "Whole proteome identification of plant candidate G-protein coupled receptors in , rice, and poplar: computational prediction and protein coupling"</p><p>http://genomebiology.com/2008/9/7/R120</p><p>Genome Biology 2008;9(7):R120-R120.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2530877.</p><p></p

Flowchart detailing our candidate GPCR identification and analysis scheme

Numbers in parentheses include redundant protein sequences. A complete list of splice variants and redundant proteins for the proteome is supplied in Additional data file 12.<p><b>Copyright information:</b></p><p>Taken from "Whole proteome identification of plant candidate G-protein coupled receptors in , rice, and poplar: computational prediction and protein coupling"</p><p>http://genomebiology.com/2008/9/7/R120</p><p>Genome Biology 2008;9(7):R120-R120.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2530877.</p><p></p

Molecular evolutionary analyses of candidate GPCRs shown to physically interact with GPA1

The , , and proteomes were subjected to BLAST analyses (e-20 cutoff) using our positive interacting candidate GPCR protein sequences (filled triangles). Multiple sequence alignments were created in ClustalX and evolutionary relationships were estimated using the neighbor joining method with 1,000 bootstrap replicates. Sequences identified by our bioinformatic pipeline as candidate GPCRs are indicated with empty triangles, with upward pointing triangles indicating those found within our high ranking candidate sets and downward pointing triangles indicating those present in the second tier. Scale bars indicate evolutionary distance as measured by residue substitutions per site. RGS1; GCR1; Cand2 and Cand8; Cand1; Cand3, 4, and 5; Cand6 and Cand7; and HHP2.<p><b>Copyright information:</b></p><p>Taken from "Whole proteome identification of plant candidate G-protein coupled receptors in , rice, and poplar: computational prediction and protein coupling"</p><p>http://genomebiology.com/2008/9/7/R120</p><p>Genome Biology 2008;9(7):R120-R120.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2530877.</p><p></p

Experimental organization and two representative results for GPA1-candidate GPCR interaction assessed by the split-ubiquitin system

Schematic showing a simplified outline of the split-ubiquitin system assay: protein A is fused to the amino-terminal half of ubiquitin as an amino- or carboxy-terminal fusion (only the amino-terminal fusion orientation is shown here); protein B is fused to the carboxy-terminal half of ubiquitin, which in turn has a fused artificial transcription factor (PLV). Interaction of protein A with protein B brings the two halves of ubiquitin into close proximity and a functional ubiquitin molecule is restored. Ubiquitin specific proteases cleave off PLV, which translocates to the nucleus and activates transcription of target genes allowing for yeast growth. Cartoon detailing the control (Nub) and test (NubG) fusion protein construct orientations for sectors 1-4 in (c). Schematic depicting the organization of the interaction assay plates in (d,e). The X represents the candidate GPCR open reading frame (ORF). Sectors 5-8 show the reciprocal assay. Representative results for the ability of candidate GPCRs to interact with GPA1, the Gα subunit, on 1 mM methionine repression media. Diploid yeast containing GPA1 fusion constructs and either candidate Cand5 (d) or TOM1 (e) fusion constructs both grow on minimal media (not shown), but Cand5 specifically interacts with GPA1 and allows growth on the repression media (d, boxed sector) while TOM1 does not (e, boxed sector).<p><b>Copyright information:</b></p><p>Taken from "Whole proteome identification of plant candidate G-protein coupled receptors in , rice, and poplar: computational prediction and protein coupling"</p><p>http://genomebiology.com/2008/9/7/R120</p><p>Genome Biology 2008;9(7):R120-R120.</p><p>Published online 31 Jul 2008</p><p>PMCID:PMC2530877.</p><p></p