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Heterotrimeric G proteins, composed of Gα, Gβ, and Gγ subunits, are a class of signal transduction complexes with broad roles in human health and agriculturally relevant plant physiological and developmental traits. In the classic paradigm, guanine nucleotide binding to the Gα subunit regulates the activation status of the complex. We sought to develop improved methods for heterologous expression and rapid purification of Gα subunits. Using GPA1, the sole canonical Gα subunit of the model plant species,Arabidopsis thaliana, we observed that, compared to conventional purification methods, rapid StrepII-tag mediated purification facilitates isolation of protein with increased GTP binding and hydrolysis activities. This allowed us to identify a potential discrepancy with the reported GTPase activity of GPA1. We also found that human GNAI1 displayed increased GTPase activity when purified using our approach, indicating our protocol is applicable to mammalian Gα subunits, potentially including those for which purification of enzymatically active protein has been historically problematic. We then utilized domain swaps of GPA1 and human GNAO1 to demonstrate that the inherent instability of GPA1 is a function of the interaction between the Ras and helical domains. Additionally, we found that GPA1-GNAO1 domain swaps uncouple the instability from the rapid nucleotide binding kinetics displayed by GPA1.

We describe a previously unreported macroscopic Arabidopsis organ, the cantil, named for its ‘cantilever’ function of holding the pedicel at a distance from the stem. Cantil development is strongest at the first nodes after the vegetative to reproductive inflorescence transition; cantil magnitude and frequency decrease acropetally. Cantils develop in wild-type Arabidopsis accessions (e.g. Col-0, Ws and Di-G) as a consequence of delayed flowering in short days; cantil formation is observed in long days when flowering is delayed by null mutation of the floral regulator FLOWERING LOCUS T. The receptor-like kinase ERECTA is a global positive regulator of cantil formation; therefore, cantils never form in the Arabidopsis strain Ler. ERECTA functions genetically upstream of heterotrimeric G proteins. Cantil expressivity is repressed by the specific heterotrimeric complex subunits GPA1, AGB1 and AGG3, which also play independent roles: GPA1 suppresses distal spurs at cantil termini, while AGB1 and AGG3 suppress ectopic epidermal rippling. These G protein mutant traits are recapitulated in long-day flowering gpa1-3 ft-10 plants, demonstrating that cantils, spurs and ectopic rippling occur as a function of delayed phase transition, rather than as a function of photoperiod per se.

Cellulose synthesis is essential for plant morphology, water transport and defense, and provides raw material for biomaterials and fuels. Cellulose is produced at the plasma membrane by Cellulose Synthase (CESA) protein complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked from the Golgi apparatus and trans-Golgi Network (TGN) to the plasma membrane. Since CESA enzymes are only active in the plasma membrane, control of CSC secretion is a critical step in the regulation of cellulose synthesis. However, the regulatory framework for CSC secretion is not clarified. In this study, we identify members of a family of seven transmembrane domain-containing proteins (7TMs) as important for cellulose production during cell wall integrity stress. 7TM proteins are often associated with guanine nucleotide-binding protein (G) protein signalling and mutants in several of the canonical G protein complex components phenocopied the 7tm mutant plants. Unexpectedly, the 7TM proteins localized to the Golgi apparatus/TGN where they interacted with the G protein complex. Here, the 7TMs and G proteins regulated CESA trafficking, but did not affect general protein secretion. Furthermore, during cell wall stress, 7TMs’ localization was biased towards small CESA-containing vesicles, specifically associated with CSC trafficking. Our results thus outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses upon exposure to cell wall integrity stress.

Cellulose is produced at the plasma membrane of plant cells by cellulose synthase (CESA) complexes (CSCs). CSCs are assembled in the endomembrane system and then trafficked to the plasma membrane. Because CESAs are only active in the plasma membrane, control of CSC secretion regulates cellulose synthesis. We identified members of a family of seven transmembrane domain-containing proteins (7TMs) that are important for cellulose production during cell wall integrity stress. 7TMs are often associated with guanine nucleotide-binding (G) protein signaling and we found that mutants affecting the Gβγ dimer phenocopied the 7tm mutants. Unexpectedly, the 7TMs localized to the Golgi/trans-Golgi network where they interacted with G protein components. Here, the 7TMs and Gβγ regulated CESA trafficking but did not affect general protein secretion. Our results outline how a G protein-coupled module regulates CESA trafficking and reveal that defects in this process lead to exacerbated responses to cell wall integrity stress.

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.

Understanding tissue-related gene expression patterns can provide important insights into gene, tissue, and organ function. Transcriptome analyses often have focused on housekeeping or tissue-specific genes or on gene coexpression. However, by analyzing thousands of single-gene expression distributions in multiple tissues of Arabidopsis thaliana, rice (Oryza sativa), human (Homo sapiens), and mouse (Mus musculus), we found that these organisms primarily operate by gene sharing, a phenomenon where, in each organism, most genes exhibit a high expression level in a few key tissues. We designed an analytical pipeline to characterize this phenomenon and then derived Arabidopsis and human gene-sharing networks, in which tissues are connected solely based on the extent of shared preferentially expressed genes. The results show that tissues or cell types from the same organ system tend to group together to form network modules. Tissues that are in consecutive developmental stages or have common physiological functions are connected in these networks, revealing the importance of shared preferentially expressed genes in conferring specialized functions of each tissue type. The networks provide predictive power for each tissue type regarding gene functions of both known and heretofore unknown genes, as shown by the identification of four new genes with functions in guard cell and abscisic acid response. We provide a Web interface that enables, based on the extent of gene sharing, both prediction of tissue-related functions for any Arabidopsis gene of interest and predictions concerning the relatedness of tissues. Common gene-sharing patterns observed in the four model organisms suggest that gene sharing evolved as a fundamental organizing principle of gene expression in diverse multicellular eukaryotes.

We have identified a family of expansin transcripts that include seven α-expansins (MjExp1 through MjExp7) and three β-expansins (MjExpB1 through MjExpB3) from Mirabilis jalapa (Nyctaginaceae) that show dramatic changes in transcript abundance during the rapid expansion and subsequent senescence of the ephemeral flowers. In general, α-expansin expression was low in small buds, high during maximal elongation of the floral tube, reduced during floral display, and upregulated during calyx infolding and collapse. Transcripts encoding auxin responsive proteins (Aux/IAA) showed a similar pattern of expression. Northern analysis using a set of overlapping probes designed to the MjExp2 transcript demonstrated a gradient of sequence conservation along its length (high to low, from the 5′ to the 3′ end of the coding region), and identified the presence of floral senescence-specific expansins. Beta expansin transcripts were found to be preferentially expressed during early floral development and sharply downregulated coincident with rapid growth. All three β-expansin transcripts are highly related, and ψMjExpB2 is an intronless pseudogene derived from MjExpB1. MjExpB3 appears to have derived from MjExpB1 in a separate gene duplication event, and is predicted to encode a truncated protein.

Protein networks and signaling cascades are key mechanisms for intra- and intercellular signal transduction. Identifying the interacting partners of a protein can provide vital clues regarding its physiological role. The bimolecular fluorescence complementation (BiFC) assay has become a routine tool for in vivo analysis of protein-protein interactions and their subcellular location. Although the BiFC system has improved since its inception, the available options for in planta analysis are still subject to very low signal-to-noise ratios, and a systematic comparison of BiFC confounding background signals has been lacking. Background signals can obscure weak interactions, provide false positives, and decrease confidence in true positives. To overcome these problems, we performed an extensive in planta analysis of published BiFC fragments used in metazoa and plants, and then developed an optimized single vector BiFC system which utilizes monomeric Venus (mVenus) split at residue 210, and contains an integrated mTurquoise2 marker to precisely identify transformed cells in order to distinguish true negatives. Here we provide our streamlined double ORF expression (pDOE) BiFC system, and show that our advance in BiFC methodology functions even with an internally fused mVenus210 fragment. We illustrate the efficacy of the system by providing direct visualization of Arabidopsis MLO1 interacting with a calmodulin-like (CML) protein, and by showing that heterotrimeric G-protein subunits Gα (GPA1) and Gβ (AGB1) interact in plant cells. We further demonstrate that GPA1 and AGB1 each physically interact with PLDα1 in planta, and that mutation of the so-called PLDα1 'DRY' motif abolishes both of these interactions.

Genomic sequencing has provided a vast resource for identifying interesting genes, but often an exact "gene-of-interest" is unknown and is only described as putatively present in a genome by an observed phenotype, or by the known presence of a conserved signaling cascade, such as that facilitated by the heterotrimeric G-protein. The low sequence similarity of G protein-coupled receptors (GPCRs) and the absence of a known ligand with an associated high-throughput screening system in plants hampers their identification by simple BLAST queries or brute force experimental assays. Combinatorial bioinformatic analysis is useful in that it can reduce a large pool of possible candidates to a number manageable by medium or even low-throughput methods. Here we describe a method for the bioinformatic identification of candidate GPCRs from whole proteomes and their subsequent in vivo analysis for G-protein coupling using a membrane based yeast two-hybrid variant (Gookin et al., Genome Biol 9:R120, 2008). Rather than present the bioinformatic process in a format requiring scripts or computer programming knowledge, we describe procedures here in a simple, biologist-friendly outline that only utilizes the basic syntax of regular expressions.

Genomic sequencing has provided a vast resource for identifying interesting genes, but often an exact "gene-of-interest" is unknown and is only described as putatively present in a genome by an observed phenotype, or by the known presence of a conserved signaling cascade, such as that facilitated by the heterotrimeric G-protein. The low sequence similarity of G protein-coupled receptors (GPCRs) and the absence of a known ligand with an associated high-throughput screening system in plants hampers their identification by simple BLAST queries or brute force experimental assays. Combinatorial bioinformatic analysis is useful in that it can reduce a large pool of possible candidates to a number manageable by medium or even low-throughput methods. Here we describe a method for the bioinformatic identification of candidate GPCRs from whole proteomes and their subsequent in vivo analysis for G-protein coupling using a membrane based yeast two-hybrid variant (Gookin et al., Genome Biol 9:R120, 2008). Rather than present the bioinformatic process in a format requiring scripts or computer programming knowledge, we describe procedures here in a simple, biologist-friendly outline that only utilizes the basic syntax of regular expressions.

G proteins, including monomeric G proteins and heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits, are molecular switches in cellular signaling. In addition to these classic G proteins, plants have several types of unconventional G proteins, such as extra-large G proteins (XLGs), developmentally regulated G proteins (DRGs), and GPCR-type G proteins (GTGs). XLGs are nuclear-localized proteins with Gα-like C-termini and large, unique, N-terminal extensions. XLGs are involved in regulation of primary root growth, root waving and skewing, and plant responses to sugars, osmotic stress, pathogens, and hormones. DRGs have all the conserved GTPase domain motifs found in conventional G proteins but do not have any other sequence similarities with conventional G proteins. The functions of DRGs in plants remain unknown. GTGs have nine predicted transmembrane domains and exhibit GTP-binding and GTPase activities, as well as ABA-binding. As one class of ABA receptors, GTGs mediate most classic ABA responses. These unconventional G proteins diversify signaling pathways mediated by G proteins in plants.

Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes. Abbreviations: ACO, 1-aminocyclopropane-1-carboxylate oxidase; CHS, chalcone synthase; PDS, phytoene desaturase; PTGS, post-transcriptional gene silencing; PVX, potato virus X; TGMV, tomato golden mosaic virus; TMV, tobacco mosaic virus; TRV, tobacco rattle virus; VIGS, virus-induced gene silencing

BackgroundIn 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.ResultsThe 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 uniquecis-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.ConclusionsOur 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.

Computational prediction and in vivo protein coupling experiments identify candidate plant G-protein coupled receptors in Arabidopsis, rice and poplar., Background The classic paradigm of heterotrimeric G-protein signaling describes a heptahelical, membrane-spanning G-protein coupled receptor that physically interacts with an intracellular Gα subunit of the G-protein heterotrimer to transduce signals. G-protein coupled receptors comprise the largest protein superfamily in metazoa and are physiologically important as they sense highly diverse stimuli and play key roles in human disease. The heterotrimeric G-protein signaling mechanism is conserved across metazoa, and also readily identifiable in plants, but the low sequence conservation of G-protein coupled receptors hampers the identification of novel ones. Using diverse computational methods, we performed whole-proteome analyses of the three dominant model plant species, the herbaceous dicot Arabidopsis thaliana (mouse-eared cress), the monocot Oryza sativa (rice), and the woody dicot Populus trichocarpa (poplar), to identify plant protein sequences most likely to be GPCRs. Results Our stringent bioinformatic pipeline allowed the high confidence identification of candidate G-protein coupled receptors within the Arabidopsis, Oryza, and Populus proteomes. We extended these computational results through actual wet-bench experiments where we tested over half of our highest ranking Arabidopsis candidate G-protein coupled receptors for the ability to physically couple with GPA1, the sole Gα in Arabidopsis. We found that seven out of eight tested candidate G-protein coupled receptors do in fact interact with GPA1. We show through G-protein coupled receptor classification and molecular evolutionary analyses that both individual G-protein coupled receptor candidates and candidate G-protein coupled receptor families are conserved across plant species and that, in some cases, this conservation extends to metazoans. Conclusion Our computational and wet-bench results provide the first step toward understanding the diversity, conservation, and functional roles of plant candidate G-protein coupled receptors.