Oryza CLIMtools: A genome-environment association resource reveals adaptive roles for heterotrimeric G proteins in the regulation of rice agronomic traits.

Modern crop varieties display a degree of mismatch between their current distributions and the suitability of the local climate for their productivity. To address this issue, we present Oryza CLIMtools (https://gramene.org/CLIMtools/oryza_v1.0/), the first resource for pan-genome prediction of climate-associated genetic variants in a crop species. Oryza CLIMtools consists of interactive web-based databases that enable the user to (1) explore the local environments of traditional rice varieties (landraces) in South-East Asia and (2) investigate the environment by genome associations for 658 Indica and 283 Japonica rice landrace accessions collected from georeferenced local environments and included in the 3K Rice Genomes Project. We demonstrate the value of these resources by identifying an interplay between flowering time and temperature in the local environment that is facilitated by adaptive natural variation in OsHD2 and disrupted by a natural variant in OsSOC1. Prior quantitative trait locus analysis has suggested the importance of heterotrimeric G proteins in the control of agronomic traits. Accordingly, we analyzed the climate associations of natural variants in the different heterotrimeric G protein subunits. We identified a coordinated role of G proteins in adaptation to the prevailing potential evapotranspiration gradient and revealed their regulation of key agronomic traits, including plant height and seed and panicle length. We conclude by highlighting the prospect of targeting heterotrimeric G proteins to produce climate-resilient crops.

Oryza CLIMtools: A Genome-Environment Association Resource Reveals Adaptive Roles for Heterotrimeric G Proteins in the Regulation of Rice Agronomic Traits.

Modern crop varieties display a degree of mismatch between their current distributions and the suitability of the local climate for their productivity. To this end, we present Oryza CLIMtools (https://gramene.org/CLIMtools/oryza_v1.0/), the first resource for pan-genome prediction of climate-associated genetic variants in a crop species. Oryza CLIMtools consists of interactive web-based databases that allow the user to: i) explore the local environments of traditional rice varieties (landraces) in South-Eastern Asia, and; ii) investigate the environment by genome associations for 658 Indica and 283 Japonica rice landrace accessions collected from georeferenced local environments and included in the 3K Rice Genomes Project. We exemplify the value of these resources, identifying an interplay between flowering time and temperature in the local environment that is facilitated by adaptive natural variation in OsHD2 and disrupted by a natural variant in OsSOC1. Prior QTL analysis has suggested the importance of heterotrimeric G proteins in the control of agronomic traits. Accordingly, we analyzed the climate associations of natural variants in the different heterotrimeric G protein subunits. We identified a coordinated role of G proteins in adaptation to the prevailing Potential Evapotranspiration gradient and their regulation of key agronomic traits including plant height and seed and panicle length. We conclude by highlighting the prospect of targeting heterotrimeric G proteins to produce crops that are climate resilient.

Influence of expression and purification protocols on Gα biochemical activity: kinetics of plant and mammalian G protein cycles.

Heterotrimeric G proteins, composed of Gα, Gß, 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. Human GNAI1 purified using our approach also displayed the expected binding and hydrolysis activities, indicating our protocol is applicable to mammalian Gα subunits, potentially including those for which purification of enzymatically active protein has been historically problematic. We subsequently 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 partially uncouple the instability from the rapid nucleotide binding kinetics displayed by GPA1.

A comparison of surgical site infections following total hip replacement and total knee replacement surgeries identified by Infection Prevention and Control and the National Surgical Quality Improvement Program in Alberta, Canada.

OBJECTIVE: To understand how the different data collections methods of the Alberta Health Services Infection Prevention and Control Program (IPC) and the National Surgical Quality Improvement Program (NSQIP) are affecting reported rates of surgical site infections (SSIs) following total hip replacements (THRs) and total knee replacements (TKRs). DESIGN: Retrospective cohort study. SETTING: Four hospitals in Alberta, Canada. PATIENTS: Those with THR or TKR surgeries between September 1, 2015, and March 31, 2018. METHODS: Demographic information, complex SSIs reported by IPC and NSQIP were compared and then IPC and NSQIP data were matched with percent agreement and Cohen's κ calculated. Statistical analysis was performed for age, gender and complex SSIs. A P value <.05 was considered significant. RESULTS: In total, 7,549 IPC and 2,037 NSQIP patients were compared. The complex SSI rate for NSQIP was higher compared to IPC (THR: 1.19 vs 0.68 [P = .147]; TKR: 0.92 vs 0.80 [P = .682]). After matching, 7 SSIs were identified by both IPC and NSQIP; 3 were identified only by IPC, and 12 were identified only by NSQIP (positive agreement, 0.48; negative agreement, 1.0; κ = 0.48). CONCLUSIONS: Different approaches to monitor SSIs may lead to different results and trending patterns. NSQIP reports total SSI rates that are consistently higher than IPC. If systems are compared at any point in time, confidence on the data may be eroded. Stakeholders need to be aware of these variations and education provided to facilitate an understanding of differences and a consistent approach to SSI surveillance monitoring over time.

A G protein-coupled receptor-like module regulates cellulose synthase secretion from the endomembrane system in Arabidopsis.

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 Gßγ 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 Gßγ 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.

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions.

The extra-large guanosine-5'-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gßγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Metabolomics of red-light-induced stomatal opening in Arabidopsis thaliana: Coupling with abscisic acid and jasmonic acid metabolism.

Environmental stimuli-triggered stomatal movement is a key physiological process that regulates CO2 uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red-light-induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red-light-stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red-light-modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red-light-modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.

Nucleotide exchange-dependent and nucleotide exchange-independent functions of plant heterotrimeric GTP-binding proteins.

Heterotrimeric guanine nucleotide-binding proteins (G proteins), which are composed of α, ß, and γ subunits, are versatile, guanine nucleotide-dependent, molecular on-off switches. In animals and fungi, the exchange of GDP for GTP on Gα controls G protein activation and is crucial for normal cellular responses to diverse extracellular signals. The model plant Arabidopsis thaliana has a single canonical Gα subunit, AtGPA1. We found that, in planta, the constitutively active, GTP-bound AtGPA1(Q222L) mutant and the nucleotide-free AtGPA1(S52C) mutant interacted with Gßγ1 and Gßγ2 dimers with similar affinities, suggesting that G protein heterotrimer formation occurred independently of nucleotide exchange. In contrast, AtGPA1(Q222L) had a greater affinity than that of AtGPA1(S52C) for Gßγ3, suggesting that the GTP-bound conformation of AtGPA1(Q222L) is distinct and tightly associated with Gßγ3. Functional analysis of transgenic lines expressing either AtGPA1(S52C) or AtGPA1(Q222L) in the gpa1-null mutant background revealed various mutant phenotypes that were complemented by either AtGPA1(S52C) or AtGPA1(Q222L). We conclude that, in addition to the canonical GDP-GTP exchange-dependent mechanism, plant G proteins can function independently of nucleotide exchange.

G protein subunit phosphorylation as a regulatory mechanism in heterotrimeric G protein signaling in mammals, yeast, and plants.

Heterotrimeric G proteins composed of Gα, Gß, and Gγ subunits are vital eukaryotic signaling elements that convey information from ligand-regulated G protein-coupled receptors (GPCRs) to cellular effectors. Heterotrimeric G protein-based signaling pathways are fundamental to human health [Biochimica et Biophysica Acta (2007) 1768, 994-1005] and are the target of >30% of pharmaceuticals in clinical use [Biotechnology Advances (2013) 31, 1676-1694; Nature Reviews Drug Discovery (2017) 16, 829-842]. This review focuses on phosphorylation of G protein subunits as a regulatory mechanism in mammals, budding yeast, and plants. This is a re-emerging field, as evidence for phosphoregulation of mammalian G protein subunits from biochemical studies in the early 1990s can now be complemented with contemporary phosphoproteomics and genetic approaches applied to a diversity of model systems. In addition, new evidence implicates a family of plant kinases, the receptor-like kinases, which are monophyletic with the interleukin-1 receptor-associated kinase/Pelle kinases of metazoans, as possible GPCRs that signal via subunit phosphorylation. We describe early and modern observations on G protein subunit phosphorylation and its functional consequences in these three classes of organisms, and suggest future research directions.

A kinase-dead version of FERONIA receptor-like kinase has dose-dependent impacts on rosette morphology and RALF1-mediated stomatal movements.

The receptor-like kinase FERONIA (FER) pleiotropically affects plant reproduction, development, and stress tolerance. We recently showed that the FER ligand RALF1 promotes stomatal closure and inhibits stomatal opening in a G-protein-dependent manner. FER responses have been designated as kinase-dependent or kinase-independent, based largely on fer complementation assays employing a kinase-dead FERK565R. Our quantification of FERK565R-GFP transcript and FERK565R-GFP protein in fer complementation lines reveal that, even within individual complementation lines, different levels of FERK565R expression prevail. FERK565R-GFP expression comparable to that of FER in Col-0 plants fail to elicit complementation of either fer rosette phenotypes or RALF1-elicited stomatal movements, whereas overexpression levels of FERK565R-GFP result in complementation. These results suggest possible alternative interpretations of previous conclusions regarding kinase-independent FER signaling.

The G Protein ß-Subunit, AGB1, Interacts with FERONIA in RALF1-Regulated Stomatal Movement.

Heterotrimeric guanine nucleotide-binding (G) proteins are composed of Gα, Gß, and Gγ subunits and function as molecular switches in signal transduction. In Arabidopsis (Arabidopsis thaliana), there are one canonical Gα (GPA1), three extra-large Gα (XLG1, XLG2, and XLG3), one Gß (AGB1), and three Gγ (AGG1, AGG2, and AGG3) subunits. To elucidate AGB1 molecular signaling, we performed immunoprecipitation using plasma membrane-enriched proteins followed by mass spectrometry to identify the protein interactors of AGB1. After eliminating proteins present in the control immunoprecipitation, commonly identified contaminants, and organellar proteins, a total of 103 candidate AGB1-associated proteins were confidently identified. We identified all of the G protein subunits except XLG1, receptor-like kinases, Ca2+ signaling-related proteins, and 14-3-3-like proteins, all of which may couple with or modulate G protein signaling. We confirmed physical interaction between AGB1 and the receptor-like kinase FERONIA (FER) using bimolecular fluorescence complementation. The Rapid Alkalinization Factor (RALF) family of polypeptides have been shown to be ligands of FER. In this study, we demonstrate that RALF1 regulates stomatal apertures and does so in a G protein-dependent manner, inhibiting stomatal opening and promoting stomatal closure in Columbia but not in agb1 mutants. We further show that AGGs and XLGs, but not GPA1, participate in RALF1-mediated stomatal signaling. Our results suggest that FER acts as a G protein-coupled receptor for plant heterotrimeric G proteins.

Heterotrimeric G proteins interact with defense-related receptor-like kinases in Arabidopsis.

Heterotrimeric G proteins (G-proteins) are versatile signaling elements conserved in Eukaryotes. In animals G-proteins relay signals from 7-transmembrane spanning G protein-coupled receptors (GPCRs) to intracellular downstream effectors; however, the existence of GPCRs in plants is controversial. Contrastingly, a surplus of receptor-like kinases (RLKs) provides signal recognition at the plant cell surface. It is established that G proteins are involved in plant defense and suggested that they relay signals from defense-related RLKs. However, it is unclear how the signaling is conducted, as physical interaction between the RLKs and G proteins has not been demonstrated. Using yeast split-ubiquitin system and Bimolecular Fluorescence Complementation assays, we demonstrate physical interaction between the Gα, Gγ1 and Gγ2 subunits, and the defense-related RD-type receptor like kinases CERK1, BAK1 and BIR1. At the same time, no interaction was detected with the non-RD RLK FLS2. We hypothesize that G-proteins mediate signal transduction immediately downstream of the pathogenesis-related RLKs.

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling.

Heterotrimeric G proteins, consisting of Gα, Gß, 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 Gßγ 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 Gßγ binding in yeast. We observed interaction of the XLGs with all three Gßγ 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-Gßγ 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.

Evidence for an unusual transmembrane configuration of AGG3, a class C Gγ subunit of Arabidopsis.

Heterotrimeric G proteins are crucial for the perception of external signals and subsequent signal transduction in animal and plant cells. In both model systems, the complex comprises one Gα, one Gß, and one Gγ subunit. However, in addition to the canonical Gγ subunits (class A), plants also possess two unusual, plant-specific classes of Gγ subunits (classes B and C) that have not yet been found in animals. These include Gγ subunits lacking the C-terminal CaaX motif (class B), which is important for membrane anchoring of the protein; the presence of such subunits gives rise to a flexible sub-population of Gß/γ heterodimers that are not necessarily restricted to the plasma membrane. Plants also contain class C Gγ subunits, which are twice the size of canonical Gγ subunits, with a predicted transmembrane domain and a large cysteine-rich extracellular C-terminus. However, neither the presence of the transmembrane domain nor the membrane topology have been unequivocally demonstrated. Here, we provide compelling evidence that AGG3, a class C Gγ subunit of Arabidopsis, contains a functional transmembrane domain, which is sufficient but not essential for plasma membrane localization, and that the cysteine-rich C-terminus is extracellular.

Fusarium oxysporum infection assays in Arabidopsis.

Increased susceptibility to Fusarium oxysporum is one of the most conspicuous characteristics of the Arabidopsis mutants lacking the heterotrimeric G protein ß and γ1 subunits. The molecular mechanisms placing these G proteins in the plant innate immunity network are yet to be discovered. However, a method to test susceptibility to and disease progression of an important plant pathogen, such as F. oxysporum, is of central importance to many plant defense studies. The optimized protocol presented here allows the routine processing and analysis of symptom progression in young Arabidopsis soil-grown seedlings and yields highly reproducible results.

Signaling specificity provided by the Arabidopsis thaliana heterotrimeric G-protein γ subunits AGG1 and AGG2 is partially but not exclusively provided through transcriptional regulation.

The heterotrimeric G-protein complex in Arabidopsis thaliana consists of one α, one ß and three γ subunits. While two of the γ subunits, AGG1 and AGG2 have been shown to provide functional selectivity to the Gßγ dimer in Arabidopsis, it is unclear if such selectivity is embedded in their molecular structures or conferred by the different expression patterns observed in both subunits. In order to study the molecular basis for such selectivity we tested genetic complementation of AGG1- and AGG2 driven by the respectively swapped gene promoters. When expressed in the same tissues as AGG1, AGG2 rescues some agg1 mutant phenotypes such as the hypersensitivity to Fusarium oxysporum and D-mannitol as well as the altered levels of lateral roots, but does not rescue the early flowering phenotype. Similarly, AGG1 when expressed in the same tissues as AGG2 rescues the osmotic stress and lateral-root phenotypes observed in agg2 mutants but failed to rescue the heat-stress induction of flowering. The fact that AGG1 and AGG2 are functionally interchangeable in some pathways implies that, at least for those pathways, signaling specificity resides in the distinctive spatiotemporal expression patterns exhibited by each γ subunit. On the other hand, the lack of complementation for some phenotypes indicates that there are pathways in which signaling specificity is provided by differences in the primary AGG1 and AGG2 amino acid sequences.

Diversity of heterotrimeric G-protein γ subunits in plants.

BACKGROUND: Heterotrimeric G-proteins, consisting of three subunits Gα, Gß and Gγ are present in most eukaryotes and mediate signaling in numerous biological processes. In plants, Gγ subunits were shown to provide functional selectivity to G-proteins. Three unconventional Gγ subunits were recently reported in Arabidopsis, rice and soybean but no structural analysis has been reported so far. Their relationship with conventional Gγ subunits and taxonomical distribution has not been yet demonstrated. RESULTS: After an extensive similarity search through plant genomes, transcriptomes and proteomes we assembled over 200 non-redundant proteins related to the known Gγ subunits. Structural analysis of these sequences revealed that most of them lack the obligatory C-terminal prenylation motif (CaaX). According to their C-terminal structures we classified the plant Gγ subunits into three distinct types. Type A consists of Gγ subunits with a putative prenylation motif. Type B subunits lack a prenylation motif and do not have any cysteine residues in the C-terminal region, while type C subunits contain an extended C-terminal domain highly enriched with cysteines. Comparative analysis of C-terminal domains of the proteins, intron-exon arrangement of the corresponding genes and phylogenetic studies suggested a common origin of all plant Gγ subunits. CONCLUSION: Phylogenetic analyses suggest that types C and B most probably originated independently from type A ancestors. We speculate on a potential mechanism used by those Gγ subunits lacking isoprenylation motifs to anchor the Gßγ dimer to the plasma membrane and propose a new flexible nomenclature for plant Gγ subunits. Finally, in the light of our new classification, we give a word of caution about the interpretation of Gγ research in Arabidopsis and its generalization to other plant species.

Gγ1+Gγ2+Gγ3=Gß: the search for heterotrimeric G-protein γ subunits in Arabidopsis is over.

In Arabidopsis, heterotrimeric G-proteins consist of one Gα (GPA1), one Gß (AGB1) and three Gγ (AGG1, AGG2 and AGG3) subunits. Gß and Gγ subunits function as obligate heterodimers, therefore any phenotypes observed in Gß-deficient mutants should be apparent in Gγ-deficient mutants. Nevertheless, the first two Gγ subunits discovered failed to explain many of the phenotypes shown by the agb1 mutants in Arabidopsis, prompting the search for additional Gγ subunits. The recent discovery of an additional, although quite atypical, Gγ subunit in Arabidopsis (AGG3) has helped to complete the picture and explains almost all of the missing agb1 'orphan' phenotypes. There is nevertheless still one unexplained phenotype, the reduction in rosette size reported for agb1, that has not been observed in any of the individual agg mutants or the double agg1agg2 mutant. We have now created a triple gamma mutant (agg1agg2agg3) in Arabidopsis and show that it recapitulates the remaining 'orphan'agb1 phenotypes. Triple agg1agg2agg3 mutants show the reduction in rosette size previously observed in agb1 mutants. In addition we show that small differences in flower and silique size observed between agb1 and agg3 mutants are also accounted for by the triple agg1agg2agg3 mutant. Our results strongly suggest that there are no additional members of the G-protein family remaining to be discovered in Arabidopsis.