Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex

Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1

Growth attenuation under saline stress is mediated by the heterotrimeric G protein complex

BackgroundPlant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity. Due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. It is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. Less is known about pathways connecting NaCl and cell proliferation and cell death. Plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric G-protein complex with glucose. Prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the Arabidopsis heterotrimeric G protein complex (AtRGS1, AtGPA1, AGB1, and AGG), we tested the hypothesis that G proteins modulate plant growth under salt stress.ResultsNa+ activates G signaling as quantitated by internalization of Arabidopsis Regulator of G Signaling protein 1 (AtRGS1). Despite being components of a singular signaling complex loss of the Gβ subunit (agb1-2 mutant) conferred accelerated senescence and aborted development in the presence of Na+, whereas loss of AtRGS1 (rgs1-2 mutant) conferred Na+ tolerance evident as less attenuated shoot growth and senescence. Site-directed changes in the Gα and Gβγ protein-protein interface were made to disrupt the interaction between the Gα and Gβγ subunits in order to elevate free activated Gα subunit and free Gβγ dimer at the plasma membrane. These mutations conferred sodium tolerance. Glucose in the growth media improved the survival under salt stress in Col but not in agb1-2 or rgs1-2 mutants.ConclusionsThese results demonstrate a direct role for G-protein signaling in the plant growth response to salt stress. The contrasting phenotypes of agb1-2 and rgs1-2 mutants suggest that G-proteins balance growth and death under salt stress. The phenotypes of the loss-of-function mutations prompted the model that during salt stress, G activation promotes growth and attenuates senescence probably by releasing ER stress

Cooperative control between AtRGS1 and AtHXK1 in a WD40-repeat protein pathway in Arabidopsis thaliana

HEXOKINASE 1 (AtHXK1) and Regulator of G-protein Signaling 1 (AtRGS1) pathways, mediate D-glucose signaling in Arabidopsis. However, it is not known the degree, if any, that these pathways overlap and how. We show modest signaling crosstalk between these pathways, albeit complex with both epistatic interactions and additive effects that may be indirect. The action of HXK1 on AtRGS1 signaling lies downstream of the primary step in G protein-mediated sugar signaling in which the WD-repeat protein, AGB1, is the propelling signaling element. RHIP1, a previously unknown protein predicted here to have a 3-stranded helical structure, interacts with both AtRGS1 and AtHXK1 in planta and is required for some glucose-regulated gene expression, providing a physical connection between these two proteins in sugar signaling. The rhip1 null mutant displays similar seedling growth phenotypes as rgs1-2 in response to glucose, further suggesting a role for RHIP1 in glucose signaling. In conclusion, glucose signaling is a complex hierarchical relationship which is specific to the target gene and sugar phenotype and suggests that there are two glycolysis-independent glucose signaling sensors: AtRGS1 and AtHXK1 that weakly communicate with each other via feed-back and feed-forward loops to fine tune the response to glucose

Predicted Functional Implications of Phosphorylation of Regulator of G Protein Signaling Protein in Plants

Heterotrimeric G proteins function in development, biotic, and abiotic stress responses, hormone signaling as well as sugar sensing. We previously proposed that discrimination of these various external signals in the G protein pathway is accomplished in plants by membrane-localized receptor-like kinases (RLKs) rather than G-protein-coupled receptors. Arabidopsis thaliana Regulator of G Signaling protein 1 (AtRGS1) modulates G protein activation and is phosphorylated by several RLKs and by WITH-NO-LYSINE kinases (WNKs). Here, a combination of in vitro kinase assays, mass spectrometry, and computational bioinformatics identified and functionally prioritized phosphorylation sites in AtRGS1. Phosphosites for two more RLKs (BRL3 and PEPR1) were identified and added to the AtRGS1 phosphorylation profile. Bioinformatics analyses revealed that RLKs and WNK kinases phosphorylate plant RGS proteins within regions that are conserved across eukaryotes and at a high frequency. Four phospho-sites among 14 identified are proximal to equivalent mammalian phosphosites that impact RGS function, including: pS437 and pT267 in GmRGS2, and pS339 and pS436 in AtRGS1. Based on these analyses, we propose that pS437 and pS436 regulate GmRGS2 and AtRGS1 protein interactions and/or localization, whereas pT267 is important for modulation of GmRGS2 GAP activity and localization. Moreover, pS339 most likely affects AtRGS1 activation

Growth attenuation under saline stress is mediated by the heterotrimeric G protein complex

BackgroundPlant growth is plastic, able to rapidly adjust to fluctuation in environmental conditions such as drought and salinity. Due to long-term irrigation use in agricultural systems, soil salinity is increasing; consequently crop yield is adversely affected. It is known that salt tolerance is a quantitative trait supported by genes affecting ion homeostasis, ion transport, ion compartmentalization and ion selectivity. Less is known about pathways connecting NaCl and cell proliferation and cell death. Plant growth and cell proliferation is, in part, controlled by the concerted activity of the heterotrimeric G-protein complex with glucose. Prompted by the abundance of stress-related, functional annotations of genes encoding proteins that interact with core components of the Arabidopsis heterotrimeric G protein complex (AtRGS1, AtGPA1, AGB1, and AGG), we tested the hypothesis that G proteins modulate plant growth under salt stress.ResultsNa+ activates G signaling as quantitated by internalization of Arabidopsis Regulator of G Signaling protein 1 (AtRGS1). Despite being components of a singular signaling complex loss of the Gβ subunit (agb1-2 mutant) conferred accelerated senescence and aborted development in the presence of Na+, whereas loss of AtRGS1 (rgs1-2 mutant) conferred Na+ tolerance evident as less attenuated shoot growth and senescence. Site-directed changes in the Gα and Gβγ protein-protein interface were made to disrupt the interaction between the Gα and Gβγ subunits in order to elevate free activated Gα subunit and free Gβγ dimer at the plasma membrane. These mutations conferred sodium tolerance. Glucose in the growth media improved the survival under salt stress in Col but not in agb1-2 or rgs1-2 mutants.ConclusionsThese results demonstrate a direct role for G-protein signaling in the plant growth response to salt stress. The contrasting phenotypes of agb1-2 and rgs1-2 mutants suggest that G-proteins balance growth and death under salt stress. The phenotypes of the loss-of-function mutations prompted the model that during salt stress, G activation promotes growth and attenuates senescence probably by releasing ER stress

Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis

Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior

A shadow detector for photosynthesis efficiency

Plants tolerate large variations in the intensity of the light environment by controlling the efficiency of solar to chemical energy conversion. To do this, plants have a mechanism to detect the intensity, duration, and change in light as they experience moving shadows, flickering light, and cloud cover. Sugars are the primary products of CO2 fixation, a metabolic pathway that is rate limited by this solar energy conversion. We propose that sugar is a signal encoding information about the intensity, duration and change in the light environment. We previously showed that the Arabidopsis heterotrimeric G protein complex including its receptor-like Regulator of G signaling protein, AtRGS1, detects both the concentration and the exposure time of sugars [Fu, et al 2014 Cell 156: 1084–1095]. This unique property, designated dose-duration reciprocity, is a behavior that emerges from the system architecture / system motif. Here, we show that another property of the signaling system is to detect large changes in light while at the same time, filtering types of fluctuation in light that do not affect photosynthesis efficiency. When AtRGS1 is genetically ablated, photosynthesis efficiency is reduced in a changing-but not a constant-light environment. Mathematical modeling revealed that information about changes in the light environment is encoded in the amount of free AtRGS1 that becomes compartmentalized following stimulation. We propose that this property determines when to adjust photosynthetic efficiency in an environment where light intensity changes abruptly caused by moving shadows on top of a background of light changing gradually from sun rise to sun set and fluctuating light such as that caused by fluttering leaves

Reciprocal Encoding of Signal Intensity and Duration in a Glucose-Sensing Circuit

Cells continuously adjust their behavior in response to changing environmental conditions. Both intensity and duration of external signals are critical factors in determining what response is initiated. To understand how intracellular signaling networks process such multidimensional information, we studied the AtRGS1-mediated glucose response system of Arabidopsis. By combining experiments with mathematical modeling, we discovered a reciprocal dose and duration response relying on the orchestrated action of three kinases (AtWNK1, AtWNK8, AtWNK10) acting on distinct time scales and activation thresholds. Specifically, we find that high concentrations of D-glucose rapidly signal through AtWNK8 and AtWNK10, whereas low, sustained sugar concentration slowly activate the pathway through AtWNK1, allowing the cells to respond similarly to transient, high-intensity signals, and sustained low-intensity signals. This “dose-duration reciprocity” allows encoding of both the intensity and persistence of glucose as an important energy resource and signaling molecule

A cyclic nucleotide-gated channel (CNGC16) in pollen is critical for stress tolerance in pollen reproductive development

Cyclic nucleotide-gated channels (CNGCs) have been implicated in diverse aspects of plant growth and development, including responses to biotic and abiotic stress, as well as pollen tube growth and fertility. Here, genetic evidence identifies CNGC16 in Arabidopsis (Arabidopsis thaliana) as critical for pollen fertility under conditions of heat stress and drought. Two independent transfer DNA disruptions of cngc16 resulted in a greater than 10-fold stress-dependent reduction in pollen fitness and seed set. This phenotype was fully rescued through pollen expression of a CNGC16 transgene, indicating that cngc16-1 and 16-2 were both loss-of-function null alleles. The most stress-sensitive period for cngc16 pollen was during germination and the initiation of pollen tube tip growth. Pollen viability assays indicate that mutant pollen are also hypersensitive to external calcium chloride, a phenomenon analogous to calcium chloride hypersensitivities observed in other cngc mutants. A heat stress was found to increase concentrations of 3′,5′-cyclic guanyl monophosphate in both pollen and leaves, as detected using an antibody-binding assay. A quantitative PCR analysis indicates that cngc16 mutant pollen have attenuated expression of several heat-stress response genes, including two heat shock transcription factor genes, HsfA2 and HsfB1. Together, these results provide evidence for a heat stress response pathway in pollen that connects a cyclic nucleotide signal, a Ca(2+)-permeable ion channel, and a signaling network that activates a downstream transcriptional heat shock response

Long-distance communication in Arabidopsis involving a self-activating G protein

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