Genetic and Systematic Approaches Toward G Protein-Coupled Abiotic Stress Signaling in Plants

Heterotrimeric G protein, composed of Gα, Gβ, and Gγ subunits, modulates plant adaptations to environmental stresses such as high salinity, drought, extreme temperatures and high light intensity. Most of these evidence were however derived solely from conventional genetics methods with which stress-associated phenotypes were compared between wild type and various G protein mutant plants. Recent advances in systematic approaches, mainly transcriptome and proteome, have contributed to in-depth understanding of molecular linkages between G proteins and environmental changes. Here, we update our knowledge on the roles of G proteins in abiotic stress responses. Furthermore, we highlight the current whole genome studies and integrated omics approach to better understand the fundamental G protein functions involved in abiotic stress responses. It is our purpose here to bridge the gap between molecular mechanisms in G protein science and stress biology and pave the way toward crop improvement researches in the future

Heterotrimeric G Protein–Coupled Signaling in Plants

Investigators studying G protein–coupled signaling—often called the best-understood pathway in the world owing to intense research in medical fields—have adopted plants as a new model to explore the plasticity and evolution of G signaling. Much research on plant G signaling has not disappointed. Although plant cells have most of the core elements found in animal G signaling, differences in network architecture and intrinsic properties of plant G protein elements make G signaling in plant cells distinct from the animal paradigm. In contrast to animal G proteins, plant G proteins are self-activating, and therefore regulation of G activation in plants occurs at the deactivation step. The self-activating property also means that plant G proteins do not need and therefore do not have typical animal G protein–coupled receptors. Targets of activated plant G proteins, also known as effectors, are unlike effectors in animal cells. The simpler repertoire of G signal elements in Arabidopsis makes G signaling easier to manipulate in a multicellular context

Gα modulates salt-induced cellular senescence and cell division in rice and maize

Highlight textThis work reveals two distinct functions of Gα in NaCl stress in rice and maize: attenuation of leaf senescence caused by sodium toxicity in leaves, and cell cycle regulation by osmotic/ionic stress.The plant G-protein network, comprising Gα, Gβ, and Gγ core subunits, regulates development, senses sugar, and mediates biotic and abiotic stress responses. Here, we report G-protein signalling in the salt stress response using two crop models, rice and maize. Loss-of-function mutations in the corresponding genes encoding the Gα subunit attenuate growth inhibition and cellular senescence caused by sodium chloride (NaCl). Gα null mutations conferred reduced leaf senescence, chlorophyll degradation, and cytoplasm electrolyte leakage under NaCl stress. Sodium accumulated in both wild-type and Gα-mutant shoots to the same levels, suggesting that Gα signalling controls cell death in leaves rather than sodium exclusion in roots. Growth inhibition is probably initiated by osmotic change around root cells, because KCl and MgSO4 also suppressed seedling growth equally as well as NaCl. NaCl lowered rates of cell division and elongation in the wild-type leaf sheath to the level of the Gα-null mutants; however there was no NaCl-induced decrease in cell division in the Gα mutant, implying that the osmotic phase of salt stress suppresses cell proliferation through the inhibition of Gα-coupled signalling. These results reveal two distinct functions of Gα in NaCl stress in these grasses: attenuation of leaf senescence caused by sodium toxicity in leaves, and cell cycle regulation by osmotic/ionic stress

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

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

Adaptive Evolution of Signaling Partners

Proteins that interact coevolve their structures. When mutation disrupts the interaction, compensation by the partner occurs to restore interaction otherwise counterselection occurs. We show in this study how a destabilizing mutation in one protein is compensated by a stabilizing mutation in its protein partner and their coevolving path. The pathway in this case and likely a general principle of coevolution is that the compensatory change must tolerate both the original and derived structures with equivalence in function and activity. Evolution of the structure of signaling elements in a network is constrained by specific protein pair interactions, by requisite conformational changes, and by catalytic activity. The heterotrimeric G protein-coupled signaling is a paragon of this protein interaction/function complexity and our deep understanding of this pathway in diverse organisms lends itself to evolutionary study. Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTP hydrolysis rate of the Gα subunit of the heterotrimeric G protein complex. An important RGS-contact site is a hydroxyl-bearing residue on the switch I region of Gα subunits in animals and most plants, such as Arabidopsis. The exception is the grasses (e.g., rice, maize, sugarcane, millets); these plants have Gα subunits that replaced the critical hydroxyl-bearing threonine with a destabilizing asparagine shown to disrupt interaction between Arabidopsis RGS protein (AtRGS1) and the grass Gα subunit. With one known exception (Setaria italica), grasses do not encode RGS genes. One parsimonious deduction is that the RGS gene was lost in the ancestor to the grasses and then recently acquired horizontally in the lineage S. italica from a nongrass monocot. Like all investigated grasses, S. italica has the Gα subunit with the destabilizing asparagine residue in the protein interface but, unlike other known grass genomes, still encodes an expressed RGS gene, SiRGS1. SiRGS1 accelerates GTP hydrolysis at similar concentration of both Gα subunits containing either the stabilizing (AtGPA1) or destabilizing (RGA1) interface residue. SiRGS1 does not use the hydroxyl-bearing residue on Gα to promote GAP activity and has a larger Gα-interface pocket fitting to the destabilizing Gα. These findings indicate that SiRGS1 adapted to a deleterious mutation on Gα using existing polymorphism in the RGS protein population

Batch Bayesian Optimization for Replicable Experimental Design

Many real-world experimental design problems (a) evaluate multiple experimental conditions in parallel and (b) replicate each condition multiple times due to large and heteroscedastic observation noise. Given a fixed total budget, this naturally induces a trade-off between evaluating more unique conditions while replicating each of them fewer times vs. evaluating fewer unique conditions and replicating each more times. Moreover, in these problems, practitioners may be risk-averse and hence prefer an input with both good average performance and small variability. To tackle both challenges, we propose the Batch Thompson Sampling for Replicable Experimental Design (BTS-RED) framework, which encompasses three algorithms. Our BTS-RED-Known and BTS-RED-Unknown algorithms, for, respectively, known and unknown noise variance, choose the number of replications adaptively rather than deterministically such that an input with a larger noise variance is replicated more times. As a result, despite the noise heteroscedasticity, both algorithms enjoy a theoretical guarantee and are asymptotically no-regret. Our Mean-Var-BTS-RED algorithm aims at risk-averse optimization and is also asymptotically no-regret. We also show the effectiveness of our algorithms in two practical real-world applications: precision agriculture and AutoML.Comment: Accepted to NeurIPS 202

Cell-free translation and purification of Arabidopsis thaliana regulator of G signaling 1 protein

Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is a protein with a predicted N-terminal 7-transmembrane (7TM) domain and a C-terminal cytosolic RGS1 box domain. The RGS1 box domain exerts GTPase activation (GAP) activity on Gα (AtGPA1), a component of heterotrimeric G protein signaling in plants. AtRGS1 may perceive an exogenous agonist to regulate the steady-state levels of the active form of AtGPA1. It is uncertain if the full length RGS1 protein exerts any atypical effects on Gα, nor has it been established exactly how AtRGS1 contributes to perception of an extracellular signal and transmits this response to a G-protein dependent signaling cascade. Such an understanding can be deduced from in vitro studies using the purified and soluble RGS1-box domain. Further studies on full-length AtRGS1 have been inhibited due to the extreme low abundance of the endogenous AtRGS1 protein in plants and lack of a suitable heterologous system to express AtRGS1. Here, we describe methods to produce full-length AtRGS1 by cell free synthesis into unilamellar liposomes and nanodiscs. The cell-free synthesized AtRGS1 exhibits GTPase activating activity on Gα and can be purified to a level suitable for biochemical analyses

A nondestructive method to estimate the chlorophyll content of Arabidopsis seedlings

Abstract Background Chlorophyll content decreases in plants under stress conditions, therefore it is used commonly as an indicator of plant health. Arabidopsis thaliana offers a convenient and fast way to test physiological phenotypes of mutations and treatments. However, chlorophyll measurements with conventional solvent extraction are not applicable to Arabidopsis leaves due to their small size, especially when grown on culture dishes. Results We provide a nondestructive method for chlorophyll measurement whereby the red, green and blue (RGB) values of a color leaf image is used to estimate the chlorophyll content from Arabidopsis leaves. The method accommodates different profiles of digital cameras by incorporating the ColorChecker chart to make the digital negative profiles, to adjust the white balance, and to calibrate the exposure rate differences caused by the environment so that this method is applicable in any environment. We chose an exponential function model to estimate chlorophyll content from the RGB values, and fitted the model parameters with physical measurements of chlorophyll contents. As proof of utility, this method was used to estimate chlorophyll content of G protein mutants grown on different sugar to nitrogen ratios. Conclusion This method is a simple, fast, inexpensive, and nondestructive estimation of chlorophyll content of Arabidopsis seedlings. This method lead to the discovery that G proteins are important in sensing the C/N balance to control chlorophyll content in Arabidopsis