Life, death and resurrection of plant GPCRs.

The activation of G-protein coupled receptors (GPCRs) by extracellular ligands constitutes the first step of heterotrimeric G-protein signalling in animals. In plants, canonical GPCRs have been known for over 25 years, often in association with agronomically important functions. But their role in plant G-protein signalling and even their annotation as GPCR was contested in the last decade, only to be revisited in the light of more recent evidences. In this first ever review on plant GPCRs, we catalogue all the plant GPCRs described to date and discuss the evidences for and against their role in plants in general and G-protein signalling in particular. We argue against writing off GPCRs and point to the missing links to be investigated to establish firm conclusions either way.

Nitrate-responsive transcriptome analysis of rice RGA1 mutant reveals the role of G-protein alpha subunit in negative regulation of nitrogen-sensitivity and use efficiency.

KEY MESSAGE: Nitrate-responsive transcriptomic, phenotypic and physiological analyses of rice RGA1 mutant revealed many novel RGA1-regulated genes/processes/traits related to nitrogen use efficiency, and provided robust genetic evidence of RGA1-regulation of NUE. Nitrogen (N) use efficiency (NUE) is important for sustainable agriculture. G-protein signalling was implicated in N-response/NUE in rice, but needed firm genetic characterization of the role of alpha subunit (RGA1). The knock-out mutant of RGA1 in japonica rice exhibited lesser nitrate-dose sensitivity than the wild type (WT), in yield and NUE. We, therefore, investigated its genomewide nitrate-response relative to WT. It revealed 3416 differentially expressed genes (DEGs), including 719 associated with development, grain yield and phenotypic traits for NUE. The upregulated DEGs were related to photosynthesis, chlorophyll, tetrapyrrole and porphyrin biosynthesis, while the downregulated DEGs belonged to cellular protein metabolism and transport, small GTPase signalling, cell redox homeostasis, etc. We validated 26 nitrate-responsive DEGs across functional categories by RT-qPCR. Physiological validation of nitrate-response in the mutant and the WT at 1.5 and 15 mM doses revealed higher chlorophyll and stomatal length but decreased stomatal density, conductance and transpiration. The consequent increase in photosynthesis and water use efficiency may have contributed to better yield and NUE in the mutant, whereas the WT was N-dose sensitive. The mutant was not as N-dose-responsive as the WT in shoot/root growth, productive tillers and heading date, but equally responsive as WT in total N and protein content. The RGA1 mutant was less impacted by higher N-dose or salt stress in terms of yield, protein content, photosynthetic performance, relative water content, water use efficiency and catalase activity. PPI network analyses revealed known NUE-related proteins as RGA1 interactors. Therefore, RGA1 negatively regulates N-dose sensitivity and NUE in rice.

Nitrate-responsive transcriptome analysis reveals additional genes/processes and associated traits viz. height, tillering, heading date, stomatal density and yield in japonica rice.

MAIN CONCLUSION: Our transcriptomic analysis expanded the repertoire of nitrate-responsive genes/processes in rice and revealed their phenotypic association with root/shoot, stomata, tiller, panicle/flowering and yield, with agronomic implications for nitrogen use efficiency. Nitrogen use efficiency (NUE) is a multigenic quantitative trait, involving many N-responsive genes/processes that are yet to be fully characterized. Microarray analysis of early nitrate response in excised leaves of japonica rice revealed 6688 differentially expressed genes (DEGs), including 2640 hitherto unreported across multiple functional categories. They include transporters, enzymes involved in primary/secondary metabolism, transcription factors (TFs), EF-hand containing calcium binding proteins, hormone metabolism/signaling and methytransferases. Some DEGs belonged to hitherto unreported processes viz. alcohol, lipid and trehalose metabolism, mitochondrial membrane organization, protein targeting and stomatal opening. 1158 DEGs were associated with growth physiology and grain yield or phenotypic traits for NUE. We identified seven DEGs for shoot apical meristem, 66 for leaf/culm/root, 31 for tiller, 70 for heading date/inflorescence/spikelet/panicle, 144 for seed and 78 for yield. RT-qPCR validated nitrate regulation of 31 DEGs belonging to various important functional categories/traits. Physiological validation of N-dose responsive changes in plant development revealed that relative to 1.5 mM, 15 mM nitrate significantly increased stomatal density, stomatal conductance and transpiration rate. Further, root/shoot growth, number of tillers and grain yield declined and panicle emergence/heading date delayed, despite increased photosynthetic rate. We report the binding sites of diverse classes of TFs such as WRKY, MYB, HMG etc., in the 1 kb up-stream regions of 6676 nitrate-responsive DEGs indicating their role in regulating nitrate response/NUE. Together, these findings expand the repertoire of genes and processes involved in genomewide nitrate response in rice and reveal their physiological, phenotypic and agronomic implications for NUE.

G-protein α-subunit (GPA1) regulates stress, nitrate and phosphate response, flavonoid biosynthesis, fruit/seed development and substantially shares GCR1 regulation in A. thaliana.

Heterotrimeric G-proteins are implicated in several plant processes, but the mechanisms of signal-response coupling and the roles of G-protein coupled receptors in general and GCR1 in particular, remain poorly understood. We isolated a knock-out mutant of the Arabidopsis G-protein α subunit (gpa1-5) and analysed its transcriptome to understand the genomewide role of GPA1 and compared it with that of our similar analysis of a GCR1 mutant (Chakraborty et al. 2015, PLoS ONE 10(2):e0117819). We found 394 GPA1-regulated genes spanning 79 biological processes, including biotic and abiotic stresses, development, flavonoid biosynthesis, transcription factors, transporters and nitrate/phosphate responses. Many of them are either unknown or unclaimed explicitly in other published gpa1 mutant transcriptome analyses. A comparison of all known GPA1-regulated genes (including the above 394) with 350 GCR1-regulated genes revealed 114 common genes. This can be best explained by GCR1-GPA1 coupling, or by convergence of their independent signaling pathways. Though the common genes in our GPA1 and GCR1 mutant datasets constitute only 26% of the GPA1-regulated and 30% of the GCR1-responsive genes, they belong to nearly half of all the processes affected in both the mutants. Thus, GCR1 and GPA1 regulate not only some common genes, but also different genes belonging to the same processes to achieve similar outcomes. Overall, we validate some known and report many hitherto unknown roles of GPA1 in plants, including agronomically important ones such as biotic stress and nutrient response, and also provide compelling genetic evidence to revisit the role of GCR1 in G-protein signalling.

Identification of extracellular matrix proteins of rice (Oryza sativa L.) involved in dehydration-responsive network: a proteomic approach.

Water-deficit or dehydration impairs almost all physiological processes and greatly influences the geographical distribution of many crop species. It has been postulated that higher plants rely mostly on induction mechanisms to maintain cellular integrity during stress conditions. Plant cell wall or extracellular matrix (ECM) forms an important conduit for signal transduction between the apoplast and symplast and acts as front-line defense, thereby playing a key role in cell fate decision under various stress conditions. To better understand the molecular mechanism of dehydration response in plants, four-week-old rice seedlings were subjected to progressive dehydration by withdrawing water and the changes in the ECM proteome were examined using two-dimensional gel electrophoresis. Dehydration-responsive temporal changes revealed 192 proteins that change their intensities by more than 2.5-fold, at one or more time points during dehydration. The proteomic analysis led to the identification of about 100 differentially regulated proteins presumably involved in a variety of functions, including carbohydrate metabolism, cell defense and rescue, cell wall modification, cell signaling and molecular chaperones, among others. The differential rice proteome was compared with the dehydration-responsive proteome data of chickpea and maize. The results revealed an evolutionary divergence in the dehydration response as well as organ specificity, with few conserved proteins. The differential expression of the candidate proteins, in conjunction with previously reported results, may provide new insight into the underlying mechanisms of the dehydration response in plants. This may also facilitate the targeted alteration of metabolic routes in the cell wall for agricultural and industrial exploitation.

GCR1 and GPA1 coupling regulates nitrate, cell wall, immunity and light responses in Arabidopsis.

G-protein signaling components have been attributed many biological roles in plants, but the extent of involvement of G-protein coupled receptor 1 (GCR1) with the Gα (GPA1) remained unknown. To address this, we have performed transcriptomic analyses on Arabidopsis gpa1-5gcr1-5 double mutant and identified 656 differentially expressed genes (DEGs). MapMan and Gene Ontology analyses revealed global transcriptional changes associated with external stimulus, cell wall organization/biogenesis and secondary metabolite process among others. Comparative transcriptomic analyses using the single and double mutants of gcr1-5 and gpa1-5 identified 194, 139 and 391 exclusive DEGs respectively, whereas 64 DEGs were common to all three mutants. Further, pair wise comparison of DEGs of double mutant with single mutants of gcr1-5 or gpa1-5 showed about one-third and over half common DEGs, respectively. Further analysis of the DEGs exclusive to the double mutant using protein-protein interaction networks revealed molecular complexes associated with nitrate and light signaling and plant-pathogen interactions among others. Physiological and molecular validation of nitrate-response revealed the sensitivity of germination to low N in the double mutant and differential expression of nitrate transporter (and nitrate reductase in all three mutants). Taken together, GCR1 and GPA1 work in partnership as well as independently to regulate different pathways.

G-protein Signaling Components GCR1 and GPA1 Mediate Responses to Multiple Abiotic Stresses in Arabidopsis.

G-protein signaling components have been implicated in some individual stress responses in Arabidopsis, but have not been comprehensively evaluated at the genetic and biochemical level. Stress emerged as the largest functional category in our whole transcriptome analyses of knock-out mutants of GCR1 and/or GPA1 in Arabidopsis (Chakraborty et al., 2015a,b). This led us to ask whether G-protein signaling components offer converging points in the plant's response to multiple abiotic stresses. In order to test this hypothesis, we carried out detailed analysis of the abiotic stress category in the present study, which revealed 144 differentially expressed genes (DEGs), spanning a wide range of abiotic stresses, including heat, cold, salt, light stress etc. Only 10 of these DEGs are shared by all the three mutants, while the single mutants (GCR1/GPA1) shared more DEGs between themselves than with the double mutant (GCR1-GPA1). RT-qPCR validation of 28 of these genes spanning different stresses revealed identical regulation of the DEGs shared between the mutants. We also validated the effects of cold, heat and salt stresses in all the 3 mutants and WT on % germination, root and shoot length, relative water content, proline content, lipid peroxidation and activities of catalase, ascorbate peroxidase and superoxide dismutase. All the 3 mutants showed evidence of stress tolerance, especially to cold, followed by heat and salt, in terms of all the above parameters. This clearly shows the role of GCR1 and GPA1 in mediating the plant's response to multiple abiotic stresses for the first time, especially cold, heat and salt stresses. This also implies a role for classical G-protein signaling pathways in stress sensitivity in the normal plants of Arabidopsis. This is also the first genetic and biochemical evidence of abiotic stress tolerance rendered by knock-out mutation of GCR1 and/or GPA1. This suggests that G-protein signaling pathway could offer novel common targets for the development of tolerance/resistance to multiple abiotic stresses.

Transcriptome analysis of Arabidopsis GCR1 mutant reveals its roles in stress, hormones, secondary metabolism and phosphate starvation.

The controversy over the existence or the need for G-protein coupled receptors (GPCRs) in plant G-protein signalling has overshadowed a more fundamental quest for the role of AtGCR1, the most studied and often considered the best candidate for GPCR in plants. Our whole transcriptome microarray analysis of the GCR1-knock-out mutant (gcr1-5) in Arabidopsis thaliana revealed 350 differentially expressed genes spanning all chromosomes. Many of them were hitherto unknown in the context of GCR1 or G-protein signalling, such as in phosphate starvation, storage compound and fatty acid biosynthesis, cell fate, etc. We also found some GCR1-responsive genes/processes that are reported to be regulated by heterotrimeric G-proteins, such as biotic and abiotic stress, hormone response and secondary metabolism. Thus, GCR1 could have G-protein-mediated as well as independent roles and regardless of whether it works as a GPCR, further analysis of the organism-wide role of GCR1 has a significance of its own.