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.

Weighted gene co-expression network analysis of nitrogen (N)-responsive genes and the putative role of G-quadruplexes in N use efficiency (NUE) in rice.

Rice is an important target to improve crop nitrogen (N) use efficiency (NUE), and the identification and shortlisting of the candidate genes are still in progress. We analyzed data from 16 published N-responsive transcriptomes/microarrays to identify, eight datasets that contained the maximum number of 3020 common genes, referred to as N-responsive genes. These include different classes of transcription factors, transporters, miRNA targets, kinases and events of post-translational modifications. A Weighted gene co-expression network analysis (WGCNA) with all the 3020 N-responsive genes revealed 15 co-expression modules and their annotated biological roles. Protein-protein interaction network analysis of the main module revealed the hub genes and their functional annotation revealed their involvement in the ubiquitin process. Further, the occurrences of G-quadruplex sequences were examined, which are known to play important roles in epigenetic regulation but are hitherto unknown in N-response/NUE. Out of the 3020 N-responsive genes studied, 2298 contained G-quadruplex sequences. We compared these N-responsive genes containing G-quadruplex sequences with the 3601 genes we previously identified as NUE-related (for being both N-responsive and yield-associated). This analysis revealed 389 (17%) NUE-related genes containing G-quadruplex sequences. These genes may be involved in the epigenetic regulation of NUE, while the rest of the 83% (1811) genes may regulate NUE through genetic mechanisms and/or other epigenetic means besides G-quadruplexes. A few potentially important genes/processes identified as associated with NUE were experimentally validated in a pair of rice genotypes contrasting for NUE. The results from the WGCNA and G4 sequence analysis of N-responsive genes helped identify and shortlist six genes as candidates to improve NUE. Further, the hitherto unavailable segregation of genetic and epigenetic gene targets could aid in informed interventions through genetic and epigenetic means of crop improvement.

Genome-Wide Urea Response in Rice Genotypes Contrasting for Nitrogen Use Efficiency.

Rice is an ideal crop for improvement of nitrogen use efficiency (NUE), especially with urea, its predominant fertilizer. There is a paucity of studies on rice genotypes contrasting for NUE. We compared low urea-responsive transcriptomes of contrasting rice genotypes, namely Nidhi (low NUE) and Panvel1 (high NUE). Transcriptomes of whole plants grown with media containing normal (15 mM) and low urea (1.5 mM) revealed 1497 and 2819 differentially expressed genes (DEGs) in Nidhi and Panvel1, respectively, of which 271 were common. Though 1226 DEGs were genotype-specific in Nidhi and 2548 in Panvel1, there was far higher commonality in underlying processes. High NUE is associated with the urea-responsive regulation of other nutrient transporters, miRNAs, transcription factors (TFs) and better photosynthesis, water use efficiency and post-translational modifications. Many of their genes co-localized to NUE-QTLs on chromosomes 1, 3 and 9. A field evaluation under different doses of urea revealed better agronomic performance including grain yield, transport/uptake efficiencies and NUE of Panvel1. Comparison of our urea-based transcriptomes with our previous nitrate-based transcriptomes revealed many common processes despite large differences in their expression profiles. Our model proposes that differential involvement of transporters and TFs, among others, contributes to better urea uptake, translocation, utilization, flower development and yield for high NUE.

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.

Comparative Transcriptomic Analyses of Nitrate-Response in Rice Genotypes With Contrasting Nitrogen Use Efficiency Reveals Common and Genotype-Specific Processes, Molecular Targets and Nitrogen Use Efficiency-Candidates.

The genetic basis for nitrogen (N)-response and N use efficiency (NUE) must be found in N-responsive gene expression or protein regulation. Our transcriptomic analysis of nitrate response in two contrasting rice genotypes of Oryza sativa ssp. Indica (Nidhi with low NUE and Panvel1 with high NUE) revealed the processes/functions underlying differential N-response/NUE. The microarray analysis of low nitrate response (1.5 mM) relative to normal nitrate control (15 mM) used potted 21-days old whole plants. It revealed 1,327 differentially expressed genes (DEGs) exclusive to Nidhi and 666 exclusive to Panvel1, apart from 70 common DEGs, of which 10 were either oppositely expressed or regulated to different extents. Gene ontology analyses revealed that photosynthetic processes were among the very few processes common to both the genotypes in low N response. Those unique to Nidhi include cell division, nitrogen utilization, cytoskeleton, etc. in low N-response, whereas those unique to Panvel1 include signal transduction, protein import into the nucleus, and mitochondria. This trend of a few common but mostly unique categories was also true for transporters, transcription factors, microRNAs, and post-translational modifications, indicating their differential involvement in Nidhi and Panvel1. Protein-protein interaction networks constructed using DEG-associated experimentally validated interactors revealed subnetworks involved in cytoskeleton organization, cell wall, etc. in Nidhi, whereas in Panvel1, it was chloroplast development. NUE genes were identified by selecting yield-related genes from N-responsive DEGs and their co-localization on NUE-QTLs revealed the differential distribution of NUE-genes between genotypes but on the same chromosomes 1 and 3. Such hotspots are important for NUE breeders.

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.

Meta-Analysis of Yield-Related and N-Responsive Genes Reveals Chromosomal Hotspots, Key Processes and Candidate Genes for Nitrogen-Use Efficiency in Rice.

Nitrogen-use efficiency (NUE) is a function of N-response and yield that is controlled by many genes and phenotypic parameters that are poorly characterized. This study compiled all known yield-related genes in rice and mined them from the N-responsive microarray data to find 1,064 NUE-related genes. Many of them are novel genes hitherto unreported as related to NUE, including 80 transporters, 235 transcription factors (TFs), 44 MicroRNAs (miRNAs), 91 kinases, and 8 phosphatases. They were further shortlisted to 62 NUE-candidate genes following hierarchical methods, including quantitative trait locus (QTL) co-localization, functional evaluation in the literature, and protein-protein interactions (PPIs). They were localized to chromosomes 1, 3, 5, and 9, of which chromosome 1 with 26 genes emerged as a hotspot for NUE spanning 81% of the chromosomes. Further, co-localization of the NUE genes on NUE-QTLs resolved differences in the earlier studies that relied mainly on N-responsive genes regardless of their role in yield. Functional annotations and PPIs for all the 1,064 NUE-related genes and also the shortlisted 62 candidates revealed transcription, redox, phosphorylation, transport, development, metabolism, photosynthesis, water deprivation, and hormonal and stomatal function among the prominent processes. In silico expression analysis confirmed differential expression of the 62 NUE-candidate genes in a tissue/stage-specific manner. Experimental validation in two contrasting genotypes revealed that high NUE rice shows better photosynthetic performance, transpiration efficiency and internal water-use efficiency in comparison to low NUE rice. Feature Selection Analysis independently identified one-third of the common genes at every stage of hierarchical shortlisting, offering 6 priority targets to validate for improving the crop NUE.

Heterotrimeric G-protein α subunit (RGA1) regulates tiller development, yield, cell wall, nitrogen response and biotic stress in rice.

G-proteins are implicated in plant productivity, but their genome-wide roles in regulating agronomically important traits remain uncharacterized. Transcriptomic analyses of rice G-protein alpha subunit mutant (rga1) revealed 2270 differentially expressed genes (DEGs) including those involved in C/N and lipid metabolism, cell wall, hormones and stress. Many DEGs were associated with root, leaf, culm, inflorescence, panicle, grain yield and heading date. The mutant performed better in total weight of filled grains, ratio of filled to unfilled grains and tillers per plant. Protein-protein interaction (PPI) network analysis using experimentally validated interactors revealed many RGA1-responsive genes involved in tiller development. qPCR validated the differential expression of genes involved in strigolactone-mediated tiller formation and grain development. Further, the mutant growth and biomass were unaffected by submergence indicating its role in submergence response. Transcription factor network analysis revealed the importance of RGA1 in nitrogen signaling with DEGs such as Nin-like, WRKY, NAC, bHLH families, nitrite reductase, glutamine synthetase, OsCIPK23 and urea transporter. Sub-clustering of DEGs-associated PPI network revealed that RGA1 regulates metabolism, stress and gene regulation among others. Predicted rice G-protein networks mapped DEGs and revealed potential effectors. Thus, this study expands the roles of RGA1 to agronomically important traits and reveals their underlying processes.

Nitrogen futures in the shared socioeconomic pathways 4.

Humanity's transformation of the nitrogen cycle has major consequences for ecosystems, climate and human health, making it one of the key environmental issues of our time. Understanding how trends could evolve over the course of the 21st century is crucial for scientists and decision-makers from local to global scales. Scenario analysis is the primary tool for doing so, and has been applied across all major environmental issues, including nitrogen pollution. However, to date most scenario efforts addressing nitrogen flows have either taken a narrow approach, focusing on a singular impact or sector, or have not been integrated within a broader scenario framework - a missed opportunity given the multiple environmental and socio-economic impacts that nitrogen pollution exacerbates. Capitalizing on our expanding knowledge of nitrogen flows, this study introduces a framework for new nitrogen-focused narratives based on the widely used Shared Socioeconomic Pathways that include all the major nitrogen-polluting sectors (agriculture, industry, transport and wastewater). These new narratives are the first to integrate the influence of climate and other environmental pollution control policies, while also incorporating explicit nitrogen-control measures. The next step is for them to be used as model inputs to evaluate the impact of different nitrogen production, consumption and loss trajectories, and thus advance understanding of how to address environmental impacts while simultaneously meeting key development goals. This effort is an important step in assessing how humanity can return to the planetary boundary of this essential element over the coming century.

Transcriptomic and network analyses reveal distinct nitrate responses in light and dark in rice leaves (Oryza sativa Indica var. Panvel1).

Nitrate (N) response is modulated by light, but not understood from a genome-wide perspective. Comparative transcriptomic analyses of nitrate response in light-grown and etiolated rice leaves revealed 303 and 249 differentially expressed genes (DEGs) respectively. A majority of them were exclusive to light (270) or dark (216) condition, whereas 33 DEGs were common. The latter may constitute response to N signaling regardless of light. Functional annotation and pathway enrichment analyses of the DEGs showed that nitrate primarily modulates conserved N signaling and metabolism in light, whereas oxidation-reduction processes, pentose-phosphate shunt, starch-, sucrose- and glycerolipid-metabolisms in the dark. Differential N-regulation of these pathways by light could be attributed to the involvement of distinctive sets of transporters, transcription factors, enriched cis-acting motifs in the promoters of DEGs as well as differential modulation of N-responsive transcriptional regulatory networks in light and dark. Sub-clustering of DEGs-associated protein-protein interaction network constructed using experimentally validated interactors revealed that nitrate regulates a molecular complex consisting of nitrite reductase, ferredoxin-NADP reductase and ferredoxin. This complex is associated with flowering time, revealing a meeting point for N-regulation of N-response and N-use efficiency. Together, our results provide novel insights into distinct pathways of N-signaling in light and dark conditions.

Nitrogen Use Efficiency Phenotype and Associated Genes: Roles of Germination, Flowering, Root/Shoot Length and Biomass.

Crop improvement for Nitrogen Use Efficiency (NUE) requires a well-defined phenotype and genotype, especially for different N-forms. As N-supply enhances growth, we comprehensively evaluated 25 commonly measured phenotypic parameters for N response using 4 N treatments in six indica rice genotypes. For this, 32 replicate potted plants were grown in the green-house on nutrient-depleted sand. They were fertilized to saturation with media containing either nitrate or urea as the sole N source at normal (15 mM N) or low level (1.5 mM N). The variation in N-response among genotypes differed by N form/dose and increased developmentally from vegetative to reproductive parameters. This indicates survival adaptation by reinforcing variation in every generation. Principal component analysis segregated vegetative parameters from reproduction and germination. Analysis of variance revealed that relative to low level, normal N facilitated germination, flowering and vegetative growth but limited yield and NUE. Network analysis for the most connected parameters, their correlation with yield and NUE, ranking by Feature selection and validation by Partial least square discriminant analysis enabled shortlisting of eight parameters for NUE phenotype. It constitutes germination and flowering, shoot/root length and biomass parameters, six of which were common to nitrate and urea. Field-validation confirmed the NUE differences between two genotypes chosen phenotypically. The correspondence between multiple approaches in shortlisting parameters for NUE makes it a novel and robust phenotyping methodology of relevance to other plants, nutrients or other complex traits. Thirty-Four N-responsive genes associated with the phenotype have also been identified for genotypic characterization of NUE.

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.

Phenotyping for Nitrogen Use Efficiency: Rice Genotypes Differ in N-Responsive Germination, Oxygen Consumption, Seed Urease Activities, Root Growth, Crop Duration, and Yield at Low N.

The biological improvement of fertilizer nitrogen use efficiency (NUE) is hampered by the poor characterization of the phenotype and genotype for crop N response and NUE. In an attempt to identify phenotypic traits for N-response and NUE in the earliest stages of plant growth, we analyzed the N-responsive germination, respiration, urease activities, and root/shoot growth of 21 Indica genotypes of rice (Oryza sativa var. indica). We found that N delays germination from 0 to 12 h in a genotype-dependent and source-dependent manner, especially with urea and nitrate. We identified contrasting groups of fast germinating genotypes such as Aditya, Nidhi, and Swarnadhan, which were also least delayed by N and slow germinating genotypes such as Panvel 1, Triguna, and Vikramarya, which were also most delayed by N. Oxygen uptake measurements in the seeds of contrasting genotypes revealed that they were affected by N source in accordance with germination rates, especially with urea. Germinating seeds were found to have endogenous urease activity, indicating the need to explore genotypic differences in the effective urea uptake and metabolism, which remain unexplored so far. Urea was found to significantly inhibit early root growth in all genotypes but not shoot growth. Field evaluation of 15 of the above genotypes clearly showed that germination rates, crop duration, and yield are linked to NUE. Slow germinating genotypes had longer crop duration and higher yield even at lower N, indicating their higher NUE, relative to fast germinating or short duration genotypes. Moreover, longer duration genotypes suffered lesser yield losses at reduced N levels as compared to short duration genotypes, which is also a measure of their NUE. Together, these results indicate the potential of germination rates, crop duration, urea utilization and its effect on root growth in the development of novel phenotypic traits for screening genotypes and crop improvement for NUE, at least in rice.

Microarray Analysis of Rice d1 (RGA1) Mutant Reveals the Potential Role of G-Protein Alpha Subunit in Regulating Multiple Abiotic Stresses Such as Drought, Salinity, Heat, and Cold.

The genome-wide role of heterotrimeric G-proteins in abiotic stress response in rice has not been examined from a functional genomics perspective, despite the availability of mutants and evidences involving individual genes/processes/stresses. Our rice whole transcriptome microarray analysis (GSE 20925 at NCBI GEO) using the G-alpha subunit (RGA1) null mutant (Daikoku 1 or d1) and its corresponding wild type (Oryza sativa Japonica Nipponbare) identified 2270 unique differentially expressed genes (DEGs). Out of them, we mined for all the potentially abiotic stress-responsive genes using Gene Ontology terms, STIFDB2.0 and Rice DB. The first two approaches produced smaller subsets of the 1886 genes found at Rice DB. The GO approach revealed similar regulation of several families of stress-responsive genes in RGA1 mutant. The Genevestigator analysis of the stress-responsive subset of the RGA1-regulated genes from STIFDB revealed cold and drought-responsive clusters. Meta data analysis at Rice DB revealed large stress-response categories such as cold (878 up/810 down), drought (882 up/837 down), heat (913 up/777 down), and salt stress (889 up/841 down). One thousand four hundred ninety-eight of them are common to all the four abiotic stresses, followed by fewer genes common to smaller groups of stresses. The RGA1-regulated genes that uniquely respond to individual stresses include 111 in heat stress, eight each in cold only and drought only stresses, and two genes in salt stress only. The common DEGs (1498) belong to pathways such as the synthesis of polyamine, glycine-betaine, proline, and trehalose. Some of the common DEGs belong to abiotic stress signaling pathways such as calcium-dependent pathway, ABA independent and dependent pathway, and MAP kinase pathway in the RGA1 mutant. Gene ontology of the common stress responsive DEGs revealed 62 unique molecular functions such as transporters, enzyme regulators, transferases, hydrolases, carbon and protein metabolism, binding to nucleotides, carbohydrates, receptors and lipids, morphogenesis, flower development, and cell homeostasis. We also mined 63 miRNAs that bind to the stress responsive transcripts identified in this study, indicating their post-transcriptional regulation. Overall, these results indicate the potentially extensive role of RGA1 in the regulation of multiple abiotic stresses in rice for further validation.

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.

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.

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.

Molecular characterization of nitrate uptake and assimilatory pathway in Arthrospira platensis reveals nitrate induction and differential regulation.

The nitrate assimilation pathway and its regulation in the high-protein neutraceutical cyanobacterium, Arthrospira (Spirulina), were studied. A complete characterization of the genes of the nitrate uptake and assimilatory pathway in Arthrospira platensis strain PCC 7345 was done including cloning, sequencing, phylogenetic analysis and expression studies. Genomic localization studies revealed that their clustering is different from the operons known in other cyanobacteria; only nrtP and narB are organized together, while nirA, glnA and gltS exist in separate genomic locations. The presence of both types of nitrate transporters (nrtP/ABC types) in A. platensis is rare, as their occurrence is usually specific to marine and freshwater microorganisms, respectively. The positive effect of nitrate on transcript accumulation of narB, nirA and nrtP genes in N-depleted and N-restored cultures confirmed nitrate induction, which is abolished by the addition of ammonium ions into the medium. Gene expression studies in response to nitrate, nitrite, ammonium and glutamine provided the first evidence of differential regulation of multiple genes of nitrate assimilatory pathway in Arthrospira.