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

ABSTRACT: 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 G? (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.

SUBMITTER: Chakraborty N

PROVIDER: S-EPMC6456573 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Project description: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.

Project description:To elucidate the effect of light intensity on the cold response (5°C; 7 days) in Arabidopsis thaliana, we compared the following parameters under standard light (150 ?mol m-2 s-1), low light (20 ?mol m-2 s-1), and dark conditions: membrane damage, photosynthetic parameters, cytokinin oxidase/dehydrogenase (CKX) activity, phytohormone levels, and transcription of selected stress- and hormone-related genes and proteome. The impact of cytokinins (CKs), hormones directly interacting with the light signaling pathway, on cold responses was evaluated using transformants overexpressing CK biosynthetic gene isopentenyl transferase (DEX:IPT) or CK degradation gene HvCKX2 (DEX:CKX) under a dexamethasone-inducible promoter. In wild-type plants, cold treatment under light conditions caused down-regulation of CKs (in shoots) and auxin, while abscisic acid (ABA), jasmonates, and salicylic acid (SA) were up-regulated, especially under low light. Cold treatment in the dark strongly suppressed all phytohormones, except ABA. DEX:IPT plants showed enhanced stress tolerance associated with elevated CK and SA levels in shoots and auxin in apices. Contrarily, DEX:CKX plants had weaker stress tolerance accompanied by lowered levels of CKs and auxins. Nevertheless, cold substantially diminished the impact from the inserted genes. Cold stress in dark minimized differences among the genotypes. Cold treatments in light strongly up-regulated stress marker genes RD29A, especially in roots, and CBF1-3 in shoots. Under control conditions, their levels were higher in DEX:CKX plants, but after 7-day stress, DEX:IPT plants exhibited the highest transcription. Transcription of genes related to CK metabolism and signaling showed a tendency to re-establish, at least partially, CK homeostasis in both transformants. Up-regulation of strigolactone-related genes in apices and leaves indicated their role in suppressing shoot growth. The analysis of leaf proteome revealed over 20,000 peptides, representing 3,800 proteins and 2,212 protein families (data available via ProteomeXchange, identifier PXD020480). Cold stress induced proteins involved in ABA and jasmonate metabolism, antioxidant enzymes, and enzymes of flavonoid and glucosinolate biosynthesis. DEX:IPT plants up-regulated phospholipase D and MAP-kinase 4. Cold stress response at the proteome level was similar in all genotypes under optimal light intensity, differing significantly under low light. The data characterized the decisive effect of light-CK cross-talk in the regulation of cold stress responses.

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

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