RRFT1 (Redox Responsive Transcription Factor 1) is involved in extracellular ATP-regulated gene expression in Arabidopsis thaliana seedlings.
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ABSTRACT:
SUBMITTER: Dong X
PROVIDER: S-EPMC7238875 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Extracellular ATP (eATP) has been reported to be involved in plant growth as a primary messenger in the apoplast. Here, roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP, showing a marked avoidance response. Roots responded similarly to ADP and bz-ATP but did not respond to AMP and GTP. The eATP avoidance response was reduced in loss-of-function mutants of heterotrimeric G protein ? subunit (G?) (gpa1-1 and gpa1-2) and enhanced in G?-over-expression (OE) lines (wG? and cG?). Ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) and Gd3+ remarkably suppressed eATP-induced root bending. ATP-stimulated Ca2+ influx was impaired in G? null mutants and increased in its OE lines. DR5-GFP and PIN2 were asymmetrically distributed in ATP-stimulated root tips, this effect was strongly suppressed by EGTA and diminished in G? null mutants. In addition, some eATP-induced genes' expression was also impaired in G? null mutants. Based on these results, we propose that heterotrimeric G?-regulated Ca2+ influx and PIN2 distribution may be key signaling events in eATP sensing and avoidance response in Arabidopsis thaliana roots.
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Project description:?-Amylases are glucan hydrolases that cleave ?-1,4-glucosidic bonds in starch. In vascular plants, ?-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for ?-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp(666)), identified as the catalytic nucleophile in other plant ?-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a ?-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and ?-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant ?-amylases, with a pH optimum of 7.5-8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how ?-amylase activity may be regulated in the chloroplast.
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Project description:Environmental stress leads to dramatic transcriptional reprogramming, which is central to plant survival. Although substantial knowledge has accumulated on how a few plant cis-regulatory elements (CREs) function in stress regulation, many more CREs remain to be discovered. In addition, the plant stress cis-regulatory code, i.e., how CREs work independently and/or in concert to specify stress-responsive transcription, is mostly unknown. On the basis of gene expression patterns under multiple stresses, we identified a large number of putative CREs (pCREs) in Arabidopsis thaliana with characteristics of authentic cis-elements. Surprisingly, biotic and abiotic responses are mostly mediated by two distinct pCRE superfamilies. In addition, we uncovered cis-regulatory codes specifying how pCRE presence and absence, combinatorial relationships, location, and copy number can be used to predict stress-responsive expression. Expression prediction models based on pCRE combinations perform significantly better than those based on simply pCRE presence and absence, location, and copy number. Furthermore, instead of a few master combinatorial rules for each stress condition, many rules were discovered, and each appears to control only a small subset of stress-responsive genes. Given there are very few documented interactions between plant CREs, the combinatorial rules we have uncovered significantly contribute to a better understanding of the cis-regulatory logic underlying plant stress response and provide prioritized targets for experimentation.
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Project description:As an apoplastic signal, extracellular ATP (eATP) is involved in plant growth and development. eATP promotes tobacco pollen germination (PG) and pollen tube growth (PTG) by stimulating Ca2+ or K+ absorption. Nevertheless, the mechanisms underlying eATP-stimulated ion uptake and their role in PG and PTG are still unclear. Here, ATP addition was found to modulate PG and PTG in 34 plant species and showed a promoting effect in most of these species. Furthermore, by using Arabidopsis thaliana as a model, the role of several signaling components involved in eATP-promoted ion (Ca2+, K+) uptake, PG, and PTG were investigated. ATP stimulated while apyrase inhibited PG and PTG. Patch-clamping results showed that ATP promoted K+ and Ca2+ influx into pollen protoplasts. In loss-of-function mutants of P2K1 (dorn1-1 and dorn1-3), heterotrimeric G protein α subunit (gpa1-1, gpa1-2), or cyclic nucleotide gated ion channel (cngc2, cngc4), eATP-stimulated PG, PTG, and ion influx were all impaired. Our results suggest that these signaling components may be involved in eATP-promoted PG and PTG by regulating Ca2+ or K+ influx in Arabidopsis pollen grains.
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Project description:Damage can be signalled by extracellular ATP (eATP) using plasma membrane (PM) receptors to effect cytosolic free calcium ion ([Ca2+ ]cyt ) increase as a second messenger. The downstream PM Ca2+ channels remain enigmatic. Here, the Arabidopsis thaliana Ca2+ channel subunit CYCLIC NUCLEOTIDE-GATED CHANNEL2 (CNGC2) was identified as a critical component linking eATP receptors to downstream [Ca2+ ]cyt signalling in roots. Extracellular ATP-induced changes in single epidermal cell PM voltage and conductance were measured electrophysiologically, changes in root [Ca2+ ]cyt were measured with aequorin, and root transcriptional changes were determined by quantitative real-time PCR. Two cngc2 loss-of-function mutants were used: cngc2-3 and defence not death1 (which expresses cytosolic aequorin). Extracellular ATP-induced transient depolarization of Arabidopsis root elongation zone epidermal PM voltage was Ca2+ dependent, requiring CNGC2 but not CNGC4 (its channel co-subunit in immunity signalling). Activation of PM Ca2+ influx currents also required CNGC2. The eATP-induced [Ca2+ ]cyt increase and transcriptional response in cngc2 roots were significantly impaired. CYCLIC NUCLEOTIDE-GATED CHANNEL2 is required for eATP-induced epidermal Ca2+ influx, causing depolarization leading to [Ca2+ ]cyt increase and damage-related transcriptional response.
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Project description:Glutathione transferase enzymes help plants to cope with biotic and abiotic stress. They mainly catalyze the conjugation of glutathione (GSH) onto xenobiotics, and some act as glutathione peroxidase. With X-ray crystallography, kinetics, and thermodynamics, we studied the impact of oxidation on Arabidopsis thaliana glutathione transferase Phi 9 (GSTF9). GSTF9 has no cysteine in its sequence, and it adopts a universal GST structural fold characterized by a typical conserved GSH-binding site (G-site) and a hydrophobic co-substrate-binding site (H-site). At elevated H2 O2 concentrations, methionine sulfur oxidation decreases its transferase activity. This oxidation increases the flexibility of the H-site loop, which is reflected in lower activities for hydrophobic substrates. Determination of the transition state thermodynamic parameters shows that upon oxidation an increased enthalpic penalty is counterbalanced by a more favorable entropic contribution. All in all, to guarantee functionality under oxidative stress conditions, GSTF9 employs a thermodynamic and structural compensatory mechanism and becomes substrate of methionine sulfoxide reductases, making it a redox-regulated enzyme.
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Project description:One of the most regulated steps of translation initiation is the recruitment of mRNA by the translation machinery. In eukaryotes, this step is mediated by the 5'end cap-binding factor eIF4E bound to the bridge protein eIF4G and forming the eIF4F complex. In plants, different isoforms of eIF4E and eIF4G form the antigenically distinct eIF4F and eIF(iso)4F complexes proposed to mediate selective translation. Using a microarray analysis of polyribosome- and non-polyribosome-purified mRNAs from 15 day-old Arabidopsis thaliana wild type [WT] and eIF(iso)4E knockout mutant [(iso)4E-1] seedlings we found 79 transcripts shifted from polyribosomes toward non-polyribosomes, and 47 mRNAs with the opposite behavior in the knockout mutant. The translationally decreased mRNAs were overrepresented in root-preferentially expressed genes and proteins from the endomembrane system, including several transporters such as the phosphate transporter PHOSPHATE1 (PHO1), Sucrose transporter 3 (SUC3), ABC transporter-like with ATPase activity (MRP11) and five electron transporters, as well as signal transduction-, protein modification- and transcription-related proteins. Under normal growth conditions, eIF(iso)4E expression under the constitutive promoter 35 S enhanced the polyribosomal recruitment of PHO1 supporting its translational preference for eIF(iso)4E. Furthermore, under phosphate deficiency, the PHO1 protein increased in the eIF(iso)4E overexpressing plants and decreased in the knockout mutant as compared to wild type. In addition, the knockout mutant had larger root, whereas the 35 S directed expression of eIF(iso)4E caused shorter root under normal growth conditions, but not under phosphate deficiency. These results indicate that selective translation mediated by eIF(iso)4E is relevant for Arabidopsis root development under normal growth conditions.
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Project description:We report here that ORS1, a previously uncharacterized member of the NAC transcription factor family, controls leaf senescence in Arabidopsis thaliana. Overexpression of ORS1 accelerates senescence in transgenic plants, whereas its inhibition delays it. Genes acting downstream of ORS1 were identified by global expression analysis using transgenic plants producing dexamethasone-inducible ORS1-GR fusion protein. Of the 42 up-regulated genes, 30 (~70%) were previously shown to be up-regulated during age-dependent senescence. We also observed that 32 (~76%) of the ORS1-dependent genes were induced by long-term (4 d), but not short-term (6 h) salinity stress (150 mM NaCl). Furthermore, expression of 16 and 24 genes, respectively, was induced after 1 and 5 h of treatment with hydrogen peroxide (H₂O₂), a reactive oxygen species known to accumulate during salinity stress. ORS1 itself was found to be rapidly and strongly induced by H₂O₂ treatment in both leaves and roots. Using in vitro binding site selection, we determined the preferred binding motif of ORS1 and found it to be present in half of the ORS1-dependent genes. ORS1 is a paralog of ORE1/ANAC092/AtNAC2, a previously reported regulator of leaf senescence. Phylogenetic footprinting revealed evolutionary conservation of the ORS1 and ORE1 promoter sequences in different Brassicaceae species, indicating strong positive selection acting on both genes. We conclude that ORS1, similarly to ORE1, triggers expression of senescence-associated genes through a regulatory network that may involve cross-talk with salt- and H₂O₂-dependent signaling pathways.
| S-EPMC3063519 | biostudies-literature
Project description:BACKGROUND:Long non-coding RNAs (lncRNAs) have emerged as new class of regulatory molecules in animals where they regulate gene expression at transcriptional and post-transcriptional level. Recent studies also identified lncRNAs in plant genomes, revealing a new level of transcriptional complexity in plants. Thousands of lncRNAs have been predicted in the Arabidopsis thaliana genome, but only a few have been studied in depth. RESULTS:Here we report the identification of Arabidopsis lncRNAs that are expressed during the vegetative stage of development in either the shoot apical meristem or in leaves. We found that hundreds of lncRNAs are expressed in these tissues, of which 50 show differential expression upon an increase in ambient temperature. One of these lncRNAs, FLINC, is down-regulated at higher ambient temperature and affects ambient temperature-mediated flowering in Arabidopsis. CONCLUSION:A number of ambient temperature responsive lncRNAs were identified with potential roles in the regulation of temperature-dependent developmental changes, such as the transition from the vegetative to the reproductive (flowering) phase. The challenge for the future is to characterize the biological function and molecular mode of action of the large number of ambient temperature-regulated lncRNAs that have been identified in this study.
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Project description:Clubroot disease of Brassicaceae is caused by an obligate biotrophic protist, Plasmodiophora brassicae. During root gall development, a strong sink for assimilates is developed. Among other genes involved in sucrose and starch synthesis and degradation, the increased expression of invertases has been observed in a microarray experiment, and invertase and invertase inhibitor expression was confirmed using promoter::GUS lines of Arabidopsis thaliana. A functional approach demonstrates that invertases are important for gall development. Different transgenic lines expressing an invertase inhibitor under the control of two root-specific promoters, Pyk10 and CrypticT80, which results in the reduction of invertase activity, showed clearly reduced clubroot symptoms in root tissue with highest promoter expression, whereas hypocotyl galls developed normally. These results present the first evidence that invertases are important factors during gall development, most probably in supplying sugars to the pathogen. In addition, root-specific repression of invertase activity could be used as a tool to reduce clubroot symptoms.
| S-EPMC6640435 | biostudies-literature