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Francisco Javier Cejudo - One of the best experts on this subject based on the ideXlab platform.
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NtrC Plays a Crucial Role in Starch Metabolism, Redox Balance, and Tomato Fruit Growth.
Plant physiology, 2019Co-Authors: Liang-yu Hou, Francisco Javier Cejudo, Matthias Ehrlich, Ina Thormählen, Martin Lehmann, Ina Krahnert, Toshihiro Obata, Alisdair R. Fernie, Peter GeigenbergerAbstract:NADPH-thioredoxin reductase C (NtrC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NtrC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NtrC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NtrC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NtrC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NtrC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NtrC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NtrC regulates the osmotic balance of developing fruits. These results indicate that NtrC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.
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Insights into the function of NADPH thioredoxin reductase C (NtrC) based on identification of NtrC-interacting proteins in vivo
Journal of experimental botany, 2019Co-Authors: Maricruz González, Víctor Delgado-requerey, Julia Ferrández, Antonio Serna, Francisco Javier CejudoAbstract:Redox regulation in heterotrophic organisms relies on NADPH, thioredoxins (TRXs), and an NADPH-dependent TRX reductase (NTR). In contrast, chloroplasts harbor two redox systems, one that uses photoreduced ferredoxin (Fd), an Fd-dependent TRX reductase (FTR), and TRXs, which links redox regulation to light, and NtrC, which allows the use of NADPH for redox regulation. It has been shown that NtrC-dependent regulation of 2-Cys peroxiredoxin (PRX) is critical for optimal function of the photosynthetic apparatus. Thus, the objective of the present study was the analysis of the interaction of NtrC and 2-Cys PRX in vivo and the identification of proteins interacting with them with the aim of identifying chloroplast processes regulated by this redox system. To assess this objective, we generated Arabidopsis thaliana plants expressing either an NtrC-tandem affinity purification (TAP)-Tag or a green fluorescent protein (GFP)-TAP-Tag, which served as a negative control. The presence of 2-Cys PRX and NtrC in complexes isolated from NtrC-TAP-Tag-expressing plants confirmed the interaction of these proteins in vivo. The identification of proteins co-purified in these complexes by MS revealed the relevance of the NtrC-2-Cys PRX system in the redox regulation of multiple chloroplast processes. The interaction of NtrC with selected targets was confirmed in vivo by bimolecular fluorescence complementation (BiFC) assays.
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NtrC-dependent redox balance of 2-Cys peroxiredoxins is needed for optimal function of the photosynthetic apparatus
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Juan Manuel Pérez-ruiz, Belén Naranjo, Valle Ojeda, Manuel Guinea, Francisco Javier CejudoAbstract:Thiol-dependent redox regulation allows the rapid adaptation of chloroplast function to unpredictable changes in light intensity. Traditionally, it has been considered that chloroplast redox regulation relies on photosynthetically reduced ferredoxin (Fd), thioredoxins (Trxs), and an Fd-dependent Trx reductase (FTR), the Fd-FTR-Trxs system, which links redox regulation to light. More recently, a plastid-localized NADPH-dependent Trx reductase (NTR) with a joint Trx domain, termed NtrC, was identified. NtrC efficiently reduces 2-Cys peroxiredoxins (Prxs), thus having antioxidant function, but also participates in redox regulation of metabolic pathways previously established to be regulated by Trxs. Thus, the NtrC, 2-Cys Prxs, and Fd-FTR-Trxs redox systems may act concertedly, but the nature of the relationship between them is unknown. Here we show that decreased levels of 2-Cys Prxs suppress the phenotype of the Arabidopsis thaliana NtrC KO mutant. The excess of oxidized 2-Cys Prxs in NtrC-deficient plants drains reducing power from chloroplast Trxs, which results in low efficiency of light energy utilization and impaired redox regulation of Calvin-Benson cycle enzymes. Moreover, the dramatic phenotype of the NtrC-trxf1f2 triple mutant, lacking NtrC and f-type Trxs, was also suppressed by decreased 2-Cys Prxs contents, as the NtrC-trxf1f2-Δ2cp mutant partially recovered the efficiency of light energy utilization and exhibited WT rate of CO2 fixation and growth phenotype. The suppressor phenotype was not caused by compensatory effects of additional chloroplast antioxidant systems. It is proposed that the Fd-FTR-Trx and NtrC redox systems are linked by the redox balance of 2-Cys Prxs, which is crucial for chloroplast function.
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An event of alternative splicing affects the expression of the NtrC gene, encoding NADPH-thioredoxin reductase C, in seed plants
Plant science : an international journal of experimental plant biology, 2017Co-Authors: Victoria A. Nájera, Juan Manuel Pérez-ruiz, María De La Cruz González, Francisco Javier CejudoAbstract:The NtrC gene encodes a NADPH-dependent thioredoxin reductase with a joint thioredoxin domain, exclusive of photosynthetic organisms. An updated search shows that although most species harbor a single copy of the NtrC gene, two copies were identified in different species of the genus Solanum, Glycine max and the moss Physcomitrella patens. The phylogenetic analysis of NtrCs from different sources produced a tree with the major groups of photosynthetic organisms: cyanobacteria, algae and land plants, indicating the evolutionary success of the NtrC gene among photosynthetic eukaryotes. An event of alternative splicing affecting the expression of the NtrC gene was identified, which is conserved in seed plants but not in algae, bryophytes and lycophytes. The alternative splicing event results in a transcript with premature stop codon, which would produce a truncated form of the enzyme. The standard splicing/alternative splicing (SS/AS) transcripts ratio was higher in photosynthetic tissues from Arabidopsis, Brachypodium and tomato, in line with the higher content of the NtrC polypeptide in these tissues. Moreover, environmental stresses such as cold or high salt affected the SS/AS ratio of the NtrC gene transcripts in Brachypodium seedlings. These results suggest that the alternative splicing of the NtrC gene might be an additional mechanism for modulating the content of NtrC in photosynthetic and non-photosynthetic tissues of seed plants.
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The chloroplast NADPH thioredoxin reductase C, NtrC, controls non-photochemical quenching of light energy and photosynthetic electron transport in Arabidopsis.
Plant Cell and Environment, 2016Co-Authors: Belén Naranjo, Francisco Javier Cejudo, Anja Krieger-liszkay, Clara Mignée, Dámaso Hornero-méndez, Lourdes Gallardo-guerrero, Marika LindahlAbstract:High irradiances may lead to photooxidative stress in plants, and non-photochemical quenching (NPQ) contributes to protection against excess excitation. One of the NPQ mechanisms, qE, involves thermal dissipation of the light energy captured. Importantly, plants need to tune down qE under light-limiting conditions for efficient utilization of the available quanta. Considering the possible redox control of responses to excess light implying enzymes, such as thioredoxins, we have studied the role of the NADPH thioredoxin reductase C (NtrC). Whereas Arabidopsis thaliana plants lacking NtrC tolerate high light intensities, these plants display drastically elevated qE, have larger trans-thylakoid ΔpH and have 10-fold higher zeaxanthin levels under low and medium light intensities, leading to extremely low linear electron transport rates. To test the impact of the high qE on plant growth, we generated an NtrC-psbs double-knockout mutant, which is devoid of qE. This double mutant grows faster than the NtrC mutant and has a higher chlorophyll content. The photosystem II activity is partially restored in the NtrC-psbs mutant, and linear electron transport rates under low and medium light intensities are twice as high as compared with plants lacking NtrC alone. These data uncover a new role for NtrC in the control of photosynthetic yield.
Robert G. Kranz - One of the best experts on this subject based on the ideXlab platform.
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Urea Utilization in the Phototrophic Bacterium Rhodobacter capsulatus Is Regulated by the Transcriptional Activator NtrC
Journal of bacteriology, 2001Co-Authors: Bernd Masepohl, Robert G. Kranz, Björn Kaiser, Nazila Isakovic, Cynthia L. Richard, Werner KlippAbstract:The phototrophic nonsulfur purple bacterium Rhodobacter capsulatus can use urea as a sole source of nitrogen. Three transposon Tn5-induced mutations (Xan-9, Xan-10, and Xan-19), which led to a Ure− phenotype, were mapped to the ureF and ureC genes, whereas two other Tn5 insertions (Xan-20 and Xan-22) were located within the NtrC and ntrB genes, respectively. As in Klebsiella aerogenes and other bacteria, the genes encoding urease (ureABC) and the genes required for assembly of the nickel metallocenter (ureD and ureEFG) are clustered in R. capsulatus (ureDABC-orf136-ureEFG). No homologues of Orf136 were found in the databases, and mutational analysis demonstrated that orf136 is not essential for urease activity or growth on urea. Analysis of a ureDA-lacZ fusion showed that maximum expression of the ure genes occurred under nitrogen-limiting conditions (e.g., serine or urea as the sole nitrogen source), but ure gene expression was not substrate (urea) inducible. Expression of the ure genes was strictly dependent on NtrC, whereas ς54 was not essential for urease activity. Expression of the ure genes was lower (by a factor of 3.5) in the presence of ammonium than under nitrogen-limiting conditions, but significant transcription was also observed in the presence of ammonium, approximately 10-fold higher than in an NtrC mutant background. Thus, ure gene expression in the presence of ammonium also requires NtrC. Footprint analyses demonstrated binding of NtrC to tandem binding sites upstream of the ureD promoter. Phosphorylation of NtrC increased DNA binding by at least eightfold. Although urea is effectively used as a nitrogen source in an NtrC-dependent manner, nitrogenase activity was not repressed by urea.
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Translational activation by an NtrC enhancer-binding protein
Journal of molecular biology, 1998Co-Authors: Paul J. Cullen, William C. Bowman, Dawn M Foster Hartnett, Sean C. Reilly, Robert G. KranzAbstract:The Rhodobacter capsulatus NtrC protein is a bacterial enhancer-binding protein that activates the transcription of at least five genes by a mechanism that does not require the RpoN RNA polymerase sigma factor. The nifR3-ntrB-NtrC operon in R. capsulatus codes for the nitrogen-sensing two component regulators NtrB and NtrC, as well as for NifR3, a protein of unknown function that is highly conserved in both prokaryotes and eukaryotes. Evidence of a unique translational control of NifR3 mediated directly by the NtrC enhancer-binding protein is reported. The nifR3-ntrB-NtrC operon is expressed from a single promoter upstream of nifR3 with the levels of transcript equivalent in wild-type and NtrC mutants under nitrogen-limited or nitrogen-sufficient conditions. LacZ reporter analyses of this operon and immunological quantitation of NifR3 and NtrC demonstrate that, unlike NtrC levels which remain constant, production of NifR3 is at least ten to 40-fold reduced in NtrC- strains. NifR3 is increased at least fivefold upon nitrogen limitation whereas NtrC production is constitutive. Surprisingly, the purified NtrC protein binds cooperatively to the nifR3 promoter region in vitro at two sets of tandem binding sites centered at +1 and -81 nucleotides relative to the transcriptional start site. Deletion analysis demonstrates that the upstream tandem sites are essential for nitrogen and NtrC-dependent production of NifR3 in vivo , but are not necessary for nifR3 transcription. These experiments indicate that NtrC stimulates the translation of the NifR3 messenger RNA while tethered to the promoter DNA. This is in contrast to five other promoters (nifA1, nifA2, glnB, mopA and anfA) in R. capsulatus which are transcriptionally activated by NtrC bound to one set of tandem binding sites that are centered greater than 100 bp upstream of the transcriptional start site.
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In Vitro Reconstitution and Characterization of the Rhodobacter capsulatus NtrB and NtrC Two-component System
The Journal of biological chemistry, 1996Co-Authors: Paul J. Cullen, William C. Bowman, Robert G. KranzAbstract:Abstract Enhancer-dependent transcription in enteric bacteria depends upon an activator protein that binds DNA far upstream from the promoter and an alternative factor () that binds with the core RNA polymerase at the promoter. In the photosynthetic bacterium Rhodobacter capsulatus, the NtrB and NtrC proteins (RcNtrB and RcNtrC) are putative members of a two-component system that is novel because the enhancer-binding RcNtrC protein activates transcription of -independent promoters. To reconstitute this putative two-component system in vitro, the RcNtrB protein was overexpressed in Escherichia coli and purified as a maltose-binding protein fusion (MBP-RcNtrB). MBP-RcNtrB autophosphorylates in vitro to the same steady state level and with the same stability as the Salmonella typhimurium NtrB (StNtrB) protein but at a lower initial rate. MBP-RcNtrBP phosphorylates the S.typhimurium NtrC (StNtrC) and RcNtrC proteins in vitro. The enteric NtrC protein is also phosphorylated in vivo by RcNtrB because plasmids that encode either RcNtrB or MBP-RcNtrB activate transcription of an NtrC-dependent nifL-lacZ fusion. The rate of phosphotransfer to RcNtrC and autophosphatase activity of phosphorylated RcNtrC (RcNtrCP) are comparable to the StNtrC protein. However, the RcNtrC protein appears to be a specific RcNtrBP phosphatase since RcNtrC is not phosphorylated by small molecular weight phosphate compounds or by the StNtrB protein. RcNtrC forms a dimer in solution, and RcNtrCP binds the upstream tandem binding sites of the glnB promoter 4-fold better than the unphosphorylated RcNtrC protein, presumably due to oligomerization of RcNtrCP. Therefore, the R. capsulatus NtrB and NtrC proteins form a two-component system similar to other NtrC-like systems, where specific RcNtrB phosphotransfer to the RcNtrC protein results in increased oligomerization at the enhancer but with subsequent activation of a -independent promoter.
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The Rhodobacter capsulatus glnB gene is regulated by NtrC at tandem rpoN-independent promoters.
Journal of bacteriology, 1994Co-Authors: D. Foster-hartnett, Robert G. KranzAbstract:Abstract The protein encoded by glnB of Rhodobacter capsulatus is part of a nitrogen-sensing cascade which regulates the expression of nitrogen fixation genes (nif). The expression of glnB was studied by using lacZ fusions, primer extension analysis, and in vitro DNase I footprinting. Our results suggest that glnB is transcribed from two promoters, one of which requires the R. capsulatus NtrC gene but is rpoN independent. Another promoter upstream of glnB is repressed by NtrC; purified R. capsulatus NtrC binds to sites that overlap this distal promoter region.
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Sequence, genetic, and lacZ fusion analyses of a nifR3–ntrB–NtrC operon in Rhodobacter capsulatus
Molecular Microbiology, 1993Co-Authors: D. Foster-hartnett, Karen K. Gabbert, Paul J. Cullen, Robert G. KranzAbstract:Summary Transcription of Rhodobacter capsulatus genes encoding the nitrogenase polypeptides (nifHDK) is repressed by fixed nitrogen and oxygen. Regulatory genes required to sense and relay the nitrogen status of the cell are gInB, ntrB (nifR2), and NtrC (nifR1). R. capsulatus nifA1 and nifA2 require NtrC for activation when fixed nitrogen is limiting. The polypeptides encoded by nifA1 and nif42 along with the alternate Sigma factor RpoN activate nifHDK and the remaining nif genes in the absence of both fixed nitrogen and oxygen. In this study we report the sequence and genetic analysis of the previously Identified nifR3–ntrB–NtrC regulatory locus. nifR3 is predicted to encode a 324-amino-acid protein with significant homology to an upstream open reading frame cotranscribed with the Escherichia coli fregulatory gene, fis. Analysis of NtrC–lacZ fusions and complementation data indicate that nifR3ntrBC constitute a single operon. nifR3–lacZfusions are expressed only when lacZ is in the proper reading frame with the predicted nifR3 gene product. Tn5, a kanamycin-resistance cassette, and miniMu insertions in nifR3 are polar on nfrBC (required for nif transcription). This gene organization suggests that the nifR3gene product may be involved in nitrogen regulation, although nifR3 is not stringently required for nitrogen fixation when ntrBC are present on a multicopy plasmid. In addition, a R. capsulatus strain with a 22-nucleotide insert in the chromosomal nifR3 gene was constructed. This nifR3 strain is able to fix nitrogen and activate nifA1 and nifA2 genes, again supporting the hypothesis that nifR3 is not stringently required for nfrC-dependent gene activation in R. capsulatus.
Sydney Kustu - One of the best experts on this subject based on the ideXlab platform.
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Mechanism of Transcriptional Activation by NtrC
Two-Component Signal Transduction, 2014Co-Authors: Susan C. Porter, Anne K. North, Sydney KustuAbstract:In the case of nitrogen regulatory protein C (NtrC), both nucleotide hydrolysis and transcriptional activation depend on phosphorylation of an aspartate residue in the N-terminal receiver domain of the protein (also called its regulatory domain). In this chapter, the authors review the evidence that the NtrC protein from enteric bacteria, which is a dimer in solution, must form an appropriate oligomer to hydrolyze nucleotide and activate transcription. Because phosphorylation of the N-terminal receiver domain of NtrC is known to increase oligomerization, effects of phosphorylation on NtrC function may be a consequence of effects on oligomerization. Recent evidence from the laboratory indicates that oligomerization determinants of NtrC are located in its central activation domain. Studies of NtrC function have been facilitated by the use of two sorts of tools: mutant forms of NtrC and derivatives of the glnA enhancer. NtrC must be phosphorylated in its receiver domain to hydrolyze ATP and activate transcription. Phosphorylation stimulates the oligomerization of NtrC, and oligomerization is, in turn, required for ATP hydrolysis and transcriptional activation. Because the phosphorylated receiver domain of NtrC functions positively, it is presumably needed for appropriate oligomerization: removing this domain by proteolysis or genetic engineering does not substitute for phosphorylation. However, the unphosphorylated protein is essentially incapable of ATP hydrolysis or transcriptional activation, even when bound to the enhancer.
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nitrogen regulatory protein c controlled genes of escherichia coli scavenging as a defense against nitrogen limitation
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Daniel P Zimmer, Eric Soupene, Haidy Lee, Volker F Wendisch, Arkady B Khodursky, Brian J Peter, Robert A Bender, Sydney KustuAbstract:Abstract Nitrogen regulatory protein C (NtrC) of enteric bacteria activates transcription of genes/operons whose products minimize the slowing of growth under nitrogen-limiting conditions. To reveal the NtrC regulon of Escherichia coli we compared mRNA levels in a mutant strain that overexpresses NtrC-activated genes [glnL(Up)] to those in a strain with an NtrC (glnG) null allele by using DNA microarrays. Both strains could be grown under conditions of nitrogen excess. Thus, we could avoid differences in gene expression caused by slow growth or nitrogen limitation per se. Rearranging the spot images from microarrays in genome order allowed us to detect all of the operons known to be under NtrC control and facilitated detection of a number of new ones. Many of these operons encode transport systems for nitrogen-containing compounds, including compounds recycled during cell-wall synthesis, and hence scavenging appears to be a primary response to nitrogen limitation. In all, ≈2% of the E. coli genome appears to be under NtrC control, although transcription of some operons depends on the nitrogen assimilation control protein, which serves as an adapter between NtrC and σ70-dependent promoters.
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Phosphorylation-induced signal propagation in the response regulator NtrC.
Journal of bacteriology, 2000Co-Authors: Jonghui Lee, Jeffrey T. Owens, Ingyu Hwang, Claude F. Meares, Sydney KustuAbstract:The bacterial enhancer-binding protein NtrC is a well-studied response regulator in a two-component regulatory system. The amino (N)-terminal receiver domain of NtrC modulates the function of its adjacent output domain, which activates transcription by the ς54 holoenzyme. When a specific aspartate residue in the receiver domain of NtrC is phosphorylated, the dimeric protein forms an oligomer that is capable of ATP hydrolysis and transcriptional activation. A chemical protein cleavage method was used to investigate signal propagation from the phosphorylated receiver domain of NtrC, which acts positively, to its central output domain. The iron chelate reagent Fe-BABE was conjugated onto unique cysteines introduced into the N-terminal domain of NtrC, and the conjugated proteins were subjected to Fe-dependent cleavage with or without prior phosphorylation. Phosphorylation-dependent cleavage, which requires proximity and an appropriate orientation of the peptide backbone to the tethered Fe-EDTA, was particularly prominent with conjugated NtrCD86C, in which the unique cysteine lies near the top of α-helix 4. Cleavage occurred outside the receiver domain itself and on the partner subunit of the derivatized monomer in an NtrC dimer. The results are commensurate with the hypothesis that α-helix 4 of the phosphorylated receiver domain of NtrC interacts with the beginning of the central domain for signal propagation. They imply that the phosphorylation-dependent interdomain and intermolecular interactions between the receiver domain of one subunit and the output domain of its partner subunit in an NtrC dimer precede—and may give rise to—the oligomerization needed for transcriptional activation.
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Unusual Oligomerization Required for Activity of NtrC, a Bacterial Enhancer-Binding Protein
Science (New York N.Y.), 1997Co-Authors: Claire Wyman, Anne K. North, Irene Rombel, Carlos Bustamante, Sydney KustuAbstract:Nitrogen regulatory protein C (NtrC) contacts a bacterial RNA polymerase from distant enhancers by means of DNA loops and activates transcription by allowing polymerase to gain access to the template DNA strand. It was shown that NtrC from Salmonella typhimurium must build large oligomers to activate transcription. In contrast to eukaryotic enhancer-binding proteins, most of which must bind directly to DNA, some NtrC dimers were bound solely by protein-protein interactions. NtrC oligomers were visualized with scanning force microscopy. Evidence of their functional importance was provided by showing that some inactive non-DNA-binding and DNA-binding mutant forms of NtrC can cooperate to activate transcription.
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Repressor Forms of the Enhancer-binding Protein NtrC: Some Fail in Coupling ATP Hydrolysis to Open Complex Formation by σ54-Holoenzyme
Journal of molecular biology, 1996Co-Authors: Anne K. North, Yehuda Flashner, David J. Weiss, Hideyuki Suzuki, Sydney KustuAbstract:Abstract NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein that activates transcription by catalyzing isomerization of closed complexes between σ54-holoenzyme and a promoter to open complexes. To catalyze this reaction, NtrC must be phosphorylated and form an appropriate oligomer so that it can hydrolyze ATP. NtrC can also repress transcription by σ70-holoenzyme. In this paper we characterize “repressor” mutant forms of NtrC fromSalmonella typhimurium, forms that have lost the ability to activate transcription by σ54-holoenzyme (in vitroactivity at least 1000-fold lower than wild-type) but retain the ability to repress transcription by σ70-holoenzyme. The amino acid substitutions in NtrCrepressorproteins that were obtained by classical genetic techniques alter residues in the central domain of the protein, the domain directly responsible for transcriptional activation. Commensurate with this, phosphorylation and the autophosphatase activities of NtrCrepressorproteins, which are functions of the amino-terminal regulatory domain of NtrC, are normal. In addition, these proteins have essentially normal DNA-binding, which is a function of the C-terminal region of NtrC and bind cooperatively to enhancers. (The NtrCG219Kprotein has “improved” DNA-binding, which is discussed.) We previously presented evidence that several NtrCrepressorproteins have impaired ATPase activity. We now show that two other repressor proteins, NtrCA216Vand NtrCA220T, have as much ATPase activity as wild-type NtrC when they are phosphorylated and bound to an enhancer and that they have considerably more activity than an unphosphorylated NtrCconstitutiveprotein, which is capable of activating transcription. These results demonstrate that NtrCA216Vand NtrCA220Tfail in a function of the central domain other than ATPase activity. Although they may fail in contact with σ54-holoenzymeper se, the fact that alanine is the amino acid normally found at these positions leads us to speculate that these proteins fail in coupling energy to a change in conformation of the polymerase.
Peter Geigenberger - One of the best experts on this subject based on the ideXlab platform.
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NtrC Plays a Crucial Role in Starch Metabolism, Redox Balance, and Tomato Fruit Growth.
Plant physiology, 2019Co-Authors: Liang-yu Hou, Francisco Javier Cejudo, Matthias Ehrlich, Ina Thormählen, Martin Lehmann, Ina Krahnert, Toshihiro Obata, Alisdair R. Fernie, Peter GeigenbergerAbstract:NADPH-thioredoxin reductase C (NtrC) forms a separate thiol-reduction cascade in plastids, combining both NADPH-thioredoxin reductase and thioredoxin activities on a single polypeptide. While NtrC is an important regulator of photosynthetic processes in leaves, its function in heterotrophic tissues remains unclear. Here, we focus on the role of NtrC in developing tomato (Solanum lycopersicum) fruits representing heterotrophic storage organs important for agriculture and human diet. We used a fruit-specific promoter to decrease NtrC expression by RNA interference in developing tomato fruits by 60% to 80% compared to the wild type. This led to a decrease in fruit growth, resulting in smaller and lighter fully ripe fruits containing less dry matter and more water. In immature fruits, NtrC downregulation decreased transient starch accumulation, which led to a subsequent decrease in soluble sugars in ripe fruits. The inhibition of starch synthesis was associated with a decrease in the redox-activation state of ADP-Glc pyrophosphorylase and soluble starch synthase, which catalyze the first committed and final polymerizing steps, respectively, of starch biosynthesis. This was accompanied by a decrease in the level of ADP-Glc. NtrC downregulation also led to a strong increase in the reductive states of NAD(H) and NADP(H) redox systems. Metabolite profiling of NtrC-RNA interference lines revealed increased organic and amino acid levels, but reduced sugar levels, implying that NtrC regulates the osmotic balance of developing fruits. These results indicate that NtrC acts as a central hub in regulating carbon metabolism and redox balance in heterotrophic tomato fruits, affecting fruit development as well as final fruit size and quality.
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NtrC links built in thioredoxin to light and sucrose in regulating starch synthesis in chloroplasts and amyloplasts
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Justyna Michalska, Francisco Javier Cejudo, Henrik Zauber, Bob B Buchanan, Peter GeigenbergerAbstract:Plants have an unusual plastid-localized NADP-thioredoxin reductase C (NtrC) containing both an NADP-thioredoxin reductase (NTR) and a thioredoxin (Trx) domain in a single polypeptide. Although NtrC is known to supply reductant for detoxifying hydrogen peroxide in the dark, its other functions are unknown. We now report that NtrC plays a previously unrecognized role in the redox regulation of ADP-glucose pyrophosphorylase (AGPase), a central enzyme of starch synthesis. When supplied NADPH, NtrC activated AGPase in vitro in a redox reaction that required the active site cysteines of both domains of the enzyme. In leaves, AGPase was activated in planta either by light or external feeding of sucrose in the dark. Leaves of an Arabidopsis NtrC KO mutant showed a decrease both in the extent of redox activation of AGPase and in the enhancement of starch synthesis either in the light (by 40–60%) or in the dark after treatment with external sucrose (by almost 100%). The light-dependent activation of AGPase in isolated chloroplasts, by contrast, was unaffected. In nonphotosynthetic tissue (roots), KO of NtrC decreased redox activation of AGPase and starch synthesis in response to light or external sucrose by almost 90%. The results provide biochemical and genetic evidence for a role of NtrC in regulating starch synthesis in response to either light or sucrose. The data also suggest that the Trx domain of NtrC and, to a lesser extent, free Trxs linked to ferredoxin enable amyloplasts of distant sink tissues to sense light used in photosynthesis by leaf chloroplasts and adjust heterotrophic starch synthesis accordingly.
Ray Dixon - One of the best experts on this subject based on the ideXlab platform.
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NtrC-Dependent Regulatory Network for Nitrogen Assimilation in Pseudomonas putida
Journal of bacteriology, 2009Co-Authors: Ana B. Hervás, Ray Dixon, Inés Canosa, Richard Little, Eduardo SanteroAbstract:Pseudomonas putida KT2440 is a model strain for studying bacterial biodegradation processes. However, very little is known about nitrogen regulation in this strain. Here, we show that the nitrogen regulatory NtrC proteins from P. putida and Escherichia coli are functionally equivalent and that substitutions leading to partially active forms of enterobacterial NtrC provoke the same phenotypes in P. putida NtrC. P. putida has only a single PII-like protein, encoded by glnK, whose expression is nitrogen regulated. Two contiguous NtrC binding sites located upstream of the N -dependent glnK promoter have been identified by footprinting analysis. In vitro experiments with purified proteins demonstrated that glnK transcription was directly activated by NtrC and that open complex formation at this promoter required integration host factor. Transcription of genes orthologous to enterobacterial codB, dppA, and ureD genes, whose transcription is dependent on 70 and which are activated by Nac in E. coli, has also been analyzed for P. putida. Whereas dppA does not appear to be regulated by nitrogen via NtrC, the codB and ureD genes have N -dependent promoters and their nitrogen regulation was exerted directly by NtrC, thus avoiding the need for Nac, which is missing in this bacterial species. Based upon these results, we propose a simplified nitrogen regulatory network in P. putida (compared to that in enterobacteria), which involves an indirect-feedback autoregulation of glnK using NtrC as an intermediary.
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effector induced self association and conformational changes in the enhancer binding protein NtrC
Molecular Microbiology, 1996Co-Authors: Esther M Farezvidal, Timothy J. Wilson, Barrie E. Davidson, Geoffrey J. Howlett, Sara Austin, Ray DixonAbstract:The Klebsiella pneumoniae nitrogen regulatory protein NtrC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of σN-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NtrC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATPγS concentration suggested an apparent dissociation constant (Kd) for ATPγS of 150 μM. In the presence of DNA, both the central and C-terminal domains of NtrC were insensitive to proteolytic cleavage, indicative of a further conformational change. NtrC S160F, a mutant form of NtrC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NtrC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NtrC displayed a monomer–dimer equilibrium with a Kd of 6 μM. In the presence of ATPγS the equilibrium was shifted towards the dimer form (Kd = 0.8 μM). A similar dissociation constant for the monomer–dimer interaction was observed with NtrC S160F in the absence of ATPγS (Kd = 0.5 μM). The addition of ATPγS induced a significant association of NtrC S160F to higher-order states with a dimer–octamer model producing a slightly, but not significantly better fit to the data than a dimer–hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.
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Effector‐induced self‐association and conformational changes in the enhancer‐binding protein NtrC
Molecular microbiology, 1996Co-Authors: M. Esther Farez-vidal, Timothy J. Wilson, Barrie E. Davidson, Geoffrey J. Howlett, Sara Austin, Ray DixonAbstract:The Klebsiella pneumoniae nitrogen regulatory protein NtrC is a response regulator which activates transcription in response to nitrogen limitation, and is a member of the family of σN-dependent enhancer-binding proteins. Using limited trypsin digestion, two domains of NtrC were detected and conformational changes within the protein in response to the binding of ligands were also observed. In the absence of ligands, the major digestion products were 42, 36 and 12.5 kDa bands corresponding to the central plus C-terminal domain, the central domain, and the N-terminal domains, respectively. Upon binding of purine but not pyrimidine nucleotides, the 36 kDa band was insensitive to further proteolysis, indicative of a conformational change in the central domain. Analysis of the dependence of this insensitivity on ATPγS concentration suggested an apparent dissociation constant (Kd) for ATPγS of 150 μM. In the presence of DNA, both the central and C-terminal domains of NtrC were insensitive to proteolytic cleavage, indicative of a further conformational change. NtrC S160F, a mutant form of NtrC that is active in the absence of phosphorylation, was more stable to proteolysis than the wild-type protein. This mutant protein is apparently locked in a conformation resembling the DNA-bound form of wild-type NtrC. The involvement of ligands in self-association was studied using sedimentation equilibrium analysis. In the absence of ligand, wild-type NtrC displayed a monomer–dimer equilibrium with a Kd of 6 μM. In the presence of ATPγS the equilibrium was shifted towards the dimer form (Kd = 0.8 μM). A similar dissociation constant for the monomer–dimer interaction was observed with NtrC S160F in the absence of ATPγS (Kd = 0.5 μM). The addition of ATPγS induced a significant association of NtrC S160F to higher-order states with a dimer–octamer model producing a slightly, but not significantly better fit to the data than a dimer–hexamer model. We propose that ligand-mediated self-association provides a common mechanism for activation of this class of transcriptional regulatory proteins.
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The prokaryotic enhancer binding protein NtrC has an ATPase activity which is phosphorylation and DNA dependent.
The EMBO journal, 1992Co-Authors: S. Austin, Ray DixonAbstract:The prokaryotic activator protein NtrC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NtrC and the reaction is ATP dependent. We find that NtrC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NtrC binding sites.
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INFLUENCE OF A MUTATION IN THE PUTATIVE NUCLEOTIDE BINDING SITE OF THE NITROGEN REGULATORY PROTEIN NtrC ON ITS POSITIVE CONTROL FUNCTION
Nucleic acids research, 1991Co-Authors: S. Austin, Craig Kundrot, Ray DixonAbstract:A mutation, serine 170 to alanine, in the proposed ATP binding site of the activator protein NtrC prevents transcriptional activation at sigma 54-dependent promoters both in vivo and in vitro. The rate of phosphorylation of the mutant protein by NTRB and the stability of mutant NtrC-phosphate were similar to those of wild-type NtrC. The phosphorylated mutant protein shows only a slight decrease in affinity (around 2-fold) for tandem NtrC binding sites in the Klebsiella pneumoniae nifL promoter suggesting that the mutation primarily influences the positive control function of NtrC. Moreover the mutant protein is trans dominant to the wild-type protein with respect to transcriptional activation at both the glnAp2 and nifL promoters. In vitro footprinting experiments reveal that the mutant protein is unable to catalyse isomerisation of closed promoter complexes between sigma 54-RNA polymerase and the nifL promoter to open promoter complexes. However, the mutant protein retains the ability to increase the occupancy of the -24, -12 region by sigma 54-RNA polymerase, forming closed complexes at the nifL promoter, which are not detectable in the absence of NtrC. These data support a model in which the activator influences the formation of closed complexes at the nifL promoter in addition to its role in catalysing open complex formation.
