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Anthony D M Glass - One of the best experts on this subject based on the ideXlab platform.
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a reevaluation of the role of arabidopsis nrt1 1 in high affinity Nitrate Transport
Plant Physiology, 2013Co-Authors: Anthony D M Glass, Zorica KoturAbstract:A reevaluation of flux data for Arabidopsis mutants reveals that Nitrate uptake through AtNRT1.1 conforms to a single low-affinity Transport system that makes virtually no contribution to high-affinity Nitrate uptake.
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Nitrate Transport capacity of the arabidopsis thaliana nrt2 family members and their interactions with atnar2 1
New Phytologist, 2012Co-Authors: Zorica Kotur, Nenah Mackenzie, Sunita A Ramesh, Stephen D Tyerman, Brent N Kaiser, Anthony D M GlassAbstract:• Interactions between the Arabidopsis Nitrate Transporter (AtNRT2.1) and Nitrate Assimilation Related protein (AtNAR2.1, also known as AtNRT3.1) have been well documented, and confirmed by the demonstration that AtNRT2.1 and AtNAR2.1 form a 150-kDa plasma membrane complex, thought to constitute the high-affinity Nitrate Transporter of Arabidopsis thaliana roots. Here, we have investigated interactions between the remaining AtNRT2 family members (AtNRT2.2 to AtNRT2.7) and AtNAR2.1, and their capacity for Nitrate Transport. • Three different systems were used to examine possible interactions with AtNAR2.1: membrane yeast split-ubiquitin, bimolecular fluorescence complementation in A. thaliana protoplasts and Nitrate uptake in Xenopus oocytes. • All NRT2s, except for AtNRT2.7, restored growth and β-galactosidase activity in the yeast split-ubiquitin system, and split-YFP fluorescence in A. thaliana protoplasts only when co-expressed with AtNAR2.1. Thus, except for AtNRT2.7, all other NRT2 Transporters interact strongly with AtNAR2.1. • Co-injection into Xenopus oocytes of cRNA of all NRT2 genes together with cRNA of AtNAR2.1 resulted in statistically significant increases of uptake over and above that resulting from single cRNA injections.
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Nitrate uptake by plant rootsthis paper is one of a selection published in a special issue comprising papers presented at the 50th annual meeting of the canadian society of plant physiologists cspp held at the university of ottawa ontario in june 2008
Botany, 2009Co-Authors: Anthony D M GlassAbstract:Nitrate is a major source of N for plant growth, particularly under aerobic agricultural conditions. This nutrient is absorbed from soil solution by both high-affinity and low-affinity Transport systems that were first defined physiologically and subsequently by molecular genetic means. This minireview presents an abbreviated history of developments in plant inorganic ion uptake, the physiology of Nitrate uptake, and recent developments in the molecular genetic characterization of Nitrate Transport systems.
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high affinity Nitrate Transport in roots of arabidopsis depends on expression of the nar2 like gene atnrt3 1
Plant Physiology, 2006Co-Authors: Mamoru Okamoto, Yaeesh M Siddiqi, Nigel M Crawford, Anshuman Kumar, Ye Wang, Anthony D M GlassAbstract:The NAR2 protein of Chlamydomonas reinhardtii has no known Transport activity yet it is required for high-affinity Nitrate uptake. Arabidopsis (Arabidopsis thaliana) possesses two genes, AtNRT3.1 and AtNRT3.2, that are similar to the C. reinhardtii NAR2 gene. AtNRT3.1 accounts for greater than 99% of NRT3 mRNA and is induced 6-fold by Nitrate. AtNRT3.2 was expressed constitutively at a very low level and did not compensate for the loss of AtNRT3.1 in two Atnrt3.1 mutants. Nitrate uptake by roots and Nitrate induction of gene expression were analyzed in two T-DNA mutants, Atnrt3.1-1 and Atnrt3.1-2, disrupted in the AtNRT3.1 promoter and coding regions, respectively, in 5-week-old plants. Nitrate induction of the Nitrate Transporter genes AtNRT1.1 and AtNRT2.1 was reduced in Atnrt3.1 mutant plants, and this reduced expression was correlated with reduced Nitrate concentrations in the tissues. Constitutive high-affinity influx was reduced by 34% and 89%, respectively, in Atnrt3.1-1 and Atnrt3.1-2 mutant plants, while high-affinity Nitrate-inducible influx was reduced by 92% and 96%, respectively, following induction with 1 mm KNO(3) after 7 d of nitrogen deprivation. By contrast, low-affinity influx appeared to be unaffected. Thus, the constitutive high-affinity influx and Nitrate-inducible high-affinity influx (but not the low-affinity influx) of higher plant roots require a functional AtNRT3 (NAR2) gene.
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apparent genetic redundancy facilitates ecological plasticity for Nitrate Transport
The EMBO Journal, 2001Co-Authors: Degen Zhou, Shiela E Unkles, Yaeesh M Siddiqi, James R Kinghorn, Anthony D M GlassAbstract:Aspergillus nidulans possesses two high-affinity Nitrate Transporters, encoded by the nrtA and the nrtB genes. Mutants expressing either gene grew normally on 1–10 mM Nitrate as sole nitrogen source, whereas the double mutant failed to grow on Nitrate concentrations up to 200 mM. These genes appear to be regulated coordinately in all growth conditions, growth stages and regulatory genetic backgrounds studied. Flux analysis of single gene mutants using 13NO3− revealed that Km values for the NrtA and NrtB Transporters were ∼100 and ∼10 μM, respectively, while Vmax values, though variable according to age, were ∼600 and ∼100 nmol/mg dry weight/h, respectively, in young mycelia. This kinetic differentiation may provide the necessary physiological and ecological plasticity to acquire sufficient Nitrate despite highly variable external concentrations. Our results suggest that genes involved in Nitrate assimilation may be induced by extracellular sensing of ambient Nitrate without obligatory entry into the cell.
Sophie Filleur - One of the best experts on this subject based on the ideXlab platform.
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atp binding to the c terminus of the arabidopsis thaliana Nitrate proton antiporter atclca regulates Nitrate Transport into plant vacuoles
Journal of Biological Chemistry, 2009Co-Authors: Alexis De Angeli, Sophie Filleur, Oscar Moran, Stefanie Wege, Genevieve Ephritikhine, Sebastien Thomine, Helene Barbierbrygoo, Franco GambaleAbstract:Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of Nitrate influx into the vacuole at cytosolic physiological Nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa Transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.
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atp binding to the c terminus of the arabidopsis thaliana Nitrate proton antiporter atclca regulates Nitrate Transport into plant vacuoles
Journal of Biological Chemistry, 2009Co-Authors: Alexis De Angeli, Sophie Filleur, Oscar Moran, Stefanie Wege, Genevieve Ephritikhine, Sebastien Thomine, Helene Barbierbrygoo, Franco GambaleAbstract:Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO3−/H+ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of Nitrate influx into the vacuole at cytosolic physiological Nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa Transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.
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Nitrate Transport in plants which gene and which control
Journal of Experimental Botany, 2002Co-Authors: Mathilde Orsel, Sophie Filleur, Vincent Fraisier, Francoise DanielvedeleAbstract:Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological level and, more recently, at the molecular level. Two classes of genes, NRT1 and NRT2, have been found to be potentially involved in the high and low affinity Nitrate Transport systems (HATS and LATS, respectively). The complexity of the molecular basis of Nitrate uptake has been enhanced by the finding that in many plants both NRT1 and NRT2 classes are represented by multigene families. Furthermore, recent studies demonstrate that the control mechanisms that lead to an active protein at the plasma membrane act on gene transcription, modulating the steady-state levels of mRNA, and on the activation of the protein, possibly by a phosphorylation/dephosphorylation process. This is a review of recent progress in the characterization of the NRT2 Nitrate Transporters, the composition of this family in Arabidopsis, their possible role in Nitrate acquisition, and some aspects of their regulation in plants.
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Nitrate Transport a key step in Nitrate assimilation
Current Opinion in Plant Biology, 1998Co-Authors: Francoise Danielvedele, Sophie Filleur, Michel CabocheAbstract:The Nitrate assimilation pathway has been the matter of intensive research during the past decade. Many genes involved in low and high affinity Nitrate uptake have been identified in fungi, algae and, more recently, in plants. The plant genes so far isolated are transcriptionally regulated; their inducibility by Nitrate seems to be a common feature, shared by their homologs in fungi and algae. A number of questions remain to be elucidated regarding the physiological roles of these Transporters and the regulation of their expression.
Anthony J. Miller - One of the best experts on this subject based on the ideXlab platform.
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identification and functional assay of the interaction motifs in the partner protein osnar2 1 of the two component system for high affinity Nitrate Transport
New Phytologist, 2014Co-Authors: Xiaoqin Liu, Anthony J. Miller, Daimin Huang, Jinyuan Tao, Xiaorong FanAbstract:A partner protein, NAR2, is essential for high-affinity Nitrate Transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and Nitrate Transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.
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Nitrate Transport and signalling
Journal of Experimental Botany, 2007Co-Authors: Anthony J. Miller, Mathilde Orsel, Xiaorong Fan, Susan J. Smith, Darren M. WellsAbstract:Physiological measurements of Nitrate (NO(3)(-)) uptake by roots have defined two systems of high and low affinity uptake. In Arabidopsis, genes encoding both of these two uptake systems have been identified. Most is known about the high affinity Transport system (HATS) and its regulation and yet measurements of soil NO(3)(-) show that it is more often available in the low affinity range above 1 mM concentration. Several different regulatory mechanisms have been identified for AtNRT2.1, one of the membrane Transporters encoding HATS; these include feedback regulation of expression, a second component protein requirement for membrane targeting and phosphorylation, possibly leading to degradation of the protein. These various changes in the protein may be important for a second function in sensing NO(3)(-) availability at the surface of the root. Another Transporter protein, AtNRT1.1 also has a role in NO(3)(-) sensing that, like AtNRT2.1, is independent of their Transport function. From the range of concentrations present in the soil it is proposed that the NO(3)(-)-inducible part of HATS functions chiefly as a sensor for root NO(3)(-) availability. Two other key NO(3)(-) Transport steps for efficient nitrogen use by crops, efflux across membranes and vacuolar storage and remobilization, are discussed. Genes encoding vacuolar Transporters have been isolated and these are important for manipulating storage pools in crops, but the efflux system is yet to be identified. Consideration is given to how well our molecular and physiological knowledge can be integrated as well to some key questions and opportunities for the future.
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a high affinity Nitrate Transport system from chlamydomonas requires two gene products
FEBS Letters, 2000Co-Authors: Jingjiang Zhou, Emilio Fernández, Aurora Galván, Anthony J. MillerAbstract:A Nitrate-regulated cluster of genes involved in Nitrate Transport and assimilation has been identified in Chlamydomonas reinhardtii. Mutant strains of the alga, which are defective in some aspect of Transport and assimilation have been used to assign functions to these genes. This analysis has suggested that two gene products are necessary to obtain a functional high affinity Nitrate system in Chlamydomonas [Quesada et al. (1994) Plant J. 5, 407-419]. In this paper we have tested this hypothesis by injecting Xenopus oocytes with mRNA prepared from these two cDNAs, Nrt2;1 and Nar2, and then assaying the oocytes for Nitrate Transport activity. Oocytes injected with single types of mRNA did not show any Nitrate Transport activity. Furthermore, Nar2 mRNA was toxic to oocytes, with nearly 60%, of the oocytes dead 3 days after the injection. However, when oocytes were injected with a mixture of two mRNAs prepared from Nrt2;1 and Nar2, a high affinity Nitrate Transport activity could be measured. However, the Km for Nitrate of this Transport system was 28 microM which is higher than the value of 1.6 microM which had been obtained by the analysis of mutant phenotypes. The pH-dependence of the Nitrate-elicited currents was consistent with a proton-coTransport mechanism. These results prove that two gene products are required to produce a functional high affinity Nitrate Transport system and that this process does not involve transcriptional regulation.
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Nitrate Transport and compartmentation in cereal root cells
Journal of Experimental Botany, 1996Co-Authors: Anthony J. Miller, Susan J. SmithAbstract:AbstractMeasurement of cytosolic Nitrate is one of the factorsrequired for the resolution of factors controlling Nitrateuptake and assimilation in plants and for identifyinglikely Nitrate Transport mechanisms at both the plasmamembrane and tonoplast. This paper reviews methodsand reported measurements of cytosolic Nitrate inhigher plants and concludes that Nitrate-selectivemicroelectrodes are the best approach. These micro-electrodes have been used to measure intracellularNitrate activitites in barley and maize root cells. Triple-barrelled electrodes, incorporating a pH-sensing barrelhave been used to identify the compartmental locationof the Nitrate-selective tip giving unequivocal estim-ates of vacuolar and cytosolic Nitrate activities. Themicroelectrode measurements are used to discuss thepossible mechanisms of Nitrate Transport at both thetonoplast and plasma membrane. The energetics ofpossible proton-coupled Transport systems aredescribed and the feasibility of the mechanism isdiscussed.Key words: Cytosol, compartmentation, Hordeum vulgareL, Nitrate, roots, Zea mays LIntroduction
Franco Gambale - One of the best experts on this subject based on the ideXlab platform.
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atp binding to the c terminus of the arabidopsis thaliana Nitrate proton antiporter atclca regulates Nitrate Transport into plant vacuoles
Journal of Biological Chemistry, 2009Co-Authors: Alexis De Angeli, Sophie Filleur, Oscar Moran, Stefanie Wege, Genevieve Ephritikhine, Sebastien Thomine, Helene Barbierbrygoo, Franco GambaleAbstract:Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of Nitrate influx into the vacuole at cytosolic physiological Nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa Transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.
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atp binding to the c terminus of the arabidopsis thaliana Nitrate proton antiporter atclca regulates Nitrate Transport into plant vacuoles
Journal of Biological Chemistry, 2009Co-Authors: Alexis De Angeli, Sophie Filleur, Oscar Moran, Stefanie Wege, Genevieve Ephritikhine, Sebastien Thomine, Helene Barbierbrygoo, Franco GambaleAbstract:Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO3−/H+ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of Nitrate influx into the vacuole at cytosolic physiological Nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa Transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.
Genevieve Ephritikhine - One of the best experts on this subject based on the ideXlab platform.
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atp binding to the c terminus of the arabidopsis thaliana Nitrate proton antiporter atclca regulates Nitrate Transport into plant vacuoles
Journal of Biological Chemistry, 2009Co-Authors: Alexis De Angeli, Sophie Filleur, Oscar Moran, Stefanie Wege, Genevieve Ephritikhine, Sebastien Thomine, Helene Barbierbrygoo, Franco GambaleAbstract:Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO(3)(-)/H(+) exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine beta-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of Nitrate influx into the vacuole at cytosolic physiological Nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa Transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.
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atp binding to the c terminus of the arabidopsis thaliana Nitrate proton antiporter atclca regulates Nitrate Transport into plant vacuoles
Journal of Biological Chemistry, 2009Co-Authors: Alexis De Angeli, Sophie Filleur, Oscar Moran, Stefanie Wege, Genevieve Ephritikhine, Sebastien Thomine, Helene Barbierbrygoo, Franco GambaleAbstract:Nitrate, one of the major nitrogen sources for plants, is stored in the vacuole. Nitrate accumulation within the vacuole is primarily mediated by the NO3−/H+ exchanger AtCLCa, which belongs to the chloride channel (CLC) family. Crystallography analysis of hCLC5 suggested that the C-terminal domain, composed by two cystathionine β-synthetase motifs in all eukaryotic members of the CLC family is able to interact with ATP. However, interaction of nucleotides with a functional CLC protein has not been unambiguously demonstrated. Here we show that ATP reversibly inhibits AtCLCa by interacting with the C-terminal domain. Applying the patch clamp technique to isolated Arabidopsis thaliana vacuoles, we demonstrate that ATP reduces AtCLCa activity with a maximum inhibition of 60%. ATP inhibition of Nitrate influx into the vacuole at cytosolic physiological Nitrate concentrations suggests that ATP modulation is physiologically relevant. ADP and AMP do not decrease the AtCLCa Transport activity; nonetheless, AMP (but not ADP) competes with ATP, preventing inhibition. A molecular model of the C terminus of AtCLCa was built by homology to hCLC5 C terminus. The model predicted the effects of mutations of the ATP binding site on the interaction energy between ATP and AtCLCa that were further confirmed by functional expression of site-directed mutated AtCLCa.