RESUMO
Glycine max symbiotic ammonium transporter 1 was first documented as a putative ammonium (NH4(+)) channel localized to the symbiosome membrane of soybean root nodules. We show that Glycine max symbiotic ammonium transporter 1 is actually a membrane-localized basic helix-loop-helix (bHLH) DNA-binding transcription factor now renamed Glycine max bHLH membrane 1 (GmbHLHm1). In yeast, GmbHLHm1 enters the nucleus and transcriptionally activates a unique plasma membrane NH4(+) channel Saccharomyces cerevisiae ammonium facilitator 1. Ammonium facilitator 1 homologs are present in soybean and other plant species, where they often share chromosomal microsynteny with bHLHm1 loci. GmbHLHm1 is important to the soybean rhizobium symbiosis because loss of activity results in a reduction of nodule fitness and growth. Transcriptional changes in nodules highlight downstream signaling pathways involving circadian clock regulation, nutrient transport, hormone signaling, and cell wall modification. Collectively, these results show that GmbHLHm1 influences nodule development and activity and is linked to a novel mechanism for NH4(+) transport common to both yeast and plants.
Assuntos
Compostos de Amônio/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Glycine max/crescimento & desenvolvimento , Glycine max/metabolismo , Nódulos Radiculares de Plantas/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/metabolismo , Proteínas de Soja/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Ligação Proteica , Nódulos Radiculares de Plantas/citologia , Nódulos Radiculares de Plantas/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Glycine max/genética , Glycine max/ultraestruturaRESUMO
The NrtA and NrtB nitrate transporters are paralogous members of the major facilitator superfamily in Aspergillus nidulans. The availability of loss-of-function mutations allowed individual investigation of the specificity and inhibitor sensitivity of both NrtA and NrtB. In this study, growth response tests were carried out at a growth-limiting concentration of nitrate (1 mM) as the sole nitrogen source, in the presence of a number of potential nitrate analogues at various concentrations, to evaluate their effect on nitrate transport. Both chlorate and chlorite inhibited fungal growth, with chlorite exerting the greater inhibition. The main transporter of nitrate, NrtA, proved to be more sensitive to chlorate than the minor transporter, NrtB. Similarly, the cation caesium was shown to exert differential effects, strongly inhibiting the activity of NrtB, but not NrtA. In contrast, no inhibition of nitrate uptake by NrtA or NrtB transporters was observed in either growth tests or uptake assays in the presence of bicarbonate, formate, malonate or oxalate (sulphite could not be tested in uptake assays owing to its reaction with nitrate), indicating significant specificity of nitrate transport. Kinetic analyses of nitrate uptake revealed that both chlorate and chlorite inhibited NrtA competitively, while these same inhibitors inhibited NrtB in a non-competitive fashion. The caesium ion appeared to inhibit NrtA in a non-competitive fashion, while NrtB was inhibited uncompetitively. The results provide further evidence of the distinctly different characteristics as well as the high specificity of nitrate uptake by these two transporters.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas de Transporte de Ânions/genética , Antifúngicos/metabolismo , Aspergillus nidulans/genética , Aspergillus nidulans/crescimento & desenvolvimento , Césio/metabolismo , Cloratos/metabolismo , Cloretos/metabolismo , Meios de Cultura/química , Proteínas Fúngicas/genética , Testes de Sensibilidade Microbiana , Nitrogênio/metabolismo , Especificidade por Substrato , Sulfitos/metabolismoRESUMO
In plants that have been deprived of nitrate for a significant length of time, a constitutive high-affinity nitrate transport system (cHATS) is responsible for initial nitrate uptake. This absorbed nitrate leads to the induction of the major nitrate transporters and enzymes involved in nitrate assimilation. By use of (13) NO3 (-) influx measurements and Blue Native polyacrylamide gel electrophoresis we examined the role of AtNRT2.5 in cHATS in wild type (WT) and various T-DNA mutants of Arabidopsis thaliana. We demonstrate that AtNRT2.5 is predominantly expressed in roots of nitrate-deprived WT plants as a 150 kDa molecular complex with AtNAR2.1. This complex represents the major contributor to cHATS influx, which is reduced by 63% compared with WT in roots of Atnrt2.5 mutants. The remaining cHATS nitrate influx in these mutants is due to a residual contribution by the inducible high-affinity transporter encoded by AtNRT2.1/AtNAR2.1. Estimates of the kinetic properties of the NRT2.5 transporter reveal that its low Km for nitrate makes this transporter ideally suited to detect and respond to trace quantities of nitrate in the root environment.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Nitratos/metabolismo , Proteínas de Transporte de Ânions/genética , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Transporte Biológico , Western Blotting , DNA Bacteriano/genética , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Hidroponia , Funções Verossimilhança , Peso Molecular , Mutagênese Insercional/genética , Mutação , Eletroforese em Gel de Poliacrilamida Nativa , Isótopos de Nitrogênio , Fenótipo , Filogenia , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismoRESUMO
Nitrite-specific plasma membrane transporters have been described in bacteria, algae and fungi, but there is no evidence of a nitrite-specific plasma membrane transporter in higher plants. We have used 13NO2(-) to characterize nitrite influx into roots of Arabidopsis thaliana. Hydroponically grown Arabidopsis mutants, defective in high-affinity nitrate transport, were used to distinguish between nitrate and nitrite uptake by means of the short-lived tracers 13NO2(-) and 13NO3(-). This approach allowed us to characterize a nitrite-specific transporter. The Atnar2.1-2 mutant, lacking a functional high-affinity nitrate transport system, is capable of nitrite influx that is constitutive and thermodynamically active. The corresponding fluxes conform to a rectangular hyperbola, exhibiting saturation at concentrations above 200 µM (Km = 185 µM and Vmax = 1.89 µmol g(-1) FW h(-1)). Nitrite influx via the putative nitrite transporter is not subject to competitive inhibition by nitrate but is downregulated after 6 h exposure to ammonium. These results signify the existence of a nitrite-specific transporter in Arabidopsis. This transporter enables Atnar2.1-2 mutants, which are incapable of sustained growth on low nitrate, to maintain significant growth on low nitrite. In wild-type plants, this nitrite flux may increase nitrogen acquisition and also participate in the induction of genes specifically induced by nitrite.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Transporte de Íons , Nitritos/metabolismo , Nitrogênio/metabolismo , Raízes de Plantas/metabolismo , Compostos de Amônio/farmacologia , Proteínas de Transporte de Ânions/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Mutação , Nitratos/metabolismoRESUMO
⢠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 ß-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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitratos/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Mutação , Isótopos de Nitrogênio/análise , Oócitos , Folhas de Planta , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Mapeamento de Interação de Proteínas , Protoplastos , RNA Complementar/genética , RNA de Plantas/genética , Proteínas Recombinantes de Fusão , Técnicas do Sistema de Duplo-Híbrido , Xenopus/genética , Xenopus/metabolismoRESUMO
High-affinity nitrite influx into mycelia of Aspergillus nidulans has been characterized by use of (13)NO(2)(-), giving average K(m) and V(max) values of 48 ± 8 µM and 228 ± 49 nmol mg(-1) dry weight (DW) h(-1), respectively. Kinetic analysis of a plot that included an additional large number of low-concentration fluxes gave an excellent monophasic fit (r(2) = 0.96), with no indication of sigmoidal kinetics. Two-dimensional (2D) and three-dimensional (3D) models of AnNitA are presented, and the possible roles of conserved asparagine residues N122 (transmembrane domain 3 ]Tm 3]), N173 (Tm 4), N214 (Tm 5), and N246 (Tm 6) are discussed.
Assuntos
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Nitritos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aspergillus nidulans/crescimento & desenvolvimento , Membrana Celular/metabolismo , Sequência Conservada , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Cinética , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
AtNRT2.1, a polypeptide of the Arabidopsis thaliana two-component inducible high-affinity nitrate transport system (IHATS), is located within the plasma membrane. The monomeric form of AtNRT2.1 has been reported to be the most abundant form, and was suggested to be the form that is active in nitrate transport. Here we have used immunological and transient protoplast expression methods to demonstrate that an intact two-component complex of AtNRT2.1 and AtNAR2.1 (AtNRT3.1) is localized in the plasma membrane. A. thaliana mutants lacking AtNAR2.1 have virtually no IHATS capacity and exhibit extremely poor growth on low nitrate as the sole source of nitrogen. Near-normal growth and nitrate transport in the mutant were restored by transformation with myc-tagged AtNAR2.1 cDNA. Membrane fractions from roots of the restored myc-tagged line were solubilized in 1.5% dodecyl-ß-maltoside and partially purified in the first dimension by blue native gel electrophoresis. Using anti-NRT2.1 antibodies, an oligomeric polypeptide (approximate molecular mass 150 kDa) was identified, but monomeric AtNRT2.1 was absent. This oligomer was also observed in the wild-type, and was resolved, using SDS-PAGE for the second dimension, into two polypeptides with molecular masses of approximately 48 and 26 kDa, corresponding to AtNRT2.1 and myc-tagged AtNAR2.1, respectively. This result, together with the finding that the oligomer is absent from NRT2.1 or NAR2.1 mutants, suggests that this complex, rather than monomeric AtNRT2.1, is the form that is active in IHATS nitrate transport. The molecular mass of the intact oligomer suggests that the functional unit for high-affinity nitrate influx may be a tetramer consisting of two subunits each of AtNRT2.1 and AtNAR2.1.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Proteínas de Transporte de Ânions/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Western Blotting , Membrana Celular/metabolismo , Eletroforese em Gel Bidimensional , Teste de Complementação Genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Complexos Multiproteicos/metabolismo , Mutação , Transportadores de Nitrato , Nitratos/metabolismo , Raízes de Plantas/genética , Ligação Proteica , Protoplastos/metabolismoAssuntos
Proteínas de Transporte de Ânions/fisiologia , Proteínas de Arabidopsis/fisiologia , Nitratos/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/genética , Cinética , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/fisiologiaRESUMO
Disruption of the Aspergillus nidulans high-affinity nitrate transporter genes (nrtA and nrtB) prevents growth on nitrate but not nitrite. We identified a distinct nitrite transporter (K(m)=4.2+/-1 microM, V(max)=168+/-21 nmolmg(-1)DW(-1)h(-1)), designated NitA. Disruption of nrtA, nrtB and nitA blocked growth on nitrite, despite low rates of nitrite depletion we ascribe to passive nitrous acid permeation. Growth of the single mutant nitA16 on nitrite was wild-type, suggesting that NrtA and/or NrtB transports nitrite as well as nitrate. Indeed, NrtA and NrtB transport nitrite at higher rates than NitA; K(m) and V(max) values were 16+/-4 microM and 808+/-67 nmolmg(-1)DW(-1)h(-1) (NrtA) and 11+/-1 microM and 979+/-17 nmolmg(-1)DW(-1)h(-1) (NrtB). We suggest that NrtA is a nitrate/nitrite transporter, NrtB absorbs nitrite in preference to nitrate and NitA is exclusively a nitrite transporter.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Proteínas de Transporte de Ânions/genética , Aspergillus nidulans/crescimento & desenvolvimento , Transporte Biológico Ativo , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos/genética , Transportadores de NitratoRESUMO
⢠We report the first use of tracer 13 NH4 + (13 N-ammonium) efflux and retention data to analyse subcellular fluxes and compartmentation of NH4 + in the leaves of a higher plant (wheat, Triticum aestivum). ⢠Leaf segments, 1-2 mm, were obtained from 8-d-old seedlings. The viability of the segments, and stability of NH 4 + acquisition over time, were confirmed using oxygen-exchange and NH 4 + -depletion measurements. Fluxes of NH 4 + and compartment sizes were estimated using tracer efflux kinetics and retention data. ⢠Influx and efflux across the plasma membrane, half-lives of exchange and cytosolic pool sizes were broadly similar to those in root systems. As the external concentration of NH 4 + ([NH 4 + ] o ) increased from 10 µ m to 10 m m , both influx and efflux greatly increased, with a sixfold increase in the ratio of efflux to influx. Half-lives were similar among treatments, except at [NH 4 + ] o = 10 m m , where they declined. Concentrations of NH 4 + in the cytosol ([NH 4 + ] c ) increased from 2.6 to 400 m m . ⢠Although [NH 4 + ] c became large as [NH 4 + ] o increased, the ratio of [NH 4 + ] c to [NH 4 + ] o decreased more than sixfold. The apparently futile cycling of NH 4 + at high [NH 4 + ] o suggested by the large fluxes of NH 4 + in both directions across the membrane indicate that leaf cells respond to potentially toxic NH 4 + concentrations in a manner similar to root cells.
RESUMO
⢠Sodium, potassium, calcium, and magnesium ion uptake physiology and tolerance to sodium and magnesium were characterized in two edaphic races (A and C) of two closely related species in the Lasthenia californica complex. ⢠Uptake rates of race A plants were 20-fold higher for Na + , and 2-fold higher for Ca 2+ and Mg 2+ than those of race C plants. Race A translocated c. 50% of absorbed Na + to the shoot compared with < 30% in race C. For Ca 2+ and Mg 2+ corresponding values for the two races were > 95% and ≤ 50%, respectively. ⢠Germination, root growth and survivorship estimates indicated greater tolerance by race A to Na + and Mg 2+ . Significant genotype treatment interactions were observed, suggesting that these races are genetically differentiated in their tolerance responses. ⢠The study suggests parallel evolution of physiological traits in populations belonging to the two species and points to intriguing correlations between the presence of sulfated flavonoids and the capacities for the uptake of and tolerance to specific ions.
RESUMO
Here, influx and efflux of (13)NO(3)(-), and net fluxes of (14)NO(3)(-) and (14)NO(2)(-), were measured in Aspergillus nidulans mutants niaD171 and niiA5, devoid of nitrate reductase (NR) and nitrite reductase (NiR) activities, respectively. Transcript and protein abundances of NrtA, the A. nidulans principal high-affinity NO(3)(-) transporter, were determined using semiquantitative reverse transcription-polymerase chain reaction and western blots, respectively. (13)NO(3)(-) influx in niaD171 was negligible relative to wild-type values, whereas efflux to influx ratios increased nine-fold. Nevertheless, NrtA mRNA and NrtA protein were expressed at levels more than two-fold and three-fold higher, respectively, in niaD171 than in the wild-type strain. This is the first demonstration of diminished high-affinity NO(3)(-) influx associated with elevated transporter levels, providing evidence that, in addition to transcriptional regulation, control of NrtA expression operates at the post-translational level. This mechanism allows for rapid control of NO(3)(-) transport at the protein level, reduces the extent of futile cycling of NO(3)(-) that would otherwise represent a significant energy drain when influx exceeds the capacity for assimilation or storage, and may be responsible for the rapid switching between the on and off state that is associated with simultaneous provision of NH(4)(+) to mycelia absorbing NO(3)(-).
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Nitratos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Aspergillus nidulans/metabolismo , Transporte Biológico Ativo , Regulação Fúngica da Expressão Gênica , Mutação , Óxidos de Nitrogênio/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Using a new Arabidopsis (Arabidopsis thaliana) mutant (Atnrt2.1-nrt2.2) we confirm that concomitant disruption of NRT2.1 and NRT2.2 reduces inducible high-affinity transport system (IHATS) by up to 80%, whereas the constitutive high-affinity transport system (CHATS) was reduced by 30%. Nitrate influx via the low-affinity transport system (LATS) was unaffected. Shoot-to-root ratios were significantly reduced compared to wild-type plants, the major effect being upon shoot growth. In another mutant uniquely disrupted in NRT2.1 (Atnrt2.1), IHATS was reduced by up to 72%, whereas neither the CHATS nor the LATS fluxes were significantly reduced. Disruption of NRT2.1 in Atnrt2.1 caused a consistent and significant reduction of shoot-to-root ratios. IHATS influx and shoot-to-root ratios were restored to wild-type values when Atnrt2.1-nrt2.2 was transformed with a NRT2.1 cDNA isolated from Arabidopsis. Disruption of NRT2.2 in Atnrt2.2 reduced IHATS by 19% and this reduction was statistically significant only at 6 h after resupply of nitrate to nitrogen-deprived plants. Atnrt2.2 showed no significant reduction of CHATS, LATS, or shoot-to-root ratios. These results define NRT2.1 as the major contributor to IHATS. Nevertheless, when maintained on agar containing 0.25 mm KNO(3) as the sole nitrogen source, Atnrt2.1-nrt2.2 consistently exhibited greater stress and growth reduction than Atnrt2.1. Evidence from real-time PCR revealed that NRT2.2 transcript abundance was increased almost 3-fold in Atnrt2.1. These findings suggest that NRT2.2 normally makes only a small contribution to IHATS, but when NRT2.1 is lost, this contribution increases, resulting in a partial compensation.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Família Multigênica , Ágar , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/genética , Transporte Biológico/fisiologia , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Nitratos/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismoRESUMO
In rice (Oryza sativa L.) OsAMT1.1 is the most active and / or most N-responsive gene responsible for high-affinity NH4+ transport (HATS) activity. We measured 13NH4+ influx and plant biomass in transgenic overexpression lines and two wild type cultivars of rice, Jarrah and Taipei, with one or more copies of OsAMT1.1. 13NH4+ influx was higher for the overexpression lines of Jarrah line when grown at 10 µm external NH4+ concentration, but not for the overexpression lines of Taipei. For seedlings grown at 2 mm external NH4+ concentration Jarrah lines 77-1 and 75-4 showed an increased influx; however, two overexpression lines of Taipei showed reduced influx rates. The biomasses of the transgenic lines grown at low and high external NH4+ concentrations were either reduced or showed no statistically significant differences compared with wild type lines. While 13NH4+ influx into roots of Jarrah line 75-4 grown at 10 µm external NH4+ concentration was significantly higher than in wild type, measurements of 13NH efflux revealed no differences, and thus net uptake of NH4+ was higher in this overexpression line.
RESUMO
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.
Assuntos
Proteínas de Transporte de Ânions/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Nitratos/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Arabidopsis/anatomia & histologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte de Íons/genética , Dados de Sequência Molecular , Família Multigênica , Mutação , Raízes de Plantas/anatomia & histologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Alinhamento de SequênciaRESUMO
All eight cysteine residues, C90, C94, C143, C147, C219, C325, C367, and C431, present in transmembrane domains of the Aspergillus nidulans NrtA nitrate transporter protein were altered individually by site-specific mutagenesis. The results indicate that six residues, C90, C147, C219, C325, C367, and C431, are not required for nitrate transport. Although alterations of C94 and C143 are less well tolerated, these residues are not mandatory and their possible role is discussed. A series of constructs, all completely devoid of cysteine residues, was generated to permit future cysteine-scanning mutagenesis. The optimum cysteine-less combination was identified as C90A, C94A, C143A, C147T, C219S, C325S, C367S, and C431S. This mutant combination yielded transformant strains with up to 40% of wild-type nitrate transport activity. Furthermore, the K(m) value and the level of protein expression were found to be similar to those of the wild-type. This cysteine-less vector should allow us to investigate in detail potentially interesting NrtA amino acids (e.g. identified from homology comparisons) which may be involved in transport, by altering these singly to cysteine and studying such residues by thiol chemistry.
Assuntos
Proteínas de Transporte de Ânions/química , Proteínas de Transporte/química , Cisteína/química , Proteínas Fúngicas/química , Aspergillus nidulans/metabolismo , Transporte Biológico , Proteínas de Transporte/genética , Mutagênese Sítio-Dirigida , Nitratos/metabolismoRESUMO
Four low-affinity (NRT1), and seven high-affinity (NRT2) nitrate transporter gene homologues have been identified in Arabidopsis thaliana. We investigated the transcript abundances of all eleven genes in shoot and root tissues in response to the provision of 1 mM NO(3)(-), using relative quantitative RT-PCR. Based upon this criterion, genes were classified as nitrate-inducible, nitrate-repressible, or nitrate-constitutive. AtNRT1.1, 2.1, and 2.2 were strongly induced by NO(3)(-), peaking at 3-12 h and subsequently declining. By contrast AtNRT2.4 showed only modest induction both in shoots and roots. Expression of AtNRT2.5, one of the nitrate-repressible genes, was strongly suppressed by nitrate provision in both roots and shoots. The last group, characterized by a constitutive expression pattern, included AtNRT1.2, 1.4, 2.3, 2.6, and 2.7. Correlation coefficients between (13)NO(3)(-) influx from 100 micro M and 5 mM [NO(3)(-)], suggest that high- and low-affinity transport systems are mediated primarily by AtNRT2.1 and AtNRT1.1, respectively. Functional roles for the other members of these families remain uncertain.
Assuntos
Proteínas de Transporte de Ânions/genética , Proteínas de Arabidopsis/genética , Arabidopsis/efeitos dos fármacos , Nitratos/farmacologia , Proteínas de Plantas , Sequência de Aminoácidos , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , Família Multigênica/genética , Transportadores de Nitrato , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de AminoácidosRESUMO
This study represents the first attempt to investigate the molecular mechanisms by which nitrate, an anion of significant ecological, agricultural, and medical importance, is transported into cells by high-affinity nitrate transporters. Two charged residues, R87 and R368, located within hydrophobic transmembrane domains 2 and 8, respectively, are conserved in all 52 high-affinity nitrate transporters sequenced thus far. Site-directed replacements of either of R87 or R368 residues by lysine were found to be tolerated, but such residue changes increased the K(m) for nitrate influx from micromolar to millimolar values. Seven other amino acid substitutions of R87 or R368 all led to loss of function and lack of growth on nitrate. No evidence was obtained of R87 or R368 forming a salt-bridge with conserved acidic residues. Remarkably, the phenotype of loss-of-function mutant R87T was found to be alleviated by an alteration to lysine of N459, present in the second copy of the nitrate signature (transmembrane domain 11), suggesting a structural or functional interplay between residues R87 and N459 in the three-dimensional NrtA protein structure. Failure of the potential reciprocal second site suppressor N168K (in the first nitrate signature copy of transmembrane domain 5) to revert R368T was observed. Taken with recent structural studies of other major facilitator superfamily proteins, the results suggest that R87 and R368 are involved in substrate binding and probably located in a region of the protein close to N459.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Arginina/metabolismo , Sequência Conservada , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Arginina/genética , Aspergillus niger/citologia , Aspergillus niger/genética , Aspergillus niger/metabolismo , Proliferação de Células , Sequência Conservada/genética , Expressão Gênica , Cinética , Mutação/genética , Transportadores de Nitrato , Nitratos/metabolismo , Sais/farmacologia , Especificidade por SubstratoRESUMO
NH(4)(+) acquisition by plant roots is thought to involve members of the NH(4)(+) transporter family (AMT) found in plants, yeast, bacteria, and mammals. In Arabidopsis, there are six AMT genes of which AtAMT1;1 demonstrates the highest affinity for NH(4)(+). Ammonium influx into roots and AtAMT1;1 mRNA expression levels are highly correlated diurnally and when plant nitrogen (N) status is varied. To further investigate the involvement of AtAMT1;1 in high-affinity NH(4)(+) influx, we identified a homozygous T-DNA mutant with disrupted AtAMT1;1 activity. Contrary to expectation, high-affinity (13)NH(4)(+) influx in the amt1;1:T-DNA mutant was similar to the wild type when grown with adequate N. Removal of N to increase AtAMT1;1 expression decreased high-affinity (13)NH(4)(+) influx in the mutant by 30% compared with wild-type plants, whereas low-affinity (13)NH(4)(+) influx (250 microM-10 mM NH(4)(+)) exceeded that of wild-type plants. In these N-deprived plants, mRNA copy numbers of root AtAMT1;3 and AtAMT2;1 mRNA were significantly more increased in the mutant than in wild-type plants. Under most growth conditions, amt1;1:T-DNA plants were indistinguishable from the wild type, however, leaf morphology was altered. However, when grown with NH(4)(+) and sucrose, the mutant grew poorly and died. Our results are the first in planta evidence that AtAMT1;1 is a root NH(4)(+) transporter and that redundancies within the AMT family may allow compensation for the loss of AtAMT1;1.
Assuntos
Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte de Cátions , Proteínas de Plantas/genética , Sulfato de Amônio/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Perfilação da Expressão Gênica , Mutação , Nitratos/farmacologia , Nitrogênio/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/fisiologia , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/fisiologia , Compostos de Potássio/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The ability to transport net nitrate was conferred upon transformant cells of the non-nitrate-assimilating yeast Pichia pastoris after the introduction of two genes, one encoding nitrate reductase and the other nitrate transport. It was observed that cells of this lower eukaryote transformed with the nitrate transporter gene alone failed to display net nitrate transport despite having the ability to produce the protein. In addition, loss-of-function nitrate reductase mutants isolated from several nitrate-assimilating fungi appeared to be unable to accumulate nitrate. Uptake assays using the tracer (13)NO(3)(-) showed that nitrate influx is negligible in cells of a nitrate reductase null mutant. In parallel studies using a higher eukaryotic plant, Arabidopsis thaliana, loss-of-function nitrate reductase strains homozygous for both NIA1 insertion and NIA2 deletion were found to have no detectable nitrate reductase mRNA or nitrate reductase activity but retained the ability to transport nitrate. The reasons for these fundamental differences in nitrate transport into the cells of representative members of these two eukaryotic kingdoms are discussed.