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Biomedical subjects

A D Glass

Publications and source records attributed to A D Glass.

At least 19 recordsLinked to original sources
PubMed2001

Apparent genetic redundancy facilitates ecological plasticity for nitrate transport.

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 K(m) values for the NrtA and NrtB transporters were approximately 100 and approximately 10 microM, respectively, while V(max) values, though variable according to age, were approximately 600 and approximately 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.

Amino Acid Sequence↗
PubMed2001

Futile transmembrane NH4(+) cycling: a cellular hypothesis to explain ammonium toxicity in plants.

Most higher plants develop severe toxicity symptoms when grown on ammonium (NH(4)(+)) as the sole nitrogen source. Recently, NH(4)(+) toxicity has been implicated as a cause of forest decline and even species extinction. Although mechanisms underlying NH(4)(+) toxicity have been extensively sought, the primary events conferring it at the cellular level are not understood. Using a high-precision positron tracing technique, we here present a cell-physiological characterization of NH(4)(+) acquisition in two major cereals, barley (Hordeum vulgare), known to be susceptible to toxicity, and rice (Oryza sativa), known for its exceptional tolerance to even high levels of NH(4)(+). We show that, at high external NH(4)(+) concentration ([NH(4)(+)](o)), barley root cells experience a breakdown in the regulation of NH(4)(+) influx, leading to the accumulation of excessive amounts of NH(4)(+) in the cytosol. Measurements of NH(4)(+) efflux, combined with a thermodynamic analysis of the transmembrane electrochemical potential for NH(4)(+), reveal that, at elevated [NH(4)(+)](o), barley cells engage a high-capacity NH(4)(+)-efflux system that supports outward NH(4)(+) fluxes against a sizable gradient. Ammonium efflux is shown to constitute as much as 80% of primary influx, resulting in a never-before-documented futile cycling of nitrogen across the plasma membrane of root cells. This futile cycling carries a high energetic cost (we record a 40% increase in root respiration) that is independent of N metabolism and is accompanied by a decline in growth. In rice, by contrast, a cellular defense strategy has evolved that is characterized by an energetically neutral, near-Nernstian, equilibration of NH(4)(+) at high [NH(4)(+)](o). Thus our study has characterized the primary events in NH(4)(+) nutrition at the cellular level that may constitute the fundamental cause of NH(4)(+) toxicity in plants.

Electrophysiology↗
PubMed2000

Isolation and characterization of HvNRT2.3 and HvNRT2.4, cDNAs encoding high-affinity nitrate transporters from roots of barley.

Two full-length cDNAs, HvNRT2.3 and HvNRT2.4, were isolated from roots of barley (Hordeum vulgare), using reverse transcriptase-PCR and RACE-PCR. The corresponding polypeptides, consisting of 507 amino acids (molecular masses of 54.6 kD), belong to the major facilitator superfamily (MFS), and are closely related (>87% identity) to those encoded by HvNRT2.1 and HvNRT2.2 (formerly BCH1 and BCH2, respectively) from roots of barley. The latter are considered to encode inducible high-affinity NO(3)(-) transporters (Trueman et al., 1996). HvNRT2 transcripts were undetectable in NO(3)(-)-deprived plants. Following exposure to either NO(3)(-) or NO(2)(-), transcript abundance and (13)NO(3)(-) influx increased to a maximum by 6 to 12 h, then declined in HvNRT2.1, HvNRT2.2, and HvNRT2.3. The pattern of HvNRT2.4 transcript abundance was different, remaining high after achieving peak abundance. When external NO(3)(-) concentrations were varied from 0 to 500 microM under steady-state conditions of NO(3)(-) supply, HvNRT2 transcript accumulation and (13)NO(3)(-) influx were highest in 50 microM NO(3)(-) -grown plants. When NH(4)(+) was provided together with NO(3)(-), transcript accumulation during the first 2 h was similar to that due to NO(3)(-) alone, but by 4 h the transcript level was significantly reduced. HvNRT2 transcript was undetectable in leaf tissues.

Amino Acid Sequence↗
PubMed2000

Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley.

To investigate the regulation of HvNRT2, genes that encode high-affinity NO(3)(-) transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mM NO(3)(-) in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH(4)(+), and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity (13)NO(3)(-) influx and HvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO(3)(-) influx by 50%, indicating that NO(3)(-) itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH(4)(+) may down-regulate HvNRT2 gene expression and high-affinity NO(3)(-) influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO(3)(-) influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2 expression, although it does not preclude a contributory effect of other amino acids.

Anion Transport Proteins↗
PubMed1999

Regulation of the hvst1 gene encoding a high-affinity sulfate transporter from Hordeum vulgare.

A cDNA, hvst1, was isolated from Hordeum vulgare by heterologous complementation in Escherichia coli. This cDNA encodes a high-affinity sulfate transporter that is 2442 bp in length and consists of 660 amino acids. Under steady-state conditions of sulfate supply during culture, sulfate influx (measured at 100 microM external sulfate concentration) and hvst1 transcript level were inversely correlated with sulfate concentrations in the culture solution. Glutathione (GSH) concentrations increased as external sulfate was increased from 2.5 to 250 microM. A time-course study, designed to investigate effects of sulfate withdrawal on the abundance of hvst1 transcript, showed a 5-fold increase of the latter within the first two hours. This was followed by a further slight increase during the next 46 h. These changes were accompanied by a parallel increase in sulfate influx and a decrease of root GSH concentrations. When plants that had been deprived of sulfate for 24 h were exposed to L-cysteine (Cys) or GSH for 3 h, GSH was the more effective down-regulator, reducing hvst1 transcript level to below that of unstarved controls. The decrease in transcript abundance induced by sulfate or Cys was partially relieved by the addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Both hvst1 transcripts and sulfate influx increased as a function of N supply to N-starved plants. Amino oxyacetate acid (AOA), an aminotransferase inhibitor, when supplied with NO3-, increased transcript abundance of hvst1, while tungstate, methionine sulfoximine (MSO) and azaserine (AZA), inhibitors of nitrate reductase, glutamine synthetase and glutamate synthase (GOGAT), respectively, were without effect. AOA decreased root concentrations of aspartate (Asp), Cys and GSH; in contrast, glutamate (Glu) concentrations remained unchanged.

Amino Acid Sequence↗
PubMed1999

Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana.

Putative high-affinity nitrate (NO3-) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by RT-PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30-73% identities with other eukaryotic high-affinity NO3- transporters. Both genes were induced by NO3-, but Nrt2;1At gene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter, Nrt2;1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression levels were strongly correlated with inducible high-affinity 13NO3- influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation of Nrt2;1At expression was mediated by NH4+, gln and other amino acids.

Anion Transport Proteins↗
PubMed1999

Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound.

Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO4(2-). Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO4(2-) uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO4(2-) restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO4(2-) transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.

Arabidopsis↗
PubMed1999

AtAMT1 gene expression and NH4+ uptake in roots of Arabidopsis thaliana: evidence for regulation by root glutamine levels.

The mechanisms involved in regulating high-affinity ammonium (NH4+) uptake and the expression of the AtAMT1 gene encoding a putative high-affinity NH4+ transporter were investigated in the roots of Arabidopsis thaliana. Under conditions of steady-state nitrogen (N) supply, transcript levels of the AtAMT1 gene and Vmax values for high-affinity 13NH4+ influx were inversely correlated with levels of N provision. Following re-supply of NH4NO3 to N-starved plants, AtAMT1 mRNA levels and 13NH4+ influx declined rapidly but remained high when the conversion of NH4+ to glutamine (Gln) was blocked with methionine sulfoximine (MSX). This result demonstrates that end products of NH4+ assimilation, rather than NH4+ itself, are responsible for regulating AtAMT1 gene expression. Consistent with this hypothesis, AtAMT1 gene expression and NH4+ influx were suppressed by provision of Gln alone, or together with NH4NO3 plus MSX. Furthermore, AtAMT1 transcript levels and 13NH4+ influx were negatively correlated with root Gln concentrations, following re-supply of N to N-starved plants. In addition to this level of control, the data suggest that high cytoplasmic [NH4+] may inhibit NH4+ influx.

Arabidopsis↗
PubMed1997

NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana. Does the CHL1-5 gene encode a low-affinity NO3- transporter?

The CHL1 gene is considered to encode a low-affinity transport system (LATS) for NO3- in Arabidopsis thaliana (Y.-F. Tsay, J.I. Schroeder, K.A. Feldmann, N.M. Crawford [1993] Cell 72: 705-713). However, the anticipated reduced NO3- uptake by the LATS associated with loss of CHL1 gene activity in chl1-5 deletion mutants was evident only when plants were grown on NH4NO3. When KNO3 was the sole N source, NO3- accumulation and short-term tracer influx (using 13NO3- and 15NO3-) in leaves and roots of wild-type and mutant plants were essentially identical. Nevertheless, root uptake of 36CIO3- by the LATS and CIO3- accumulation in roots and shoots of mutant plants were significantly lower than in wild-type plants when grown on KNO3. One explanation for these results is that a second LATS is able to compensate for the chl1-5 deficiency in KNO3-grown plants. Growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in NO3- uptake becomes apparent.

Arabidopsis↗
PubMed1993

Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2.

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.

Ammonia↗
PubMed1992

Effects of nitrite, chlorate, and chlorite on nitrate uptake and nitrate reductase activity.

Effects of NO(2) (-), ClO(3) (-), and ClO(2) (-) on the induction of nitrate transport and nitrate reductase activity (NRA) as well as their effects on NO(3) (-) influx into roots of intact barley (Hordeum vulgare cv Klondike) seedlings were investigated. A 24-h pretreatment with 0.1 mol m(-3) NO(2) (-) fully induced NO(3) (-) transport but failed to induce NRA. Similar pretreatments with ClO(3) (-) and ClO(2) (-) induced neither NO(3) (-) transport nor NRA. Net ClO(3) (-) uptake was induced by NO(3) (-) but not by ClO(3) (-) itself, indicating that NO(3) (-) and ClO(3) (-) transport occur via the NO(3) (-) carrier. At the uptake step, NO(2) (-) and ClO(2) (-) strongly inhibited NO(3) (-) influx; the former exhibited classical competitive kinetics, whereas the latter exhibited complex mixed-type kinetics. ClO(3) (-) proved to be a weak inhibitor of NO(3) (-) influx (K(i) = 16 mol m(-3)) in a noncompetitive manner. The implications of these findings are discussed in the context of the suitability of these NO(3) (-) analogs as screening agents for the isolation of mutants defective in NO(3) (-) transport.

Journal Article↗
PubMed1992

De Novo Synthesis of Plasma Membrane and Tonoplast Polypeptides of Barley Roots during Short-Term K Deprivation : In Search of the High-Affinity K Transport System.

[(35)S]Methionine labeling of intact barley roots (Hordeum vulgare cv Klondike) after short (6-12 h) and longer (18-24 and 90-96 h) periods of K(+) deprivation revealed that several membrane polypeptides were synthesized in significantly increased amounts following withdrawal of K(+) from nutrient solutions. One of these, a 43-kD polypeptide localized in plasma membrane- and tonoplast-enriched fractions, accounted for a large part of (35)S incorporation into membranes when [(35)S]methionine was administered for 6 h following 6 h of K(+) deprivation. With increasing duration of K(+) deprivation, (35)S incorporation into this 43-kD polypeptide decreased. This polypeptide, referred to as KR43, was not synthesized when NO(3) (-) or inorganic phosphate was removed or when Rb(+) was substituted for K(+). However, it was synthesized when K(+) was removed and replaced by an equivalent concentration of Na(+). The intrinsic nature of this polypeptide and the time course of changes in its expression, which correspond with changes of K(+)((86)Rb) influx associated with K(+) deprivation, provide evidence that this polypeptide may form part of the high-affinity K(+) transport system in barley roots. A possible role for this polypeptide is discussed in the context of changes in the subcellular distribution of K(+) in barley roots following interruption of K(+) supply. A 45-kD microsomal polypeptide, identified in earlier studies as a response to K(+) deprivation, is suggested to be an extrinsic protein, readily displaced from membranes by exposure to ethylenediaminetetraacetate.

Journal Article↗
PubMed1992

Studies of the Uptake of Nitrate in Barley : IV. Electrophysiology.

Transmembrane electrical potential differences (Deltapsi) of epidermal and cortical cells were measured in intact roots of barley (Hordeum vulgare L. cv Klondike). The effects of exogenous NO(3) (-) on Deltapsi (in the concentration range from 100 micromolar to 20 millimolar) were investigated to probe the mechanisms of nitrate uptake by the high-affinity (HATS) and low-affinity (LATS) transport systems for NO(3) (-) uptake. Both transport systems caused depolarization of Deltapsi, demonstrating that the LATS (like the HATS) for NO(3) (-) uptake is probably mediated by an electrogenic cation (H(+)?) cotransport system. Membrane depolarization by the HATS was "inducible" by NO(3) (-), and saturable with respect to exogenous [NO(3) (-)]. By contrast, depolarization by the LATS was constitutive, and first-order in response to external [NO(3) (-)]. H(+) fluxes, measured in 200 micromolar and in 5 millimolar Ca(NO(3))(2) solutions, failed to alkalinize external media as anticipated for a 2 H(+):1 NO(3) (-) symport. However, switching from K(2)SO(4) solutions (which were strongly acidifying) to KNO(3) solutions at the same K(+) concentration caused marked reductions in H(+) efflux. These observations are consistent with NO(3) (-) uptake by the HATS and the LATS via 2 H(+):1 NO(3) (-) symports. These observations establish that the HATS for nitrate uptake by barley roots is essentially similar to those reported for Lemna and Zea mays by earlier workers. There are, nevertheless, distinct differences between barley and corn in their quantitative responses to external NO(3) (-).

Journal Article↗
PubMed1992

Immunofluorescent Localization of Plasma Membrane H-ATPase in Barley Roots and Effects of K Nutrition.

The plasma membrane H(+)-ATPase (PM-H(+)-ATPase) of barley (Hordeum vulgare L. cv Klondike) roots was assayed by cross-reaction on western blots and cryosections with an antibody against the PM-H(+)-ATPase from corn roots. Under conditions of reduced K availability, which have previously been shown to increase K influx by greater than 25-fold, there were only minor changes detected in PM-H(+)-ATPase levels. Antibody labeling of cryosections showed the relative distribution of PM-H(+)-ATPase among cell types in root tips and mature roots. Epidermal cells, both protoderm and mature root epidermis, including root hairs, had high levels of antibody binding. In mature roots, the stelar tissue showing the highest antibody binding was the companion cells of the phloem, followed by pericycle, xylem parenchyma, and endodermis.

Journal Article↗
PubMed1992

Studies of the uptake of nitrate in barley : v. Estimation of root cytoplasmic nitrate concentration using nitrate reductase activity-implications for nitrate influx.

The cytoplasmic NO(3) (-) concentration ([NO(3) (-)](c)) was estimated for roots of barley (Hordeum vulgare L. cv Klondike) using a technique based on measurement of in vivo nitrate reductase activity. At zero external NO(3) (-) concentration ([NO(3) (-)](o)), [NO(3) (-)](c) was estimated to be 0.66 mm for plants previously grown in 100 mum NO(3) (-). It increased linearly with [NO(3) (-)](o) between 2 and 20 mm, up to 3.9 mm at 20 mm [NO(3) (-)](o). The values obtained are much lower than previous estimates from compartmental analysis of barley roots. These observations support the suggestion (MY Siddiqi, ADM Glass, TJ Ruth [1991] J Exp Bot 42: 1455-1463) that the nitrate reductase-based technique and compartmental analysis determine [NO(3) (-)](c) for two separate pools; an active, nitrate reductase-containing pool (possibly located in the epidermal cells) and a larger, slowly metabolized storage pool (possibly in the cortical cells), respectively. Given the values obtained for [NO(3) (-)](c) and cell membrane potentials of -200 to -300 mV (ADM Glass, JE Schaff, LV Kochian [1992] Plant Physiol 99: 456-463), it is very unlikely that passive influx of NO(3) (-) is possible via the high-concentration, low-affinity transport system for NO(3) (-). This conclusion is consistent with the suggestion by Glass et al. that this system is thermodynamically active and capable of transporting NO(3) (-) against its electrochemical potential gradient.

Journal Article↗
PubMed1990

Potassium-dependent changes in the expression of membrane-associated proteins in barley roots : I. Correlations with k(rb) influx and root k concentration.

Barley (Hordeum vulgare L. cv Halcyon) seedlings which had been grown in full strength complete inorganic nutrient media (containing 6 millimolar K(+)) had high internal K(+) concentrations and low values of K(+) ((86)Rb(+)) influx when influx was measured from solutions containing 100 micromolar K(+). Transfer of these plants to solutions lacking K(+) resulted in significant reductions of root and shoot K(+) concentrations and values of K(+) ((86)Rb(+)) influx increased by greater than 10-fold within 3 days. When plants treated in this way were returned to complete solutions, containing K(+), the changes induced by K(+) deprivation were reversed. Parallel studies of microsomal membranes by means of SDS-PAGE demonstrated that the expression of a group of polypeptides increased or decreased in parallel with changes of K(+) ((86)Rb(+)) influx. Most prominent of these were 45 and 34 kilodalton polypeptides which specifically responded to K(+) status of the barley plants; their expression was not enhanced by N or P deprivation. The 45 kilodalton polypeptide was susceptible to degradation by a membrane associated protease when microsomes were washed in buffer containing 0.2 millimolar PMSF. This loss was prevented by increasing PMSF concentration to 2 millimolar.

Journal Article↗
PubMed1990

Studies of the Uptake of Nitrate in Barley: I. Kinetics of NO(3) Influx.

(13)NO(3) (-) was used to investigate patterns of NO(3) (-) influx into roots of barley plants (Hordeum vulgare L. cv Klondike) previously grown with (;induced') or without (;uninduced') a source of external NO(3) (-) ([NO(3) (-)](0)). In both induced and uninduced plants, (13)NO(3) (-) influx was biphasic in the range from 0.005 to 50 moles per cubic meter [NO(3) (-)](0). In the low concentration range (<1 mole per cubic meter for induced plants and <0.3 mole per cubic meter for uninduced plants), influx was saturable and V(max) and K(m) values for influx either increased or decreased according to NO(3) (-) pretreatment. By contrast, (13)NO(3) (-) influx in the high concentration range revealed a strictly linear concentration dependence. These fluxes appeared to be mediated by a constitutive, rather than an inducible, transport system.

Journal Article↗