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Background: Alzheimer's disease (AD) is the most prevalent form of dementia worldwide and is characterized by progressive memory loss and cognitive impairment. Our understanding of AD pathogenesis is limited and no effective disease-modifying treatment is available. Mitochondria are cytoplasmic organelles critical to the homeostatic regulation of glucose and energy in the cell., Methods: Mitochondrial abnormalities are found early in the course of AD and dysfunctional mitochondria are involved in AD progression. The resulting respiratory chain impairment, neuronal apoptosis, and generation of reactive oxygen species are highly damaging to neurons. Restoration of mitochondrial function may provide a novel therapeutic strategy for AD., Results: This review discusses the specifics of mitochondrial fragmentation, imbalances in fission and fusion, and DNA damage seen in AD and the contribution of compromised mitochondrial activity to AD etiopathogenesis. It explores how an understanding of the processes underlying mitochondrial failure may lead to urgently needed treatment innovations. It considers individual mitochondrial proteins that have emerged as promising drug targets and evaluates neuroprotective agents that could improve the functional state of mitochondria in the setting of AD., Conclusions: There is great promise in exploring original approaches to preserving mitochondrial viability as a means to achieve breakthroughs in treating AD., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Prostate cancer is the second most common form of cancer in men. For advanced, high risk prostate cancer, androgen deprivation therapy (ADT) is the preferred treatment and can induce remission, but resistance to ADT brings biochemical recurrence and progression of cancer. ADT brings adverse effects such as erectile dysfunction, decreased libido, and diminished physical strength. It is estimated that between 25 and 50% of men on ADT manifest some form of cognitive dysfunction that may be self-reported or reported by a family member. There is concern that impaired cognitive function with ADT is due to loss of testosterone support. Testosterone and its metabolites are known to possess neuroprotective properties. While a direct causal relationship between ADT and cognitive decline in prostate cancer patients has not been established, this review describes the controversy surrounding the possible connection between ADT and neurocognitive deterioration. The cellular and molecular mechanisms believed to underlie the protection of neuronal integrity by androgens are discussed. Results from animal models and human clinical studies are presented. Finally, we call attention to lifestyle modifications that may minimize cognitive issues in prostate cancer patients., (© 2021. The Author(s), under exclusive licence to Federación de Sociedades Españolas de Oncología (FESEO).)

Apolipoprotein (apo) B, the critical structural protein of the atherogenic lipoproteins, has two major isoforms: apoB48 and apoB100. ApoB48 is found in chylomicrons and chylomicron remnants with one apoB48 molecule per chylomicron particle. Similarly, a single apoB100 molecule is contained per particle of very-low-density lipoprotein (VLDL), intermediate density lipoprotein, LDL and lipoprotein(a). This unique one apoB per particle ratio makes plasma apoB concentration a direct measure of the number of circulating atherogenic lipoproteins. ApoB levels indicate the atherogenic particle concentration independent of the particle cholesterol content, which is variable. While LDL, the major cholesterol-carrying serum lipoprotein, is the primary therapeutic target for management and prevention of atherosclerotic cardiovascular disease, there is strong evidence that apoB is a more accurate indicator of cardiovascular risk than either total cholesterol or LDL cholesterol. This review examines multiple aspects of apoB structure and function, with a focus on the controversy over use of apoB as a therapeutic target in clinical practice. Ongoing coronary artery disease residual risk, despite lipid-lowering treatment, has left patients and clinicians with unsatisfactory options for monitoring cardiovascular health. At the present time, the substitution of apoB for LDL-C in cardiovascular disease prevention guidelines has been deemed unjustified, but discussions continue.

Background: The goal of slowing or halting the development of Alzheimer disease (AD) has resulted in the huge allocation of resources by academic institutions and pharmaceutical companies to the development of new treatments. The etiology of AD is elusive, but the aggregation of amyloid-β and tau peptide and oxidative processes are considered critical pathologic mechanisms. The failure of drugs with multiple mechanisms to meet efficacy outcomes has caused several companies to decide not to pursue further AD studies and has left the field essentially where it has been for the past 15 years. Efforts are underway to develop biomarkers for detection and monitoring of AD using genetic, imaging, and biochemical technology, but this is of minimal use if no intervention can be offered., Review Summary: In this review, we consider the natural progression of AD and how it continues despite present attempts to modify the amyloid-related machinery to alter the disease trajectory. We describe the mechanisms and approaches to AD treatment targeting amyloid, including both passive and active immunotherapy as well as inhibitors of enzymes in the amyloidogenic pathway., Conclusion: Lessons learned from clinical trials of amyloid reduction strategies may prove crucial for the leap forward toward novel therapeutic targets to treat AD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease with high mortality that commonly occurs in middle-aged and older adults. IPF, characterized by a decline in lung function, often manifests as exertional dyspnea and cough. Symptoms result from a fibrotic process driven by alveolar epithelial cells that leads to increased migration, proliferation, and differentiation of lung fibroblasts. Ultimately, the differentiation of fibroblasts into myofibroblasts, which synthesize excessive amounts of extracellular matrix proteins, destroys the lung architecture. However, the factors that induce the fibrotic process are unclear. Diagnosis can be a difficult process; the gold standard for diagnosis is the multidisciplinary conference. Practical biomarkers are needed to improve diagnostic and prognostic accuracy. High-resolution computed tomography typically shows interstitial pneumonia with basal and peripheral honeycombing. Gas exchange and diffusion capacity are impaired. Treatments are limited, although the anti-fibrotic drugs pirfenidone and nintedanib can slow the progression of the disease. Lung transplantation is often contraindicated because of age and comorbidities, but it improves survival when successful. The incidence and prevalence of IPF has been increasing and there is an urgent need for improved therapies. This review covers the detailed cellular and molecular mechanisms underlying IPF progression as well as current treatments and cutting-edge research into new therapeutic targets., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest directly relevant to the content of this paper., (Copyright © 2020 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative brain disorder associated with relentlessly progressive cognitive impairment and memory loss. AD pathology proceeds for decades before cognitive deficits become clinically apparent, opening a window for preventative therapy. Imbalance of clearance and buildup of amyloid β and phosphorylated tau proteins in the central nervous system is believed to contribute to AD pathogenesis. However, multiple clinical trials of treatments aimed at averting accumulation of these proteins have yielded little success, and there is still no disease-modifying intervention. Here, we discuss current knowledge of AD pathology and treatment with an emphasis on emerging biomarkers and treatment strategies., Competing Interests: Competing interests: None declared., (© American Federation for Medical Research 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Cardiovascular disease (CVD) remains the leading cause of death worldwide, despite multiple treatment options. In addition to elevated lipid levels, oxidative stress and inflammation are key factors driving atherogenesis and CVD. New strategies are required to mitigate risk and most urgently for statin-intolerant patients. The neuropeptide hormone oxytocin, synthesized in the brain hypothalamus, is worthy of consideration as a CVD ancillary treatment because it moderates factors directly linked to atherosclerotic CVD such as inflammation, weight gain, food intake and insulin resistance. Though initially studied for its contribution to parturition and lactation, oxytocin participates in social attachment and bonding, associative learning, memory and stress responses. Oxytocin has shown promise in animal models of atherosclerosis and in some human studies as well. A number of properties of oxytocin make it a candidate CVD treatment. Oxytocin not only lowers fat mass and cytokine levels, but also improves glucose tolerance, lowers blood pressure and relieves anxiety. Further, it has an important role in communication in the gut-brain axis that makes it a promising treatment for obesity and type 2 diabetes. Oxytocin acts through its receptor which is a class I G-protein-coupled receptor present in cells of the vascular system including the heart and arteries. While oxytocin is not used for heart disease at present, residual CVD risk remains in a substantial portion of patients despite multidrug regimens, leaving open the possibility of using the endogenous nonapeptide as an adjunct therapy. This review discusses the possible role for oxytocin in human CVD prevention and treatment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (© 2014 John Wiley & Sons Ltd.)

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.

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.

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.

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-β-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., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

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.

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.

This review article discusses the historical origin of our continuously evolving model of the etiology of atherosclerotic cardiovascular disease. The basic molecular biologic concepts underlying the development of coronary artery disease and the dynamic connection between the immune system and arterial integrity are explored. Emphasis is placed on the role of inflammation as a driving force in the process of atherosclerosis and vascular endothelium as a modulating factor in the pathogenesis of coronary artery disease.

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.

With about 200,000 phytochemicals in existence, identifying those of biomedical significance is a mammoth task. In the postgenomic era, relating metabolite fingerprints, abundances, and profiles to genotype is also a large task. Ion analysis using Fourier transformed ion cyclotron resonance mass spectrometry (FT-ICR-MS) may provide a high-throughput approach to measure genotype dependency of the inferred metabolome if reproducible techniques can be established. Ion profile inferred metabolite fingerprints are coproducts. We used FT-ICR-MS-derived ion analysis to examine gdhA (glutamate dehydrogenase (GDH; EC 1.4.1.1)) transgenic Nicotiana tabacum (tobacco) carrying out altered glutamate, amino acid, and carbon metabolisms, that fundamentally alter plant productivity. Cause and effect between gdhA expression, glutamate metabolism, and plant phenotypes was analyzed by (13) NH(4)(+) labeling of amino acid fractions, and by FT-ICR-MS analysis of metabolites. The gdhA transgenic plants increased (13)N labeling of glutamate and glutamine significantly. FT-ICR-MS detected 2,012 ions reproducible in 2 to 4 ionization protocols. There were 283 ions in roots and 98 ions in leaves that appeared to significantly change abundance due to the measured GDH activity. About 58% percent of ions could not be used to infer a corresponding metabolite. From the 42% of ions that inferred known metabolites we found that certain amino acids, organic acids, and sugars increased and some fatty acids decreased. The transgene caused increased ammonium assimilation and detectable ion variation. Thirty-two compounds with biomedical significance were altered in abundance by GDH including 9 known carcinogens and 14 potential drugs. Therefore, the GDH transgene may lead to new uses for crops like tobacco.

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.

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.

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.

The NR72.1 gene codes for a high-affinity nitrate transporter in Arabidopsis thaliana. To examine the regulation of NRT2.1 gene expression, we used a promoter-beta-glucuronidase (GUS) fusion and found that the NRT2.1 promoter directs expression to the epidermal, cortical and endodermal cell layers of mature root parts. The gene appeared to be expressed essentially in roots, but was also present in the leaf hydathodes. Investigation of NRT2.1 expression pattern during the plant developmental cycle showed that it increased rapidly during early vegetative growth, peaked prior to floral stem emergence, and decreased to very low levels in flowering and silique-bearing plants. Experiments with various nitrogen supply regimes demonstrated the induction of NRT2.1 expression by nitrate and repression by amino acids. Amino acid analysis showed that this repression was specifically related to increased internal glutamine, suggesting a role for this particular amino acid in nitrogen signalling responsible for nitrate uptake regulation. Taken together, our results support the hypothesis that the NRT2.1 gene codes for a major component of the inducible high-affinity transport system for nitrate, which is spatially and developmentally controlled at the transcriptional level. Surprisingly, NRT2.1 was not expressed in younger root parts, although a similar rate of nitrate influx was observed in both young and old root samples. This lack of correlation between nitrate influx and NRT2.1 expression suggests that another high-affinity nitrate transporter operates in root tips.

In order to investigate the molecular basis of high-affinity ammonium absorption by roots of rice plants (Oryza sativa subspecies indica) the expression patterns of three members of the AMT1 family of genes in rice seedling roots in response to altered nitrogen provision and diurnal changes in irradiance were examined. The 13NH4+ influx and transcript levels of OsAMT1.1 in roots decreased several fold within 48 h when plants acclimated to 10 micro m external NH4+ for 3 weeks were transferred to 10 mm NH4+. Likewise when plants acclimated in 10 mm NH4+ were transferred to 10 micro m NH4+, there was an equally rapid up-regulation of OsAMT1.1 and 13NH4+ influx in the roots. Changes in transcript abundance of OsAMT1.2 following these treatments were approximately 50% less than in OsAMT1.1, and changes of OsAMT1.3 expression were even less. By contrast, in response to the diurnal changes of irradiance, root transcript abundance of OsAMT1.3 and 15NH4+ influx increased approximately three-fold late in the photoperiod, whereas OsAMT1.1 and OsAMT1.2 exhibited only modest changes. The present results suggest that high-affinity NH4+ influx is differentially regulated at the transcriptional level through the expression of three members of the OsAMT1 family of genes in roots of rice seedlings in response to changes of N status and daily irradiance. In general, these findings are in agreement with earlier observations in Arabidopsis and tomato.

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.

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.

Inorganic nitrogen concentrations in soil solutions vary across several orders of magnitude among different soils and as a result of seasonal changes. In order to respond to this heterogeneity, plants have evolved mechanisms to regulate and influx. In addition, efflux analysis using (13)N has revealed that there is a co-ordinated regulation of all component fluxes within the root, including biochemical fluxes. Physiological studies have demonstrated the presence of two high-affinity transporter systems (HATS) for and one HATS for in roots of higher plants. By contrast, in Arabidopsis thaliana there exist seven members of the NRT2 family encoding putative HATS for and five members of the AMT1 family encoding putative HATS for. The induction of high-affinity transport and Nrt2.1 and Nrt2.2 expression occur in response to the provision of, while down-regulation of these genes appear to be due to the effects of glutamine. High-affinity transport and AMT1.1 expression also appear to be subject to down-regulation by glutamine. In addition, there is evidence that accumulated and may act post-transcriptionally on transporter function. The present challenge is to resolve the functions of all of these genes. In Aspergillus nidulans and Chlamydomonas reinhardtii there are but two high-affinity transporters and these appear to have undergone kinetic differentiation that permits a greater efficiency of absorption over the wide range of concentration normally found in nature. Such kinetic differentiation may also have occurred among higher plant transporters. The characterization of transporter function in higher plants is currently being inferred from patterns of gene expression in roots and shoots, as well as through studies of heterologous expression systems and knockout mutants.

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.

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.

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.

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.

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.

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.

NO3- uptake by plant roots is rapidly inhibited by exposure to NH4+. The rapidity of the effect has led to the presumption that the inhibition results from the direct effects of NH4+ at the plasma membrane. The mechanism of this inhibition, however, has been in contention. In the present study we used the radiotracer 13N to determine the relative effects of short-term exposures to NH4+ on the 13NO3- influx, efflux, and partitioning of absorbed 13N in barley (Hordeum vulgare) roots. Plants were grown without NO3- or NO2- (uninduced for NO3- uptake), or with 0.1, 1.0, 10 mM NO3-, or 0. 1 mM NO2- (to generate plant roots induced for NO3- uptake). Exposure to 1 mM NH4+ strongly reduced influx; the effect was most pronounced in plants induced for NO3- uptake when NO3- absorption was measured at low external NO3-. At higher [NO3-] and in uninduced plants the inhibitory effect was much diminished, indicating that NH4+ inhibition of influx was mediated via effects on the inducible high-affinity transport system rather than on the constitutive high-affinity transport system or the low-affinity transport system. Exposure to NH4+ also caused increased NO3- efflux; the largest effect was at low external [NO3-] in uninduced plants. In absolute terms, the reduction of influx made the dominant contribution to the observed reduction of net uptake of NO3-. Differences in response between plants induced with NO3- and those induced with NO2- indicate that NO2- may not be an appropriate analog for NO3- under all conditions.

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.

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.

Many reports have shown that plant growth and yield is superior on mixtures of NO3- and NH4+ compared with provision of either N source alone. Despite its clear practical importance, the nature of this N-source synergism at the cellular level is poorly understood. In the present study we have used the technique of compartmental analysis by efflux and the radiotracer 13N to measure cellular turnover kinetics, patterns of flux partitioning, and cytosolic pool sizes of both NO3- and NH4+ in seedling roots of rice (Oryza sativa L. cv IR72), supplied simultaneously with the two N sources. We show that plasma membrane fluxes for NH4+, cytosolic NH4+ accumulation, and NH4+ metabolism are enhanced by the presence of NO3-, whereas NO3- fluxes, accumulation, and metabolism are strongly repressed by NH4+. However, net N acquisition and N translocation to the shoot with dual N-source provision are substantially larger than when NO3- or NH4+ is provided alone at identical N concentrations.

High-affinity K+ uptake in plant roots is rapidly up-regulated when K+ is withheld and down-regulated when K+ is resupplied. These processes make important contributions to plant K+ homeostasis. A cDNA coding for a high-affinity K+ transporter, HKT1, was earlier cloned from wheat (Triticum aestivum L.) roots and functionally characterized. We demonstrate here that in both barley (Hordeum vulgare L.) and wheat roots, a rapid and large up-regulation of HKT1 mRNA levels resulted when K+ was withdrawn from growth media. This effect was specific for K+; withholding N caused a modest reduction of HKT1 mRNA levels. Up-regulation of HKT1 transcript levels in barley roots occurred within 4 h of removing K+, which corresponds to the documented increase of high-affinity K+ uptake in roots following removal of K+. Increased expression of HKT1 mRNA was evident before a decline in total root K+ concentration could be detected. Resupply of 1 mM K+ was sufficient to strongly reduce HKT1 transcript levels. In wheat root cortical cells, both membrane depolarizations in response to 100 &mgr;M K+, Cs+, and Rb+, and high-affinity K+ uptake were enhanced by K+ deprivation. Thus, in both plant systems the observed physiological changes associated with manipulating external K+ supply were correlated with levels of HKT1 mRNA expression. Implications of these findings for K+ sensing and regulation of the HKT1 mRNA levels in plant roots are discussed.

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.

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.

[(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.

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.

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.

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.

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) (-).

(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.

Q(10) values for (13)NO(3) (-) influx were determined in ;uninduced' (NO(3) (-)-starved) and ;induced' (NO(3) (-)-pretreated) roots of barley (Hordeum vulgare L.) plants at various concentrations of external NO(3) (-) ([NO(3) (-)](0)). At 0.02 mole per cubic meter [NO(3) (-)](0), Q(10) values for influx were from 3 to 4 between 5 and 10 degrees C. As [NO(3) (-)](0) increased Q(10) values decreased, reaching values of 1.2 and 2.0, respectively, at 20 moles per cubic meter in uninduced and induced plants. The metabolic dependence of (13)NO(3) (-) influx at low and high [NO(3) (-)](0) (0.1 and 20.0 moles per cubic meter, respectively) in uninduced and induced plants was probed by the use of various inhibitors. These experiments confirmed the findings of the Q(10) studies, demonstrating that at low [NO(3) (-)](0) (13)NO(3) (-) influx was extremely sensitive to metabolic inhibition. By contrast, at high [NO(3) (-)](0), influx was relatively insensitive to the presence of inhibitors.

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.

Potassium influx isotherms were obtained for 10 cultivars of barley using plants which had been grown with or without potassium (high K(+) and low K(+) plants, respectively), and the cultivars ranked with respect to K(m) or V(max) values for influx with a view to using these rankings as a predictive measure of long term performance under conditions of potassium-limited growth. Analyses of these rankings revealed significant differences between cultivars. Net uptake rates for low K(+) plants, determined over a 24-hour period, confirmed the differences between upper (Herta) and lower (Conquest) ranked cultivars, and established similar differences in the rates of translocation to the shoot. Efflux analyses showed no differences in potassium efflux from the cytoplasm or from the vacuole for these cultivars. Growth rate studies under different conditions of potassium limitation indicated, with some exceptions, strong positive correlations between ranks accorded cultivars on the basis of influx kinetics and those based upon growth rates.

Plasmalemma influx isotherms for K(+) were measured in the system I concentration range (0.01-0.32 mm), for barley (Hordeum vulgare L.) roots of varying internal K(+) concentration, and Km values for influx calculated. In plants grown for several days in CaSO(4) or in CaSO(4) plus KCl solutions, as well as in plants grown in CaSO(4) for several days and then rapidly loaded with KCl during a pretreatment period, Michaelis constant values were positively correlated with internal K(+) concentrations. Influx of K(+) is shown to be sigmoidally related to internal K(+) concentration and Hill plots of influx data give linear transformations with n = 4. This information is taken as support for an allosteric model for the regulation of K(+) influx in which the "carrier" is envisaged as possessing a single external binding site for K(+) as well as four internal sites for allosteric control of influx.