Mouse strain-specific acute respiratory effects of nicotine unrelated to nicotine metabolism.

Plethysmograph measurement of respiratory phenotypes provides a highly sensitive means to study nicotine response in experimental model animals. We measured average respiratory frequency, tidal volume, minute volume and inspiratory time in C3H/HeJ and C57BL/6J mice subcutaneously administered 0.35 and 0.70 mg/kg nicotine. Both mouse strains showed significantly altered respiratory and locomotion phenotypes relative to saline-injected controls when administered the higher dose, but only C57BL/6J responded to the lower nicotine dose. Respiratory and locomotion phenotypes rarely differed significantly by sex. To investigate whether the strain-specific differences in nicotine sensitivity were related to differences in clearance, we followed up by measuring nicotine clearance in C3H/HeJ and C57BL/6J mice (0.35 mg/kg subcutaneous) and found sex differences in both strains, but no difference between strains.

Nicotine dependence is associated with functional variation in FMO3, an enzyme that metabolizes nicotine in the brain.

A common haplotype of the flavin-containing monooxygenase gene FMO3 is associated with aberrant mRNA splicing, a twofold reduction in in vivo nicotine N-oxidation and reduced nicotine dependence. Tobacco remains the largest cause of preventable mortality worldwide. CYP2A6, the primary hepatic nicotine metabolism gene, is robustly associated with cigarette consumption but other enzymes contribute to nicotine metabolism. We determined the effects of common variants in FMO3 on plasma levels of nicotine-N-oxide in 170 European Americans administered deuterated nicotine. The polymorphism rs2266780 (E308G) was associated with N-oxidation of both orally administered and ad libitum smoked nicotine (P⩽3.3 × 10-5 controlling for CYP2A6 genotype). In vitro, the FMO3 G308 variant was not associated with reduced activity, but rs2266780 was strongly associated with aberrant FMO3 mRNA splicing in both liver and brain (P⩽6.5 × 10-9). Surprisingly, in treatment-seeking European American smokers (n=1558) this allele was associated with reduced nicotine dependence, specifically with a longer time to first cigarette (P=9.0 × 10-4), but not with reduced cigarette consumption. As N-oxidation accounts for only a small percentage of hepatic nicotine metabolism we hypothesized that FMO3 genotype affects nicotine metabolism in the brain (unlike CYP2A6, FMO3 is expressed in human brain) or that nicotine-N-oxide itself has pharmacological activity. We demonstrate for the first time nicotine N-oxidation in human brain, mediated by FMO3 and FMO1, and show that nicotine-N-oxide modulates human α4ß2 nicotinic receptor activity in vitro. These results indicate possible mechanisms for associations between FMO3 genotype and smoking behaviors, and suggest nicotine N-oxidation as a novel target to enhance smoking cessation.

Wheat leaves emit nitrous oxide during nitrate assimilation.

Nitrous oxide (N(2)O) is a key atmospheric greenhouse gas that contributes to global climatic change through radiative warming and depletion of stratospheric ozone. In this report, N(2)O flux was monitored simultaneously with photosynthetic CO(2) and O(2) exchanges from intact canopies of 12 wheat seedlings. The rates of N(2)O-N emitted ranged from <2 pmol x m(-2) x s(-1) when NH(4)(+) was the N source, to 25.6 +/- 1.7 pmol x m(-2) x s(-1) (mean +/- SE, n = 13) when the N source was shifted to NO(3)(-). Such fluxes are among the smallest reported for any trace gas emitted by a higher plant. Leaf N(2)O emissions were correlated with leaf nitrate assimilation activity, as measured by using the assimilation quotient, the ratio of CO(2) assimilated to O(2) evolved. (15)N isotopic signatures on N(2)O emitted from leaves supported direct N(2)O production by plant NO(3)(-) assimilation and not N(2)O produced by microorganisms on root surfaces and emitted in the transpiration stream. In vitro production of N(2)O by both intact chloroplasts and nitrite reductase, but not by nitrate reductase, indicated that N(2)O produced by leaves occurred during photoassimilation of NO(2)(-) in the chloroplast. Given the large quantities of NO(3)(-) assimilated by plants in the terrestrial biosphere, these observations suggest that formation of N(2)O during NO(2)(-) photoassimilation could be an important global biogenic N(2)O source.

Nitrogen balance for wheat canopies (Triticum aestivum cv. Veery 10) grown under elevated and ambient CO2 concentrations.

We examined the hypothesis that elevated CO2 concentration would increase NO3- absorption and assimilation using intact wheat canopies (Triticum aestivum cv. Veery 10). Nitrate consumption, the sum of plant absorption and nitrogen loss, was continuously monitored for 23 d following germination under two CO2 concentrations (360 and 1000 micromol mol-1 CO2) and two root zone NO3- concentrations (100 and 1000 mmol m3 NO3-). The plants were grown at high density (1780 m-2) in a 28 m3 controlled environment chamber using solution culture techniques. Wheat responded to 1000 micromol mol-1 CO2 by increasing carbon allocation to root biomass production. Elevated CO2 also increased root zone NO3- consumption, but most of this increase did not result in higher biomass nitrogen. Rather, nitrogen loss accounted for the greatest part of the difference in NO3- consumption between the elevated and ambient [CO2] treatments. The total amount of NO3(-)-N absorbed by roots or the amount of NO3(-)-N assimilated per unit area did not significantly differ between elevated and ambient [CO2] treatments. Instead, specific leaf organic nitrogen content declined, and NO3- accumulated in canopies growing under 1000 micromol mol-1 CO2. Our results indicated that 1000 micromol mol-1 CO2 diminished NO3- assimilation. If NO3- assimilation were impaired by high [CO2], then this offers an explanation for why organic nitrogen contents are often observed to decline in elevated [CO2] environments.

Chlorate as a Transport Analog for Nitrate Absorption by Roots of Tomato.

Several studies have indicated that chlorate (ClO3-) and nitrate (NO3-) may share a common transport system in higher plants. Here, we compared the interactions between ClO3- and NO3-uptake by roots of intact tomato (Lycopersicon esculentum cv T5) plants. Exposure to ClO3- for more than 2 h inhibited both net ClO3- and K+ uptake, presumably because of ClO3- toxicity; consequently, subsequent measurements were conducted after short exposures to ClO3-. The apparent affinity and apparent maximum rate of absorption for net ClO3- and NO3- uptake were very similar. Interactions between ClO3- and NO3- transport were complex; 50 [mu]M NO3- acted as a mixed inhibitor of net ClO3- uptake, but 50 [mu]M ClO3- had no significant effect on net NO3- uptake, and 500 [mu]M ClO3- had no significant effect on 15NO3- influx. If the two ions share a single common high-affinity transport system, it is much more selective for NO3- than would be suggested by the similarity of net NO3- and ClO3- uptake kinetics. Our results indicate that, although NO3- may interfere with root ClO3- uptake, ClO3- is not a useful analog for the root high-affinity NO3- transport system.

Nitrogen dynamics in plant growth systems.

The predominant nitrogen source for the plants in closed environmental systems is the mineral nitrogen (i.e., nitrate and/or ammonium) in the nutrient medium. The following focuses on the processes through which plants obtain nitrate and ammonium from the rhizosphere and on the influences that each form has upon plant performance. Most plant species can sustain full growth at nitrate or ammonium concentrations that are over two orders of magnitude lower than those provided in most plant growth systems. Under the high concentrations (mM) normally used, root nitrogen absorption is downregulated: a) both the affinity and capacity of the transport systems for ammonium or nitrate are diminished, b) efflux of either ion becomes a significant percentage of influx, and c) root growth is inhibited. High concentrations also promote accumulation of ammonium or borate in plant tissues to potentially deleterious levels and foster microbial outbreaks. Several lines of evidence argue that roots in natural soils are normally exposed to lower concentrations (micromoles) of nitrate or ammonium: models of root nutrient absorption indicate that roots deplete rhizosphere nitrate and ammonium to such levels; the high-affinity transport systems for nitrate and ammonium have optimal control in this range; and root growth and development is maximized under such conditions. The high-affinity transport systems are distinct for nitrate and ammonium. In general, the affinity of the nitrate system for nitrate is less than the ammonium system for ammonium. Nitrate absorption is induced by the presence of ammonium or nitrate. Roots most rapidly absorb nitrate in the zone where root hairs emerge and ammonium in the zone of division near the apex. Nitrate absorption tends to alkalinize the rhizosphere, whereas ammonium absorption acidifies the rhizosphere. The energy requirements for absorption and assimilation of nitrate are several fold higher than those of ammonium. Root growth and elongation are more extensive when ammonium is provided as the sole nitrogen source, perhaps as a consequence of the lower energy requirements or the increased rhizosphere acidity.

Methylammonium as a Transport Analog for Ammonium in Tomato (Lycopersicon esculentum L.).

Methylammonium (CH3NH3+) has been widely used as an analog of ammonium (NH4+) for examining transport in bacteria and fungi. We compared the kinetics of root CH3NH3+ and NH4+ uptake from solution culture in intact tomato (Lycopersicon esculentum cv T5) plants. Efflux of NH4+ and CH3NH3+ was negligible. The apparent maximum rate of absorption (apparent Vmax) was similar for NH4+ and CH3NH3+, but the apparent affinity (apparent Km) was about 10-fold greater for NH4+ than for CH3NH3+. In characterizing the interaction between NH4+ and CH3NH3+ transport, we used [15N]NH4+ and [14C]CH3NH3+ as well as improved methods for analysis of nonisotopic CH3NH3+ and NH4+. CH3NH3+ acted as an inhibitor of NH4+ influx. Relatively low concentrations of NH4+ strongly inhibited CH3NH3+ influx. Treatments with 1 mM methionine sulfoximine that blocked NH4+ assimilation had little influence on NH4+ inhibition of CH3NH3+ influx. These results suggest that the two ions share a common transport system in tomato, but because this transport system has a much greater affinity for NH4+, CH3NH3+ may be used as a transport analog only when ambient concentrations of NH4+ are very low.

Root respiration associated with ammonium and nitrate absorption and assimilation by barley.

We examined nitrate assimilation and root gas fluxes in a wild-type barley (Hordeum vulgare L. cv Steptoe), a mutant (nar1a) deficient in NADH nitrate reductase, and a mutant (nar1a;nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the nar1a mutation influences assimilation only in the shoot and that exposure to NO(3) (-) induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient-the ratio of carbon dioxide evolved to oxygen consumed-did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and nar1a mutant, but had negligible effects on root gas fluxes in the nar1a;nar7w mutant. These results indicate that, under NH(4) (+) nutrition, 14% of root carbon catabolism is coupled to NH(4) (+) absorption and assimilation and that, under NO(3) (-) nutrition, 5% of root carbon catabolism is coupled to NO(3) (-) absorption, 15% to NO(3) (-) assimilation, and 3% to NH(4) (+) assimilation. The additional energy requirements of NO(3) (-) assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH(4) (+) and NO(3) (-) absorption and assimilation constitute a significant portion of root respiration.

CO(2) Inhibits Respiration in Leaves of Rumex crispus L.

Curly dock (Rumex crispus L.) was grown from seed in a glasshouse at an ambient CO(2) partial pressure of about 35 pascals. Apparent respiration rate (CO(2) efflux in the dark) of expanded leaves was then measured at ambient CO(2) partial pressure of 5 to 95 pascals. Calculated intercellular CO(2) partial pressure was proportional to ambient CO(2) partial pressure in these short-term experiments. The CO(2) level strongly affected apparent respiration rate: a doubling of the partial pressure of CO(2) typically inhibited respiration by 25 to 30%, whereas a decrease in CO(2) elicited a corresponding increase in respiration. These responses were readily reversible. A flexible, sensitive regulatory interaction between CO(2) (a byproduct of respiration) and some component(s) of heterotrophic metabolism is indicated.

Effects of Exposure to Ammonium and Transplant Shock upon the Induction of Nitrate Absorption.

In barley (Hordeum vulgare L. cv Steptoe) seedlings, the time course for induction of root nitrate absorption varied significantly with pretreatment. Net nitrate uptake of nitrogen-deprived plants more than doubled during the 12 hours after first exposure to nitrate. For these plants, gentle physical disturbance of the roots inhibited net nitrate absorption for more than 6 hours and potassium absorption for 2 hours. Pretreatment with ammonium appeared sufficient to induce nitrate absorption; plants either grown for 2 weeks on or exposed for only 10 hours to a medium containing ammonium as a sole nitrogen source showed high rates of net nitrate uptake when first shifted to a medium containing nitrate. Gentle physical manipulation of these plants inhibited nitrate absorption for 2 hours and potassium absorption for more than 12 hours. These results indicate (a) that experimental protocols should avoid physical manipulation of the roots when-ever possible and (b) that ammonium or a product of ammonium assimilation can induce nitrate absorption.

Measurement of net fluxes of ammonium and nitrate at the surface of barley roots using ion-selective microelectrodes.

Neutral carrier-based liquid membrane ion-selective microelectrodes for NH(4) (+) and NO(3) (-) were developed and used to investigate inorganic nitrogen acquisition in two varieties of barley, Hordeum vulgare L. cv Olli and H. vulgare L. cv Prato, originating in cold and warm climates, respectively. In the present paper, the methods used in the fabrication of ammonium- and nitrate-selective microelectrodes are described, and their application in the study of inorganic nitrogen uptake is demonstrated. Net ionic fluxes of NH(4) (+) and NO(3) (-) were measured in the unstirred layer of solution immediately external to the root surface. The preference for the uptake of a particular ionic form was examined by measuring the net flux of the predominant form of inorganic nitrogen, with and without the alternative ion in solution. Net flux of NH(4) (+) into the cold-adapted variety remained unchanged when equimolar concentrations (200 micromolar) of NH(4) (+) and NO(3) (-) were present. Similarly, net flux of NO(3) (-) into the warm-adapted variety was not affected when NH(4) (+) was also present in solution. The high temporal and spatial resolution afforded by ammonium- and nitrate-selective microelectrodes permits a detailed examination of inorganic nitrogen acquisition and its component ionic interactions.

Oxygen and carbon dioxide fluxes from barley shoots depend on nitrate assimilation.

A custom oxygen analyzer in conjunction with an infrared carbon dioxide analyzer and humidity sensors permitted simultaneous measurements of oxygen, carbon dioxide, and water vapor fluxes from the shoots of intact barley plants (Hordeum vulgare L. cv Steptoe). The oxygen analyzer is based on a calciazirconium sensor and can resolve concentration differences to within 2 microliters per liter against the normal background of 210,000 microliters per liter. In wild-type plants receiving ammonium as their sole nitrogen source or in nitrate reductase-deficient mutants, photosynthetic and respiratory fluxes of oxygen equaled those of carbon dioxide. By contrast, wild-type plants exposed to nitrate had unequal oxygen and carbon dioxide fluxes: oxygen evolution at high light exceeded carbon dioxide consumption by 26% and carbon dioxide evolution in the dark exceeded oxygen consumption by 25%. These results indicate that a substantial portion of photosynthetic electron transport or respiration generates reductant for nitrate assimilation rather than for carbon fixation or mitochondrial electron transport.

Root excision decreases nutrient absorption and gas fluxes.

The roots of barley plants (Hordeum vulgare L. cv Steptoe) were monitored before and after excision for net uptake of carbon dioxide, oxygen, ammonium, potassium, nitrate, and chloride and for their content of sucrose, glucose, fructose, and malic acid. All fluxes began to attenuate within 2 hours after excision. Net potassium uptake returned to control levels 6 hours after excision, but carbon dioxide, oxygen, ammonium, and nitrate fluxes continued to diminish for the remainder of the observation period. The addition of 0.1 molar glucose or 0.1 molar sucrose to excision medium had no significant effect on these changes in ion and gas fluxes. Net chloride uptake was negligible for all treatments. Sugar and malic acid content of the root declined after excision. Sucrose and glucose levels remained depressed for the entire observation period, whereas fructose and malic acid returned to control levels after 9 hours. These results indicate that excision has profound, adverse effects on root respiration and the absorption of mineral nitrogen.

The influence of ammonium and chloride on potassium and nitrate absorption by barley roots depends on time of exposure and cultivar.

Net uptakes of K(+) and NO(3) (-) were monitored simultaneously and continuously for two barley (Hordeum vulgare) cultivars, Prato and Olli. The cultivars had similar rates of net K(+) and NO(3) (-) uptake in the absence of NH(4) (+) or Cl(-). Long-term exposure (over 6 hours) to media which contained equimolar mixtures of NH(4) (+), K(+), Cl(-), or NO(3) (-) affected the cultivars very differently: (a) the presence of NH(4) (+) as NH(4)Cl stimulated net NO(3) (-) uptake in Prato barley but inhibited net NO(3) (-) uptake in Olli barley; (b) Cl(-) inhibited net NO(3) (-) uptake in Prato but had little effect in Olli; and (c) NH(4) (+) as (NH(4))(2)SO(4) inhibited net K(+) uptake in Prato but had little effect in Olli. Moreover, the immediate response to the addition of an ion often varied significantly from the long-term response; for example, the addition of Cl(-) initially inhibited net K(+) uptake in Olli barley but, after a 4 hour exposure, it was stimulatory. For both cultivars, net NH(4) (+) and Cl(-) uptake did not change significantly with time after these ions were added to the nutrient medium. These data indicate that, even within one species, there is a high degree of genotypic variation in the control of nutrient absorption.

Plant economics.

Plants and small business firms face similar difficulties in their struggle for existence. Survival is most tenuous during the juvenile stages, seedlings and new businesses having the highest mortality rates. Successful establishment in either an ecological or a business community requires carving out a niche. Proliferation of a plant or business (the major strategy for ensuring persistence) depends on productivity which in turn depends on efficient acquisition and allocation of resources. A pattern of resource acquisition and allocation that is efficient in one climate may prove disastrous in another. Businesses must engage in long-term as well as short-term planning, and they base such planning on microeconomic theory; this theory has also provided insights into the responses of higher plants to different selective pressures.

Relationship between Mineral Nitrogen Influx and Transpiration in Radish and Tomato.

Net ammonium and nitrate influx were independent of transpiration rate for intact seedlings of both a wild species of radish (Raphanus raphanistrum) and a wilty tomato mutant (Lycopersicon esculentum Mill. cv RR flacca).

Differences in steady-state net ammonium and nitrate influx by cold- and warm-adapted barley varieties.

A flowing nutrient culture system permitted relatively rapid determination of the steady-state net nitrogen influx by an intact barley (Hardeum vulgare L. cv Kombar and Olli) plant. Ion-selective electrodes monitored the depletion of ammonium and nitrate from a nutrient solution after a single pass through a root cuvette. Influx at concentrations as low as 4 micromolar was measured. Standard errors for a sample size of three plants were typically less than 10% of the mean.When grown under identical conditions, a variety of barley bred for cold soils had higher nitrogen influx rates at low concentrations and low temperatures than one bred for warm soils, whereas the one bred for warm soils had higher influx rates at high concentrations and high temperatures. Ammonium was more readily absorbed than nitrate by both varieties at all concentrations and temperatures tested. Ammonium and nitrate influx in both varieties were equally inhibited by low temperatures.

Diurnal Ion Fluctuations in the Mesophyll Tissue of the Crassulacean Acid Metabolism Plant Mesembryanthemum crystallinum.

Both laboratory- and field-grown Mesembryanthemum crystallinum plants exhibited large scale diurnal ion fluctuations. In mesophyll tissue, potassium and sodium levels varied in conjunction with acid levels while chloride levels varied in opposition. Thus, dark CO(2) fixation in this Crassulacean acid metabolism species seems analogous to the common plant process of malate synthesis to balance cation surplus. Sodium levels in the epidermis appeared to fluctuate in opposition to those in the mesophyll. It is proposed that inorganic cations cycle between mesophyll and epidermal tissue to balance malate accumulation and to produce stomatal opening in the dark.

Salt Requirement for Crassulacean Acid Metabolism in the Annual Succulent, Mesembryanthemum crystallinum.

In experiments with the facultative Crassulacean acid metabolism (CAM) species, Mesembryanthemum crystallinum, only plants which received high levels of inorganic salts fixed substantial amounts of CO(2) by the CAM pathway. Equivalent osmolarities of polyethylene glycol 6000 did not yield any CAM fixation. Plant water potential and turgor pressure had no detectable influence on the amount of CAM fixation. These observations rule out the possibility that the inorganic ions were acting as osmotic agents.Carbon dioxide and water exchange analysis showed that when water supply was not limiting, salt-deprived plants sustained higher reductive pentose phosphate cycle carbon fixation rates than salt-treated plants. Under water stress conditions, salt-deprived plants using only the reductive pentose phosphate cycle pathway assimilated less carbon and were less efficient in their water use than salt-treated plants using predominately the CAM pathway. These results support the hypothesis that the ability to use the CAM pathway reduces the capacity for reductive pentose phosphate cycle fixation but permits higher productivity in water-limited environments.