Humic acid differentially improves nitrate kinetics under low‐ and high‐affinity systems and alters the expression of plasma membrane H+‐ATPases and nitrate transporters in rice

Humic acids (HAs) have a major effect on nutrient uptake, metabolism, growth and development in plants. Here, we evaluated the effect of HA pretreatment applied with a nutrient solution on the uptake kinetics of nitrate nitrogen (N‐NO₃^?) and the metabolism of nitrogen (N) in rice under conditions of high and low NO₃^? supply. In addition, the kinetic parameters of NO₃^? uptake, N metabolites, and nitrate transporters (NRTs) and the plasma membrane (PM) H⁺‐ATPase gene expression were examined. The plants were grown in a growth chamber with modified Hoagland and Arnon solution until 21 days after germination (DAG), and they were then transferred to a solution without N for 48 h and then to another solution without N and with and without the addition of HAs for another 48 h. After this period of N deprivation, the plants received new nutrient solutions containing 0.2 and 2.0 mM N‐NO₃^?. Treatment of rice plants with HA promoted the induction of the genes OsNRT2.1‐2.2/OsNAR2.1 and some isoforms PM H⁺‐ATPase in roots. The application of HAs differentially modified the parameters of the uptake kinetics of NO₃^? under both concentrations. When grown with 0.2 mM NO₃^?, the plants pretreated with HA had lower K_m and C_min values as well as a higher V_max/K_m ratio. When grown with 2 mM NO₃^?, the plants pretreated with HA had a higher V_max value, a greater root and shoot mass, and a lower root/shoot ratio. The N fractions were also altered by pretreatment with HA, and a greater accumulation of NO₃^? and N‐amino was observed in the roots and shoots, respectively, of plants pretreated with HA. The results suggest that pretreatment with HA modifies root morphology and gene expression of PM H⁺‐ATPases and NO₃^? transporters, resulting in a greater efficiency of NO₃^? acquisition by high‐ and low‐affinity systems.     

Assessing the sources and magnitude of diurnal nitrate variability in the San Joaquin River (California) with an in situ optical nitrate sensor and dual nitrate isotopes

1. We investigated diurnal nitrate (NO₃⁻) concentration variability in the San Joaquin River using an in situ optical NO₃⁻ sensor and discrete sampling during a 5‐day summer period characterized by high algal productivity. Dual NO₃⁻ isotopes (δ¹⁵N_NO3 and δ¹⁸O_NO3) and dissolved oxygen isotopes (δ¹⁸O_DO) were measured over 2 days to assess NO₃⁻ sources and biogeochemical controls over diurnal time‐scales. 2. Concerted temporal patterns of dissolved oxygen (DO) concentrations and δ¹⁸O_DO were consistent with photosynthesis, respiration and atmospheric O₂ exchange, providing evidence of diurnal biological processes independent of river discharge. 3. Surface water NO₃⁻ concentrations varied by up to 22% over a single diurnal cycle and up to 31% over the 5‐day study, but did not reveal concerted diurnal patterns at a frequency comparable to DO concentrations. The decoupling of δ¹⁵N_NO3 and δ¹⁸O_NO3 isotopes suggests that algal assimilation and denitrification are not major processes controlling diurnal NO₃⁻ variability in the San Joaquin River during the study. The lack of a clear explanation for NO₃⁻ variability likely reflects a combination of riverine biological processes and time‐varying physical transport of NO₃⁻ from upstream agricultural drains to the mainstem San Joaquin River. 4. The application of an in situ optical NO₃⁻ sensor along with discrete samples provides a view into the fine temporal structure of hydrochemical data and may allow for greater accuracy in pollution assessment.     

COMPARISONS OF NITRATE UPTAKE, STORAGE, AND REDUCTION IN MARINE DIATOMS AND FLAGELLATES

Diatoms, but not flagellates, have been shown to increase rates of nitrogen release after a shift from a low growth irradiance to a much higher experimental irradiance. We compared NO₃ ^? uptake kinetics, internal inorganic nitrogen storage, and the temperature dependence of the NO₃ ^? reduction enzymes, nitrate (NR) and nitrite reductase (NiR), in nitrogen‐replete cultures of 3 diatoms (Chaetoceros sp., Skeletonema costatum, Thalassiosira weissflogii) and 3 flagellates (Dunaliella tertiolecta, Pavlova lutheri, Prorocentrum minimum) to provide insight into the differences in nitrogen release patterns observed between these species. At NO₃ ^? concentrations <40 μmol‐N·L ^{? 1}, all the diatom species and the dinoflagellate P. minimum exhibited saturating kinetics, whereas the other flagellates, D. tertiolecta and P. lutheri, did not saturate, leading to very high estimated K _s values. Above ～60 μmol‐N·L ^{? 1}, NO₃ ^? uptake rates of all species tested continued to increase in a linear fashion. Rates of NO₃ ^? uptake at 40 μmol‐N·L ^{? 1}, normalized to cellular nitrogen, carbon, cell number, and surface area, were generally greater for diatoms than flagellates. Diatoms stored significant amounts of NO₃ ^? internally, whereas the flagellate species stored significant amounts of NH₄ ⁺ . Half‐saturation concentrations for NR and NiR were similar between all species, but diatoms had significantly lower temperature optima for NR and NiR than did the flagellates tested in most cases. Relative to calculated biosynthetic demands, diatoms were found to have greater NO₃ ^? uptake and NO₃ ^? reduction rates than flagellates. This enhanced capacity for NO₃ ^? uptake and reduction along with the lower optimum temperature for enzyme activity could explain differences in nitrogen release patterns between diatoms and flagellates after an increase in irradiance.     

The nitrogen balance of Raphanus sativus X raphanistrum plants. I. Daily nitrogen use under high nitrate supply

Abstract Growth-chamber cultivated Raphanus plants accumulate nitrate during their vegetative growth. After 25 days of growth at a constant supply to the roots of 1 mol m^?3 (NO^?₃) in a balanced nutrient solution, the oldest leaves (eight-leaf stage) accumulated 2.5% NO^?₃-nitrogen (NO₃-N) in their lamina, and almost 5% NO₃-N in their petioles on a dry weight basis. This is equivalent to approximately 190 and 400 mol^?3 m^?3 concentration of NO^?₃ in the lamina and the petiole, respectively, as calculated on a total tissue water content basis. Measurements were made of root NO^?₃ uptake, NO^?₃ fluxes in the xylem, nitrate uptake by the mesophyll cells, and nitrate reduction as measured by an in vivo test. NO^?₃ uptake by roots and mesophyll cells was greater in the light than in the dark. The NO^?₃ concentration in the xylem fluid was constant with leaf age, but showed a distinct daily variation as a result of the independent fluxes of root uptake, transpiration and mesophyll uptake. NO^?₃ was reduced in the leaf at a higher rate in the light than in the dark. The reduction was inhibited at the high concentrations calculated to exist in the mesophyll vacuoles, but reduction continued at a low rate, even when there was no supply from the incubation medium. Sixty-four per cent of the NO^?₃ influx was turned into organic nitrogen, with the remaining NO^?₃ accumulating in both the light and the dark.     

A comparative kinetic analysis of nitrate and ammonium influx in two early-successional tree species of temperate and boreal forest ecosystems

Root NO₃ ^? and NH₄ ⁺ influx systems of two early‐successional species of temperate (trembling aspen: Populus tremuloides Michx.) and boreal (lodgepole pine: Pinus contorta Dougl. ex Loud. var. latifolia Engelm.) forest ecosystems were characterized. NO₃ ^? and NH₄ ⁺ influxes were biphasic, consisting of saturable high‐affinity (HATS) and constitutive non‐saturable low‐affinity transport systems (LATS) that were evident at low and relatively high N concentrations, respectively. NO₃ ^? influx via HATS was inducible (IHATS); nitrate pre‐treatment resulted in 8–10‐fold increases in the V_max for influx in both species. By contrast, HATS for NH₄ ⁺ were entirely constitutive. In both species, V_max values for NH₄ ⁺ influx were higher than those for NO₃ ^? uptake; the differences were larger in pine (6‐fold) than aspen (1·8‐fold). In aspen, the K_m for NH₄ ⁺ influx by HATS was approximately 3‐fold higher than for IHATS NO₃ ^? influx, while in pine the K_m for IHATS NO₃ ^? influx was approximately 3‐fold higher than for NH₄ ⁺ influx. The aspen IHATS for NO₃ ^? influx appeared to be more efficient than that of pine (V_max values for aspen being approximately 10‐fold higher and K_m values being approximately 13‐fold lower than for pine). By contrast, only small differences in values for the NH₄ ⁺ HATS were evident between the two species. The kinetic parameters observed here probably result from adaptations to the N availabilities in their respective natural habitats; these may contribute to the distribution and niche separation of these species.     

Iodate Reduction by Shewanella oneidensis Does Not Involve Nitrate Reductase

Microbial iodate (IO₃^?) reduction is a major component of iodine biogeochemical cycling and is the basis of alternative strategies for remediation of iodine-contaminated environments. The molecular mechanism of microbial IO₃^? reduction, however, is not well understood. In several microorganisms displaying IO₃^? and nitrate (NO₃^?) reduction activities, NO₃^? reductase is postulated to reduce IO₃^? as alternate electron acceptor. In the present study, whole genome analyses of 25 NO₃^?-reducing Shewanella strains identified various combinations of genes encoding one assimilatory (cytoplasmic Nas) and three dissimilatory (membrane-associated Nar and periplasmic Napα and Napβ) NO₃^? reductases. Shewanella oneidensis was the only Shewanella strain whose genome encoded a single NO₃^? reductase (Napβ). Terminal electron acceptor competition experiments in S. oneidensis batch cultures amended with both NO₃^? and IO₃^? demonstrated that neither NO₃^? nor IO₃^? reduction activities were competitively inhibited by the presence of the competing electron acceptor. The lack of involvement of S. oneidensis Napβ in IO₃^? reduction was confirmed via phenotypic analysis of an in-frame gene deletion mutant lacking napβA (encoding the NO₃^?-reducing NapβA catalytic subunit). S. oneidensis ΔnapβA was unable to reduce NO₃^?, yet reduced IO₃^? at rates higher than the wild-type strain. Thus, NapβA is required for dissimilatory NO₃^? reduction by S. oneidensis, while neither the assimilatory (Nas) nor dissimilatory (Napα, Napβ, and Nar) NO₃^? reductases are required for IO₃^? reduction. These findings provide the first genetic evidence that IO₃^? reduction by S. oneidensis does not involve nitrate reductase and indicate that S. oneidensis reduces IO₃^? via an as yet undiscovered enzymatic mechanism.     

Nitrate assimilation in leaf blades of wheat of different age

A field experiment on wheat (Triticum aestivum L.) ev. Shera grown at 120 kg N ha^?1 was conducted. Half of the dose of fertilizer N was applied at the pre-sowing stage and the other half when the seedlings were one month old. The leaf blades were examined for their NO₃^? content and NO₃^? assimilatory activity at various stages of growth and development. Soil nitrate level at 50 cm depth was determined throughout the wheat growing season in terms of cencentration (μg/ml) and total amount (kg ha^?1). The upper leaf blades were examined for their capacity to assimilate NO₃^?. Highly significant correlation between NR (nitrate reductase) activity and NO₃^? content in the leaf blades. NR activity and soil NO₃^?, and between soil NO₃^? and leaf blade NO₃^? was observed. Findings on low soil NO₃^? status during the reproductive phase and the capacity of the upper leaf blades to assimilate additional amounts of NO₃^?, point to the need for developing a programme of soil fertilizer application whereby all the leaf blades can utilize the NO₃^? optimally and thus result in greater N harvest.     

RAPID AMMONIUM‐ AND NITRATE‐INDUCED PERTURBATIONS TO CHL a FLUORESCENCE IN NITROGEN‐STRESSED DUNALIELLA TERTIOLECTA (CHLOROPHYTA)1

When NH₄ ⁺ or NO₃ ^? was supplied to NO₃ ^? ‐stressed cells of the microalga Dunaliella tertiolecta Butcher, immediate transient changes in chl a fluorescence were observed over several minutes that were not seen in N‐replete cells. These changes were predominantly due to nonphotochemical fluorescence quenching. Fluorescence changes were accompanied by changes in photosynthetic oxygen evolution, indicating interactions between photosynthesis and N assimilation. The magnitude of the fluorescence change showed a Michaelis‐Menten relationship with half‐saturation concentration of 0.5 μM for NO₃ ^? and 10 μM for NH₄ ⁺ . Changes in fluorescence responses were characterized in D. tertiolecta both over 5 days of N starvation and in cells cultured at a range of NO₃ ^? ‐limited growth rates. Variation in responses was more marked in starved than in limited cells. During N starvation, the timing and onset of the fluorescence responses were different for NO₃ ^? versus NH₄ ⁺ and were correlated with changes in maximum N uptake rate during N starvation. In severely N‐starved cells, the major fluorescence response to NO₃ ^? disappeared, whereas the response to NH₄ ⁺ persisted. N‐starved cells previously grown with NH₄ ⁺ alone showed fluorescence responses with NH₄ ⁺ but not NO₃ ^? additions. The distinct responses to NO₃ ^? and NH₄ ⁺ may be due to the differences between regulation of the uptake mechanisms for the two N sources during N starvation. This method offers potential for assessing the importance of NO₃ ^? or NH₄ ⁺ as an N source to phytoplankton populations and as a diagnostic tool for N limitation.     

ISOLATION AND PARTIAL CHARACTERIZATION OF NITRATE ASSIMILATION MUTANTS OF DUNALIELLA TERTIOLECTA (CHLOROPHYCEAE)1

Fifteen nitrate assimilation-deficient mutants of the euryhaline green alga, Dunaliella tertiolecta Butcher were selected by their chlorate resistance. Ten mutants, unable to grow on NO₃^? but able to grow on NO₂^?, had no detectable nitrate reductase activity. Five mutants, unable to grow on either NO₃^? or NO₂^?, had depressed levels of both nitrate and nitrite reductase. A method for assaying methyl viologen-nitrate reductase in the presence of nitrite reductase is described.     

Photosynthetic energy supply for NO3− assimilation in Scenedesmus

NO₃^?-dependent O₂ in synchronous Scenedesmus obtusiusculus Chod. in the absence of CO₂ is stoichiometric with NH₄⁺ excretion, indicating a close coupling of NO₃^? reduction to non-cyclic electron flow. Also in the presence of CO₂, NO₃^? stimulates O₂ evolution as manifested by an increase in the O₂/CO₂ ratio from 0.96 to 1.11. This quotient was increased to 1.36 by addition of NO₂^?, without competitive interaction with CO₂ fixation, indicating that the capacity for non-cyclic electron transport at saturating light is non-limiting for simultaneous reduction of NO₃^? and CO₂ at high rates. During incubation with NO₃^?+ CO₂, no NH₄⁺ is released to the outer medium, whereas during incubation with NO₂^?+ CO₂, excess NH₄⁺ is formed and excreted. NO₃^? uptake is stimulated by CO₂, and this stimulation is also significant when the cellular energy metabolism is restricted by moderate concentrations of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, whereas NO₃^? uptake in the absence of CO₂ is severely inhibited by the uncoupler. Also under energy-restricted conditions NO₃^? uptake is not competitive with CO₂ fixation. Antimycin A is inhibitory for NO₃^? uptake in the absence of CO₂, and there is no enhancement of NO₃^? uptake by CO₂ in the presence of antimycin A. It is assumed that the energy demand for NO₃^? uptake is met by energy fixed as triosephosphates in the Calvin cycle. Antimycin A possibly affects the transfer of reduced triose phosphates from the chloroplast to the cytoplasm. Active carbon metabolism also seems to exert a control effect on NO₃^? assimilation, inducing complete incorporation of all NO₃^? taken up into amino acids. This control effect is not functional when NO₂^? is the nitrogen source. Active carbon metabolism thus seems to be essential both for provision of energy for NO₃^? uptake and for regulation of the process.     

Nitrogen regulation of the xyl genes of Pseudomonas putida mt‐2 propagates into a significant effect of nitrate on m‐xylene mineralization in soil

The nitrogen species available in the growth medium are key factors determining expression of xyl genes for biodegradation of aromatic compounds by Pseudomonas putida. Nitrogen compounds are frequently amended to promote degradation at polluted sites, but it remains unknown how regulation observed in the test tube is propagated into actual catabolism of, e.g. m‐xylene in soil, the natural habitat of this bacterium. To address this issue, we have developed a test‐tube‐to‐soil model system that exposes the end‐effects of remediation practices influencing gene expression of P. putida mt‐2. We found that NO₃^? compared with NH₄⁺ had a stimulating effect on xyl gene expression in pure culture as well as in soil, and that this stimulation was translated into increased m‐xylene mineralization in soil. Furthermore, expression analysis of the nitrogen‐regulated genes amtB and gdhA allowed us to monitor nitrogen sensing status in both experimental systems. Hence, for nitrogen sources, regulatory patterns that emerge in soil reflect those observed in liquid cultures. The current study shows how distinct regulatory traits can lead to discrete environmental consequences; and it underpins that attempts to improve bioremediation by nitrogen amendment should integrate knowledge on their effects on growth and on catabolic gene regulation under natural conditions.     

Interaction between atmospheric and pedospheric nitrogen nutrition in spruce (Picea abies L. Karst) seedlings

The effect of NO₂ fumigation on root N uptake and metabolism was investigated in 3-month-old spruce (Picea abics L. Karst) seedlings. In a first experiment, the contribution of NO₂ to the plant N budget was measured during a 48 h fumigation with 100mm³m^?3 NO₂. Plants were pre-treated with various nutrient solutions containing NO₂ and NH₄⁺, NO₃^? only or no nitrogen source for 1 week prior to the beginning of fumigation. Absence of NH₄⁺ in the solution for 6d led to an increased capacity for NO₃^? uptake, whereas the absence of both ions caused a decrease in the plant N concentration, with no change in NO₃^? uptake. In fumigated plants, NO₂ uptake accounted for 20–40% of NO₃^? uptake. Root NO₃^? uptake in plants supplied with NH₄⁺plus NO₃^? solutions was decreased by NO₂ fumigation, whereas it was not significantly altered in the other treatments. In a second experiment, spruce seedlings were grown on a solution containing both NO₂ and NH₄⁺ and were fumigated or not with 100mm³m^?3 NO₂ for 7 weeks. Fumigated plants accumulated less dry matter, especially in the roots. Fluxes of the two N species were estimated from their accumulations in shoots and roots, xylem exudate analysis and ¹⁵N labelling. Root NH₄⁺ uptake was approximately three times higher than NO₃^? uptake. Nitrogen dioxide uptake represented 10–15% of the total N budget of the plants. In control plants, N assimilation occurred mainly in the roots and organic nitrogen was the main form of N transported to the shoot. Phloem transport of organic nitrogen accounted for 17% of its xylem transport. In fumigated plants, neither NO₃^? nor NH₄⁺ accumulated in the shoot, showing that all the absorbed NO₂ was assimilated. Root NO₃^? reduction was reduced whereas organic nitrogen transport in the phloem increased by a factor of 3 in NO₂-fimugated as compared with control plants. The significance of the results for the regulation of whole-plant N utilization is discussed.     

Relations between uptake and utilization of NO−3 in Pisum growing exponentially under nitrogen limitation

The utilization and translocation of nitrogen was investigated in exponentially growing, nitrogen-limited Pisum sativum L. cv. Marma. The plants were given N daily at exponentially increasing, although suboptimal, relative nitrogen addition rates (R_N) calculated to yield a relative increment in N of 0.06 day^?1 and 0.12 day^?1. After 10 days of NO^?₃ additions (26 days after sowing), the relative growth rate more or less equaled R_N. Uptake of NO^?₃ was several-fold higher than the N requirement for the growth rate set by R_N. The daily addition of NO^?₃ was taken up after 7 to 8 h, resulting in a cyclic behaviour in the NO^?₃ utilization. During the phase of net NO^?₃ influx, the filling phase (0 to 8 h), in vitro nitrate reductase activity (NR activity) and intracellular levels of soluble N in the root increased. In the phase of no net influx of NO^?₃ the depletion phase (8 to 24 h), the plants were entirely dependent on stored N. During this phase both in vitro NR activity and intracellular levels of soluble N decreased. Also the calculated actual rate of NO^?₃ reduction was high in the filling phase, while it was close to zero in the depletion phase. The pattern of these fluctuations indicates that the regulation of NO^?₃ utilization involves an interplay between transmembrane fluxes of NO^?₃, the cytosolic NO^?₃ concentration and NR activity. Cyclic fluctuations in N-containing compounds were also found in the xylem. Nitrogen was mainly transported as amino acids. The pattern of NO^?₃ transport in the xylem and the fluctuations in the shoot of in vitro NR activity indicate that a reasoning similar to that for the regulation of NO^?₃ assimilation in the root also applies for the shoot. The results also indicate a substantial supply of amino acids to the xylem through recirculation from the shoot.