External Nitrate Concentration Research Articles

Dynamics of hemoglobins during nodule development, nitrate response, and dark stress in Lotus japonicus.

Legume nodules express multiple leghemoglobins (Lbs) and non-symbiotic hemoglobins (Glbs), but how they are regulated is unclear. Here, we study the regulation of all Lbs and Glbs of Lotus japonicus in different physiologically relevant conditions and mutant backgrounds. We quantified hemoglobin expression, localized reactive oxygen species (ROS) and nitric oxide (NO) in nodules, and deployed mutants deficient in Lbs and in the transcription factors NLP4 (associated with nitrate sensitivity) and NAC094 (associated with senescence). Expression of Lbs and class 2 Glbs was suppressed by nitrate, whereas expression of class 1 and 3 Glbs was positively correlated with external nitrate concentrations. Nitrate-responsive elements were found in the promoters of several hemoglobin genes. Mutant nodules without Lbs showed accumulation of ROS and NO and alterations of antioxidants and senescence markers. NO accumulation occurred by a nitrate-independent pathway, probably due to the virtual disappearance of Glb1-1 and the deficiency of Lbs. We conclude that hemoglobins are regulated in a gene-specific manner during nodule development and in response to nitrate and dark stress. Mutant analyses reveal that nodules lacking Lbs experience nitro-oxidative stress and that there is compensation of expression between Lb1 and Lb2. They also show modulation of hemoglobin expression by NLP4 and NAC094.

Physio-molecular traits of contrasting bread wheat genotypes associated with 15N influx exhibiting homeolog expression bias in nitrate transporter genes under different external nitrate concentrations.

The high affinity nitrate transport system is a potential target for improving nitrogen use efficiency of bread wheat growing either under optimal or limiting nitrate concentration. Nitrate uptake is one of the most important traits to take into account to improve nitrogen use efficiency in wheat (Triticum aestivum L.). In this study, we aimed to gain an insight into the regulation of NO3- -uptake and translocation systems in two contrasting wheat genotypes [K9107(K9) vs. Choti Lerma (CL)]. Different conditions, such as NO3--uptake rates, soil-types, N-free solid external media, and external NO3- levels at the seedling stage, were considered. We also studied the contribution of homeolog expression of five genes encoding two nitrate transporters in the root tissue, along with their overall transcript expression levels relative to specific external nitrate availability. We observed that K9107 had a higher 15N influx than Choti Lerma under both limiting as well as optimum external N conditions in vermiculite-perlite (i.e., N-free solid) medium, with the improved translocation efficiency in Choti Lerma. However, in different soil types, different levels of 15N-enrichment in both the genotypes were found. Our results also demonstrated that the partitioning of dry matter in root and shoot was different under these growing conditions. Moreover, K9107 showed significantly higher relative expression of TaNRT2.1 at the lowest and TaNPF6.1 and TaNPF6.2 at the highest external nitrate concentrations. We also observed genotype-specific and nitrate starvation-dependent homeolog expression bias in all five nitrate transporter genes. Our data suggest that K9107 had a higher NO3- influx capacity, involving different nitrate transporters, than Choti Lerma at the seedling stage.

New insights into the role of chrysanthemum calcineurin B\u2013like interacting protein kinase CmCIPK23 in nitrate signaling in Arabidopsis roots

Nitrate is an important source of nitrogen and also acts as a signaling molecule to trigger numerous physiological, growth, and developmental processes throughout the life of the plant. Many nitrate transporters, transcription factors, and protein kinases participate in the regulation of nitrate signaling. Here, we identified a gene encoding the chrysanthemum calcineurin B-like interacting protein kinase CmCIPK23, which participates in nitrate signaling pathways. In Arabidopsis, overexpression of CmCIPK23 significantly decreased lateral root number and length and primary root length compared to the WT when grown on modified Murashige and Skoog medium with KNO3 as the sole nitrogen source (modified MS). The expression of nitrate-responsive genes differed significantly between CmCIPK23-overexpressing Arabidopsis (CmCIPK23-OE) and the WT after nitrate treatment. Nitrate content was significantly lower in CmCIPK23-OE roots, which may have resulted from reduced nitrate uptake at high external nitrate concentrations (≥ 1 mM). Nitrate reductase activity and the expression of nitrate reductase and glutamine synthase genes were lower in CmCIPK23-OE roots. We also found that CmCIPK23 interacted with the transcription factor CmTGA1, whose Arabidopsis homolog regulates the nitrate response. We inferred that CmCIPK23 overexpression influences root development on modified MS medium, as well as root nitrate uptake and assimilation at high external nitrate supply. These findings offer new perspectives on the mechanisms by which the chrysanthemum CBL interacting protein kinase CmCIPK23 influences nitrate signaling.

The Effect of Nutrient Availability and Light Conditions on the Growth and Intracellular Nitrogen Components of Land-Based Cultivated Saccharina latissima (Phaeophyta)

With the increasing interest and activities regarding seaweed cultivation in Europe, it is becoming crucial to utilize the opportunities that lies within environmental resources, such as light and nutrients, to produce biomass of a high yield and quality. The chemical composition of seaweed varies between seasons and depths as an effect of resource supply and environmental conditions. These factors are particularly important to assess for economically feasible species like the kelp Saccharina latissima. This paper examines how differences in light conditions and nutrient availability affect the growth and intracellular nitrogen in S. latissima. This was done through cultivating sporophytes in land-based tanks with four different combinations of high/low light and high/low nutrient supply over an experimental period of 20 days, with measurements of growth rate and subsequent analysis of tissue nitrogen metabolites. The results revealed that the mean growth rate and the intracellular nitrogen components of the sporophytes were positively related to the external nitrate concentration during the experimental period, indicating that S. latissima requires high nutrient concentration to maintain a rapid growth.

Combined Allosteric Responses Explain the Bifurcation in Non-Linear Dynamics of 15N Root Fluxes Under Nutritional Steady-State Conditions for Nitrate.

With regard to thermodynamics out of equilibrium, seedlings are open systems that dissipate energy towards their environment. Accordingly, under nutritional steady-state conditions, changes in external concentrations of one single ion provokes instability and reorganization in the metabolic and structure/architecture of the seedling that is more favorable to the fluxes of energy and matter. This reorganization is called a bifurcation and is described in mathematics as a non-linear dynamic system. In this study, we investigate the non-linear dynamics of 15N fluxes among cellular compartments of B. napus seedlings in response to a wide range of external concentrations (from 0.05 to 20 mM): this allows to determine whether any stationary states and bifurcations could be found. The biphasic behavior of the root uptake rate (vin) was explained by the combined cooperative properties between the vapp (N uptake, storage and assimilation rate) and vout (N translocation rate) 15N fluxes that revealed a unique and stable stationary state around 0.28 mM nitrate. The disappearance of this stationary state around 0.5 mM external nitrate concentrations provokes a dramatic bifurcation in 15N flux pattern. This bifurcation in the vin and vout 15N fluxes fits better with the increase of BnNPF6.3/NRT1.1 expression than BnNRT2.1 nitrate transporter genes, confirming the allosteric property of the BnNPF6/NRT1.1 transporter, as reported in the literature between low and high nitrate concentrations. Moreover, several statistically significant power-law equations were found between variations in the shoots tryptophan concentrations (i.e., IAA precursor) with changes in the vapp and vout 15N fluxes as well as a synthetic parameter of plant N status estimated from the root/shoot ratio of total free amino acids concentrations. These relationships designate IAA as one of the major biological parameters related to metabolic and structural-morphological reorganization coupled with the N and water fluxes induced by nitrate. The results seriously challenge the scientific grounds of the concept of high- and low-affinity of nitrate transporters and are therefore discussed in terms of the ecological significance and physiological implications on the basis of recent agronomic, physiological and molecular data of the literature.

Root architecture traits variation and nitrate-influx responses in diverse wheat genotypes under different external nitrogen concentrations

In order to identify the genetic variations in root system architecture traits and their probable association with high- and low-affinity nitrate transport system, we performed several experiments on a genetically diverse set of wheat genotypes grown under two external nitrogen levels (optimum and limited nitrate conditions) at two growth points of the seedling stage. Further, we also examined the nitrate uptake and its transport under different combinations of nitrate availability in the external media using 15N-labelled N-source (15NO3−), and gene expression pattern of different high- and low-affinity nitrate transporters. We observed that nitrate starvation invariably increases the total root size in all genotypes. However, the variation of component traits of total root size under nitrate starvation is genotype-specific at both stages. Further, we also observed genotypic variation in both nitrate uptake and translocation depending on the growth stage, external nitrate concentration and growing conditions. The expression of the TaNRT2.1 gene was invariably up-regulated under low external nitrate concentration; however, it gets reduced after a longer period (21 days) of starvation than the early stage (14 days). Among the four NRT1.1 orthologs, TaNPF6.3 and TaNPF6.4 consistently showed higher expression than TaNPF6.1 and TaNPF6.2 at higher nitrate concentration at both the growth stages. TaNPF6.3 and TaNPF6.4 apparently showed a feature of typical low-affinity nitrate transporter gene at higher external nitrate concentration at 14 and 21 days growth stages, respectively. The present study reveals the complex root system of wheat that has genotype-specific N-foraging along with highly coordinated high- and low-affinity nitrate transport systems for nitrate uptake and transport.

Modelling Nitrogen Uptake in Plants and Phytoplankton: Advantages of Integrating Flexibility into the Spatial and Temporal Dynamics of Nitrate Absorption

Under field conditions, plants need to optimize nutrient ion and water acquisition in their fluctuating environment. One of the most important variables involved in variations of ion uptake processes is temperature. It modifies the thermodynamic processes of root uptake and ion diffusion in soil throughout day–night and ontogenetic cycles. Yet, most models of nitrogen (N) uptake in plants are built from set values of microscopic kinetic parameters, Vm and Km, derived from a Michaelis–Menten (MM) interpretation of nutrient isotherms. An isotherm is a curve depicting the response of root nitrate influx to external nitrate concentrations at a given temperature. Models using the MM formalism are based on several implicit assumptions that do not always hold, such as homothetic behavior of the kinetic parameters between the different root biological scales, i.e., the epidermis cell, root segments, root axes, and the whole root system. However, in marine phytoplankton, it has been clearly demonstrated that the macroscopic behavior in the nutrient uptake of a colony cannot be confounded with the microscopic behavior of individual cells, due to the cell diffusion boundary layer. The same is also true around plant root segments. Improved N uptake models should either take into account the flexibility of the kinetic parameters of nitrate uptake at the cellular level (porter–diffusion approach) or use the more realistic macroscopic kinetic parameters proposed by the flow–force approach. Here we present recent solutions proposed in marine phytoplankton and plant nutrient uptake models to make a more flexible description of the nutrient ion uptake process. Use of the mechanistic porter–diffusion approach developed in marine phytoplankton introduces more flexibility in response to cell characteristics and physical processes driven by temperature (diffusion and convection). The thermodynamic flow–force interpretation of plant-based nutrient uptake isotherms introduces more flexibility in response to environmental cues and root aging. These two approaches could help solve many problems that modelers encounter in these two research areas.

Changes in 15NO3 - Availability and Transpiration Rate Are Associated With a Rapid Diurnal Adjustment of Anion Contents as Well as 15N and Water Fluxes Between the Roots and Shoots.

Background and Aims: Understanding interactions between water and nitrate fluxes in response to nitrate availability and transpiration rate is crucial to select more efficient plants for the use of water and nitrate.Methods: Some of these interactions were investigated in intact Brassica napus plants by combining a non-destructive gravimetric device with 15NO3- labeling. The set-up allowed high-resolution measurement of the effects of a cross-combination of two concentrations of KNO3 or KCl (0.5 and 5 mM) with two different rates of transpiration controlled by the relative humidity during a day–night cycle.Key Results: Results show that (1) high external nitrate concentrations increased root water uptake significantly whatever the transpiration rate, (2) nitrate translocation depended both on the rate of nitrate uptake and loading into xylem (3) dilution-concentration effect of nitrate in the xylem was mainly modulated by both external nitrate availability and transpiration rate, (4) dynamic changes in 15N translocation in the xylem modified shoot growth and capacitance, and (5) variations in tissue concentrations of NO3- induced by the experimental conditions were balanced by changes in concentrations of chloride and sulfate ions. These effects were even more amplified under low transpiration condition and 0.5 mM external nitrate concentration.Conclusion: Taken together, these results highlight the fine and rapid adjustment of anion contents, nitrate and water flows to changes in transpiration rate and nitrate availability during a day–night cycle. The use of this non-invasive gravimetric device is therefore a powerful tool to assess candidates genes involved in nitrogen and water use efficiency.

External Nitrogen and Carbon Source-Mediated Response on Modulation of Root System Architecture and Nitrate Uptake in Wheat Seedlings

Nitrogen uptake efficiency is an important component trait that could be targeted for improving nitrogen use efficiency of crop plants. To understand the responses of different nitrate transport systems and the influence of root system architecture on nitrate uptake under limited nitrate conditions in wheat (Triticum aestivum L.) at the seedling stage, we studied nitrate uptake, root system architecture, and expression of different nitrate transporter genes in induced and non-induced wheat seedlings. Further, effects of inclusion of sucrose and two amino acids (glutamine and asparagine) in induction medium on these parameters were also studied. We observed that the induced wheat root system took up more nitrate as compared to non-induced root system in a dose-dependent manner. Gene expression of both high- and low-affinity nitrate transporter gene showed differential expression in the induced root tissues, as compared to non-induced tissues, depending on the concentration of nitrate present in induction medium. External nutrient media containing sucrose, glutamine, and asparagine reduce nitrate concentration in both root and shoot tissues and also influence the gene expression of these transporters. Our observations indicate that upon induction with milder external nitrate concentrations, the root architecture is modulated by changing overall lateral root size and 1st order lateral root numbers along with activation of nitrate transporters which acquire and transport nitrate in roots and shoots, respectively, depending on the carbon and nitrogen source available to seedlings.

Knock-Down of CsNRT2.1, a Cucumber Nitrate Transporter, Reduces Nitrate Uptake, Root length, and Lateral Root Number at Low External Nitrate Concentration.

Nitrogen (N) is a macronutrient that plays a crucial role in plant growth and development. Nitrate () is the most abundant N source in aerobic soils. Plants have evolved two adaptive mechanisms such as up-regulation of the high-affinity transport system (HATS) and alteration of the root system architecture (RSA), allowing them to cope with the temporal and spatial variation of . However, little information is available regarding the nitrate transporter in cucumber, one of the most important fruit vegetables in the world. In this study we isolated a nitrate transporter named CsNRT2.1 from cucumber. Analysis of the expression profile of the CsNRT2.1 showed that CsNRT2.1 is a high affinity nitrate transporter which mainly located in mature roots. Subcellular localization analysis revealed that CsNRT2.1 is a plasma membrane transporter. In N-starved CsNRT2.1 knock-down plants, both of the constitutive HATS (cHATS) and inducible HATS (iHATS) were impaired under low external concentration. Furthermore, the CsNRT2.1 knock-down plants showed reduced root length and lateral root numbers. Together, our results demonstrated that CsNRT2.1 played a dual role in regulating the HATS and RSA to acquire effectively under N limitation.

Identification and molecular characterization of the Brachypodium distachyon NRT2 family, with a major role of BdNRT2.1.

The small monocot plant Brachypodium distachyon is rapidly emerging as a powerful model system to study questions unique to the monocot crops. An extensive BLAST search was carried to identify putative orthologues of the Arabidopsis NRT2 genes in the fully sequenced Brachypodium genome. Seven genes encoding putative high-affinity nitrate transporters (BdNRT2) were identified. Transcriptional analysis of individual BdNRT2 gene under various nitrogen sources and levels in the wild-type and a T-DNA mutant of BdNRT2.1 were performed. A transgenic approach was taken to complement the bdnrt2.1 mutant. BdNRT2.1 and BdNRT2.2 were strongly induced by nitrate resupply to nitrogen-starved plants and were classified as inducible genes. BdNRT2.5 was found to be repressed by nitrate resupply whereas other members were constitutively expressed in the root. Interestingly, higher ammonium concentrations also triggered similar gene expression regulation, suggesting BdNRT2 gene expression was also governed by internal nitrogen status, not just external nitrate concentrations. In bdnrt2.1 mutant, the high-affinity transporter system (HATS) was reduced by 30% and BdNRT2.2 and BdNRT2.6 were differentially regulated. This pioneering research demonstrates that genes in the BdNRT2 family have diverse roles, differing from the Arabidopsis AtNRT2 family, in response to various nitrogen conditions. BdNRT2.1 serves as a key member of the family.

The δ15N response and nitrate assimilation of Orychophragmus violaceus and Brassica napus plantlets in vitro during the multiplication stage cultured under different nitrate concentrations

Natural nitrogen isotope composition (δ15N) is an indicator of nitrogen sources and is useful in the investigation of nitrogen cycling in organisms and ecosystems. δ15N is also used to study assimilation of inorganic nitrogen. However, the foliar δ15N of intact plants, which is a consequence of nitrate assimilation occurring in the roots and shoots, is not suited for studying nitrate assimilation in cases where nitrate is the sole nitrogen source. In this study, Orychophragmus violaceus (Ov) and Brassica napus (Bn) plantlets, in which nitrate assimilation occurred in the leaves, were used to study the relationship between foliar δ15N and nitrate assimilation. The plantlets were grown in vitro in culture media with different nitrate concentrations, and no root formation occurred for the plantlets during the multiplication stage. Nitrogen isotope fractionation occurred in both the Ov and the Bn plantlets under all treatments. Furthermore, the foliar nitrogen content of both the Ov and Bn plantlets increased with increasing nitrate concentration. Foliar nitrogen isotope fractionation was negatively correlated with foliar nitrogen content for both the Ov and Bn plantlets. Our results suggest that the foliar nitrogen isotope fractionation value could be employed to evaluate nitrate assimilation ability and leaf nitrate reductase activity. Moreover, high external nitrate concentrations could contribute to improved foliar nitrogen content and enhanced nitrate assimilation ability.

Dietary Nitrate and the Epidemiology of Cardiovascular Disease: Report From a National Heart, Lung, and Blood Institute Workshop.

In view of continuing unanswered questions regarding the geographical and demographic distribution of cardiovascular disease, and recent discoveries about the effects of dietary nitrate on cardiovascular physiology, the National Heart, Lung, and Blood Institute (NHLBI) convened a workshop to

Responses of Ulva prolifera to short-term nutrient enrichment under light and dark conditions

To define responses of short-term nutrient uptake in Ulva prolifera, we measured uptake rates, enzyme activity, and tissue nutrient content in lab experiments where we manipulated nutrient supply and irradiation. Nitrate uptake of U. prolifera was significantly impacted by the external nitrate concentrations, and ammonium uptake was mainly determined by the light availability. The measured nitrogen contents in tissues were higher than the calculated values from the uptake of dissolve inorganic nitrogen, indicating that U. prolifera might use multiple nitrogen sources. High external phosphate concentrations and sufficient light can accelerate the phosphate uptake of U. prolifera, while the measured phosphorus contents in tissues were lower than the calculated values from the uptake of phosphate, suggesting a possibility of internal phosphorus release. The enzymatic activities of nitrate reductase (NR), acid phosphatase (AcP) and alkaline phosphatase (AP) showed little changes, indicating that enzymatic activity might not a direct factor determining the short-term nutrient uptake of U. prolifera.

Breaking conceptual locks in modelling root absorption of nutrients: reopening the thermodynamic viewpoint of ion transport across the root.

The top-down analysis of nitrate influx isotherms through the Enzyme-Substrate interpretation has not withstood recent molecular and histochemical analyses of nitrate transporters. Indeed, at least four families of nitrate transporters operating at both high and/or low external nitrate concentrations, and which are located in series and/or parallel in the different cellular layers of the mature root, are involved in nitrate uptake. Accordingly, the top-down analysis of the root catalytic structure for ion transport from the Enzyme-Substrate interpretation of nitrate influx isotherms is inadequate. Moreover, the use of the Enzyme-Substrate velocity equation as a single reference in agronomic models is not suitable in its formalism to account for variations in N uptake under fluctuating environmental conditions. Therefore, a conceptual paradigm shift is required to improve the mechanistic modelling of N uptake in agronomic models. An alternative formalism, the Flow-Force theory, was proposed in the 1970s to describe ion isotherms based upon biophysical 'flows and forces' relationships of non-equilibrium thermodynamics. This interpretation describes, with macroscopic parameters, the patterns of N uptake provided by a biological system such as roots. In contrast to the Enzyme-Substrate interpretation, this approach does not claim to represent molecular characteristics. Here it is shown that it is possible to combine the Flow-Force formalism with polynomial responses of nitrate influx rate induced by climatic and in planta factors in relation to nitrate availability. Application of the Flow-Force formalism allows nitrate uptake to be modelled in a more realistic manner, and allows scaling-up in time and space of the regulation of nitrate uptake across the plant growth cycle.

An updated model for nitrate uptake modelling in plants. II. Assessment of active root involvement in nitrate uptake based on integrated root system age: measured versus modelled outputs

Background and Aims An updated version of a mechanistic structural-functional model was developed to predict nitrogen (N) uptake throughout the growth cycle by a crop of winter oilseed rape, Brassica napus, grown under field conditions. Methods The functional component of the model derives from a revisited conceptual framework that combines the thermodynamic Flow-Force interpretation of nitrate uptake isotherms and environmental and in planta effects on nitrate influx. Estimation of the root biomass (structural component) is based upon a combination of root mapping along the soil depth profile in the field and a relationship between the specific root length and external nitrate concentration. The root biomass contributing actively to N uptake was determined by introduction of an integrated root system age that allows assignment of a root absorption capacity at a specific age of the root. Key Results Simulations were well matched to measured data of N taken up under field conditions for three levels of N fertilization. The model outputs indicated that the two topsoil layers (0-30 and 30-60 cm) contained 75-88 % of the total root length and biomass, and accounted for 90-95 % of N taken up at harvest. Conclusions This conceptual framework provides a model of nitrate uptake that is able to respond to external nitrate fluctuations at both functional and structural levels.

Integrating fluctuating nitrate uptake and assimilation to robust homeostasis

Nitrate is an important nitrogen source used by plants. Despite of the considerable variation in the amount of soil nitrate, plants keep cytosolic nitrate at a homeostatic controlled level. Here we describe a set of homeostatic controller motifs and their interaction that can maintain robust cytosolic nitrate homeostasis at fluctuating external nitrate concentrations and nitrate assimilation levels. The controller motifs are divided into two functional classes termed as inflow and outflow controllers. In the presence of high amounts of environmental nitrate, the function of outflow controllers is associated to efflux mechanisms removing excess of nitrate from the cytosol that is taken up by low-affinity transporter systems (LATS). Inflow controllers on the other hand maintain homeostasis in the presence of a high demand of nitrate by the cell relative to the amount of available environmental nitrate. This is achieved by either remobilizing nitrate from a vacuolar store, or by taking up nitrate by means of high-affinity transporter systems (HATS). By combining inflow and outflow controllers we demonstrate how nitrate uptake, assimilation, storage and efflux are integrated to a regulatory network that maintains cytosolic nitrate homeostasis at changing environmental conditions.

Effect of high nitrate concentration on PHB storage in sequencing batch reactor under anoxic conditions

The study investigated effect of high influent nitrate concentration on poly-β-hydroxybutyrate, (PHB), storage in a sequencing batch reactor, (SBR), under anoxic conditions. Acetate was fed as pulse during anoxic phase, sustained with external nitrate feeding. SBR operation involved three runs at steady state with COD/N ratios of 3.84, 2.93 and 1.54 gCOD/gN, where external nitrate concentrations gradually increased from 50 mg N/l to 114 mg N/l and 226 mg N/l, in 1st, 2nd and 3rd runs, respectively. In 1st run, acetate was fully converted into PHB with the storage yield value of 0.57–0.59 gCOD/gCOD, calculated both in terms of PHB formation and NO X utilization, confirming storage was the sole substrate utilization mechanism. In the following runs, PHB formation was reduced and the storage yield based on PHB dropped down to 0.40 and 0.33 gCOD/gCOD with increasing influent nitrate concentrations, indicating that higher portions of acetate were diverted to simultaneous direct growth. The observations suggested that nitrite accumulation detected at low COD/N ratios was responsible for inhibition of PHB storage.

Molecular analysis of physiological responses to changes in nitrogen in a marine macroalga, Porphyra yezoensis (Rhodophyta)

The rhodophyte seaweed Porphyra yezoensis, known more commonly world-wide as "nori", is an important commercial crop in Japan. Cultivation of nori in Japan is often affected by outbreaks of "iroochi", a discoloration of the thalli due to a decrease in inorganic nutrients in the culture area that in turn decreases the amount of photosynthetic pigments in the thalli. Treating thalli with inorganic nitrogen can reverse iroochi. In this paper, we report on the characterization of three P. yezoensis genes, a nitrate transporter (PyNRT2) and two urea transporters (PyUT1 and PyUT2), which may be involved in reversing iroochi. The predicted length of the PyNRT2 protein was 479 amino acids (AA), while PyUT1 and PyUT2 were 740 and 680 AA, respectively. PyNRT2 was more similar to NRT2 from a chromophyte than to NRTs from Chlamydomonas and higher plants. The two P. yezoensis UTs had 56% AA identity to each other, and showed the closest relationship to higher plant and yeast DUR3 proteins which formed a subfamily of the sodium-solute symporter protein family. Hydrophobicity plots of the AA sequences showed that the PyNRT2, PyUT1, and PyUT2 included 12, 15, and 16 transmembrane domains, respectively. Southern blot analysis indicated that the P. yezoensis genome has a single NRT2-encoding gene and at least four UT-encoding genes. Expression analysis of PyNRT2 and PyUT genes showed that the messenger RNA level of the PyNRT2 gene reached a maximum after 48 h in the nitrate starvation condition and was then restored to the constitutive level, while expression of the PyUT genes was induced in proportion to treatment times in the nitrate starvation condition. These results suggest that the PyNRT2 and PyUT are responsible for the high-affinity nitrate/urea transport systems that operate under low external nitrate concentrations.

The effect of nitrate and plant size on nitrate uptake and in vitro nitrate reductase activity in strawberry ( Fragaria × ananassa cv. Selva)

Nitrogen plays a major role in the growth and yield of strawberry. For optimizing nitrogen fertilizer application, it is necessary to understand the response of strawberry to nitrogen supply. The objective of this study was to determine the effect of nitrate supply and plant size on strawberry nitrate uptake and nitrate reductase activity (NRA). Strawberry plants cv. Selva were grown under growth chamber conditions in nutrient solutions containing 0, 0.1, 0.25, 0.7 or 4 mM nitrate. Cumulative nitrate uptake increased with the increase in nitrate supply. Increasing nitrate concentrations from 0 to 0.25 mM decreased leaf NRA; however, further increases to 4 mM nitrate restored NRA to activities observed at 0 mM nitrate. The activity of NADH- and NADPH-dependent NR was similar at every external nitrate concentration. Activity of NR was greater in the smallest plants and decreased as plant size increased. However, increasing external nitrate concentration increased nitrate uptake, but had not the same effect on nitrate reductase activity, showing that NRA and nitrate uptake are not necessarily correlated.