Effect of amino compounds on nitrate utilization by roots of dwarf bean

Amino compounds (1 mM, pH 5) were given prior to, together with, or after the addition of nitrate to study their effect on nitrate uptake and in vivo nitrate reductase activity (NRA) in roots of Phaseolus vulgaris. The effect of amino compounds varied with the amino species, the nitrate status of the plant (induced vs uninduced) and the aspect of nitrate utilization. Cysteine inhibited the nitrate uptake rate and root NRA under all conditions tested. NRA in uninduced roots was stimulated by tryptophan, and arginine inhibited NRA under all conditions tested. Uptake was inhibited by aspartate and glutamate and stimulated by leucine when these amino compounds were given prior to or after completion of the apparent induction of nitrate uptake. In the presence of β-alanine and tryptophan, induction of uptake was accelerated.     

Role of sugars in nitrate utilization by roots of dwarf bean

Nitrate uptake and in vivo, nitrate reductase activity (NRA) in roots of Phaseolus vulgaris, L. cv. Witte Krombek were measured in nitrogen-depleted plants of varying sugar status, Variation in sugar status was achieved at the start of nitrate nutrition by excision, ringing, darkness or administration of sugars to the root medium. The shape of the apparent induction pattern of nitrate uptake was not influenced by the sugar status of the absorbing tissue. When measured after 6 h of nitrate nutrition (0.1 mol m^?3), steady state nitrate uptake and root NRA were in the order intact>dark>ringed>excised. Exogenous sucrose restored NRA in excised roots to the level of intact plants. The nitrate uptake rate of excised roots, however, was not fully restored by sucrose (0.03–300 mol m^?3). When plants were decapitated after an 18 h NO₃^? pretreatment, the net uptake rate declined gradually to become negative after three hours. This decline was slowed down by exogenous fructose, whilst glucose rapidly (sometimes within 5 min) stimulated NG^?₃ uptake. Presumably due to a difference in NO₃^? due to a difference in NO₃^? uptake, the NRA of excised roots was also higher in the presence of glucose than in the presence of fructose after 6 h of nitrate nutrition. The sugar-stimulation of, oxygen consumption as well as the release of ¹⁴CO₂ from freshly absorbed (U-¹⁴C) sugar was the same for glucose and fructose. Therefore, we propose a glucose-specific effect on NO₃^? uptake that is due to the presence of glucose rather than to its utilization in root respiration. A differential glucose-fructose effect on nitrate reductase activity independent of the effect on NO₃^? uptake was not indicated. A constant level of NRA occurred in roots of NO₃^? induced plants. Removal of nutrient nitrate from these plants caused an exponential NRA decay with an approximate half-life of 12 h in intact plants and 5.5 h in excised roots. The latter value was also found in roots that were excised in the presence of nitrate, indicating that the sugar status primarily determines the apparent rate of nitrate reductase decay in excised roots.     

Utilization of nitrite and nitrate by dwarf bean

The uptake and conversion of NO ₂ ^- and the effect of NO ₂ ^- on the uptake and reduction of NO ₃ ^- were examined in N-depleted Phaseolus vulgaris L. Nitrite uptake at 0.1 mmol dm^-3 was against an electrochemical gradient and became constant after one or two initial phases. Steadystate uptake declined with increasing ambient NO ₂ ^- concentration (0–0.7 mmol dm^-3). In this concentration range root oxygen consumption was unaffected by NO ₂ ^- , indicating that the decrease of NO ₂ ^- uptake was not related to respiration. After 6 h NO ₂ ^- supply, about one-third of the absorbed NO ₂ ^- had accumulated, mainly in the root system. Oxidation of NO ₂ ^- to NO ₃ ^- was not observed. The apparent induction period for NO ₃ ^- uptake was about 6 h in control plants and 3.5 h in plants that were pretreated for 18 h with NO ₂ ^- . In contrast, the time course of NO ₂ ^- uptake was unaffected by pretreatment with NO ₃ ^- . Steadystate NO ₃ ^- uptake was less affected by NO ₂ ^- than was steady-state NO ₂ ^- uptake by NO ₃ ^- . Nitrate reductase activity (NRA) in leaves and roots was induced by both NO ₃ ^- and NO ₂ ^- . In roots, induction with NO ₂ ^- was faster than with NO ₃ ^- , but there was no difference in NRA after 5 h. Nitrite inhibited NRA in the roots of NO ₃ ^- -induced plants and thus seems to stimulate the induction, but not the activity of induced nitrate reductase. In view of the observed differences in time course and mutual competition, a common uptake mechanism for NO ₂ ^- and NO ₃ ^- seems unlikely. Expression of the NO ₂ ^- effect on the induction of NO ₃ ^- uptake required more time than the induction itself. We therefore conclude that NO ₂ ^- is not the physiological inducer of NO ₃ ^- uptake.Abbreviations NR(A) nitrate reductase (activity) - BM basal medium     

The effect of endogenous and externally supplied nitrate on nitrate uptake and reduction in sugarbeet seedlings

The pericarp of the dormant sugarbeet fruit acts as a storage reservoir for nitrate, ammonium and -amino-N. These N-reserves enable an autonomous development of the seedling for 8–10 d after imbibition. The nitrate content of the seed (1% of the whole fruit) probably induces nitrate-reductase activity in the embryo enclosed in the pericarp. Nitrate that leaks out of the pericarp is reabsorbed by the emerging radicle. Seedlings germinated from seeds (pericarp was removed) without external N-supply are able to take up nitrate immediately upon exposure via a low-capacity uptake system (v_max = 0.8 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.12 mM). We assume that this uptake system is induced by the seed nitrate (10 nmol/seed) during germination. Induction of a high-capacity nitrate-uptake system (v_max = 3.4 mol NO ₃ ^- ·(g root FW)^–1·h^–1; K_s = 0.08 mM) by externally supplied nitrate occurs after a 20-min lag and requires protein synthesis. Seedlings germinated from whole fruits absorb nitrate via a highcapacity uptake mechanism induced by the pericarp nitrate (748 nmol/pericarp) during germination. The uptake rates of the high-capacity system depend only on the actual nitrate concentration of the uptake medium and not on prior nitrate pretreatments. Nitrate deprivation results in a decline of the nitrate-uptake capacity (t_1/2 of v_max = 5 d) probably caused by the decay of carrier molecules. Small differences in K_s but significant differences in v_max indicate that the low- and high-capacity nitrate-uptake systems differ only in the number of identical carrier molecules.Abbreviations NR nitrate reductase - pFPA para-fluorophenylalanine This work was supported by a grant from Bundesministerium für Forschung und Technologie and by Kleinwanzlebener Saatzucht AG, Einbeck.     

Photosynthetic photon flux effects on bean response to nitrogen dioxide

Phaseolus vulgaris L. cv. Kinghorn Wax seedlings, supplied with nutrient solution containing either 0 or 5 mM nitrate as sole N source, were exposed to 0.25 μl/l NO₂ for 6 hr each day for 10 days at continuous photosynthetic photon flux (PPF) of 100, 300, 500 or 700 μmol m⁻² sec⁻¹. There was a significant interaction of PPF and nitrate. Shoot and root dry weights increased with increasing PPFs only when nitrate was supplied. The main effects of NO₂ on plant growth were significant; none of the interactions involving NO₂ were significant. Exposure to NO₂ decreased shoot and root dry weight in both the presence and absence of nutrient N and at all PPF levels. All interactions were significant for in vitro leaf nitrate reductase activity (NRA), which increased markedly at PPFs above 100 μmol m⁻² sec⁻¹ when nitrate was supplied. Treatment with NO₂ strongly inhibited enzyme activity in the presence of nitrate, particularly at the 300 μmol m⁻² sec⁻¹ PPF level. These experiments demonstrated that PPF level does not modify the effect of NO₂ on growth but does have a major effect on NRA and on NO₂ effects on NRA in the presence of nutrient nitrate.     

Cloning and expression of a gene encoding a root specific nitrate reductase in bean (Phaseolus vulgaris)

A structural gene encoding nitrate reductase (NR) in the legume Phaseolus vulgaris cv. Saxonia has been cloned and sequenced. The NR gene encodes a protein of 881 amino acids with a MW of 99.2 kDa. The coding sequence is interrupted by three introns, which are located in the molybdopterin cofactor binding domain. In the flanking regions the signals of a functional eukaryotic gene are present. The gene is the smallest NR gene so far identified in higher plants. Comparison to other NRs shows homology ranging from 65 to 74% at the amino acid level. The similarity is highest for the three functional domains, and lowest in the N-terminal end of the protein. mRNA studies demonstrate that the gene is nitrate inducible and expressed exclusively in the roots of bean. Southern blot analysis indicates the presence of a second NR gene in bean. This gene may encode a NR enzyme expressed in leaves.     

Optimized nitrate reductase assay predicts the rate of nitrate utilization in the halotolerant microalga Dunaliella viridis

An in situ method for measuring nitrate reductase (NR) activity in Dunaliella viridis was optimized in terms of incubation time, concentration of KNO₃, permeabilisers (1-propanol and toluene), pH, salinity, and reducing power (glucose and NADH). NR activity was measured by following nitrite production and was best assayed with 50 mM KNO₃, 1.2 mM NADH, 5% 1-propanol (v/v), at pH 8.5. The estimated half-saturation constant (K_s) for KNO₃ was 5 mM. Glucose had no effect as external reducing power source, and NADH concentrations >1.2 mM inhibited NR activity. Nitrite production was linear up to 20 min; longer incubation did not lead to higher nitrate reduction. The use of the optimized assay predicted the rate of NO ₃ ⁻ removal from the external medium by D. viridis with high degree of precision. This revised version was published online in September 2006 with corrections to the Cover Date.     

Molecular approaches to the analysis of nitrate assimilation

Abstract. The application of molecular approaches such as mutant analysis and recombinant DNA technology, in conjunction with immunology, are set to revolutionize our understanding of the nitrate assimilation pathway. Mutant analysis has already led to the identification of genetic loci encoding a functional nitrate reduction step and is expected to lead ultimately to the identification of genes encoding nitrate uptake and nitrite reduction. Of particular significance would be identification of genes whose products contribute to regulatory networks controlling nitrogen metabolism. Recombinant DNA techniques are particularly powerful and have already allowed the molecular cloning of the genes encoding the apoprotein of nitrate reductase and nitrite reductase. These successes allow for the first lime the possibility to study directly the role of environmental factors such as type of nitrogen source (NO₃⁻ or NH₄⁺) available to the plant, light, temperature water potential and CO₂ and O₂ tensions on nitrate assimilation gene expression and its regulation at the molecular level. This is an important advance since our current understanding of the regulation of nitrate assimilation is based largely on changes of activity of the component steps. The availability of mutants, cloned genes, and gene transfer systems will permit attempts to manipulate the nitrate assimilation pathway.     

Identification and characterization of a nitrate reductase gene from bean (Phaseolus vulgaris) containing four introns

A structural gene encoding nitrate reductase (NR) in bean ( Phaseolus vulgaris ) has been cloned and sequenced. The NR gene encodes a protein of 890 amino acids with a molecular mass of 100 kDa. Comparison to the other known NR gene from bean reveals 76% amino acid identity and comparison to NRs from other species shows amino acid identities ranging from 67 to 77%. At three positions the amino acid sequence displays differences from residues conserved in all other known NR proteins. The coding sequence is interrupted by four introns. Three of them are located at conserved positions in the region encoding the molybdenum cofactor-binding domain. The fourth intron is located in the hinge region between the heme and the FAD domain. This is the only example in which more than three introns have been found in a higher plant NR gene. The mRNA cap site was identified as an adenosine 79 nucleotides (nt) upstream of the ATG translation start codon. Northern analysis shows that the gene is nitrate inducible and highly expressed in trifoliolate leaves of 20-day-old bean plants and only weakly expressed in roots. The gene is also induced by light and sucrose in leaves of dark-adapted plants. The mRNA displays diurnal oscillation under the control of a circadian rhythm. Putative conserved GATA motifs in the promoter are discussed.     

Identification of chitinase mRNA in abscission zones from bean (Phaseolus vulgaris Red Kidney) during ethylene-induced abscission

Abstract. Total RNA was extracted from bean leaf abscission zones at different times after the induction of abscission by ethylene. The RNA was translated in the wheat germ system and the products analysed by SDS-PAGE. Products of molecular weight (raw) 42, 32 and 17 kD were seen to accumulate substantially during the induction. An attempt was made to establish that the mRNA species which produced the 32 kD product, which was coded for the ethylene-regulated enzyme chitinase. Mature chitinase (30 kD) was purifed from ethylene-treated abscission zones and used to raise monospecific antibodies in rabbits. These antibodies recognized the 32 kD product and mature chitinase. The 2 kD difference in molecular weight was due to the presence of the signal sequence which could be removed by microsomal membranes. Chitinase was also detected by enzymatic assay and immunoblotting of crude homogenates from ethylene-treated abscission zones. Chitinase appears to be ubiquitous in bean plants and probably does not have a direct role in abscission.     

Nitrogen fixation and nodule occupancy by native strains of Rhizobium on different cultivars of common bean (Phaseolus vulgaris L.)

A field experiment under rainfed conditions was conducted in Durango, México, to assess N2-fixation of three cultivars of common bean (Phaseolus vulgaris L.) using ¹⁵N-methodology. In addition, diversity of rhizobial isolates obtained from nodules of the different plant genotypes was evaluated by intrinsic antibiotic resistance (IAR), PCR using enterobacterial repetitive intergenic consensus (ERIC) primers, PCR-RFLP analysis of the 16S rRNA gene and multilocus enzyme electrophoresis (MLEE). Selected isolates were used to determine acetylene reduction and competitive ability under greenhouse conditions. The three cultivars tested did not show high variation in N2-fixation, the %Ndfa values ranged from 19 to 26%. Variability in N2-fixation efficiency among various native rhizobial isolates was very high and our results indicate that differences in competitive abilitiy exist also. PCR-RFLP of the 16S rRNA gene and MLEE revealed that most of the isolates belong to the species Rhizobium etli. Intrinsic antibiotic resistance analysis and ERIC-PCR showed high diversity among isolates. In contrast, our results using MLEE show low genetic diversity (H = 0.105).     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

Seasonal changes in nitrate use by three woody species: the importance of the leaf-expansion period

Seasonal changes in plant NO₃ ⁻-N use were investigated by measuring leaf nitrate reductase activity (NRA), leaf N concentration, and leaf expansion in one evergreen woody species (Quercus glauca Thunb.) and two deciduous woody species [Acer palmatum Thunb. and Zelkova serrata (Thunb.) Makino]. Leaf N concentration was highest at the beginning of leaf expansion and decreased during the expansion process to a steady state at the point of full leaf expansion in all species. The leaf NRA of all species was very low at the beginning of leaf expansion, followed by a rapid increase and subsequent decrease. The highest leaf NRA was observed in the middle of the leaf-expansion period, and the lowest leaf NRA occurred in summer for all species. Significant positive correlations were detected between leaf NRA and leaf expansion rates, while leaf N concentrations were negatively correlated with leaf area. In the evergreen Q. glauca, the N concentration in current buds increased before leaves opened; concurrently, the N concentration in 1-year-old leaves decreased by 25%. Our results show that the leaf-expansion period is the most important period for NO₃ ⁻-N assimilation by broadleaf tree species, and that decreases in leaf N concentration through the leaf-expansion period are at least partly compensated for by newly assimilated NO₃ ⁻-N in current leaves.     

Effect of nitrogen (NH4 NO3) supply on absorption of ammonium and nitrate by conventional and hybrid rice during reproductive growth

Although NH₄ ⁺ has generally been accepted as the preferred N source for fertilising rice, some workers have concluded tha NO₃ ^- is as effective as NH₄ ⁺. The present glasshouse study exmined the relative uptake of NH₄ ⁺ and NO₃ ^- from solution and cultures containing 5–120 mg N/L supplied as NH₄NO₃ by a hybrid rice (India) and a conventional rice cultivar (Japonica). At all levels of N supply, the hybrid rice had higher leaf area and higher rates of uptake of total N than the conventional cultivar. Net photosynthesis rates were similar for both cultivars at the highest rates of N supply, but were lower at 5–40 mg N/L for the hybrid cultivar than for the conventional cultivar. At all levels of N supply, the conventional rice cultivar absorbed more NH₄ ⁺ than NO₃ ^-. In contrast, the hybrid rice absorbed more NH₄ ⁺ than NO₃ ^- at the low levels of N supply (5–40 mg N/L), but more NO₃ ^- than NH₄ ⁺ at the high levels of at 80 and 120 mg N/L. It is concluded that the uptake of N by rice is under genetic control and also dependent on levels of N supply. Thus the appropriate form of N fertiliser for rice may depend on cultivar and rates of N supply.     

Induction of a high-capacity nitrate-uptake mechanism in barley roots prompted by nitrate uptake through a constitutive low-capacity mechanism

Roots of nitrate-starved and nitrate-pretreated seedlings of Hordeum vulgare were used to investigate the induction of a high-capacity uptake mechanism for nitrate. When exposed to 0.2 mmol·l^-1KNO₃, nitrate-starved roots took up nitrate at a rate of approx. 1 mol·(g FW)^-1·h^-1; K⁺ was absorbed at a rate ten-times higher. Nitrate uptake accelerated after a lag of about 1 h, until it matched the rate of K⁺ uptake about 4 h later. p-Fluorophenylalanine (FPA), which prevents the synthesis of functioning proteins, suppressed the development of the high-capacity mechanism. Pretreatment of the roots with 0.2 mmol·l^-1 Ca(NO₃)₂ for 24 h established the high-capacity mechanism. Pretreated roots were able to absorb nitrate at high rates immediately upon exposure to 0.2 mmol·l^-1KNO₃, in the absence or presence of FPA. The high-capacity mechanism, once established, appeared to have a protein turnover as slow as that of the low-capacity mechanism or that of the mechanism involved in the uptake of K⁺. In contrast, the mechanisms for the transport of nitrate and K⁺ into the xylem vessels were completely blocked by FPA within 1 h of application, confirming earlier evidence for a rapid turnover of the transport proteins in the xylem parenchyma.Nitrate reduction proceeded at rates which were roughly one-tenth as large as the rates of the respective nitrate-uptake processes, indicating that nitrate-reductase activity was determined by the rate of nitrate uptake and not vice versa.We conclude that the formation of a high-capacity nitrate-uptake mechanism in barley roots occurs in response to nitrate uptake through a constitutive mechanism of low capacity which appears to function as a sensing mechanism for nitrate in the environment of the roots.Abbreviation FPA p-fluorophenylalanine     

Effects of genetic modification of nitrate reductase expression on 15NO3– uptake and reduction in Nicotiana plants

The physiological consequences for NO₃^– utilization by the plant of underexpression and overexpression of nitrate reductase (NR) were investigated in nine transformants of Nicotiana tabacum and Nicotiana plumbaginifolia. The in vitro NR activities (NRAs) in both roots and leaves of low- and high-NR tobacco transformants ranged from 5–10% to 150–200%, respectively, of those measured in wild-type plants. The level of NR expression markedly affected the NO₃^– reduction efficiency in detached leaves and intact plants. In both species, ¹⁵NO₃^– reduction ranged from 15–45% of ¹⁵NO₃^– uptake in the low-NR plants, to 40–80% in the wild-type, and up to 95% in high-NR plants. In the high-NR genotypes, however, total ¹⁵NO₃^– assimilation was not significantly increased when compared with that in wild-type plants, because the higher ¹⁵NO₃^– reduction efficiency was offset by lower ¹⁵NO₃^– uptake by the roots. The inhibition of NO₃^– uptake appeared to be the result of negative feedback regulation of NO₃^– influx, and is interpreted as an adjustment of NO₃^– uptake to prevent excessive amino acid synthesis. In genotypes underexpressing NR, the low ¹⁵NO₃^– reduction efficiency also was generally associated with a decrease in net ¹⁵NO₃^– uptake as compared with the wild type. Thus, underexpression of NR resulted in an inhibition of reduced ¹⁵N synthesis in the plant, although the effect was much less pronounced than that expected from the very low NRAs. The restricted NO₃^– uptake in low-NR plants emphasizes the point that the products of NO₃^– assimilation are not the only factors responsible for down-regulation of the NO₃^– uptake system.