Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Nitrate use by tobacco cells in response to N-stress and ammonium nutrition

Summary Characterization of NO ₃ ^– use by suspension cultured tobacco cells during a culture cycle is needed to take advantage of cell cultures for further study of the biochemical regulation of NO ₃ ^– uptake induction and decay processes. Tobacco (Nicotiana tabacum L., cv. Ky14) cells were cultured with media containing different N sources. Cells cultured with a mixture of NO ₃ ^– and NH ₄ ⁺ (40 mM NO ₃ ^– plus 20 mM NH ₄ ⁺ , in Murashige and Skoog media) initially grew slightly faster but attained the same maximum cell culture density as those cultured with 40 mM NO ₃ ^– only. Cells subcultured with N-free media grew at a similar rate for the first 3 d as those cells grown with N, then ceased further growth. The cessation of growth of cells subcultured with N-free media coincided with depletion of cell NO ₃ ^– . The NO ₃ ^– influx of cells subcultured with N-free media increased eleven-fold and those grown with N increased four- to five-fold before declining. Maximal NO ₃ ^– influx rates occurred at the onset of the stationary growth phase for N-stressed cells, while cells grown with N reached maximums prior to the stationary phase of cell growth. Cells grown with a mixture of NO ₃ ^– and NH ₄ ⁺ had lower NO ₃ ^– reductase (NR) activity and higher cell NO ₃ ^– levels than those of cells grown with NO ₃ ^– only. The NR activity of cells subcultured with N-free media peaked within 1 d after subculture before declining to a constitutive level when cell NO ₃ ^– was depleted. The level of cell NO ₃ ^– plays a critical role in the expression of the NO ₃ ^– uptake and reduction processes. The transitions in the expression of NO ₃ ^– uptake and reduction activities of tobacco cell suspension cultures should prove valuable for further study of the biochemical and molecular basis for the regulation of these processes.Abbreviations DTT DL-dithiothreitol - EDTA ethylenediamine tetraacetate - FW fresh weight - MS media Murashige & Skoog media - NADH ß-nicotinamide adenine dinucleotide reduced form - PMSF phenylmethyl-sulfonyl fluoride     

The ability of several high arctic plant species to utilize nitrate nitrogen under field conditions

The ability to utilize NO _inf3 ^sup– in seven high arctic plant species from Truelove Lowland, Devon Island, Canada was investigated, using an in vivo assay of maximum potential nitrate reductase (NR) activity and applications of ¹⁵N. Plant species were selected on the basis of being characteristic of nutrient-poor and nutrient-rich habitats. In all species leaves were the dominant site of NR activity. Root NR activity was negligible in all species except Saxifraga cernua. NO _inf3 ^sup– availability per se did not appear to limit NR activity of the species typically found on nutrient-poor sites (Dryas integrifolia, Saxifraga oppositifolia, and Salix arctica), or in Cerastium alpinum, as leaf NR activities remained low, even after NO _inf3 ^sup– addition. ¹⁵NO _inf3 ^sup– uptake was limited in D. integrifolia and Salix arctica. However, the lack of field induction of NR activity in C. alpinum and Saxifraga oppositifolia was not due to restricted nitrate uptake, as ¹⁵NO _inf3 ^sup– labelled NO _inf3 ^sup– entered the roots and shoots of both species. Leaf NR activity rates were low in three of the species typical of nutrient-rich habitats (O. digyna, P. radicatum and Saxifraga cernua), sampled from a site containing low soil NO _inf3 ^sup– . Additions of NO _inf3 ^sup– significantly increased leaf NR activity in these latter species, suggesting that potential NR activity was limited by the availability of NO _inf3 ^sup– . ¹⁵N labelled NO _inf3 ^sup– was taken up by O. digyna. P. radicatum and Saxifraga cernua. Although two species (D. integrifolia and Salix arctica) showed little utilization of NO _inf3 ^sup– , we concluded that five of the seven selected high arctic plant species (C. alpinum, O. digyna, P. radicatum, Saxifraga cernua and Saxifraga oppositifolia) do have the potential to utilize NO _inf3 ^sup– as a nitrogen source under field conditions, with the highest potential to utilize NO _inf3 ^sup– occurring in three of the species typically found on fertile habitats.     

Effects of light quantity and quality and soil nitrogen status on nitrate reductase activity in rainforest species of the genus Piper

Summary We studied nitrate reductase (NR) activity in six species of the genus Piper (Piperaceae) growing under a broad range of light availabilities. Field measurements were made on plants growing naturally in rainforest at the Los Tuxtlas Tropical Biological Preserve, Veracruz, Mexico at high- and lowlight extremes for each species. Foliar nitrogen on an area basis was positively related to the average daily photosynthetically active photon flux density (PFD) received by the leaf (r=0.76, p<0.01). In vivo NR activity was highly correlated with PFD (r=0.95, p<0.001) and less so with total leaf nitrogen (r=0.68, p<0.05). In vivo NR activity was always higher in high-light plants than in low-light plants within a species. Similarly, gap species such as P. auritum had much higher in vivo NR activities than shade species such as P. aequale. Soil NO ₃ ^– and NH ₄ ⁺ pools and nitrogen-mineralization rates at Los Tuxtlas were similar between high- and low-light sites, indicating that the elevated NR activities in high-light plants were not the result of higher NO ₃ ^– availabilities in high-light microsites. We performed additional experiments at Stanford, California, USA on Piper plants grown at high- and low-light. Foliar NR was highly inducible by nitrate in the gap species (auritum) but not in the generalist (hispidum) or shade (aequale) species. Root NR activities were, in general, an order of magnitude lower than foliar activities. In total, these studies suggest that Piper gap species are inherently more competent to assimilate NO ₃ ^– and are better able to respond to sudden increases in NO ₃ ^– availability than are shade species.CJWDPB publication # 1097     

Resource limitations to nitric oxide emissions from a sagebrush-steppe ecosystem

We monitored soil emissions of NO, NO₂, N₂O, and CO₂ throughout the summer dry season at a remote North American sagebrush-steppe ecosystem following application of several resources, including water, NH ₄ ⁺ , NO ₃ ^– and sucrose. Despite low levels of soil NH ₄ ^– (5.60±0.95 mg NH ₄ ^– -N per kg soil, mean ± S.E.), and NO ₃ ^– -N (1.34±0.20 mg NO ₃ ^– -N per kg soil), NO emissions ranged from about 0.2 to 2.8 ng NO-N m^–2 s^–1, comparable to rates measured from many agricultural, tropical, and other undisturbed ecosystems. Soil wetting increased NO emissions as much as 400-fold when initial gravimetric soil moisture contents were less than about 50 mg kg _soil ^–1 and soil temperature was greater than or equal to 20 °C. Wetting treatments with 20 mg NH ₄ ⁺ -N kg _soil ^–1 raised NO emission rates to a level that was nearly an order of magnitude higher than that observed after water addition alone. Wetting treatments with 20 mg NO ₃ ^– -N kg _soil ^–1 , 240 mg sucrose-C kg _soil ^–1 , or NO ₃ ^– plus sucrose had no statistically significant effect upon NO emissions. Soil denitrifying enzyme activity was low at this site, and N₂O emissions in the field were below detection limits. Soil nitrifying enzyme activity was extremely high at this site, indicating that the NH ₄ ⁺ released by ammonification would be consumed at least once every 1.7 days. These observations indicate that NO emissions from this undisturbed ecosystem were likely a consequence of high nitrification activity, and that sagebrush-steppe ecosystems may be a more important NO source than has been previously assumed.     

Induction of nitrate reductase, nitrite reductase, and glutamine synthetase isoforms in sunflower cotyledons as affected by nitrate, light, and plastid integrity

Summary We investigated the inducibility of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), and glutamine synthetase (GS; EC 6.3.1.2) isoforms in cotyledons of 7-day-old seedlings of sunflower (Helianthus annuus L.) in relation to light, nitrogen source (NO ₃ ^– , NO ₂ ^– or NH ₄ ⁺ ), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO ₂ ^– or NH ₄ ⁺ the synthesis of NiR and chloroplastic GS (GS₂) over the constitutive levels present in N-free-grown seedlings. In vivo inhibition of NR activity by tungsten application to seedlings and measurements of tissue NO ₃ ^– concentration indicate that NO ₃ ^– -dependent enzyme induction is elicited by NO ₃ ^– per se and not by a product of its assimilatory reduction, e.g., NO ₂ ^– or NH ₄ ⁺ . In the presence of NO ₃ ^– , light remarkably enhanced the appearance of NR, NiR, and GS₂, while the activity of the cytosolic GS isoform (GS₁) was adversely affected. Cycloheximide suppressed much more efficiently than chloramphenicol the light- and NO ₃ ^– -dependent increase of GS₂ activity, indicating that sunflower chloroplastic GS is synthesized on cytoplasmic 80S ribosomes. When the plastids were damaged by photooxidation in cotyledons made carotenoid-free by application of norflurazon, the positive action of light and NO ₃ ^– on the appearance of NR, NiR, and GS₂ isoform was greatly abolished. Therefore, it is suggested that intact chloroplasts are required for the inductive effect of light and NO ₃ ^– and/or for the accumulation of newly formed enzymes in the organelle.Abbreviations CAP chloramphenicol - CHX cycloheximide - GS glutamine synthetase - GS₁ cytosolic GS - GS₂ plastidic (chloroplastic) GS - NF norflurazon - NiR nitrite reductase - NR nitrate reductase     

Nitrogen nutrition of rice plants under salinity

Two rice (Oryza sativa L.) cultivars, Koshihikari and Pokkali, were grown in solution culture at three concentrations of NaCl or Na₂SO₄ [0 (S0), 50 (S1), and 100 (S2) mmol dm^–3] and three N contents [0.7 (N1), 7 (N2) and 14 (N3) mmol dm^–3]. Salinity significantly decreased dry matter of both cultivars. Pokkali had better growth than Koshihikari under both saline and non-saline conditions. Applications of N enhanced development of shoot dry mass under S0 and S1 treatments up to N2. Under S2, N application had no effect on shoot dry mass of both cultivars. Root dry mass of both cultivars decreased with increasing N application at S1 and S2. Shoot and root NO₃-N content in both rice cultivars increased with increasing N concentration in the nutrient solutions. The absorption of NO₃-N was less in Koshihikari than Pokkali plants, and also was much less in Cl^– than SO₄ ^2– salinity suggesting the antagonism between Cl^– and NO₃ ^–. In addition a significant negative correlation between concentrations of NO₃-N and Cl^– in the shoots or roots was observed in both cultivars     

Quantification of N2-fixation and survival of inoculated diazotrophs associated with roots of Kallar grass

White clover plants were grown for 97 days under two temperature regimes (20/15°C and 8/5°C day/night temperatures) and were supplied with either small amounts (a total of 80 mg N pot^–1) of ammonium (NH ₄ ⁺ ) or nitrate (NO ₃ ^– ) nitrogen, or received no mineral N and relied on N₂ fixation. Greatest growth and total leaf area of clover plants occurred in N₂ fixing and NO ₃ ^– -fed plants grown at 20/15°C and poorest growth occurred in NH ₄ ⁺ -fed plants grown at 8/5°C. Nodule mass per plant was greater at 8/5°C due to increased nodule numbers rather than increased dry weight per nodule. This compensated to some extent for the reduced N₂-fixing activity per unit dry weight of nodule tissue found at the low growth temperature up to 116 d after sowing, but thereafter both activity per nodule dry weight and activity per plant were greater at the low temperature. Highest nitrate reductase activity (NRA) per g fresh weight and total activity per leaf, petiole or root occurred in NO ₃ ^– -fed plants at 8/5°C. Low growth temperature resulted in a greater partitioning of total plant NRA to the roots of NO ₃ ^– -fed plants. The results are considered in relation to the use of N fertiliser in the spring under field conditions.     

Accumulation and reduction of nitrate in cereal plants dependent on N supply

Summary In pot experiments the NO ₃ ^– accumulation and the occurrence of nitrate reductase (NR) capacity of wheat plants were investigated depending on late N applications at tillering, shooting and heading. NO ₃ ^– is preferentially accumulated in the stems, while NR dominates in the leaves. NO ₃ ^– accumulation is enhanced by late N treatments especially if N supply at seeding is sufficient. NR capacity of the plants is stimulated by late nitrogen supply, but its increment rates decrease with increasing NO ₃ ^– accumulation.     

Soybean mutants lacking constitutive nitrate reductase activity : I. Selection and initial plant characterization

The objectives of this study were to select and initially characterize mutants of soybean (Glycine max L. Merr. cv Williams) with decreased ability to reduce nitrate. Selection involved a chlorate screen of approximately 12,000 seedlings (progeny of mutagenized seed) and subsequent analyses for low nitrate reductase (LNR) activity. Three lines, designated LNR-2, LNR-3, and LNR-4, were selected by this procedure.

In growth chamber studies, the fully expanded first trifoliolate leaf from NO₃⁻-grown LNR-2, LNR-3, and LNR-4 plants had approximately 50% of the wild-type NR activity. Leaves from urea-grown LNR-2, LNR-3, and LNR-4 plants had no NR activity while leaves from comparable wild-type plants had considerable activity; the latter activity does not require the presence of NO₃⁻ in the nutrient solution for induction and on this basis is tentatively considered as a constitutive enzyme. Summation of constitutive (urea-grown wild-type plants) and inducible (NO₃⁻-grown LNR-2, LNR-3, or LNR-4 plants) leaf NR activities approximated activity in leaves of NO₃⁻-grown wild-type plants. Root NR activities were comparable in wild-type and mutant plants grown on NO₃⁻, and roots of both plant types lacked constitutive NR activity when grown on urea. In both growth chamber- and field-grown plants, oxides of nitrogen [NO_(x)] were evolved from young leaves of wild-type plants, but not from leaves of LNR-2 plants, during in vivo NR assays. Analysis of leaves from different canopy locations showed that constitutive NR activity was confined to the youngest three fully expanded leaves of the wild-type plant and, therefore, on a total plant canopy basis, the NR activity of LNR-2 plants was approximately 75% that of wild-type plants. It is concluded that: (a) the NR activity in leaves of NO₃⁻-grown wild-type plants includes both constitutive and inducible activity; (b) the missing NR activity in LNR-2, LNR-3, and LNR-4 leaves is the constitutive component; and (c) the constitutive NR activity is associated with NO_(x) evolution and occurs only in physiologically young leaves.

     

Temperature dependence of inorganic nitrogen uptake and assimilation in Antarctic sea-ice microalgae

Summary The influence of temperature on NO ₃ ^- and NH ₄ ⁺ uptake, and the activity of the assimilatory enzyme NO ₃ ^- reductase (NR) was compared to inorganic C uptake (photosynthesis) in natural assemblages of Antarctic sea-ice microalgae. NO ₃ ^- and NH ₄ ⁺ uptake reached a maximum between 0.5°–2.0°C and 2.0°–3.0°C, respectively, which was close to that for photosynthesis (2.5°–3.0°C). NR showed a distinctly higher temperature maximum (10.0°–12.0°C) and a lower Q₁₀ value than inorganic N and C transport. Our data imply that, owing to differential temperature characteristics between N transport and N assimilation at in situ temperature (-1.9°C), the incorporation of extracellular NO ₃ ^- into cellular macromolecules, may be limited by transport of NO ₃ ^- into the cell rather than the intracellular reduction of NO ₃ ^- to NH ₄ ⁺ . Despite differences in temperature maxima between N transport and N assimilation, the overall low temperature maxima of inorganic N metabolism characterizes Antarctic sea-ice microalgae as psychrophilic. Our study is the first to examine the temperature dependence of inorganic N uptake and assimilation in sea-ice microbial communities.     

Post hoc assessment of an operational biocontrol program: efficacy of the flea beetle Aphthona lacertosa Rosenhauer (Chrysomelidae: Coleoptera), an introduced biocontrol agent for leafy spurge

Mixed populations of Aphthona lacertosa and Aphthona czwalinae were released at more than 50 locations in Alberta in 1997. Two and 3 years post-release, beetle populations were primarily A. lacertosa, with A. czwalinae forming less than 0.5% of the sampled populations. Beetle densities were moderate (10–70 beetles per m²) or high (>70 beetles per m²) at 14% and more than 60% of the sampled sites in 1999 and 2000, respectively. Larger beetles had greater instantaneous egg loads (r²=0.424,P=0.003). In 2000, the largest beetles were found at moderate density sites and there was a significant negative relationship between beetle size and the time taken to accumulate a degree day threshold of 1230 (for females: r²=0.678,P=0.001). Sites with the most rapid accumulation of degree days have the greatest potential for beetle population growth based on potential fecundity. Changes in leafy spurge percent cover, stem density, and canopy height from 1997 to 2000 were assessed across sites with low (<10 beetles per m²), moderate, and high beetle densities in 2000. Sites with high beetle densities had significantly greater reductions of leafy spurge within 5 m of the release point than sites with low beetle densities (P<0.017). Damage caused by the beetles at high-density sites was often visible as a halo-shaped patch of dead leafy spurge stems. The significant overall reduction of leafy spurge within release patches makes A. lacertosa a promising biocontrol agent for leafy spurge in Alberta.     

Changes in amino acid content of an algal feed species (Navicula sp.) and their effect on growth and survival of juvenile abalone (Haliotis rubra)

The growth and survival of juvenile Haliotis rubra, when fed with the diatom Navicula sp. cultured in f/2 medium containing combined nitrogen at 24.71 mg NO₃-N L^–1 (high), 12.35 mg NO₃-N L^–1 (standard) or 2.47 mg NO₃-N L^–1 (low), were compared in a 33-day trial. The alga in the low nitrogen medium contained 37% less total amino acid than that in the high and standard nitrogen media. There was a slightly greater reduction in essential amino acids (40%) compared to non-essential amino acids (35%). Juvenile abalone feeding on Navicula grown in medium with low nitrate and lower total amino acid content grew more slowly than when fed on the same species grown in standard or higher nitrogen medium with a higher amino acid content. The growth rate of juveniles was highest (43 m d^–1) in the high nitrate treatment followed (40 m d^–1) by the standard nitrate treatment and lowest (31 m d^–1) in the low nitrate treatment. The survival of the juveniles was also effected by the diet. Survival was better in the high and standard nitrogen media (88%) than the low nitrogen medium (75%). The results suggest that in order to achieve uniformity in nutritional quality of diatoms and good growth of abalone juveniles in commercial abalone nurseries, the nitrogen concentration in tanks should be monitored and additional nitrate added to provide an optimum concentration of between 2 and 12 mg NO₃-N L^–1.     

Short-term modulation of nitrate reductase activity by exogenous nitrate in Nicotiana plumbaginifolia and Zea mays leaves

Maize (Zea mays L.) grown on low (0.8 mM) NO ₃ ^- , as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO ₃ ^- on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg²⁺. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO ₃ ^- did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO ₃ ^- content and the NR activation state. Similarly, the NR activation state did not respond to NO ₃ ^- . Since the NR activation state determined from the degree of Mg²⁺-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO ₃ ^- -induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO ₃ ^- . We conclude that NO ₃ ^- does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C₃ species) or maize (a C₄ species). The short-term regulation of NR therefore differs from the NO ₃ ^- -mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl chlorophyll - MC microcystin-LR - PEP-Case phosphoenolpyruvate carboxylase - SPS sucrose-phosphate synthase We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency.     

Organogenesis in cell cultures of leafy spurge (Euphorbiaceae) accessions from Europe and North America

Plants were regenerated from leafy spurge (Euphorbia esula L.) cell suspensions obtained from stem callus. A North Dakota accession was highly regenerable, but two accessions from Oregon and Austria formed only a few plantlets. Organogenesis occurred in media without growth regulators, under fluorescent lights (30 to 90 E m^–2 s^–1, 14 h photoperiod). Organogenesis was greatest in larger size clumps subcultured during maximum cell growth into media containing a reduced:oxidized nitrogen ratio of 33:67. Roots formed first and some clumps produced shoots. Organogenic suspension cultures also were initiated from hypocotyl and root segments of germinated seedlings, directly in liquid medium. Plantlets of the North Dakota accession formed in vitro adapted to greenhouse conditions. They were phenotypically similar to the parent plants.     

The culture of rat myeloma and rat hybridoma cells in a protein-free medium

Y0 is a rat x rat hybridoma cell line, which does not secrete immunoglobulin, produced using a fusion partner derived from the Y3 (Y3,Ag.1.2.3) rat myoloma cell line. Y0 and Y3 have both been widely used as fusion partners in the production of rat x rat hybridomas. Y0 has also been used in recombinant gene technology. Y0 cells grown in shake flask culture, using RPMI 1640 medium with 4mM l-glutamine and 5% foetal bovine serum, reached a maximal cell density of 1.5×10⁶ cells ml^–1 with 86% viability. Y0 cells which has been adapted to grow in ABC protein-free medium reached a maximal density, in shake flask culture, of 8.75×10⁵ cells ml^–1 with 79% viability. An improved protein-free medium, designated W38 medium, was developed. In shake flask culture, W38 medium supported Y0 cell growth to a density of 2.02×10⁶ cells ml^–1 with 96% viability. Two Y3 hybridomas, YID 13.9.4 cells and SAM 618 cells were adapted to growth in W38 medium. For both hybridomas, cell growth and product yield in shake flask culture using W38 medium was superior to that obtained with serum-containing RPMI 1640 medium.Abbreviations F12 Hams F12 medium - DMEM Dulbeccos medium - RPMI RPMI 1640 medium - FBS foetal bovine serum     

Inducible nitrate reductase of rice plants as a possible indicator for nitrification in water-logged paddy soils

When following the pattern of the disappearance of NH ₄ ⁺ –N from ammonium sulfate applied to the flooded soil-rice plant system (field and greenhouse experiments) during a growing season, it was observed that the lowest NH ₄ ⁺ –N level coincided with the highest value of NR activity in the leaves. Nitrate was detected in both the root and shoot systems of the rice plants and autotrophic nitrifiers (Nitrosomonas and Nitrobacter) were particularly abundant. Since it was also demonstrated in this work that the NR activity of rice plants grown with nitrate fertilization (growth chamber culture experiments) was inducible by its substrate, it can be assumed that NH ₄ ⁺ –N oxidation takes place in the water-logged soil studied. Therefore, the occurrence of the nitrification process following NH ₄ ⁺ –N fertilizer application can be predicted by thein vitro orin situ evaluation of the NR activity of the rice leaf as an indicator.     

Effects of putrescine and inhibitors of putrescine biosynthesis on organogenesis inEuphorbia esula L.

Summary Exogenous putrescine (&#x2264;5 mM) had little effect on root or shoot formation in aseptically isolated hypocotyl segments of leafy spurge (Euphorbia esula L.) grown on full-strength B5 medium. Unexpectedly, putrescine inhibited root and shoot formation in hypocotyl segments grown on B5 medium diluted 10-fold. In the full-strength medium, root and shoot formation were inhibited by 0.5 mM concentrations ofdl-&#x03B1;-difluoromethylornithine (DFMO) anddl-&#x03B1;-difluoromethylarginine (DFMA). DFMO and DFMA are inhibitors of the ornithine decarboxylase and arginine decarboxylase pathways, respectively, of putrescine biosynthesis in plants. Exogenous putrescine (0.5 to 5 mM) did not reverse either the DFMO-or DFMA-induced inhibition of shoot formation. However, the DFMA-induced inhibition of root formation was partially reversed by exogenous putrescine. The auxin, indole-3-acetic acid (IAA), reduced the inhibitory effects of DFMO+DFMA (applied together) on both roots and shoots. In the first few days of culture, the endogenous levels of putrescine and spermidine, but not of spermine, increased in the presence of IAA. The levels of putrescine and spermidine in the tissues did not correlate well with either root or shoot production in the later stages of organ formation; especially in tissues treated with IAA. These results show that there were no obvious correlations between polyamine levels and organogenesis in leafy spurge hypocotyl segments, although residual putrescine or spermidine or both in the tissues at the time of excision may be indirectly involved in the early stages of root formation.     

The role of nitrate and ammonium ions and light on the induction of nitrate reductase in maize leaves

Corn seedlings (Zea mays cv W64A × W182E) were grown hydroponically, in the presence or absence of NO₃⁻, with or without light and with NH₄Cl as the only N source. In agreement with earlier results nitrate reductase (NR) activity was found only in plants treated with both light and NO₃⁻. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer of the proteins to nitrocellulose paper and reaction with antibodies prepared against a pure NR showed that crude extracts prepared from light-grown plants had a polypeptide of approximately 116 kilodaltons (the subunit size for NR) when NO₃⁻ was present in the growth medium. Crude extracts from plants grown in the dark did not have the 116 kilodalton polypeptide, although smaller polypeptides, which reacted with NR-immunoglobulin G, were sometimes found at the gel front. When seedlings were grown on Kimpack paper or well washed sand, NR activity was again found only when the seedlings were exposed to light and NO₃⁻. Under these conditions, however, a protein of about 116 kilodaltons, which reacted with the NR antibody was present in light-grown plants whether NO₃⁻ was added to the system or not. The NR antibody cross-reacting protein was also seen in hydroponically grown plants when NH₄Cl⁻ was the only added form of nitrogen. These results indicate that the induction of an inactive NR-protein precursor in corn is mediated either by extremely low levels of NO₃⁻ or by some other unidentified factor, and that higher levels of NO₃⁻ are necessary for converting the inactive NR cross-reacting protein to a form of the enzyme capable of reducing NO₃⁻ to NO₂⁻.     

Complementation in somatic hybrids indicates four types of nitrate reductase deficient lines in Nicotiana plumbaginifolia

Summary Allelism of nine nitrate reductase deficient (NR^–) Nicotiana plumbaginifolia cell lines was tested by complementation after protoplast fusion. Complementation was recognized by the appearance of somatic hybrid colonies growing on a selective NH₄ ⁺/NO₃ ^– medium which cannot support the growth of NR^– lines. All five apoenzyme defective (NA) lines were non-complementing and therefore allelic. The apoenzyme and the cofactor defective (NX) lines were complementing, as expected, and gave somatic hybrids with restored nitrate reductase activity. The four cofactor defective lines were found to belong to three complementation groups (NX1 and NX9; NX21; NX24). Two of these (NX21 and NX24) are of new types which have not been previously described in flowering plants. In the somatic hybrids restoration of NR activity was accompanied by the restoration of plant regeneration ability. On leave from: Instituto di Mutagenesi e Differenziamento CNR, Via Svezia, 10, 56100, Pisa, Italy