J. M. Maldonado

Nitrate and nitrite uptake and reduction by intact sunflower plants.

Nitrogen-starved sunflower plants (Helianthus annuus L. cv. Peredovic) cannot absorb NO3−or NO2−upon initial exposure to these anions. Ability of the plants to take up NO3−and NO2−at high rates from the beginning was induced by a pretreatment with NO3−. Nitrite also acted as inducer of the NO2−-uptake system. The presence of cycloheximide during NO3−-pretreatment prevented the subsequent uptake of NO3−and NO2−, indicating that both uptake systems are synthesized de novo when plants are exposed to NO3−. Cycloheximide also suppressed nitrate-reductase (EC 1.6.6.1) and nitrite-reductase (EC 1.7.7.1) activities in the roots. The sulfhydryl-group reagent N-ethylmaleimide greatly inhibited the uptake of NO3−and NO2−. Likewise, N-ethylmaleimide promoted in vivo the inactivation of nitrate reductase without affecting nitrite-reductase activity. Rates of NO3−and NO2−uptake as a function of external anion concentration exhibited saturation kinetics. The calculated Km values for NO3−and NO2−uptake were 45 and 23 μM, respectively. Rates of NO3−uptake were four to six times higher than NO3−-reduction rates in roots. In contrast, NO2−-uptake rates, found to be very similar to NO3−-uptake rates, were much lower (about 30 times) than NO2−-reduction rates. Removal of oxygen from the external solution drastically suppressed NO3−and NO2−uptake without affecting their reduction. Uptake and reduction were also differentially affected by pH. The results demonstrate that uptake of NO3−and NO2−into sunflower plants is mediated by energy-dependent inducible-transport systems distinguishable from the respective enzymatic reducing systems.

Differential effects of ammonium and tungsten on nitrate and nitrite uptake and reduction by sunflower plants

Abstract Net NO 3 − uptake by intact sunflower plants ( Helianthus annuus L. cv Peredovic) was substantially decreased by the presence of NH 4 + in the external solution. The simultaneous addition of the glutamine synthetase (GS)-inhibitor methionine sulphoximine (MSO) did not relieve the inhibitory effect of NH 4 + . The addition of MSO alone also resulted in a substantial inhibition of NO 3 − uptake. Hence, glutamine or another immediate product of NH 4 + assimilation did not appear to be involved in the NH 4 + effect. By contrast to NO 3 − , NO 2 − uptake was not affected by the presence of NH 4 + or MSO. Addition of NH 4 + and/or MSO also promoted a decrease of root nitrate reductase (NR) activity without affecting nitrite reductase (NiR) activity. Furthermore, NH 4 + - and MSO-treatments led to accumulation of both NH 4 + and NO 3 − in root tissue. It is suggested that the long-term inhibitory effect of NH 4 + on NO 3 − uptake is the result of a decreased rate of NO 3 − reduction. On the other hand, pretreatment of the plants with tungsten suppressed NO 3 − uptake but not NO 2 − uptake. Besides, tungsten fully inhibited root NR activity without affecting NiR activity. Inhibition of NO 3 − uptake by tungsten led to a decrease of internal NO 3 − levels. On the basis of these results and of recent observations by other authors, it is suggested that the NR complex from roots might also be involved in the uptake of NO 3 − by sunflower plants.

Glutamine-synthetase isoforms appearing in sunflower cotyledons during germination

Ion-exchange chromatography has been used to separate the isoforms of glutamine synthetase (GS; EC 6.3.1.2) appearing in sunflower (Helianthus annuus L. cv. Peredovic) cotyledons during seedling growth under different light and nitrogen conditions. Both in dry and imbibed seeds, only a single form of GS (GSs) was detected. Upon seed germination, the GSs isoform was gradually replaced by cytosolic (GS1) and plastidic (GS2) isoforms. Light and nitrate decreased the levels of GS1. In contrast, the appearance of GS2 was greatly stimulated by light. Nitrate also had a positive effect, particularly in the light. Light and nitrate acted synergistically on the appearance of GS2. The GS2:GS1 ratio in cotyledons of 9-d-old seedlings ranged from about 2, in darkness and nitrate-deprivation conditions, to 16 under light and nitrate application. The possible physiological roles of the distinct GS isoforms appearing in the epigeal cotyledons of sunflower during germination, and their differential regulation by light and nitrate, are discussed.

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

SummaryWe 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 (NO3−, NO2− or NH4+), and the involvement of plastids. Nitrate was absolutely (and specifically) required for NR induction, and stimulated more effectively than NO2− or NH4+ 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 NO3− concentration indicate that NO3−-dependent enzyme induction is elicited by NO3− per se and not by a product of its assimilatory reduction, e.g., NO2− or NH4+. In the presence of NO3−, 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 NO3−-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 NO3− 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 NO3− and/or for the accumulation of newly formed enzymes in the organelle.

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO3-in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH4+or urea as nitrogen sources. Moreover, the presence of NH4+did not abolish the NO3--dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO3-but not by NH4+or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.

Organic nitrogen content and nitrate and nitrite reductase activities in tritordeum and wheat grown under nitrate or ammonium

Tritordeum is a fertile amphiploid derived from durum wheat (Triticum turgidum L. conv. durum) × a wild barley (Hordeum chilense Roem. et Schultz.). The organic nitrogen content of tritordeum grain (34 mg g-1 DW) was significantly higher than that of its wheat parent (25 mg g-1 DW). Leaf and root nitrogen content became higher in tritordeum than in wheat after four weeks of growth, independently of the nitrogen source (either NO3- or NH4+). Under NO3- nutrition, tritordeum generally exhibited higher levels of nitrate reductase (NR) activity than wheat. Nitrite reductase (NiR) levels were however lower in tritordeum than in its wheat parent. In NH4+-grown plants, both NR and NiR activities progressively decreased in the two species, becoming imperceptible after 3 to 5 weeks of growth. Results indicate that, in addition to a higher rate of NO3- reduction, other physiological factors must be responsible for the greater accumulation of organic nitrogen in tritordeum grain.

Isoforms of glutamine synthetase in cotyledons, leaves and roots of sunflower plants

Summary Isoforms of glutamine synthetase (GS) have been isolated from different tissues of sunflower plants by ion-exchange chromatography. Two forms (GS 1 and GS 2 ) of GS were identified in leaves and only one form (GS r ) was identified in roots. The relative proportions of GS 1 and GS 2 in leaves were 5% and 95% of the total GS activity, respectively. Green cotyledons also contained both GS 1 (21 %) and GSz (79 %). In etiolated cotyledons, the activity level of the GSz isoform greatly decreased while GS 2 activity slightly increased.

In vitro Stabilization and Tissue Distribution of Nitrogena-ssimilating Enzymes in Sunflower

Summary Nitrate reductase (NR), glutamine synthetase (GS) and glutamate dehydrogenase (GDH)activities were examined both in cotyledons and embryonic axes of 7-day-old seedlings and roots of 15-day-old plants of sunflower ( Helianthus annuus ) cv. Peredovic and cv. Sungro 380. Very low activities were observed in crude extracts prepared with a basal extraction medium consisting of 0.1 M Tris-HCI buffer, pH 7.5, 1 mM EDTA, and 511M FAD. Addition to this medium of either 10 mM cysteine, 1 % (w/v) polyvinylpolypyrrolidone or 2.5 % (w/v) casein substantially increased the recovery of the enzyme activities. Notwithstanding, NR activities in vitro were still much lower than those observed in vivo . These results indicate that NR, GS, and GDH of sunflower are susceptible to inactivation by phenolics during tissue disruption. In addition, NR appears to be inactivated during extraction by some inactivating factor existing in the tissues. The activities of NR and GS were higher in cotyledons and leaves than in embryonic axesand roots, respectively, whereas the opposite occurred for GDH. On the other hand, GS activity, more than GDH, was correlated with NR level in different leaves. These observations suggest that cotyledons and leaves are major sites of nitrogen assimilation in sunflower plants. Moreover, the correlation of GS and NR is consistent with a function of GS, more than GDH, in the assimilation of ammonia derived from nitrate reduction.

Effect of Light Quality on Growth, Contents of Carbohydrates, Protein and Pigments, and Nitrate Reductase Activity in Soybean Plants

Summary Soybean ( Glycine max L. cv. Williams) plants were grown from germination with nitrate as the nitrogen source under a 16-h photoperiod supplied by white, blue or red fluorescent lamps at a photon fluence rate of 46 µmol m -2 s -1 . As compared with white-light-grown plants, blue light inhibited stem elongation whereas red light had a stimulatory effect. By contrast, leaf growth was increased by blue light and reduced by red light. The high initial concentrations of carbohydrates and protein in the cotyledons gradually declined during seedling growth, irrespective of the light quality. Both in primary and first trifoliate leaves, the levels of carbohydrates and protein progressively increased up to leaf maturation. The accumulation of protein was higher in the plants grown under blue light, especially in the first trifoliate leaves. On the contrary, red light stimulated carbohydrate accumulation. The levels of nitrate reductase (EC 1.6.6.1) activity in leaves were found to be higher under red than under blue light. Accordingly, the greater protein concentration occurring in the soybean plants illuminated with blue light cannot be ascribed to a blue light activation of nitrate reductase leading to a higher availability of ammonia for the synthesis of amino acids and proteins, as has been previously suggested. Red light was more effective than blue light in stimulating the synthesis of chlorophyll and carotenoids in leaves.

Rhythmic variations of ferredoxin-glutamate synthase activity in sunflower (Helianthus annuus) leaves

Summary Changes in the activity of ferredoxin-glutamate synthase (Fd-GOGAT; EC 1.4.7.1) in response to lightdark transitions were monitored in sunflower ( Helianthus annuus L. cv. Peredovic) leaves. Plants grown in a controlled environment with nitrate as nitrogen source and a daily 16-h light period were subjected to different light-dark conditions. When plants were transferred either to continuous light or to continuous darkness for 30 h, Fd-GOGAT activity displayed rhythmic fluctuations with a period of 4–5 h. Similar rhythmic variations were observed in plants under 16 h – 8 h, 6 h – 6 h, and 3 h – 3 h light-dark cycles. These results indicate that Fd-GOGAT activity in sunflower leaves shows an ultradian rhythmicity.