Effects of Nitrate Nutrition on Nitrogen Metabolism in Cassava

Two experiments were conducted independently with plants of cassava (Manihot esculenta Crantz) growing in sand with nutrient solutions with four nitrate concentrations (0.5, 3, 6 or 12 mM). In leaves, nitrate-N was undetectable at the low nitrate applications; total-N, ammonium-N, amino acid-N, reduced-N and insoluble-N all increased linearly, while soluble proteins did it curvilinearly, with increasing nitrate supply. In contrast, soluble-N did not respond to N treatments. Total-N and soluble proteins, but not nitrate-N or ammonium-N, were much higher in leaves than in roots. Plants grown under severe N deficiency accumulated ammonium-N and amino acid-N in their roots. Further, plants were exposed to either 3 or 12 mM nitrate-N, and leaf activities of key N-assimilating enzymes were evaluated. Activities of nitrate reductase, glutamine synthetase, glutamate synthase and glutamate dehydrogenase were considerably lower in low nitrate supply than in high one. Despite the low nitrate reductase activity, cassava leaves showed an ability to maintain a large proportion of N in soluble proteins.     

The Relationship of Abscisic Acid to Nitrate Reductase Activity in the Potato Plant

Abscisic acid (ABA) at 3.8 µM suppressed both in vivoand in vitro nitrate reductase activity in roots, stems andleaves of potato plants grown in solution culture. Suppressionwas maximal between 24 and 48 h, followed by recovery of activityat 72 h in roots and leaves and at 96 h in stems. Removal from ABA after 24 h resulted in complete recovery ofnitrate reductase activity in roots by 24 h and partial recoveryin leaves. ABA treatment enhanced nitrate accumulation in roots,decreased that of leaves, but had no effect on stem nitratecontent. ABA enhanced decay of the enzyme following nitrate removal;by 7 h activity in roots was 22.5% of the initial value comparedto 55% in the control. ABA showed a less drastic effect on lossof activity in leaves and stems. These results indicate thatABA suppression of nitrate reductase activity is not dependenton nitrate uptake, and although it reduced leaf nitrate contentthere was no clear relationship between tissue nitrate levelsand the ABA response. (Received September 13, 1984; Accepted July 1, 1985)     

Triadimenol Increases Nitrate Levels and Nitrate Reductase Activity in Canola Leaves

Nitrate reductase activity in the first true leaves of canola(Brassica napus L.) seedlings grown in one-quarter strengthHoagland's solution from seeds pretreated with triadimenol (0.3or 30 g (a.i.) kg^–1 of seed) was higher than controlsduring the growth period of 15 to 25 d after planting. Triadimenolalso increased chlorophyll levels, the increase being more pronouncedat its lower concentration. The treatment also increased theweight and nitrate content of the leaves. When seedlings weregrown in nutrient solution containing 1 to 20 mM nitrate, theincrease in nitrate reductase activity by triadimenol was higherat lower rather than at higher nitrate concentrations. The nitratelevels and Kjeldahl nitrogen in the triadimenol-treated leaveswas higher than the controls at concentrations of added nitrateabove 2 mM. Addition of nitrate to plants grown in ammonium,increased nitrate reductase activity more in plants grown fromtriadimenol-treated seeds than controls. However, addition of10µM triadimenol for 24 h to ammonium-grown plants hadlittle effect on enzyme activity, both in the absence as wellas the presence of nitrate. This study demonstrates that triadimenolincreases nitrate reductase activity and nitrate accumulationin the leaves and at least part of the increased enzyme activityis independent of nitrate accumulation. Key words: Triazoles, nitrate content, nitrate reductase activity     

Changes in the activities of nitrogen assimilation enzymes ofLolium perenne L. during regrowth after cutting

The activities of key enzymes involved in N assimilation were investigated after defoliation of 6-week-old ryegrass plants grown in water culture conditions. In a first experiment, nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities were measured in roots, stubble and leaves on the day of cutting and at 7-day intervals over the following 5-week period of regrowth. Ammonia assimilation enzymes showed little change whereas the nitrate reductase activity sharply decreased 2 weeks after clipping. In a second experiment, the nitrate reductase activity was measured at 2- or 3-day intervals 1 week before and 3 weeks after clipping.In vivo andin vitro assays both showed an increasing activity in leaves up to 8 days after cutting while root activity decreased. The opposite changes then occurred and both organs recovered their initial nitrate reductase activity levels after 12–14 days of regrowth. These fluctuations in nitrate reductase activity were considered to be related to the capacity for C assimilation and the nitrate availability.     

Nitrate and Carbohydrate Effects on Nodulation and Nitrogen Fixation (Acetylene Reduction) Activity of Lentil (Lens esculenta Moench)

Lentils (Lens esculenta Moench, cv. Tekoas) grown in a nutrient solution containing 15 millimolar nitrate had 84% fewer nodules than lentils grown in nitrate-free nutrient solution. Nodules from the nitrate-grown plants weighed 71% less than nodules from the nitrate-free plants. Nitrate-grown plants also fixed much less nitrogen (measured by acetylene reduction) than the nitrate-free plants. When lentils were grown in a solution containing 15 millimolar nitrate and 75 millimolar fructose, glucose, or sucrose, however, the nitrogen fixation activity of their nodules was similar to that of nodules from nitrate-free plants. Leaves of lentils grown in the nitrate-sugar solutions had only about 7% as much nitrate reductase activity and accumulated only 10% as much nitrate as leaves from lentils grown in the nitrate solution alone. Roots of lentils grown in the nitrate-sugar solutions had similar nitrate reductase activity but accumulated only 17 to 25% as much nitrate as roots from lentils grown in the nitrate solution. The results indicate that the added sugars alleviated the inhibitory effects of nitrate on symbiotic nitrogen fixation not only by increasing the carbohydrate supply so lentils could support both nitrogen fixation and nitrate reduction but also by inhibiting the accumulation of nitrate and, hence, lowering nitrate reductase activity in the leaves.     

Partitioning of inorganic nitrogen assimilation between the roots and shoots of cerrado and forest trees of contrasting plant communities of South East Brasil

Summary Woody plants growing in cerrado and forest communities of south-east Brasil were found to have low levels of nitrate reductase activity in their leaves suggesting that nitrate ions are not an important nitrogen source in these communities. Only in the leaves of species growing in areas of disturbance, such as gaps and forest margins, were high levels of nitrate reductase present. When pot-grown plants were supplied with nitrate, leaves and roots of almost all species responded by inducing increased levels of nitrate reductase. Pioneer or colonizing species exhibited highest levels of nitrate reductase and high shoot: root nitrate reductase activities. Glutamine synthetase, glutamate synthase and glutamate dehydrogenase were present in leaves and roots of the species examined.¹⁵N-labelled nitrate and ammonium were used to compare the assimilatory characteristics of two species:Enterolobium contortisiliquum, with a high capacity to reduce nitrate, andCalophyllum brasiliense, of low capacity. The rate of nitrate assimilation in the former was five times that of the latter. Both species had similar rates of ammonium assimilation. Results for eight species of contrasting habitats showed that leaf nitrogen content increased in parallel with xylem sap nitrogen concentrations, suggesting that the ability of the root system to acquire, assimilate or export nitrate determines shoot nitrogen status. These results emphasise the importance of nitrogen transport and metabolism in roots as determinants of whole plant nitrogen status.     

Growth of Zinc-Deficient Rats During Intra-Gastric Tube Feeding

The main aim of the study was to determine the role of cerium in the amelioration of calcium-deficiency effects in spinach plants. Spinach plants were cultivated in Hoagland’s solution. They were subjected to calcium-deficiency and to cerium chloride administered in the calcium-present Hoagland’s media and calcium-deficient Hoagland’s media. Within 3weeks, young leaves developed distinct calcium-deficient symptoms, and plant growth significantly inhibited to calcium deprivation as would be expected; cerium-treated groups grown in the same conditions did not develop calcium-deficient symptoms; fresh weight, dry weight and chlorophyll content of spinach plants were increased by 35.9, 45 and 64.05% compared to those of plants cultivated in calcium-deficient media. In addition, calcium deprivation in spinach plants caused the reduction of photosynthetic rate, oxygen evolution rate and ribulose-1,5-bisphosphate carboxylase/oxygenase activity. The reduction of activities of nitrate reductase, glutamate dehydrogenase, glutamate synthase and glutamic-pyruvic transaminase was observed under calcium-deficient media. However, cerium treatment under calcium-deficient media could significantly improve photosynthesis and nitrogen metabolism of spinach plants. This is viewed as evidence that cerium added to calcium-deficient media in the spinach plants could substitute for calcium and improve spinach growth.     

Effect of applied nitrate on enzymes of ammonia assimilation in nodules ofCicer arietinum L.

Summary The relationship between N₂-fixation, nitrate reductase and various enzymes of ammonia assimilation was studied in the nodules and leaves ofC. arietinum. In the nodules of the plants growing on atmospheric nitrogen, maximum activities of glutamine synthetase (GS), glutamate synthase (GOGAT), glutamate dehydrogenase (GDH), asparagine synthetase (AS) and aspartate aminotransferase (AAT) were recorded just prior to maximum activity of nitrogenase. In nitrate fed plants, the first major peak of GDH and AS coincided with that of nitrate reductase in the nodules. With the exception of AS, application of nitrate decreased the activities of all these enzymes in nodules but not in leaves. Activities of GS, GOGAT and AAT were affected to much greater extent than that of GDH. On comparing the plants grown without nitrate and those with nitrate, the ratios of the activities of GDH/GS and GDH/GOGAT in nitrate given plants, increased by 4 and 12 fold, respectively. The results presented in this paper suggest that in nodules of nitrate fed plants, assimilation of ammonia via GDH assumes much greater importance.     

Influence of fruit load and environmental factors on nitrate reductase activity and on concentration of nitrate and carbohydrates in leaves of eggplant (Solanum melongena)

Both the in vivo (+ nitrate) nitrate reductase (NR) activity (leaf disks incubated in the presence of KNO₃) and the in vivo (? nitrate) NR activity (leaf disks incubated without KNO₃) in leaves of eggplant (Solanum melongena L. cv. Bonica) were affected by rapidly growing fruits. Plants with a fruit load showed more pronounced diurnal variation in (+ nitrate) NR activity and higher (? nitrate) NR activity than plants without fruit. The higher (? nitrate) NR activity was accompanied by higher nitrate and lower sucrose and starch contents of leaves. The more pronounced diurnal changes in (+ nitrate) NR activity were paralleled by more pronounced diurnal variation in carbohydrate content of leaves. Fruit removal led to a decrease in both (? nitrate) NR activity and nitrate concentration in leaves, while the carbohydrate content increased. Plants supplied with ammonium instead of nitrate showed only slightly lower (+ nitrate) but no (? nitrate) NR activity. As for plants treated with nitrate, diurnal changes in (+ nitrate) NR activity were most pronounced in leaves of plants with fruit and this again was paralleled by a more pronounced diurnal variation in the carbohydrate concentration in the leaves. Increasing the oxygen level of the atmosphere to 50% led to a dramatic decrease in the (+ nitrate) NR activity and to an increase in both (? nitrate) NR activity and nitrate concentration, which was accompanied by decreasing carbohydrate contents of the leaves. Low light intensities and extended dark periods caused similar changes in NR activity and nitrate and carbohydrate concentrations in leaves. Increasing the nitrate concentration in the nutrient solution led to a rise in (+ nitrate) and (? nitrate) NR activity, but only the (? nitrate) NR activity paralleled the nitrate concentration in the leaves. This increase in the nitrate concentration was accompanied by a decrease in the carbohydrate content of the leaves. It is concluded that the level of and the diurnal changes in both (+ nitrate) and (? nitrate) NR activity and the concentration of nitrate in the leaves are dependent upon their carbohydrate status.     

The effect of NO3- and NH4 + ions on enzymes involved in nitrogen assimilation inPisum sativum L

Nitrate reductase level in leaves of pea plants is higher than in roots despite of the lower content of endogenous nitrate. Addition of ammonium ions to nutrient solution containing nitrate decreases nitrate reductase level in leaves estimatedin vivo while its level estimatedin vitro is increased. Glutamine synthetase (GS) level in roots decreases during short (24 and 48 h) and long (14 d) term cultivation of seedlings in solutions containing ammonium ions. This decrease occurs in leaves only after the long term influence of ammonium ions. Level of this enzyme is higher in plants grown in the presence of nitrogen (ammonium and nitrate) as compared to those grown without the nitrogen. Level of glutamate dehydrogenase in roots is increased after both short and long term cultivation of plants in the presence of ammonium ions.     

Nitrogen Assimilation in Barley (Hordeum vulgare L. cv. Mazurka) in Response to Nitrate and Ammonium Nutrition

Barley plants (Hordeum vulgare L. cv. Mazurka) were grown inaerated solution cultures with 2 mM or 8 mM inorganic nitrogensupplied as nitrate alone, ammonium alone or 1:1 nitrate+ammonium.Activities of the principal inorganic nitrogen assimilatoryenzymes and nitrogen transport were measured. Activities ofnitrate and nitrite reductases, glutamine synthetase and glutamatesynthase were greater in leaves than in roots but glutamatedehydrogenase was most active in roots. Only nitrate and nitritereductases changed notably (4–10 times) in response tothe different nitrogen treatments. Nitrate reductase appearedto be rate-limiting for nitrate assimilation to glutamate inroots and also in leaves, where its total in vitro activitywas closely related to nitrate flux in the xylem sap and wasslightly in excess of that needed to reduce the transportednitrate. Xylem nitrate concentration was 13 times greater thanthat in the nutrient solution. Ammonium nitrogen was assimilatedalmost completely in the roots and the small amount releasedinto the xylem sap was similar for the nitrate and the ammoniumtreatments. The presence of ammonium in the nutrient decreasedboth export of nitrate to the xylem and its accumulation inleaves and roots. Nitrate was stored in stem bases and was releasedto the xylem and thence to the leaves during nitrogen starvation.In these experiments, ammonium was assimilated principally inthe roots and nitrate in the leaves. Any advantage of this divisionof function may depend partly on total conversion of inorganicnitrogen to amino acids when nitrate and ammonium are givenin optimal concentrations. Hordeum vulgare L., barley, nitrate, ammonium, nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, glutamate dehydrogenase, nitrogen transport     

Increased Arginine Biosynthesis during Phosphorus Deficiency : A Response to the Increased Ammonia Content of Leaves

The accumulation of arginine in leaves of four citrus rootstock cultivars during P deficiency has been demonstrated to be due to increased de novo synthesis rather than decreased catabolism or increased protein degradation (E Rabe, CJ Lovatt, 1984, Plant Physiol 76: 747-752). In this report, we provide evidence (a) that the increased activity of the arginine biosynthetic pathway observed for citrus rootstocks grown under P-deficient conditions for 7 months is due to an increase in the concentration of ammonia in leaves of P-deficient plants and (b) that ammonia accumulation and removal through arginine systhesis are early responses to phosphorus deficiency for both a woody perennial, rough lemon (Citrus limon), and an herbaceous annual, summer squash (Cucurbita pepo). Transferring 5-day-old squash plants to a phosphorus-deficient nutrient solution for only 10 days resulted in a 2-fold increase in the concentration of nitrate in the youngest fully expanded leaves (YFE). Concomitantly, the specific activity of nitrate reductase doubled and the ammonia content of P-deficient YFE leaves increased to a concentration significantly greater that of leaves from healthy control plants (P < 0.05). Consistent with increased availability of ammonia, the incorporation of NaH¹⁴CO₃ into arginine plus urea doubled during phosphorus deficiency and arginine accumulated. Despite the accumulation of nitrate and ammonia in YFE leaves during phosphorus deficiency, the total nitrogen content of these leaves was less than that of the healthy control plants. Similar results were obtained for rough lemon. Nitrate content of the YFE leaves increased 1.5- and 3.0-fold in plants deprived of phosphorus for 6 and 12 weeks, respectively. Ammonia content of the leaves increased as P deficiency progressed to 1.4 ± 0.08 mg (± se, n = 4) per gram dry weight, a level 1.8-fold greater than that of the P-sufficient control plants. During P deficiency de novo arginine biosynthesis in rough lemon increased 10-fold. Immersing the petiole of YFE leaves from P-sufficient squash and rough lemon plants in 50 millimolar NH₄⁺ for 3 hours resulted in the accumulation of ammonia in the leaves, and a 4-fold increase in the incorporation of NaH¹⁴CO₃ into arginine plus urea. Taken together, these results provide strong evidence that the accumulation of nitrate and ammonia in leaves is an early response of both woody and herbaceous plants to P deprivation. The data are consistent with the hypothesis that increased de novo arginine biosynthesis in leaves during P deficiency is in response to ammonia content of the leaves.     

Reversibility of the effects of cadmium on the growth and nitrogen metabolism in the tomato(Lycopersicon esculentum)

In order to better understand the effects of heavy metals on the growth of plants, we decided to perform recovering experiments by following both chemical and physiological parameters in cadmium pre-stressed tomato seedlings after cadmium had been removed from the nutrient solution. The work shows that cadmium suppression results in resumption of growth activity. The biomass of leaves and stems rose steadily. The increase in root biomass exceeded those of leaves and stems. At the same time, nitrate content was increased to reach the level obtained with unstressed controls. In all the organs studied, the activities of the enzymes involved in the anabolic nitrogen primary assimilation pathways (nitrate reductase (NR), nitrite reductase (NiR) and glutamine synthetase (GS) soared after that cadmium had been removed. While NAD(+)-dependent glutamate dehydrogenase (GDH-NAD+) activity also rose progressively during the recovering time, the cognate NADH-dependent glutamate dehydrogenase (GDH-NADH) activity decreased. This result allows us to propose that the ammonia produced by the stress-induced protein catabolism is detoxified and re-assimilated by the GDH-NADH isoenzyme. On the basis of these results, we will discuss the ability of the plant to dilute the effects of pollutants during the recovering period. An important outcome of this work is that a transient contamination of the culture medium by pollutants is not necessarily followed by a significant depreciation in product yield or quality.     

Tobacco plants that lack expression of functional nitrate reductase in roots show changes in growth rates and metabolite accumulation.

When tobacco is provided with a high nitrate supply, only a small amount of the nitrate taken up by the roots is immediately assimilated inside the roots, while the majority is transported to the leaves where it is reduced to ammonium. To elucidate the importance of root nitrate assimilation, tobacco plants have been engineered that showed no detectable nitrate reductase activity in the roots. These plants expressed the nitrate reductase structural gene nia2 under control of the leaf-specific potato promoter ST-LS1 in the nitrate reductase-mutant Nia30 of Nicotiana tabacum. Homozygous T2-transformants grown in sand or hydroponics with 5.1 mM nitrate had approximately 55-70% of wild-type nitrate reductase acivity in leaves, but lacked nitrate reductase acivity in roots. These plants showed a retarded growth as compared with wild-type plants. The activation state of nitrate reductase was unchanged; however, diurnal variation of nitrate reductase acivity was not as pronounced as in wild-type plants. The transformants had higher levels of nitrate in the leaves and reduced amounts of glutamine both in leaves and roots, while roots showed higher levels of hexoses (3-fold) and sucrose (10-fold). It may be concluded that the loss of nitrate reductase acivity in the roots changes the allocation of reduced nitrogen compounds and sugars in the plant. These plants will be a useful tool for laboratories studying nitrate assimilation and its interactions with carbon metabolism.     

Nitrate Reductase Activity of Wheat Seedlings during Exposure to and Recovery from Water Stress and Salinity

Nitrate reductase activity was inhibited as a result of reduced soil moisture potentials or application of NaCI to nutrient solutions. The decrease in enzyme activity of wheat seedlings exposed to salinity, was found 24 hours after exposure to stress. The effect of stress on nitrate reductase was found in cell-free extracts as well as in riro in assays of intact leaf sections. A recovery in enzyme activity was found after irrigation or after removal of seedlings from salinity. While relative water content of the leaves was restored within 3 hours after removal of stress, full recovery of enzyme activity occurred only after 24 hours. Cycloheximide and chloramphenicol suppressed the activity of nitrate reductase in non-stressed seedlings, but had no effect on the activity of plants exposed to salinity. However, during removal of stress, cycloheximide prevented completely the recovery of nitrate reductase, while chloramphenicol did not interfere with the recovery of the inhibited enzyme activity. It is concluded that a fraction of nitrate reductase may be located in the cytoplasm and lost activity during stress, probably due to inhibited protein synthesis. Another fraction which may be associated with chloroplasts, was inhibited by stress due to conformational changes or partial denaturation.     

Physiological responses of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> to different nitrogen forms

The responses of carbon and nitrogen metabolisms in the medical plant Catharanthus roseus to the nitrogen solutions (N1, N2 and N3) containing different ratio of nitrate to ammonium (1:0, N1; 1:1, N2; 1:3, N3) were investigated here. The plants in N3 nitrogen solution were strongly inhibited in photosynthetic gas exchange and carbohydrate accumulation, reflecting the toxicity symptom of excess ammonium continuously accumulated in plants. The treatment with N2 nitrogen solution, however, displayed an obviously synergistic effect on plant growth and metabolisms in contrast to nitrate as the sole source. The short-term (7 days) exposure of plants to N2 nitrogen solution resulted in an increased shoot/root ratio, leaf mass ratio, and Pn, as well as the elevated levels of sucrose, glutamate, aspartate, proline and threonine. The plants in N2 nitrogen solution accumulated twofold catharanthine and vinblastine than did the plants in N1 or N3 nitrogen solution after the long-term incubation. Internal nitrate had an increased accumulation in the plants in N2 nitrogen solution compared to the counterparts. The supply of N2 or N3 nitrogen solution to plants for 7 days induced an over tenfold increase of ammonium in leaves as compared to the case using N1 nitrogen solution. The increased ammonium ion promoted the activities of NADH-dependent glutamate dehydrogenase (NADH-GDH) both in the leaf and root of plants. Under the ammonium-containing solution (N2 and N3 nitrogen solutions), there was a significantly increased activity for glutamine synthase (GS) in the root during experiment and for nitrate reductase (NR) in the leaf and root only after 21 days of treatment. The performed correlation analysis revealed a negative relation between soluble sugars and internal ammonium, whereas a positive correlation of alkaloid production with glutamate and aspartate.     

Nitrate reductase,nitrite reductase and glutamate dehydrogenase levels in roots and leaves of maize seedlings

Total activities of nitrate and nitrite reductases were higher in 4 to 20 day old maize plants in the leaves than in the roots. The ratio of activities found in the leaves and in the roots respectively was much higher in the case of nitrate reductase than in the case of nitrite reductase. On the other hand higher glutamate dehydrogenase activity in the roots than in the leaves clearly indicates that the roots play a more important role in the assimilation of ammonium than in the assimilation of nitrate. When comparing the distribution of seminal and nodal adventitious roots of maize seedlings with the assimilation of inorganic nitrogen on the basis of enzyme levels, it could be deduced that during the first 20 days of seedling growth seminal roots were more involved in the assimilation of nitrate whereas nodal adventitious roots were more active in ammonium assimilation.