Nitrate-grown Plants Research Articles

Plant Primary Metabolism Regulated by Nitrogen Contributes to Plant-Pathogen Interactions.

Nitrogen contributes to plant defense responses by the regulation of plant primary metabolism during plant-pathogen interactions. Based on biochemical, physiological, bioinformatic and transcriptome approaches, we investigated how different nitrogen forms (ammonium vs. nitrate) regulate the physiological response of cucumber (Cucumis sativus) to Fusarium oxysporum f. sp. cucumerinum (FOC) infection. The metabolic profile revealed that nitrate-grown plants accumulated more organic acids, while ammonium-grown plants accumulated more amino acids; FOC infection significantly increased levels of both amino acids and organic acids in the roots of ammonium-grown plants. Transcriptome analysis showed that genes related to carbon metabolism were mostly up-regulated in plants grown with nitrate, whereas in ammonium-grown plants the up-regulated genes were mostly those that were related to primary nitrogen metabolism. Root FOC colonization and disease incidence were positively correlated with levels of root amino acids and negatively correlated with levels of root organic acids. In conclusion, organic acid metabolism and expression of related genes increased under nitrate, whereas ammonium increased the level of amino acids and expression of related genes; these altered levels of organic acids and amino acids resulted in different tolerances to FOC infection depending on the nitrogen forms supplied.

The occurrence and control of nitric oxide generation by the plant mitochondrial electron transport chain.

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Qi -site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Qo -site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate-grown wild-type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium-grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate-grown plants. Hence the ETC, likely the Q-cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non-energy-conserving electron sink upstream of Complex III.

Variation in fluxes estimated from nitrogen isotope discrimination corresponds with independent measures of nitrogen flux in Populus balsamifera L.

Acquisition of mineral nitrogen by roots from the surrounding environment is often not completely efficient, in which a variable amount of leakage (efflux) relative to gross uptake (influx) occurs. The efflux/influx ratio (E/I) is, therefore, inversely related to the efficiency of nutrient uptake at the root level. Time-integrated estimates of E/I and other nitrogen-use traits may be obtainable from variation in stable isotope ratios or through compartmental analysis of tracer efflux (CATE) using radioactive or stable isotopes. To compare these two methods, Populus balsamifera L. genotypes were selected, a priori, for high or low nitrogen isotope discrimination. Vegetative cuttings were grown hydroponically, and E/I was calculated using an isotope mass balance model (IMB) and compared to E/I calculated using (15) N CATE. Both methods indicated that plants grown with ammonium had greater E/I than nitrate-grown plants. Genotypes with high or low E/I using CATE also had similarly high or low estimates of E/I using IMB, respectively. Genotype-specific means were linearly correlated (r = 0.77; P = 0.0065). Discrepancies in E/I between methods may reflect uncertainties in discrimination factors for the assimilatory enzymes, or temporal differences in uptake patterns. By utilizing genotypes with known variation in nitrogen isotope discrimination, a relationship between nitrogen isotope discrimination and bidirectional nitrogen fluxes at the root level was observed.

Involvement of nitrite in the nitrate-mediated modulation of fermentative metabolism and nitric oxide production of soybean roots during hypoxia

It is widely accepted that nitrate but not ammonium improves tolerance of plants to hypoxic stress, although the mechanisms related to this beneficial effect are not well understood. Recently, nitrite derived from nitrate reduction has emerged as the major substrate for the synthesis of nitric oxide (NO), an important signaling molecule in plants. Here, we analyzed the effect of different nitrogen sources (nitrate, nitrite and ammonium) on the metabolic response and NO production of soybean roots under hypoxia. Organic acid analysis showed that root segments isolated from nitrate-cultivated plants presented a lower accumulation of lactate and succinate in response to oxygen deficiency in relation to those from ammonium-cultivated plants. The more pronounced lactate accumulation by root segments of ammonium-grown plants was followed by a higher ethanol release in the medium, evidencing a more intense fermentation under oxygen deficiency than those from nitrate-grown plants. As expected, root segments from nitrate-cultivated plants produced higher amounts of nitrite and NO during hypoxia compared to ammonium cultivation. Exogenous nitrite supplied during hypoxia reduced both ethanol and lactate production and stimulated cyanide-sensitive NO emission by root segments from ammonium-cultivated plants, independent of nitrate. On the other hand, treatments with a NO donor or a NO scavenger did not affect the intensity of fermentation of soybean roots. Overall, these results indicate that nitrite participates in the nitrate-mediated modulation of the fermentative metabolism of soybean roots during oxygen deficiency. The involvement of mitochondrial reduction of nitrite to NO in this mechanism is discussed.

Interaction of Sulfur and Nitrogen Nutrition in Tobacco (Nicotiana tabacum) Plants: Significance of Nitrogen Source and Root Nitrate Reductase

The significance of root nitrate reductase for sulfur assimilation was studied in tobacco (NICOTIANA TABACUM) plants. For this purpose, uptake, assimilation, and long-distance transport of sulfur were compared between wild-type tobacco and transformants lacking root nitrate reductase, cultivated either with nitrate or with ammonium nitrate. A recently developed empirical model of plant internal nitrogen cycling was adapted to sulfur and applied to characterise whole plant sulfur relations in wild-type tobacco and the transformant. Both transformation and nitrogen nutrition strongly affected sulfur pools and sulfur fluxes. Transformation decreased the rate of sulfate uptake in nitrate-grown plants and root sulfate and total sulfur contents in root biomass, irrespective of N nutrition. Nevertheless, glutathione levels were enhanced in the roots of transformed plants. This may be a consequence of enhanced APR activity in the leaves that also resulted in enhanced organic sulfur content in the leaves of the tranformants. The lack of nitrate reductase in the roots in the transformants caused regulatory changes in sulfur metabolism that resembled those observed under nitrogen deficiency. Nitrate nutrition reduced total sulfur content and all the major fractions analysed in the leaves, but not in the roots, compared to ammonium nitrate supply. The enhanced organic sulfur and glutathione levels in ammonium nitrate-fed plants corresponded well to elevated APR activity. But foliar sulfate contents also increased due to decreased re-allocation of sulfate into the phloem of ammonium nitrate-fed plants. Further studies will elucidate whether this decrease is achieved by downregulation of a specific sulfate transporter in vascular tissues.

Accumulation of ammonium in Norway spruce (Picea abies) seedlings measured by in vivo 14N-NMR

(14)N-NMR and (31)P-NMR have been used to monitor the in vivo pH in roots, stems, and needles from seedlings of Norway spruce, a typical ammonium-tolerant plant. The vacuolar and cytoplasmic pH measured by (31)P-NMR was found to be c. pH 4.8 and 7.0, respectively, with no significant difference between plants growing with ammonium or nitrate as the N-source. The (1)H-coupled (14) NH 4+ resonance is pH-sensitive: at alkaline pH it is a narrow singlet line and below pH 4 it is an increasing multiplet line with five signals. The pH values in ammonium-containing compartments measured by (14)N-NMR ranged from 3.7 to 3.9, notably lower than the estimated pH values of the P(i) pools. This suggests that, in seedlings of Norway spruce, ammonium is stored in vacuoles with low pH possibly to protect the seedlings against the toxic effects of ammonium ( NH 4+) or ammonia (NH3). It was also found that concentrations of malate were 3-6 times higher in stems than in roots and needles, with nitrate-grown plants containing more malate than plants grown with ammonium.

Effect of Nitrogen Form and Root-zone pH on Growth and Nitrogen Uptake of Tea ( Camellia sinensis ) Plants

Tea (Camellia sinensis) is considered to be acid tolerant and prefers ammonium nutrition, but the interaction between root zone acidity and N form is not properly understood. The present study was performed to characterize their interaction with respect to growth and mineral nutrition. Tea plants were hydroponically cultured with NH4+, NO3- and NH(4+) + NO3-, at pH 4.0, 5.0 and 6.0, which were maintained by pH stat systems. Plants supplied with NO3- showed yellowish leaves resembling nitrogen deficiency and grew much slower than those receiving NH4+ or NH(4+) + NO3- irrespective of root-zone pH. Absorption of NH4+ was 2- to 3.4-fold faster than NO3- when supplied separately, and 6- to 16-fold faster when supplied simultaneously. Nitrate-grown plants had significantly reduced glutamine synthetase activity, and lower concentrations of total N, free amino acids and glucose in the roots, but higher concentrations of cations and carboxylates (mainly oxalate) than those grown with NH4+ or NH(4+) + NO3-. Biomass production was largest at pH 5.0 regardless of N form, and was drastically reduced by a combination of high root-zone pH and NO3-. Low root-zone pH reduced root growth only in NO(3-)-fed plants. Absorption of N followed a similar pattern as root-zone pH changed, showing highest uptake rates at pH 5.0. The concentrations of total N, free amino acids, sugars and the activity of GS were generally not influenced by pH, whereas the concentrations of cations and carboxylates were generally increased with increasing root-zone pH. Tea plants are well-adapted to NH(4+)-rich environments by exhibiting a high capacity for NH4+ assimilation in their roots, reflected in strongly increased key enzyme activities and improved carbohydrate status. The poor plant growth with NO3- was largely associated with inefficient absorption of this N source. Decreased growth caused by inappropriate external pH corresponded well with the declining absorption of nitrogen.

Sugar exudation by roots of kallar grass [Leptochloa fusca (L.) Kunth] is strongly affected by the nitrogen source.

Exudation of sugars (glucose, fructose and sucrose) and that of cations and anions from intact roots of kallar grass [Leptochloa fusca (L.) Kunth] grown hydroponically with ammonium or nitrate (3 mM) as N source was investigated. In different experiments, plants grown on ammonium had slightly higher sugar contents than nitrate-grown plants, but their total sugar exudation during a 2-h period was up to 79-fold higher than under nitrate nutrition. Relative root exudation of inorganic anions and cations and that of amino acids (as a percentage of the internal contents exuded per time) was either similar or slightly higher from ammonium-grown than from nitrate-grown plants. Analysis of root architectural parameters revealed that ammonium-grown plants had a higher number of root tips/side roots per gram root fresh weight than nitrate-grown plants, whereas other root parameters, viz. length, diameter, volume and surface area were similar under the two N sources. A majority of the fine roots having diameter up to 0.4 mm represented up to 86% of the total root length, 64% of the total root surface area, and 35% of the total root volume; the root length and surface area per root system of that major root population were similar in ammonium- and nitrate-grown plants. Apparently, root architecture was not responsible for the different exudation rates. Within 12-24 h after shifting ammonium-grown plants to nitrate nutrition, root sugar levels and visible root architecture remained unchanged, yet the sugar exudation rate was reduced 30-fold. Short-term uptake of [14C]glucose (10 microM) from the rooting medium was similar for ammonium- and nitrate-grown plants. Thus, the very different sugar exudation rates were neither related to internal root sugar concentration, nor to the different root architecture, nor to differential resorption of sugars by ammonium- versus nitrate-grown plants. Increased external Ca2+ did not alter sugar exudation, and decreased external pH (4.5) only slightly increased sugar exudation from roots of nitrate-grown plants kept at pH 6.5. It is suggested that the much higher sugar exudation in response to ammonium may facilitate the ecologically and economically important association of diazotrophs with kallar grass roots.

Heterogeneity of Maize Root Mitochondria from Plants Grown in the Presence of Ammonium

Mitochondria isolated from root tissue of maize plants grown on a modified Knop solution containing 10.9 mM nitrate ± 7.2 mM ammonium were purified on the discontinuous Percoll density gradient with polyvinylpyrrolidone (PVP) added. The presence of PVP allowed separation of several mitochondrial fractions of a different density. Contrary to mitochondria isolated from plants grown in the presence of nitrate alone, revealing only two fractions, the mitochondria from NH4 +/NO3 −-plants were distributed in four fractions. Total amount of mitochondria, as well as specific activities of some nitrogen metabolism enzymes and tricarboxylic acid (TCA) cycle enzymes of all mitochondrial fractions, and respiratory activities of two lower density fractions isolated from plants grown on mixed nitrogen were higher in comparison to mitochondria from nitrate-grown plants.

Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max).

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 microM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots.

Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity

The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium. Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.

Changes in the Morphology of Roots and Leaves of Carob Seedlings Induced by Nitrogen Source and Atmospheric Carbon Dioxide

Abstract Carob seedlings were grown hydroponically for 9 weeks under 360 and 800 μl l -1 CO 2 . One of two nitrogen sources, nitrate or ammonium, was added to the nutrient medium at concentrations of 3 mol m -3 . Root systems of the developing plants supplied with nitrate compared to those supplied with ammonium were characterized by: (a) more biomass on the lower part of the root; (b) fewer lateral roots of first and second order; (c) longer roots; (d) higher specific root length; (e) a smaller root diameter. The morphology of the root systems of nitrate-fed plants changed in the presence of elevated carbon dioxide concentrations, resembling, more closely, that of ammonium-fed plants. Total leaf area was higher in ammonium- than in nitrate-fed plants. Nitrate-fed plants had greater total leaf area in the presence of high carbon dioxide than in normal CO 2 , due to an increase in epidermal cell size that led to development of larger leaflets with lower stomatal frequency. The observed changes in the morphology of roots and shoots agreed with the results observed for total biomass production. Nitrate-fed plants increased their biomass production by 100% in the presence of elevated CO 2 compared to 15% in ammonium-fed plants, indicating that the response of carob to high CO 2 concentrations is very dependent on the nitrogen source. Under elevated CO 2 , nitrate-grown plants had a larger content of sucrose in both roots and shoots, while no significant difference was observed in the content of sucrose in ammonium-grown plants, whether in ambient or enriched carbon dioxide. Hence, the differences in soluble carbohydrate contents can, at least partly, account for differences in root and shoot morphology.

Diurnal modulation of phosphoenolpyruvate carboxylation in pea leaves and roots as related to tissue malate concentrations and to the nitrogen source.

Phosphoenolpyruvate (PEP) carboxylation was measured as dark 14CO2 fixation in leaves and roots (in vivo) or as PEP carboxylase (PEPCase) activity in desalted leaf and roof extracts (in vitro) from Pisum sativum L. cv. Kleine Rheinländerin. Its relation to the malate content and to the nitrogen source (nitrate or ammonium) was investigated. In tissue from nitrate-grown plants, PEP carboxylation varied diurnally, showing an increase upon illumination and a decrease upon darkening. Diurnal variations in roots were much lower than in leaves. Fixation rates in leaves remained constantly low in continuous darkness or high in continuous light. Dark CO2 fixation of leaf slices also decreased when leaves were preilluminated for 1 h in CO2-free air, suggesting that the modulation of dark CO2 fixation was related to assimilate availability in leaves and roots. Phosphoenolpyruvate carboxylase activity was also measured in vitro. However, no difference in maximum enzyme activity was found in extracts from illuminated or darkened leaves, and the response to substrate and effectors (PEP, malate, glucose-6-phosphate, pH) was also identical. The serine/threonine protein kinase inhibitors K252b, H7 and staurosporine, and the protein phosphatase 2A inhibitors okadaic acid and cantharidin, fed through the leaf petiole, did not have the effects on dark CO2 fixation predicted by a regulatory system in which PEPCase is modulated via reversible protein phosphorylation. Therefore, it is suggested that the diurnal modulation of PEP carboxylation in vivo in leaves and roots of pea is not caused by protein phosphorylation, but rather by direct allosteric effects. Upon transfer of plants to ammonium-N or to an N-free nutrient solution, mean daily malate levels in leaves decreased drastically within 4-5 d. At that time, the diurnal oscillations of PEP carboxylation in vivo disappeared and rates remained at the high light-level. The coincidence of the two events suggests that PEPCase was de-regulated because malate levels became very low. The drastic decrease of leaf malate contents upon transfer of plants from nitrate to ammonium nutrition was apparently not caused by increased amino acid or protein synthesis, but probably by higher decarboxylation rates.

Effects of elevated CO2 , nitrogen form and concentration on growth and photosynthesis of a fast- and slow-growing grass.

Growth and photosynthesis of Agrostis capillaris L. and Nardus stricta L. were measured for plants grown under ambient and elevated concentrations of CO2 (340 and 550 μl CO2 l(-1) respectively) and a range of nitrogen concentrations (0.01, 0.1, 1 and 5 mg N l(-1) ) supplied as either ammonium sulphate or sodium nitrate. After 42 d of growth for A. capillaris and 49 d of growth for N. stricta, the higher nitrogen concentrations resulted in stimulation of growth. The form of nitrogen did not affect the total dry weight attained by A. capillaris. However, ammonium-grown N. stricta attained a greater total dry weight than did nitrate-grown plants. Nitrogen form influenced leaf area ratio, which was greater in nitrate-grown A. capillaris and in ammonium-grown N. stricta. At the two lowest nitrogen concentrations there was no effect of elevated CO2 on total dry weight in either species, whilst at the two highest nitrogen concentrations positive growth responses to elevated C02 were observed. Photosynthetic capacity and carboxylation efficiency were lower in plants grown in elevated CO2 at the two lowest nitrogen concentrations, and were associated with greater leaf soluble carbohydrate content and lower foliar nitrogen concentrations. By contrast, the CO2 treatment did not affect these parameters at the two highest nitrogen concentrations employed.

Ammonia emission from young barley plants: influence of N source, light/dark cycles and inhibition of glutamine synthetase

Barley (Hordeum vulgare L. cv. Golf) plants were grown at two different relative addition rates ; 0.1 and 0.2 d -1 of nitrate. Three to five days before measurements started the plants were transferred to a nutrient solution with 2 mM nitrate or ammonium. The ammonium-grown plants showed increased ammonium levels in both shoots and roots and also increased ammonium concentrations in xylem sap. Ammonia emission measured in cuvettes connected to an automatic NH 3 monitor was close to zero for nitrate-grown plants but increased to 0.59 and 0.88 nmol NH 3 m -2 s -1 for plants transferred to ammonium after growing at RA = 0.2 and 0.1 d -1 , respectively. In darkness, NH 3 emission decreased together with photosynthesis and transpiration, but increased rapidly when the light was turned on again. Addition of 0.5 mM methionine sulphoximine (MSO) to the plants caused an almost complete inhibition of both root and shoot glutamine synthetase (GS) activity after 24 h. Ammonia emission increased dramatically and photosynthesis and transpiration decreased in both nitrate- and ammonium-grown plants as a result of the GS inhibition. At the same time plant tissue and xylem sap ammonium concentrations increased, indicating the importance of GS in controlling plant ammonium levels and thereby NH 3 emission from the leaves.

Solute content and energy status of roots of barley plants cultivated at different pH on nitrate- or ammonium-nitrogen.

Barley seedlings (Hordeum vulgare cv. 'Gaulois') were precultured hydroponically for 4d on nutrient solutions with nitrate (3 mM) as N-source at pH 4.7, and were then transferred to solutions with ammonium or nitrate as N-source (3 mM), buffered and adjusted to different pH (4.0, 5.5, 6.8). Variations in shoot and root growth and solute contents were examined and grouped into pH-effects, ammonium effects and interactions. Shoot biomass was not affected under all conditions. Root fresh weight was insensitive to the external pH when nitrate was the N-source, but was drastically affected by a combination of ammonium and low pH. In contrast, root length was negatively affected by low pH per se. In nitrate-grown plants, ammonium levels in roots and shoots were low (0.5 to 1 mM). After transfer of plants to ammonium solution, roots accumulated ammonium within 24 h about sixfold (18 mM) above the external concentration. At pH 5.5 or 6.8, but not at pH 4, root ammonium contents decreased afterwards to a lower steady state value (10 mM). Leaves also accumulated ammonium, especially at the most acidic pH. Concentrations of major inorganic cations in roots were markedly but differentially affected by acidic pH and ammonium. The magnesium content of roots was drastically decreased (from 18 to 2 mM) by ammonium nutrition, and this was independent of the external pH. In contrast, calcium levels in roots were decreased by low external pH, independent of the N-source. Potassium levels in roots were rather insensitive to both low pH and ammonium. The pH of crude root homogenates was measured in order to obtain at least crude information about trends in possible cellular pH changes. At an external pH of 4.0 and 5.5, the pH of the homogenates was 5.8, and it increased to 6.3 when the external pH was 6.8. The pH of the homogenates was not affected by the N-source. In spite of the drastic effects of the N-source on the concentration of ammonium and magnesium in root tissues, ATP/ADP-ratios were not affected. Also, sugar levels were unchanged or even increased. Thus, growth impairment could not be traced back to impaired carbohydrate or ATP-supply, which might occur as a consequence of ammonium accumulation or Mg2+ -deficiency.

Responses of greening bean seedling leaves to nitrogen dioxide and nutrient nitrate supply

Phaseolus vulgaris cv. Kinghorn Wax seedlings grown in darkness at 25°C for 7 days with half strength Hoagland's nutrient solution containing no nitrogen, were transferred to lit continouus stirred tank reactors (CSTRs) in atmospheres containing 0 or 0·3 ppm NO 2 and irrigated with a nutrient solution containing 0 or 5 m m nitrate as sole nitrogen source and allowed to grow for a period of up to 5 days in a 14 h photoperiod. Exposure to NO 2 increased total Kjeldahl nitrogen in the leaves. Further, the exposure to NO 2 increased chlorophyll content from day 3 onwards and inhibited the leaf dry weight substantially on days 4 and 5. The primary leaves of the seedlings exposed to 0·3 ppm NO 2 and supplied with nitrate accumulated some nitrite after 5 days of exposure. Some of the seedlings were returned from CSTRs to growth chambers and allowed to grow for a further period of 5 days in a 14 h photoperiod without NO 2. The growth which developed after the NO 2 exposure growth period, as measured by fresh and dry weights of the leaves, was significantly less in NO 2-exposed plants than in nitrate-grown plants. The experiments demonstrate that the leaves of greening seedlings are able to assimilate NO 2 and that a reduction in leaf dry weight by prolonged NO 2 exposure in the presence of nutrient nitrate can be associated with nitrite accummulation, and that NO 2 has a carry-over effect beyond the duration of NO 2 exposure. It is apparent that NO 2 induces some durable biochemical or cytological aberration in the presence of nutrient nitrate, which adversely affects subsequent leaf growth.

Inward and outward transport of ammonium in roots of maize and sorghum: contrasting effects of methionine sulphoximine

To examine the adaptive responses of ammonium efflux of nitrate-grown plants during first exposure to ammonium nutrition, isotopic flows of ammonium in and out of root tissue of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) were delineated. Appreciable efflux of 14 N-ammonium occurred during the first 2 min of exposure, suggesting that ammonium efflux is characteristic of plants grown with nitrate as the sole source of nitrogen. Treatment with methionine sulphoximine (MSX) enhanced the initial 14 N-ammonium efflux 5-fold in maize but less than 2-fold in sorghum. Increasing ambient acidity from pH 6 to pH 4 had relatively little effect on 14 N-ammonium efflux in either species

Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant−1·h−1. Bypassing CO2 fixation and sugar formation, the 14C label from [14C]sucrose was directly applied to leaf blades to study sugar translocation. The 14C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the 14C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all 14C translocated from [U-14C]sucrose applied to wheat leaves, and 14C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of β-fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 μmol ethylene formed·mg−1 Chl·h−1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth.

Effects of Different Nitrogen Sources and Concentrations on CAM Photosynthesis in Kalanchoe blossfeldiana

When Kalanchoe blossfeldiana Poelln. cv. Hikan plants were cultured in solutions containing 0-2, 1-0, 5-0 or 10 mM of nitrate or ammonium under a long-day photoperiod, some criteria of CAM (Crassulacean acid metabolism) photosynthesis (diurnal changes of C02 uptake, titratable acidity and malate content in leaves) were examined. The plants absorbed 90 to 100% of C02 during the light phase regardless of the supplied nitrogen. Nitrate-grown plants absorbed about 10% of C02 during the dark phase regardless of the supplied concentration, whereas in ammonium-grown plants the nocturnal C02 uptake occurred at 0 2 mM, at which the plants depleted nitrogen and no uptake was observed at the higher concentrations. Changes of nocturnal increase in titratable acidity and malate content almost corresponded with the changes in the amount of nocturnal C02 uptake. Also K. daigremontiana plants supplied with 10 mM of ammonium had a less CAM-like pattern of diurnal C02 uptake than the plants supplied with 10 mM of nitrate. These results suggest that a sufficient supply of ammonium depresses CAM photosynthesis.