Denitrification in lucerne nodules is not involved in nitrite detoxification

Dissimilatory reduction of ionic nitrogen oxides to gaseous forms such as nitrous oxide or nitrogen can be carried out by free living or symbiotic forms of some strains of Rhizobium meliloti. In this paper we investigate whether bacteroid denitrification plays a role in the alleviation of the inhibitory effects of nitrate on nitrogen fixation both in bacteroid incubations as in whole nodules. The presence of a constitutive nitrate reductase (NR) activity in isolated bacteroids caused nitrite accumulation in the incubation medium, and acetylene reduction activity in these bacteroids was progressively inhibited, since nitrite reductase (NiR) activity was unable to reduce all the nitrite produced by NR and denitrification occurred slowly. Even nodules infiltrated with nitrate and nitrite failed to increase gaseous forms of nitrogen substantially, indicating that nitrite availability was not limiting denitrification by bacteroids. In spite of the low rates of bacteroidal denitrification, the effect of nodule denitrification on the inhibition of nitrogen fixation by nitrate in whole plants was tested. For that purpose, lucerne plants (Medicago sativa L. cv. Aragon) were inoculated with two Rhizobium meliloti strains: 102-F-65 (non denitrifying) and 102-F-51 (a highly denitrifying strain). After a seven days nitrate treatment, both strains showed the same pattern of inhibition, and it occurred before any nitrate or nitrite accumulation within the nodules could be detected. This observation, together with the lack of alleviation of the ARA inhibition in the denitrifying strain, and the limited activity of dissimilatory nitrogen reduction present in these bacteroids, indicate a role other than nitrite detoxification for denitrification in nodules under natural conditions.     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

Metabolism of poly-\-hydroxybutyric acid in bacteroids ofRhizobium lupini in connection with nitrogen fixation and photosynthesis

Summary The darkening of lupin plants grown in a sand culture on a nitrogen-free medium at a stage of initial flowering led to a sharply decreased nitrogen fixation intensity which eventually ceased. Decreased intensity of nitrogen fixation in bacteroids was accompanied by an accumulation of poly--hydroxybutyric acid (PHB); in the course of 10–20 h (depending upon temperature) its content increased by 2.5–3.0 times. If, following darkening, the plants were once again exposed to light, an abrupt increase of nitrogen fixation intensity was observed and a simultaneous decrease of PHB content.It has been shown that lupin's exposure to light in¹⁴CO₂ atmosphere lasting 19 h resulted in the latter's incorporation into PHB, bacteroids and into the entire nodule; these processes developed almost in parallel.During the early period of vegetation growth prior to flowering, the PHB content of bacteroids decreased from 13–14 to 3–4% of dry weight, whereas the intensity of nitrogen fixation was raised. Concurrently increase of the activity of some enzymes connected with the PHB metabolism (acetoacetyl-CoA-reductase, acetyl-CoA-acetyl transferase, PHB-depolymerase, CoA-transferase of 3-ketoacids) occured. The plants' subsequent ageing and reduction of nitrogen fixation intensity led to a noticeable increase of PHB content and a decrease of the above mentioned enzymes' activity. The specific activity of \-hydroxybutyric dehydrogenase involved with PHB catabolism was high and was maintained at a constant level throughout the entire vegetative period.     

Nitrogen nutrition and the development and senescence of nodules on cowpea seedlings

Cowpea (Vigna unguiculata (L.) Walp cv. Vita 3) seedlings inoculated with Rhizobium strain CB756 were cultured with their root systems maintained in air or in Ar: O₂ (80:20, v/v) during early nodule development (up to 24 d after sowing). Compared with those in air, seedlings in Ar:O₂ showed progressive N deficiency with inhibited shoot growth, reduced ribulose-1,5-bisphosphate carboxylase and total protein levels and loss of chlorophyll in the leaves. Nodule initiation, differentiation of infected and uninfected nodule tissues and the ultrastructure of bacteriod-containing cells were similar in the air and Ar: O₂ treatments up to 16 d after sowing. Thereafter the Ar: O₂ treatment caused cessation of growth and development of nodules, reduced protein levels in bacteroids and nodule plant cells, and progressive degeneration of nodule ultrastructure leading to premature senescence of these organs. Provision of NO ₃ ^- (0.1–0.2 mM) to Ar: O₂-grown seedlings overcame the abovementioned consequences of N₂ deficiency on nodule and plant growth, but merely delayed the degenerative effects of Ar: O₂ treatment on nodule structure and senescence. Treatment of Ar: O₂-grown seedlings with NO ₃ ^- greatly increased the protein level of nodules but the increase was largely restricted to the plant cell fraction as opposed to the bacteroids. By contrast, NO ₃ ^- treatment of air-grown seedlings increased protein of bacteroid and host nodule fractions to the same relative extents when compared with air-grown plants not supplemented with NO ₃ ^- . These findings, taken together with studies of the distribution of N in nodules of symbiotically effective plants grown from ¹⁵N-labeled seed, indicate that direct incorporation of fixation products by bacteroids may be a critical feature in the establishment and continued growth of an effective symbiosis in the cowpea seedling.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase     

Differentiation of nodules of Glycine max

Plants of Glycine max var. Caloria, infected as 14 d old seedlings with a defined titre of Rhizobium japonicum 3Il b85 in a 10 min inoculation test, develop a sharp maximum of nitrogenase activity between 17 and 25 d after infection. This maximum (14±3 nmol C₂H₄ h^-1 mg nodule fresh weight^-1), expressed as per mg nodule or per plant is followed by a 15 d period of reduced nitrogen fixation (20–30% of peak activity). 11 d after infection the first bacteroids develop as single cells inside infection vacuoles in the plant cells, close to the cell wall and infection threads. As a cytological marker for peak multiplication of bacteroids and for peak N₂-fixation a few days later the association of a special type of nodule mitochondria with amyloplasts is described. 20 d after inoculation, more than 80% of the volume of infected plant cells is occupied by infection vacuoles, mostly containing only one bacteroid. The storage of poly--hydroxybutyrate starts to accumulate at both ends of the bacteroids. Non infected plant cells are squeezed between infected cells (25d), with infection vacuoles containing now more than two (up to five) bacteroids per section. Bacteroid development including a membrane envelope is also observed in the intercellular space between plant cells. 35 d after infection, more than 50% of the bacteroid volume is occupied by poly--hydroxybutyrate. The ultrastructural differentiation is discussed in relation to some enzymatic data in bacteroids and plant cell cytoplasm during nodule development.     

Nitrate reductase activity of free-living and symbiotic uptake hydrogenase-positive and uptake hydrogenase-negative strains of Bradyrhizobium japonicum

The nitrate reductase activity (NR) of selected uptake hydrogenase-positive (hup ⁺) and uptake hydrogenase-negative (hup ^-) strains of Bradyrhizobium japonicum were examined both in free-living cells and in symbioses with Glycine max L. (Marr.) cv. Williams. Bacteria were cultured in a defined medium containing either 10 mM glutamate or nitrate as the sole nitrogen source. Nodules and bacteriods were isolated from plants that were only N₂-dependent or grown in the presence of 2 mM KNO₃. Rates of activity in nodules were determined by an in vivo assay, and those of cultured cells and bacteriods were assayed after permeabilization of the cells with alkyltrimethyl ammonium bromide. All seven strains examined expressed NR activity as free-living cells and as symbiotic forms, regardless of the hup genotype of the strain used for inoculation. Although the presence of nitrate increased nitrate reduction by cultures cells and nodules, no differences in NR activity were observed between bacteroids isolated from nodules of plants fed with nitrate or grown on N₂-fixation exclusively. Cultured cells, nodules and bacteriods of strains with hup ^- genotype (USDA 138, L-236, 3. 15B3 and PJ17) had higher rates of NR activity than those with hup ⁺ genotype (USDA 110, USDA 122 DES and CB1003). These results suggest that NR activity is reduced in the presence of a genetic determinant associated with the hup region of B. japonicum.Abbreviations EDTA ethylene-diamine tetraacetic acid - Hup hydrogen uptake - MOPS 3-(N-morpholino)-propane sulfonic acid - NR nitrate reductase - PVP polyvinyl-polypyrrolidone - Tris Tris(hydroxymethyl)-aminomethane     

Nitrate reductase activity of two leafy vegetables as affected by nickel and different nitrogen forms

The author studied the effect of different nickel concentrations (0, 0.4, 40 and 80 μM Ni) on the nitrate reductase (NR) activity of New Zealand spinach (Tetragonia expansa Murr.) and lettuce (Lactuca sativa L. cv. Justyna) plants supplied with different nitrogen forms (NO₃ ⁻–N, NH₄ ⁺–N, NH₄NO₃). A low concentration of Ni (0.4 μM) did not cause statistically significant changes of the nitrate reductase activity in lettuce plants supplied with nitrate nitrogen (NO₃ ⁻–N) or mixed (NH₄NO₃) nitrogen form, but in New Zealand spinach leaves the enzyme activity decreased and increased, respectively. The introduction of 0.4 μM Ni in the medium containing ammonium ions as a sole source of nitrogen resulted in significantly increased NR activity in lettuce roots, and did not cause statistically significant changes of the enzyme activity in New Zealand spinach plants. At a high nickel level (Ni 40 or 80 μM), a significant decrease in the NR activity was observed in New Zealand spinach plants treated with nitrate or mixed nitrogen form, but it was much more marked in leaves than in roots. An exception was lack of significant changes of the enzyme activity in spinach leaves when plants were treated with 40 μM Ni and supplied with mixed nitrogen form, which resulted in the stronger reduction of the enzyme activity in roots than in leaves. The statistically significant drop in the NR activity was recorded in the aboveground parts of nickel-stressed lettuce plants supplied with NO₃ ⁻–N or NH₄NO₃. At the same time, there were no statistically significant changes recorded in lettuce roots, except for the drop of the enzyme activity in the roots of NO₃ ⁻-fed plants grown in the nutrient solution containing 80 μM Ni. An addition of high nickel doses to the nutrient solution contained ammonium nitrogen (NH₄ ⁺–N) did not affect the NR activity in New Zealand spinach plants and caused a high increase of this enzyme in lettuce organs, especially in roots. It should be stressed that, independently of nickel dose in New Zealand spinach plants supplied with ammonium form, NR activity in roots was dramatically higher than that in leaves. Moreover, in New Zealand spinach plants treated with NH₄ ⁺–N the enzyme activity in roots was even higher than in those supplied with NO₃ ⁻–N.     

Denitrification of nitrate to nitrogen gas by washed cells ofRhizobium japonicum and by bacteroids fromGlycine max

Nitrate, nitrite and nitrous oxide were denitrified to N₂ gas by washed cells ofRhizobium japonicum CC706 as well as by bacteroids prepared from root nodules ofGlycine max (L.) Merr. (CV. Clark 63). Radiolabelled N₂ was produced from either K¹⁵NO₃ or Na¹⁵NO₂ by washed cells ofRh. japonicum CC705 grown with either nitrate only (5 mM) or nitrate (5 mM) plus glutamate (10 mM). Nitrogen gas was also produced from N₂O. Similar results were obtained with bacteroids ofG. max. The stoichiometry for the utilization of¹⁵NO ₃ ^- or¹⁵NO ₂ ^- and the produciton of¹⁵N₂ was 2:1 and for N₂O utilization and N₂ production it was 1:1. Some of the¹⁵N₂ gas produced by denitrification of¹⁵NO ₃ ^- in bacteroids was recycled via nitrogenase into cell nitrogen.     

Carbon metabolism and bacteroid respiration in nodules of chick-pea (Cicer arietinum L.) plants grown under saline conditions

ABSTRACT

The present work investigates the relationships between nitrogen fixation, carbon metabolism and oxygen consumption by bacteroids of Mesorhizobium ciceri in root nodules of chick-pea plants. Its aim was to establish whether some of the compounds which accumulate under salt stress may be used as respiratory substrates by bacteroids to fuel their own metabolism and nitrogenase activity. Plants were grown in a growth chamber, and salt stress was induced by adding 50 mM NaCl to the nutrient solution at sowing. The data presented here show a rise in fermentative metabolism in nodules of chick-pea plants exposed to high salinity, and suggest that proline, lactate or ethanol, may play an important role as energy-yielding substrates for bacteroids in this plant species. The bacteroids could utilize glucose as a respiratory substrate both under control and saline conditions, while malate did not appear to be the preferred substrate in the presence of salt.     

Nitrate reductase and nitrite reductase activity in free-living cells and bacteroids of Rhizobium loti

Cells of Rhizobium loti strains T1 and U226 cultured in defined medium with glutamate as the only nitrogen source and bacteroids isolated from root nodules of Lotus corniculatus, L. pedunculatus and L. tenuis did not show constitutive (non-nitrate induced) nitrate reductase activity (NRA). In contrast, nitrite reductase activity (NiRA) was present in both free-living cells and bacteroids of either strain T1 or U226. Constitutive NRA and NiRA were detected in the cytosol fraction from nodules of all three symbioses examined. An induced NRA was expressed in bacteroids after a 10 h incubation in the presence of nitrate.     

The role of formate metabolism in nitrogen fixation in Rhizobium Spp.

Formate metabolism supported nitrogen-fixation activity in free-living cultures of Rhizobium japonicum. However, formate0dependent nitrogense activity was observed only in the presence of carbon sources such as glutamate, ribose or aspartate which by themselves were unable to support nitrogenase activity. Formate-dependent nitrogenase activity was not detected in the presence of carbon sources such as malate, gluconate or glycerol which by themselves supported nitrogenase activity. A mutant strain of R. japonicum was isolated that was unable to utilise formate and was shown to lack formate dehydrogenase activity. This mutant strain exhibited no formate-dependent nitrogenase activity. Both the wild-type and mutant strains nodulated soybean plants effectively and there were no significant differences in the plant dry weight or total nitrogen content of the respective plants. Furthermore pea bacteroids lacked formate dehydrogenase activity and exogenously added formate had no stimulatory effect on the endogenous oxygen uptake rate. The role of formate metabolism in symbiotic nitrogen fixation is discussed.Abbreviation FDH formate dehydrogenase     

Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth ofCeanothus griseus varhorizontalis

Rooted cuttings ofCeanothus griseus varhorizontalis were irrigated with 0, 10, 20, 50, 75 or 100ppm nitrogen as NH₄NO₃ for eight weeks prior to inoculation with infectiveFrankia. After inoculation, half of the plants for each treatment nitrogen level continued to be irrigated with the preconditioning nitrogen level and half were given no more supplemental nitrogen. For plants continuously receiving nitrogen, nodule initiation (nodule number) was inversely correlated with increasing supplemental nitrogen levels, and suppressed above 50 ppm N. Leaf nitrogen above 2% in continuous-N plants correlated with greatly reduced or suppressed nodulation. Plants maintained after inoculation without supplemental nitrogen showed influence of the prior nitrogen treatment on nodulation. Preconditioning at 50 ppm and above greatly reduced the number of nodules formed. The evidence suggests that stored internal nitrogen can regulate nodulation.Plant biomass accumulated maximally when nodulation was suppressed, at 75 and 100 ppm supplemental N applied continuously. Internode elongation during the nodulation period occurred only on nodulated plants, or in the presence of supplemental N (10 ppm and above).     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

Effect of substrate nitrogen on the performance ofin vitro propagatedAlnus glutinosa clones inoculated with Sp+ and Sp− Frankia strains

The addition of combined nitrogen to substrate at an appropriate rate can stimulate N₂-fixation thus inreasing the efficiency of the Alnus-Frankia symbiosis. To examine how nitrogen additions can effect the peformance of different pairs of symbionts, growth and time course of N₂-fixation were studied in plants supplied with NH₄NO₃. Two cloned ofAlnus glutinosa (L.) Gaertn., propagatedin vitro, were inoculated with two strains ofFrankia (AVP3d and ACN14a) and grown in a greenhouse. Calcined montmorillonite (Totfaice^R) was used as growth substrate. Six N treatments were made up of varied amounts of NH₄NO₃ supplied in one single addition shortly before inoculation. Weekly measurements of shoot height and repeated measurements of nitrogenase activity (acetylene reduction) performed on intact root systems were used to monitor the development of the symbioses. Nitrogen treatments containing from 0.10 to 0.68 mg N g⁻¹ dry substrate stimulated N₂-fixation as well as growth. The relative performance of the two clones was different according to N treatment; one clone showed a greater benefit from the nitrogen input. Our results support the recommendation that selection of symbionts according to performance should be carried out with an input of combined nitrogen. This can provide optimum conditions for the development of each pair of symbionts.     

Growth and nitrogen fixation inAlnus glutinosa (L.) Gaertn. under carbon dioxide enrichment of the root atmosphere

The effects of aeration of the N-free rooting medium with elevated CO₂ on (a) acetylene reduction by perlite-grown plants and (b) N₂-fixation and long-term growth of nutrient solution-grown plants were determined for nodulatedAlnus glutinosa (L.) Gaertn. In the former experiments, roots of intact plants were incubated in acetylene in air in darkened glass jars for 3 hr, followed by a further 3 hr incubation period in air enriched with CO₂ (0–5%). During incubation, the CO₂ content of the jars increased by 0.17% per hour due to respiration of the root system, so that the CO₂ content at 3 hr was 0.5%. Additional enrichment of the rooting medium gas-phase with CO₂ equivalent to 1.1% and 1.75% CO₂ of the gas volume significantly increased nitrogenase activity (ethylene production) by 55% and 50% respectively, while enrichment with greater than 2.5% CO₂ decreased activity. In contrast, ethylene production by control plants, where CO₂ was not added to the assay jars, decreased by 8% over the assay period. In long-term growth experiments, nodulated roots of intactAlnus glutinosa plants were sealed into jars containing N-free nutrient solution (pH 6.3) and aerated with air, or air containing elevated levels of CO₂ (1.5% and 5%). Comparison of the appearance of CO₂-treated with air treated plants suggested that 1.5% CO₂ stimulated plant growth. However, at harvest after 5 or 6 weeks variability between plants masked the significance of differences in plant dry weight. A significant increase of 33% in total nitrogen of plants aerated with 1.5% CO₂, compared with air-treated plants, was demonstrated, broadly in line with the short-term increase in acetylene reducing activity observed following incubations with similar CO₂ concentrations. Shoot dry weight was not affected significantly by long-term exposure to 5% CO₂, the main effect on growth being a 20% reduction in dry weight of the root system, possibly through inhibition of root system respiration. However, in contrast to the inhibitory effects of high CO₂ on acetylene reduction there was no significant effect on the amounts of N₂ fixed.     

Effects of combined nitrogen on anapleurotic carbon assimilation and bleeding sap composition in Phaseolus vulgaris L.

Dwarf french beans, Phaseolus vulgaris L., were grown with or without inoculation with rhizobia (strain 3644), and with or without a combined nitrogen source (nitrate or ammonium ions). The distribution of radioactivity into products of dark ¹⁴CO₂ assimilation was studied in roots or nodules from these plants. A detailed study was also made of the distribution and rates of excretion of nitrogen in xylem bleeding sap in 28 day old plants grown on the various sources of nitrogen. Whereas detached nodules accumulated radioactive glycine, serine and glutamate when incubated with ¹⁴CO₂, bleeding sap from plants root fed ¹⁴CO₂ contained low levels of radioactivity in these compounds but higher levels in allantoin. Chemical analysis showed allantoin to be the major compound transported in the xylem of nodulated plants, whether or not they were fed on combined nitrogen. In contrast uninoculated plants accumulated mainly amino acids in the bleeding sap, the amount and chemical composition of which depended on the combined nitrogen source.Abbreviations PEP phosphoenol pyruvate - OAA oxaloacetate     

Cadmium in white lupin nodules: Impact on nitrogen and carbon metabolism

The aims of this work were to investigate the microlocalisation of cadmium (Cd) in Lupinus albus L. cv. Multolupa nodules, and to determine its effects on carbon and nitrogen metabolism. Nodulated white lupin plants were grown in a growth chamber with or without Cd (150 μM). Energy-dispersive X-ray microanalysis showed the walls of the outer nodule cortex cells to be the main area of Cd retention, helping to reduce the harmful effect Cd might have on the amount of N₂ fixed by the bacteroids. Sucrose synthase activity declined by 33% in the nodules of the Cd-treated plants, and smaller reductions were recorded in glutamine synthetase, aspartate aminotransferase, alkaline invertase and NADP-dependent isocitrate dehydrogenase activities. The Cd treatment also sharply reduced nodule concentrations of malate, succinate and citrate, while that of starch doubled, but that of sucrose experienced no significant change. In summary, the present results show that white lupins accumulate significant amounts of Cd in their root nodules. However, the activity of some enzymes involved in ammonium assimilation did decline, promoting a reduction in the plant N content. The downregulation of sucrose synthase limits the availability of carbon to the bacteroids, which might interfere with their respiration. Carbon metabolism therefore plays a primary role in the impaired function of the white lupin root nodule caused by Cd, while N metabolism appears to have a more secondary involvement.     

Effects of multiple stresses on radish growth and resource allocation

Summary Acclimation of wild radish plants to a simultaneous combination of SO₂ fumigation and decreasing nitrate availability was investigated. Plants were grown for 24 d under continuous daytime (10h) exposure to 0 or 0.4 ppm SO₂ and were grown in a nutrient solution with stable nitrate concentrations of 100 M for the first 15 d, 50 M from day 15 to day 19, and 25 M from day 19 to day 24. Analysis of relative growth rates (RGR) showed that radish plants responded rapidly to changes in nitrate availability and that SO₂ treatment affected those responses. Shoot RGR of plants from both treatments and root RGR of control plants showed rapid declines and subsequent recoveries in response to decreasing nitrate availability. Root RGR of SO₂-treated plants declined rapidly in response to decreased nitrate availability, but did not recover as quickly or completely as root RGR of control plants. Analysis of specific leaf weights and tissue nitrogen concentrations showed that control plants had significantly higher amounts of nitrogen in tissues after nitrate availability was lowered, and had higher rates of nitrate uptake in comparison to SO₂-treated plants; especially when nitrate availability was highest. Furthermore, control plants had temporarily higher rates of root respiration in comparison to SO₂-treated plants, suggesting that control plants temporarily allocated more resources to physiological processes occurring in roots, such as nutrient uptake. Although SO₂-induced changes in growth and resource allocation of plants were relatively small, it was probable that SO₂ treatment of radish plants affected plant nitrogen balance, and subsequently affected the ability of plants to respond to decreased nitrate availibility, by affecting resource partitioning to nitrate uptake and root growth.     

Nodulation and growth response ofAllocasuarina andCasuarina species to phosphorus fertilization

To examine how soil phosphorus status affects nitrogen fixation by the Casuarinaceae —Frankia symbiosis,Casuarina equisetifolia and two species ofAllocasuarina (A. torulosa andA. littoralis) inoculated or fertilized with KNO₃ were grown in pots in an acid soil at 4 soil phosphate levels. InoculatedC. equisetifolia nodulated well by 12 weeks after planting and the numbers and weight of nodules increased markedly with phosphorus addition. Growth ofC. equisetifolia dependent on symbiotically fixed nitrogen was more sensitive to low levels of phosphorus (30 mg kg^–1 soil) than was growth of seedings supplied with combined nitrogen; at higher levels of phosphorus, the growth response curves were similar for both nitrogen fertilized and inoculated plants. The interaction between phosphorus and nitrogen treatments (inoculated and nitrogen fertilized) demonstrated that there was a greater requirement of phosphorus for symbiotic nitrogen fixation than for plant growth when soil phosphorus was low.WithAllocasuarina species, large plant to plant variation in nodulation occurred both within pots and between replicates. This result suggests genetic variation in nodulation withinAllocasuarina species. Nodulation ofAllocasuarina species did not start until 16 weeks after planting and no growth response due toFrankia inoculation was obtained at the time of harvest. Addition of nitrogen starter is suggested to boost plant growth before the establishment of the symbiosis. Growth ofAllocasuarina species fertilized with nitrogen responded to increasing levels of phosphorus up to 90 mg P/kg soil after which it declined by 69% forA. littoralis. The decrease in shoot weight ofA. littoralis, A. torulosa, C. equisetifolia andC. cunninghamiana at high phosphorus was confirmed in a sand culture experiment, and may be atributable to phosphorus toxicity.     

Sagebrush and grasshopper responses to atmospheric carbon dioxide concentration

Summary Seed- and clonally-propagated plants of Big Sagebrush (Artemisia tridentata var.tridentata) were grown under atmospheric carbon dioxide regimes of 270, 350 and 650 μl l⁻¹ and fed toMelanoplus differentialis andM. sanguinipes grasshoppers. Total shrub biomass significantly increased as carbon dioxide levels increased, as did the weight and area of individual leaves. Plants grown from seed collected in a single population exhibited a 3–5 fold variation in the concentration of leaf volatile mono- and sesquiterpenes, guaianolide sesquiterpene lactones, coumarins and flavones within each CO₂ treatment. The concentration of leaf allelochemicals did not differ significantly among CO₂ treatments for these seed-propagated plants. Further, when genotypic variation was controlled by vegetative propagation, allelochemical concentrations also did not differ among carbon dioxide treatments. On the other hand, overall leaf nitrogen concentration declined significantly with elevated CO₂. Carbon accumulation was seen to dilute leaf nitrogen as the balance of leaf carbon versus nitrogen progressively increased as CO₂ growth concentration increased. Grasshopper feeding was highest on sagebrush leaves grown under 270 and 650 μl l⁻¹ CO₂, but varied widely within treatments. Leaf nitrogen concentration was an important positive factor in grasshopper relative growth but had no overall effect on consumption. Potential compensatory consumption by these generalist grasshoppers was apparently limited by the sagebrush allelochemicals. Insects with a greater ability to feed on chemically defended host plants under carbon dioxide enrichment may ultimately consume leaves with a lower nitrogen concentration but the same concentration of allelochemicals. Compensatory feeding may potentially increase the amount of dietary allelochemicals ingested for each unit of nitrogen consumed.