Carbon Costs Associated with N2 Fixation in Vicia faba L and Pisum sativum 1. over a 14-Day Period

Abstract: Long-term (14 days) carbon costs of N₂ fixation were studied in pot trials. For this purpose the CO₂ release from the root space of nodulated and non-nodulated (urea nourished) Vicia faba L. and Pisum sativum L. plants was compared and related to the amount of fixed or assimilated N. Additional measurements of shoot CO₂ exchange and dry matter increment were carried out in order to calculate the overall carbon balance. The carbon costs for N₂ fixation in Vicia faba 1. (2.87 mg C/mg N_fiX) were higher than in Pisum sativum L. (2.03 mg C/mg N_fix). However, the better carbon efficiency in Pisum sativum 1. did not lead to a better growth performance compared to Vicia faba L. Vicia faba L. compensated for the carbon and energy expenditure by more intensive photosynthesis in the N₂-fixing treatment. This was not the case with Pisum sativum L., where the carbon balance indicates that the carbon costs of N₂ fixation restricted root growth. It is proposed that low carbon costs for N₂ fixation indicate an adaptation to a critical carbon supply of roots and nodules, e.g., during the pod-filling of grain legumes.     

Maintenance and growth respiration in shoots and roots of sunflower plants grown at different root temperatures

Continuous measurements of CO₂-evolution and dry matter accumulation were carried out on shoots and roots separately of intact Helianthus annuus L. cv. Autumn Beauty plants grown in nutrient solution at different root temperatures. The data were used to distinguish between growth and maintenance components of respiration. The maintenance and growth coefficients were higher in the root system than in the shoots. The overall efficiency of assimilate utilization was within the range reported in the literature. An increase in root temperature increased the maintenance part of root respiration and, to a lesser degree, also shoot maintenance respiration. Neither root nor shoot growth respiration coefficients were affected by root temperature. It is concluded that the study of whole-plant respiration masks differences in energy utilization between shoots and roots.     

The role of phosphorus in nitrogen fixation by young pea plants (Pisum sativum)

The influence of P on N₂ fixation and dry matter production of young pea ( Pisum sativum L. cv. Bodil) plants grown in a soil-sand mixture was investigated in growth cabinet experiments. Nodule dry weight, specific C₂H₂ reduction and P concentration in shoots responded to P addition before any growth response could be observed. The P concentration in nodules responded only slightly to P addition. A supply of P to P-deficient plants increased both the nodule dry weight, specific C₂H₂ reduction and P concentration in shoots relatively faster than it increased shoot dry weight and P concentration in nodules. Combined N applied to plants when N₂ fixation had commenced, increased shoot dry weight only at the highest P levels. This indicates that the smaller plant growth at the low P levels did not result from N deficiency. The reduced nodulation and N₂ fixation in P-deficient plants seem to be caused by impaired shoot metabolism and not by a direct effect of P deficiency of the nodules.     

Differences in nitrogen metabolism of Faidherbia albida and other N2-fixing tropical woody acacias reflect habitat water availability

The activities of nitrate reductase and glutamine synthetase were evaluated in young plants of Faidherbia albida , a tropical woody legume, fed with different N sources under hydroponic conditions. Results showed that assimilation of both NO₃⁻ and NH₄⁺ preferentially took place in shoots. A basal amount of nitrate reductase activity was detected in shoots of plants grown with an NO₃⁻-free solution or placed under N₂-fixing conditions, and also in nodules of N₂-fixing plants. This strongly suggests that constitutive nitrate reductase activity is present in these organs. Analyses of the soluble nitrogenous content showed that the major form of N in the different organs was α-amino acids (particularly amides), irrespective of the N status of the culture conditions. The same result was obtained for nodulated plants grown in local sandy soil. In this case, amide-N generally accounted for more than 40% of the total soluble N. This was especially true in nodules. Ureide-N never exceeded 9% of the total soluble N and did not appear to increase with increasing nodule nitrogenase activity. Amides were also predominant in three N₂-fixing Sahelian acacias ( Acacia seyal , A. nilotica and A. tortilis ), showing that F. albida does not differ from Sahelian Acacia in terms of the metabolism of fixed N. However, like another Sahelian acacia growing preferentially near water ( A. nilotica ), F. albida can be distinguished from acacias growing strictly in arid zones ( A. seyal and A. tortilis ) in terms of initial growth, water and nitrate management.     

Genetic factors in Rhizobium affecting the symbiotic carbon costs of N2 fixation and host plant biomass production

The effect of genetic factors in Rhizobium on host plant biomass production and on the carbon costs of N₂ fixation in pea root nodules was studied. Nine strains of Rhizobium leguminosarum were constructed, each containing one of three symbiotic plasmids in combination with one of three different genomic backgrounds. The resulting strains were tested in symbiosis with plants of Pisum sativum using a flow-through apparatus in which nodule nitrogenase activity and respiration were measured simultaneously under steady state conditions. Nodules formed by strains containing the background of JI6015 had the lowest carbon costs of N₂ fixation (7.10–8.10 μmol C/μmol N₂), but shoot dry weight of those plants was also smaller than that of plants nodulated by strains with the background of B151 or JI8400. Nodules formed by these two strain types had carbon costs of N₂ fixation varying between 11.26 and 13.95 μmol C/μmol N₂. The effect of symbiotic plasmids on the carbon costs was relatively small. A time-course experiment demonstrated that nodules formed by a strain derived from JI6015 were delayed in the onset of nitrogenase activity and had a lower rate of activity compared to nodules induced by a strain with the background of B151. The relationship between nitrogenase activity, carbon costs of N₂ fixation and host plant biomass production is discussed.     

Effects of root restriction on the growth and physiology of cucumber plants

Cucumber plants ( Cucumis sativus L. cv. Athene F1) were grown with four treatments: unrestricted root volume fruiting (UF); unrestricted root volume non-fruiting (UN); restricted root volume fruiting (RF); and restricted root volume non-fruiting (RN). Restricting root volume to 40 ml reduced leaf area, and by day 60 leaf area was only 20% that of unrestricted plants. Leaf area reduction in restricted plants was due to a combination of smaller and fewer leaves. Root restriction strongly depressed total dry matter production in both root and shoot. Significant differences of treatments in shoot and root growth rates were akpparent 30 days after sowing. RN plants had a 70% lower net photosynthesis (P_n), stomatal conductance (g_s) and transpiration rate (E) measured on day 50, while root restriction had no effect on P_n in fruiting plants, although g_s and E were significantly decreased due to restriction. Respiration capacity of restricted roots decreased sharply after day 24 compared with unrestricted root systems. Initially, O₂ may have been the limiting resource and root respiration capacity a major factor involved in root restriction, since it causes imbalances in root growth substances and related hormones that alter the plant's morphology.     

Ammonium and nitrate nutrition inPlantago lanceolata andPlantago major L. ssp.major

P. lanceolata andP. major were grown in culture solutions with nitrate or ammonium as the nitrogen source. Dry matter accumulation in the shoot was faster with nitrate than with ammonium, whilst that of the roots was not affected by the nitrogen source. As a consequence, the shoot-to-root ratio was lower with ammonium than with nitrate. InP. lanceolata, dry matter percentage of shoot and root tissue was lower with nitrate nutrition, suggesting better elongation growth than with ammonium. However, in shoot tissue ofP. major the opposite was found. The rate of root respiration declined with time, and this was almost completely due to a declining activity of the alternative path, which amounted to about 30–60% of total root respiration. Respiration via the cytochrome path was for a part of time slightly increased by ammonium, whereas the activity of the alternative path was strongly enhanced. The concentration of ethanol-soluble carbohydrates (SC) in the roots of both species was higher when nitrate was used, but no difference in the concentration of starch was found. When the plants were transferred from one nitrogen source to the other, many parameters, including the concentration of nitrate and chloride, and the shoot to root ratio, adjusted to the new situation in both species. Grassland Species Research Group, Publication no. 116.     

The effect of nitrogen source on photosynthesis of carob at high CO2 concentrations

Carob seedlings ( Ceratonia siliqua L. cv. Mulata), fed with nitrate or ammonium, were grown in growth chambers containing two levels of CO₂ (360 or 800 μl l⁻¹), three root temperatures (15, 20 or 25°C), and the same shoot temperature (20/24°C, night/day temperature). The response of the plants to CO₂ enrichment was affected by environmental factors such as the type of inorganic nitrogen in the medium and root temperature. Increasing root temperature enhanced photosynthesis rate more in the presence of nitrate than in the presence of ammonium. Differences in photosynthetic products were also observed between nitrate- and ammonium-fed carob seedlings. Nitrate-grown plants showed an enhanced content of sucrose, while ammonium led to enhanced storage of starch. Increase in root temperature caused an increase in dry mass of the plants of similar proportions in both nitrogen sources. The enhancement of the rates of photosynthesis by CO₂ enrichment was proportionally much larger than the resulting increases in dry mass production when nitrate was the nitrogen source. Ammonium was the preferred nitrogen source for carob at both ambient and high CO₂ concentrations. The level of photosynthesis of a plant is limited not only by atmospheric CO₂ concentration but also by the nutritional and environmental conditions of the root.     

Inorganic nitrogen regulation of glutamate uptake in the cyanobacterium Nostoc muscorum

In Nostoc muscorum (Anabaena ATCC 27893) glutamate was not metabolised as a fixed nitrogen source, rather it functioned as an inhibitor of growth. The latter effect was nitrogen source specific and occurred in N₂-fixing cultures but not in cultures assimilating nitrate or ammonium. NO₃^--grown cultures lacked heterocysts and nitrogenase activity and showed a nearly 50% reduction in glutamate uptake rates, as well as in the final extent of glutamate taken up, compared to N₂-fixing or nitrogen-limited control cultures. NH₄⁺-grown cultures showed a similar response, except that the reduction in glutamate uptake rates and the final exten of glutamate taken up was over 80%. The present results suggest a relation between nitrate/ammounium nitrogen-dependent inhibition of glutamate uptake, probably via repression of the glutamate transport system, and glutamate toxicity.     

Respiratory energy requirements of roots vary with the potential growth rate of a plant species

The rates of growth, net rate of nitrate uptake and root respiration of 24 wild species were compared under conditions of optimum nutrient supply. The relative growth rate (RGR)of the roots of these species varied between 110 and 370 mg g^-1 day^-1 and the net rate of nitrate uptake between 1 and 7 mmol (g root dry weight)^-1 day^-1. The rate of root respiration was positively correlated with the RGR of the roots. Root respiration was also calculated from the measured rate of growth and nitrate uptake, using previously determined values for the costs of maintenance, growth and ion uptake of two slow-growing species. The calculated rate of respiration was slightly lower than the measured one for slow-growing species, but twice as high as measured rates for rapid-growing species. This discrepancy was not due to a relatively smaller electron flow through the alternative pathway and, consequently, a more efficient ATP production in the fast-growing species. Neither could variation in specific costs for root growth or maintenance explain these differences. Therefore, we conclude that fast-growing species have lower specific respiratory costs for ion uptake than slow-growing ones. Due partly to these lower specific costs of nutrient uptake, the fraction of respiration that rapid-growing species spend on anion uptake is lower than that of slow-growing species, in spite of the much higher rate of ion uptake of the fast-growing ones.     

Pea seedling growth and development regulated by cotyledons and modified by irradiance

Growth and development of hydroponically grown pea seedlings ( Pisum sativum L. cv. Alaska) were measured using stem and root length as well as number of leaves and lateral roots. The growth was dependent on the presence of cotyledons and was modulated by the irradiance. All plants were grown in a full nutrient solution. If grown at low irradiance (73 μmol m^-2s^-1) they depended more and for a longer time on the cotyledons than plants grown at high irradiance (220 μmol m^-2s^-1). Low irradiance caused stem elongation but decreased root length and number of lateral roots as compared to plants grown at high irradiance. The dark respiration of the leaves was measured as oxygen uptake. In plants grown at the low irradiance, excision of the cotyledons caused the rate of oxygen uptake to increase by a factor of three, and the increase was sensitive to cyanide. Decotyledonized plants showed a high respiration rate and a diminished leaf growth for their entire life cycle. CO₂ fixation also increased in decotyledonized pea seedlings grown at either irradiance. The mobilization of food reserves from the seeds was positively correlated to seed dry weight, but only if the plants were grown at 73 μmol m^-2s^-1. Increasing dry weight of the seed enhanced top growth, whereas root growth was depressed, so that top and root responds differently with regard to that part of growth which depends on mobilization of reserves from the seed.     

Effect of respiratory homeostasis on plant growth in cultivars of wheat and rice

Some plants have the ability to maintain similar respiratory rates (measured at the growth temperature), even when grown at different temperatures, a phenomenon referred to as respiratory homeostasis. The underlying mechanisms and ecological importance of this respiratory homeostasis are not understood. In order to understand this, root respiration and plant growth were investigated in two wheat cultivars (Triticum aestivum L. cv. Stiletto and cv. Patterson) with a high degree of homeostasis, and in one wheat cultivar (T. aestivum L. cv. Brookton) and one rice cultivar (Oryza sativa L. cv. Amaroo) with a low degree of homeostasis. The degree of homeostasis (H) is defined as a quantitative value, which occurs between 0 (no acclimation) and 1 (full acclimation). These plants were grown hydroponically at constant 15 or 25 °C. A good correlation was observed between the rate of root respiration and the relative growth rates (RGR) of whole plant, shoot or root. The plants with high H showed a tendency to maintain their RGR, irrespective of growth temperature, whereas the plants with low H grown at 15 °C showed lower RGR than those grown at 25 °C. Among several parameters of growth analysis, variation in net assimilation rate per shoot mass (NAR_m) appeared to be responsible for the variation in RGR and rates of root respiration in the four cultivars. The plants with high H maintained their NAR_m at low growth temperature, but the plants with low H grown at 15 °C showed lower NAR_m than those grown at 25 °C. It is concluded that respiratory homeostasis in roots would help to maintain growth rate at low temperature due to a smaller decrease in net carbon gain at low temperature. Alternatively, growth rate per se may control the demand of respiratory ATP, root respiration rates and sink demands of photosynthesis. The contribution of nitrogen uptake to total respiratory costs was also estimated, and the effects of a nitrogen leak out of the roots and the efficiency of respiration on those costs are discussed.     

Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Response of Potato to Phosphorus Deficiency

Morphological and biochemical interactions between a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus fasciculatum [Thaxt. sensu Gerdemann] Gerdemann and Trappe) and potato (Solanum tuberosum L.) plants during the development of P deficiency were characterized. Nonmycorrhizal (NM) plants grown for 63 d with low abiotic P supply (0.5 mM) produced 34, 52, and 73% less root, shoot, and tuber dry matter, respectively, than plants grown with high P (2.5 mM). The total leaf area and the leaf area:plant dry weight ratio of low-P plants were substantially lower than those of high-P plants. Moreover, a lower shoot:root dry weight ratio and tuber:plant dry weight ratio in low-P plants than in high-P plants characterized a major effect of P deficiency stress on dry matter partitioning. In addition to a slower rate of growth, low-P plants accumulated nonreducing sugars and nitrate. Furthermore, root respiration and leaf nitrate reductase activity were lower in low-P plants than in high-P plants. Low abiotic P supply also induced physiological changes that contributed to the greater efficiency of P acquisition by low-P plants than by high-P plants. For example, allocation of dry matter and P to root growth was less restricted by P deficiency stress than to shoot and tuber growth. Also, the specific activities of root acid phosphatases and vanadate-sensitive microsomal ATPases were enhanced in P-deficient plants. The establishment of a VAM symbiosis by low-P plants was essential for efficient P acquisition, and a greater root infection level for P-stressed plants indicated increased compatibility to the VAM fungus. By 63 d after planting, low-P VAM plants had recovered 42% more of the available soil P than low-P NM plants. However, the VAM fungus only partially alleviated P deficiency stress and did not completely compensate for inadequate abiotic P supply. Although the specific activities of acid phosphatases and microsomal ATPases were only marginally influenced by VAM infection, VAM roots characteristically had a higher protein concentration and, consequently, enhanced microsomal ATPase and acid phosphatase activities on a fresh weight basis compared with NM roots. Morphological and ultrastructural details of VAM plants are discussed in relation to the influence of the VAM symbiosis on P nutrition of potato.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

In vivo nitrate reductase activities in different organs of lucerne plants: Effects of combined nitrogen during first vegetative growth and after shoot harvest

Nitrate reductase (EC 1.6.6.1–3; NR) activity was evaluated in nodulated lucerne ( Medicago sativa L. cv. Europe) grown aeroponically in both the presence and absence of applied nitrogen. Determination of in vivo NR activity was done with organ pieces in 0.1 M K⁺-phosphate, pH 7.5, 0.1 M KNO₃ and 1% n -propanol. NR activity was detected in all plant parts. Leaves accounted for 40% of the whole plant activity. Root activity was as high as leaf activity. Stem NR activity accounted for 14 to 20% of the total plant activity. NR activity was also detected in symbolically dependent plants grown without combined nitrogen. Nodule NR in symbolically dependent plants accounted for 17% of the tolal plant aclivity. When nitrate was present in the nulrienl medium, NR increased 5-fold as compared lo N₂-dependenl plants. Varying levels of nitrale (1.65 to 4 m M ) had no influence on leaf or stem activities. However, root NR activity seemed to be related to the nitrale concentration in the nulrient medium. Throughoul inilial vegelative growth, in vivo NR and nitrogenase (acelylene reduction) increased simultaneously. After shoot harvest, nitrogenase (acetylene reduction) aclivity drastically decreased with reduction of photosynthate supply, whereas NR increased in all organs, especially in N₂-dependenl plants.     

Growth comparisons of a supernodulating soybean (Glycine max) mutant and its wild-type parent

The growth of a supernodulating, nitrate-tolerant soybean [ Glycine max (L.) Merr.] mutant nts 382 (nitrate-tolerant symbiosis) was compared to that of its wild-type parent, cv. Bragg, over the first 50 days after sowing. Plants were grown either inoculated in the absence of an external nitrogen source or uninoculated in the presence of 5 m M KNO₃. For both treatments, nts 382 growth up to 13 days after planting was faster than that of cv. Bragg. Thereafter, supernodulation of inoculated nts 382 occurred and growth of cv. Bragg was faster; shoot and root dry weight increments and leaf area were greater in cv. Bragg, but the N content of nts 382 was higher. Relative growth and net assimilation rates were lower in nts 382, which had faster shoot and root respiration rates. Shoot growth of uninoculated plants was similar for both mutant and wild-type but roots of nts 382 were slightly smaller than those of cv. Bragg. Total plant N content was similar in uninoculated cv. Bragg and nts 382 but the latter had a higher leaf N content. Early lateral root formation (prior to nodule emergence) was greater in nts 382 regardless of whether rhizobia or KJNO₃ were present. We conclude that nts 382 has some inherent differences from its parent but that supernodulation significantly retards plant growth.     

Regulation of nitrate reductase cellular levels in the cyanobacteria Anabaena variabilis and Synechocystis sp.

Abstract The effect of the nitrogen source on the cellular activity level of assimilatory nitrate reductase in the cyanobacteria Anabaena variabilis (ATCC29413) and Synechocystis sp. (PCC6714) has been examined. In the filamentous N₂-fixing A. variabilis , nitrate behaved as a nutritional inducer of nitrate reductase, with ammonium acting (via products of its assimilation) as an antagonist with regard to nitrate. Ammonium-promoted repression of nitrate reductase was also evident in the unicellular non-nitrogen fixer Synechocystis , but in this strain nitrate was not required as an obligatory inducer.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Characterization of Na+ exclusion mechanisms of salt-tolerant reed plants in comparison with salt-sensitive rice plants

Salt-tolerant reed plants ( Phragmites communis Trinius) and salt-sensitive rice plants ( Oryza sativa L. cv. Kinmaze) were grown in salinized nutrient solutions up to 50 m M NaCl, and growth, Na⁺ contents and kinetics of ²²Na⁺ uptake and translocation were compared between the species to characterize the salt tolerance mechanisms operating in reed plants. When both plants were grown under the same salinity, Na⁺ contents of the shoots were lower in reed plants, although those of the roots were quite similar. The shoot base region of both species accumulated Na⁺ more than the leaf blades did. Sodium-22 uptake and pulse-chase experiments suggested that the lower Na⁺ transport rate from root to shoot could limit excessive Na⁺ accumulation in the reed shoot. There was a possibility that the apparently lower ²²Na⁺ transport rate to the shoot of reed plants was due to net downward Na⁺ transport from shoot base to root.     

Suppression of ammonium uptake by nitrogen supply and its relief during nitrogen limitation

Barley ( Hordeum vulgare , cv. Triumph), wheat ( Triticum aestivum , cv. Kleiber) and oat ( Avena sativa , cv. Tarok) were grown until day 20 in nitrate-containing solutions or in nitrogen-free solutions for periods up to 8 days immediately prior to day 20. They then were exposed for 4 h to complete, nitrate-free solutions containing 0.5 or 2.0 mM ammonium (98 atom%¹⁵N). In all 3 species in 2 experiments, net ammonium uptake was low in plants grown continuously in nitrate, and increased 3 to 4-fold with increasing nitrogen deprivation. Charge balance during net ammonium uptake was largely maintained by the sum of net potassium and net proton efflux. Variations in root ammonium concentration at the time of exposure to the ammonium solutions revealed no consistent pattern with net ammonium uptake, implying that a product of ammonium assimilation may serve as a negative effector for the uptake process. In nitrogen-replete plants, and in those deprived of nitrogen for 2 days, the amounts of endogenous ¹⁴N-ammonium recovered in the ambient ¹⁵N-ammonium solution during the 4-h uptake period were greater than the initial amounts of ¹⁴N-ammonium present in the root tissue. Significant generation of ¹⁴N-ammonium from endogenous organic nitrogen sources was thus evident in all 3 species.