Exploration of the nitrogen transport system of a nodulated legume using 15N

Summary Feeding experiments using ¹⁵N₂ or ¹⁵NO₃ are described investigating the transport of nitrogen in the field pea (Pisum arvense L.). Nitrogen assimilated by root or nodules moves preferentially upwards to the shoot through the xylem. Parts of the root below or distal to a region of assimilation can benefit from this nitrogen but do so to a much greater extent when the shoot is left attached than when it has been removed. A considerable proportion of the nitrogen received by a shoot from the root or nodules is apparently returned to the root in the translocation stream, this cycled nitrogen being especially important in the nutrition of outlying parts of nodulated roots growing in media lacking combined nitrogen.Nitrogen from nitrate fed to a mature leaf is exported in quantity to all parts of the plant except older regions of the shoot. Leaf and stem segments immediately above the fed leaf, and the root and its nodules receive large shares of this nitrogen, although the root's share declines noticeably as the plant ages.The root appears to be extremely inactive in transferring nitrogen from the downward translocation stream across to the stream of nitrogen leaving the root in the xylem. This may act as a major obstacle to the free circulation and mixing of nitrogen within the plant body.A scheme is proposed embracing the main quantitative features of the transport system for nitrogen in the species.     

Modeling the transport and utilization of carbon and nitrogen in a nodulated legume

An empirical modeling technique was developed for depicting quantitatively the transport and partitioning of photosynthetically fixed C and symbiotically fixed N during 10-day intervals of a 40-day period in the growth of nodulated plants of white lupin (Lupinus albus L. cv. Ultra). Model construction utilized data for C and N consumption of plant parts and C:N weight ratios of the xylem and phloem fluids serving specific plant organs. Formulas were derived from calculating the net transport of C and N between plant parts in xylem and phloem. The models provided quantitative information on the dependence of growing organs on xylem and phloem for their supply of C and N, the cycling of N through leaflets and of C through nodules, the extent of direct incorporation of fixed N into growing nodules, and the involvement of N from shoot translocate in the nutrition of the nodulated root. Stem plus petioles abstracted considerably more N from xylem than expected from their transpirational activity. Xylem to phloem transfer of recently fixed N in mature stem and petioles was substantiated by the models, being depicted as a device for dispensing N to growing parts of the shoot extra to that attracted transpirationally in xylem or received as translocate from leaflets.     

Seasonal patterns of 13C partitioning between shoots and nodulated roots of N2- or nitrate-fed Pisum sativum L

The effect of nitrogen source (N(2) or nitrate) on carbon assimilation by photosynthesis and on carbon partitioning between shoots and roots was investigated in pea (Pisum sativum L. 'Baccara') plants at different growth stages using (13)C labelling. Plants were grown in the greenhouse on different occasions in 1999 and 2000. Atmospheric [CO(2)] and growth conditions were varied to alter the rate of photosynthesis. Carbon allocation to nodulated roots was unaffected by N source. At the beginning of the vegetative period, nodulated roots had priority for assimilates over shoots; this priority decreased during later stages and became identical to that of the shoot during seed filling. Carbon allocation to nodulated roots was always limited by competition with shoots, and could be predicted for each phenological stage: during vegetative and flowering stages a single, negative exponential relationship was established between sink intensity (percentage of C allocated to the nodulated root per unit biomass) and net photosynthesis. At seed filling, the amount of carbon allocated to the nodulated root was directly related to net photosynthesis. Respiration of nodulated roots accounted for more than 60 % of carbon allocated to them during growth. Only at flowering was respiration affected by N supply: it was significantly higher for strictly N(2)-fixing plants (83 %) than for plants fed with nitrate (71 %). At the vegetative stage, the increase in carbon in nodulated root biomass was probably limited by respiration losses.     

Photosynthesis and photosynthate partitioning in n(2)-fixing soybeans

Leaf area, chlorophyll content, net CO₂ photoassimilation, and the partitioning of fixed carbon between leaf sucrose and starch and soluble protein were examined in Glycine max (L) Merr. cv Williams grown under three different nitrogen regimes. One group (Nod+/+) was inoculated with Bradyrhizobium and watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. A second set (Nod+/−) was inoculated and had N₂ fixation as its sole source of nitrogen. A third group (Nod⁻) was not inoculated and was watered daily with a nutrient solution containing 6 millimolar NH₄NO₃. The mean net micromole CO₂ uptake per square decimeter per hour of the most recently matured source leaves was similar among the three groups of plants, being about 310. Mean leaf area of the source leaves, monitored for net photosynthesis was also similar. However, the mean milligram of chlorophyll per square decimeter of Nod+/− test leaves was about 50% lower than the other groups' leaves and indicated nitrogen deficiency. Thus, Nod+/− utilized their chlorophyll more efficiently for photosynthetic CO₂ uptake than the plants of the other treatments. The ratio of foliar carbohydrate:protein content was high in Nod+/− but low in the plants from the other two treatments. This inverse relationship between foliar protein and carbohydrate content suggests that more fixed carbon is diverted to the synthesis of protein when nitrogen availability is high. It was also found that Nod+/− sequestered more storage protein in their paraveinal mesophyll than plants of the other treatments. This study indicates that when inorganic nitrogen regimes are used to control photosynthate partitioning, then both leaf carbohydrate and leaf protein must be considered as end products of carbon assimilate allocation.     

Transport of organic solutes in Phloem and xylem of a nodulated legume

Collections of xylem exudate of root stumps or detached nodules, and of phloem bleeding sap from stems, petioles, and fruits were made from variously aged plants of Lupinus albus L. relying on nodules for their N supply. Sucrose was the major organic solute of phloem, asparagine, glutamine, serine, aspartic acid, valine, lysine, isoleucine, and leucine, the principal N solutes of both xylem and phloem. Xylem sap exhibited higher relative proportions of asparagine, glutamine and aspartic acid than phloem sap, but lower proportions of other amino acids. Phloem sap of petioles was less concentrated in asparagine and glutamine but richer in sucrose than was phloem sap of stem and fruit, suggesting that sucrose was unloaded from phloem and amides added to phloem as translocate passed through stems to sinks of the plant. Evidence was obtained of loading of histidine, lysine, threonine, serine, leucine and valine onto phloem of stems but the amounts involved were small compared with amides. Analyses of petiole phloem sap from different age groups of leaves indicated ontogenetic changes and effects of position on a shoot on relative rates of export of sucrose and N solutes. Diurnal fluctuations were demonstrated in relative rates of loading of sucrose and N solutes onto phloem of leaves. Daily variations in the ability of stem tissue to load N onto phloem streams were of lesser amplitude than, or out of phase with fluctuations in translocation of N from leaves. Data were related to recent information on C and N transport in the species.     

Metabolism and translocation of fixed nitrogen in the nodulated legume

Summary Nitrogen (N₂) fixed by Rhizobium bacteroids in the legume nodule is excreted as ammonia to the surrounding host cell where it is efficiently assimilated into the amide group of glutamine. Generally glutamine is a minor exported solute of nitrogen, being further metabolised to asparagine in temperate species and to the ureides, allantoin and allantoic acid in tropical species. These solutes serve as the principal translocated forms of nitrogen in xylem. Compartmentalisation of the pathways of nitrogen metabolism and the role of ammonia in regulation of their activity is examined in nodules of both asparagine-forming (Lupinus albus L.) and ureide-forming (Vigna unguiculata L. Walp) symbioses.     

Effects of water deficit on N2(C2H2) fixation in cowpea and groundnut

The effect of water deficit on nodulation, N₂ fixation, photosynthesis, and total soluble sugars and leghemoglobin in nodules was investigated in cowpea and groundnut. Nitrogenase activity completely ceased in cowpea with a decrease in leaf water potential ( _leaf) from –0.4 MPa to –0.9 MPa, while in groundnut it continued down to –1.7 MPa. With increasing water stress, the acetylene reduction activity (ARA) declined very sharply in cowpea, but ARA gradually decreased in groundnut. Even with mild water stress ( _leaf of 0.2 MPa), nodule fresh weight declined 50% in cowpea partly due to a severe nodule shedding whereas nodule fresh weight declined in groundnut only when _leaf decreased by 1.0 MPa. No nodule shedding was noticed even at a higher stress level in groundnut. Photosynthesis and stomatal conductance were also more stable in groundnut than in cowpea under water stress. There was a sharp increase in total soluble sugars and leghemoglobin in the nodules of groundut with water stress, but no definite trend could be found in cowpea.     

Effects of elevated CO2 and N addition on growth and N2 fixation of a legume subshrub (Caragana microphylla Lam.) in temperate grassland in China

It is well demonstrated that the responses of plants to elevated atmospheric CO(2) concentration are species-specific and dependent on environmental conditions. We investigated the responses of a subshrub legume species, Caragana microphylla Lam., to elevated CO(2) and nitrogen (N) addition using open-top chambers in a semiarid temperate grassland in northern China for three years. Measured variables include leaf photosynthetic rate, shoot biomass, root biomass, symbiotic nitrogenase activity, and leaf N content. Symbiotic nitrogenase activity was determined by the C(2)H(2) reduction method. Elevated CO(2) enhanced photosynthesis and shoot biomass by 83% and 25%, respectively, and the enhancement of shoot biomass was significant only at a high N concentration. In addition, the photosynthetic capacity of C. microphylla did not show down-regulation under elevated CO(2). Elevated CO(2) had no significant effect on root biomass, symbiotic nitrogenase activity and leaf N content. Under elevated CO(2), N addition stimulated photosynthesis and shoot biomass. By contrast, N addition strongly inhibited symbiotic nitrogenase activity and slightly increased leaf N content of C. microphylla under both CO(2) levels, and had no significant effect on root biomass. The effect of elevated CO(2) and N addition on C. microphylla did not show interannual variation, except for the effect of N addition on leaf N content. These results indicate that shoot growth of C. microphylla is more sensitive to elevated CO(2) than is root growth. The stimulation of shoot growth of C. microphylla under elevated CO(2) or N addition is not associated with changes in N(2)-fixation. Additionally, elevated CO(2) and N addition interacted to affect shoot growth of C. microphylla with a stimulatory effect occurring only under combination of these two factors.     

Effects of magnesium deficiency on photosynthesis and carbohydrate partitioning

Magnesium nutrition is often forgotten, while its absence adversely affects numerous functions in plants. Magnesium deficiency is a growing concern for crop production frequently observed in lateritic and leached acid soils. Competition with other cations (Ca²⁺, Na⁺, and K⁺) is also found to be an essential factor, inducing magnesium deficiency in plants. This nutrient is required for chlorophyll formation and plays a key role in photosynthetic activity. Moreover, it is involved in carbohydrate transport from source-to-sink organs. Hence, sugar accumulation in leaves that results from the impairment of their transport in phloem is considered as an early response to Mg deficiency. The most visible effect is often recorded in root growth, resulting in a significant reduction of root/shoot ratio. Carbohydrate accumulation in source leaves is attributed to the unique chemical proprieties of magnesium. As magnesium is a nutrient with high mobility in plants, it is preferentially transported to source leaves to prevent severe declines in photosynthetic activity. In addition, Mg is involved in the source-to-sink transport of carbohydrates. Hence, an inverse relationship between Mg shortage and sugar accumulation in leaves is often observed. We hereby review all these aspects with a special emphasis on the role of Mg in photosynthesis and the structural and functional effects of its deficiency on the photosynthetic apparatus.     

Difference in delta(15)N signatures between nodulated roots and shoots of soybean is indicative of the contribution of symbiotic N(2) fixation to plant N

Symbiotic N(2) fixation has a variable effect on the (15)N abundance of different parts of legumes. Increases in fixation result in (15)N enrichment of nodules, while decreases, in combination with an increased uptake of mineral N, result in (15)N depletion of the root system. The difference between soybean shoot and below-ground delta(15)N (Deltadelta(15)N=delta(15)N(shoot)-delta(15)N(belowground)) was assessed in hydroponic culture over a range of rates of supply of mineral N. The fractional contribution of N(2) fixation to N uptake (%Ndfa) was determined using the natural abundance (NA) technique with ryegrass as a reference plant. Deltadelta(15)N and %Ndfa were highly correlated, and the relationship was tested using the same soybean cultivar grown in pots in N-rich soil. Estimates of %Ndfa derived from the NA method and from the Deltadelta(15)N approach yielded near-identical values. A literature survey showed similar relationships between %Ndfa and Deltadelta(15)N with different growth stages of soybeans grown under glasshouse and field conditions, different cowpea (Vigna unguiculata) cultivars in the field, and tagasaste (Chamaecytisus proliferus) in hydroponic culture. Possible confounding and species-specific (either plant or Rhizobium spp.) influences are discussed. The difference in delta(15)N signatures between nodulated roots and shoots is confirmed as a robust means of quantifying %Ndfa: it is independent of reference plants and offers the possibility of estimating %Ndfa in soils where the isotope composition of mineral N closely matches that of atmospheric N(2).     

Partitioning of carbon and nitrogen and the nutrition of root and shoot apex in a nodulated legume

Empirically based models depicting exchanges of C, N, and H₂O in phloem and xylem among organs of nodulated white lupin (Lupinus albus cv Ultra) were constructed for the interval 51 to 58 days after sowing. Information was incorporated on the economy of C, N, and H₂O in plant parts, the solute composition of transport fluids collected at selected sites on the plant, and the photosynthetic inputs, transpirational losses, and translocatory activities of different age groups of leaflets and stem + petiole segments of the shoot. Partitioning of C and N showed preferential transfer of N to the shoot apex, which imported 13 milligrams C per milligram N, compared with 54 milligrams C per milligram N for the nodulated root. Leaves translocated assimilates at a C:N weight ratio of 43 to 59, and older leaves serving the roots produced the translocate most rich in N relative to C. The shoot apex was enriched with N, additional to its intake from leaves, by direct uptake of xylem fluid (C:N ratio, 2.4) and receipt of nitrogenous solutes transferred from xylem to upward-moving phloem streams in upper regions of the stem. The models for flow of N and H₂O indicated that xylem streams passing to leaves were substantially less rich in N than the adjacent stream moving through the body of the stem and that a progressive increase in concentration of N occurred within stem xylem elements from base to top of the shoot. This apparently resulted from an abstraction of N from xylem of departing leaf traces, possibly by xylem transfer cells, and a subsequent feedback of this N to xylem streams passing on up the shoot. Upper leaves and shoot apex, therefore, acquired more N from xylem per unit of H₂O transpired than lower parts of the shoot.     

Nitrogen nutrition in nodulated field plants of the shrub tea legume Aspalathus linearis assessed using 15N natural abundance

Provision of N, P, and Ca to field plants of A. linearis markedly (P<0.05) increased growth and N nutrition in a very acidic nutrient-poor soil. Application of P and Ca promoted a significant increase in %N derived from fixation and amounts of N fixed compared to those receiving no nutrients. N₂ fixation measured under field conditions ranged from 3.8 g N plant^-1 in unfertilized control to 7.1 g N plant^-1 in fertilized plants. Overall, about 85% increase in N₂ fixation was observed with P supply. The high N₂-fixing activity in P-treated plants was confirmed by their lower (more negative) ∂¹⁵N values. Age of plants also influenced growth and symbiotic activity as the ∂¹⁵N values, %N derived from fixation, and N fixed were lower in 1- and 2-year-old plants compared to 3-year-old. The contribution of symbiotic fixation in unfertilized A. linearis to the N economy of the ecosystem ranged from 105 kg N ha^-1 in 1-year-old plants to 128 kg N ha^-1 in 3-year-old plants, clearly indicating the remarkable adaptation of this symbiosis to the very nutrient-poor, low pH conditions of Cedarberg soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of phosphate deficiency on assimilate partitioning in barley seedlings

Photosynthesis and assimilate partitioning were measured in barley (Hordeum vulgare L.) seedlings raised from seed with mineral nutrient solution containing either 0 or 1 mmolar orthophosphate (P_i). Carbon assimilation rates and leaf extension rates were inhibited about 40% by low P_i treatment. Starch and sucrose levels were similar in P_i-depleted compared to control leaves during most of the photoperiod. These results indicated that low P_i treatment inhibited carbon export from the primary leaf. Acid extractable P_i decreased about 60% in P_i-depleted barley leaves after a dark to light transition. In similar experiments with control barley and tobacco (Nicotiana tabacum L.) leaves acid extractable P_i decreased about 25% and 15%, respectively. These findings provided preliminary evidence for a diurnal variation in leaf P_i content. Photosynthetic intermediate levels were lower and extractable enzyme activities were the same or higher in low P_i compared to control leaves. These observations suggested that photosynthetic intermediate pools turnover more quickly in low P_i than in P_i-sufficient leaves.     

Methods for the continuous measurement of O2 consumption and H2 production by nodulated legume root systems

The details of two systems for measuring O2 uptake and H2 production in flow-through gas systems used to study nodule physiology and biochemistry are presented here. Both are constructed from commercially available fuel cells. Oxygen uptake measurements are based upon the differential signal from paired detectors exposed to sample and blank gas streams. Because of the very small signal:background ratio needed to detect O2 uptake against atmospheric O2 concentration, these detectors were mounted in thermally controlled aluminium blocks designed to avoid changes in back pressure. Also to avoid apparent concentration differences arising because of pressure differences at the detectors it is important that sample, control and calibration gas flow rates are the same. Hydrogen detectors were mounted in an aluminium block similar to that used for O2, although the requirements for temperature and back pressure control are much less stringent for this application.The limit for differential detection of O2 uptake in air was about 25 ppm (1 mol 1^-l) and for H2 production 1 ppm (0l04 mol l^-1). Linearity checks for the two detectors over the range 4-90% O2 and 15-750 ppm H2 gave regression coefficients of >0.999 and neither detector was significantly affected by changing the background mixing gas from N2 to Ar or He provided that flow rates and back pressures remained constant. Water vapour had no effect on the H2 detectors, but caused small baseline shifts during O2 uptake measurements which were obviated with silica gel drying filters. Changes in gas stream pO2 produced small baseline shifts with the H2 detectors, but did not effect the magnitude of the H2 signal.Two examples are provided of the use of these detectors together with the soyabean/USDA 16 symbiosis in a flow-through system furnished with an infrared gas analyser (IRGA) to measure CO2 production.     

Homozygosity for the IgG2 subclass allotype G2M(n) protects against severe infection in hereditary C2 deficiency

Homozygous C2 deficiency (C2D) is the most common deficiency of the classical complement pathway in Western countries. It is mostly found in patients with autoimmune disease or susceptibility to bacterial infections and in healthy persons. We wished to assess to what extent other immunological factors might explain differences of susceptibility to infections in C2D. For this reason, 44 Swedish patients with C2D were stratified with regard to the severity of documented infections. Investigations of IgG subclass levels, IgG subclass-specific GM allotypes, concentrations of factor B, properdin, and factor H, and polymorphisms of mannan-binding lectin and the Fc receptors FcgammaRIIa and FcgammaRIIIb were performed. Homozygosity for the G2M*n allele, which is known to promote Ab responses to polysaccharide Ags, was strongly associated with the absence of severe infections (p < 0.001) in the patients, suggesting a major protective role. The combination of mannan (or mannose)-binding lectin and C2 deficiency was found to be a minor susceptibility factor for invasive infection (p = 0.03). Low concentrations of IgG2 and factor B might sometimes contribute to susceptibility to infection. Other factors investigated did not appear to be important. In conclusion, the findings indicated that efficient Ab responses to polysaccharides are protective against severe infection in C2D. Implications with regard to vaccination should be considered.     

Nitrate additions enhance the photosynthetic sensitivity of a nodulated South African Mediterranean-climate legume (Podalyria calyptrata) to elevated UV-B

Nitrate deposition from anthropogenic activities into nutrient impoverished soils of Mediterranean ecosystems represents a significant income to their N economy, which may potentially increase the sensitivity of those typically UV-B (280–315 nm) resilient plants from these ecosystems with superior photosynthetic rates especially to increases in solar UV-B flux due to ozone depletion. This proposal was examined by exposing nodulated Podalyria calyptrata seedlings for 6-months in nitrate deficient and nitrate replete sand culture to biologically effective UV-B (UV-B_BE) supplements approximating 40 and 77% above clear-sky background (control) at an outdoor site. Leaf photosynthesis and chemical composition of purely symbiotic plants were unaffected by increased UV-B_BE. Conversely, nodulated plants that received nitrate supplements displayed a linear reduction in stomatal conductance (g_S) but non-linear asymptotic reductions in light-saturated net CO₂ assimilation rate (A_sat), apparent carboxylation efficiency (ACE) and nitrogen use efficiency (NUE) with increased UV-B_BE. RuBP regeneration limitations on CO₂ assimilation were not apparent, since A_sat at saturating internal leaf CO₂ concentrations displayed insignificant depressions with increased UV-B_BE. Nor was there any suggestion of diminished light absorption capacity of antenna complexes, or of photosynthetic inhibition due to starch accumulation, since leaf chlorophyll and carotenoid contents and non-structural carbohydrate concentrations were insignificantly altered by increased UV-B_BE. Also, there seemed unlikely photosynthetic inhibition due to reduced allocation of N to Rubisco with increased UV-B_BE in the nitrate-fed plants, since both ACE and A_sat were negatively correlated with leaf N content, much of which probably constituted assimilated nitrate according to the less negative δ¹⁵N values. We suggest that the molecular processes that rendered P. calyptrata plants receiving nitrate supplements more sensitive to photosynthetic inhibition by increased UV-B_BE may be related indirectly to their more active metabolic state, this apparent from their elevated respiratory and photosynthetic rates, rather than to any direct UV-B effects on CO₂ uptake and fixation.     

Impact of arbuscular mycorrhizal fungi (AMF) and atmospheric CO2 concentration on the biomass production and partitioning in the forage legume alfalfa

The influence of mycorrhizal symbiosis, atmospheric CO₂ concentration and the interaction between both factors on biomass production and partitioning were assessed in nodulated alfalfa (Medicago sativa L.) associated or not with arbuscular mycorrhizal fungi (AMF) and grown in greenhouse at either ambient (392 μmol?mol^?1) or elevated (700 μmol?mol^?1) CO₂ air concentrations. Measurements were performed at three stages of the vegetative period of plants. Shoot and root biomass achieved by plants at the end of their vegetative period were highly correlated to the photosynthetic rates reached at earlier stages, and there was a significant relationship between CO₂ exchange rates and total nodule biomass per plant. In non-mycorrhizal alfalfa, the production of leaves, stems and nodules biomass significantly increased when plants had been exposed to elevated CO₂ concentration in the atmosphere for 4 weeks. Regardless CO₂ concentration at which alfalfa were cultivated, mycorrhizal symbiosis improved photosynthetic rates and growth of alfalfa at early stages of the vegetative period and then photosynthesis decreased, which suggests that AMF shortened the vegetative period of the host plants. At final stages of the vegetative period, AMF enhanced both area and biomass of leaves as well as the leaves to stems ratio when alfalfa plants were cultivated at ambient CO₂. The interaction of AMF with elevated CO₂ improved root biomass and slightly increased the leaves to stems ratio at the end of the vegetative growth. Therefore, AMF may favor both the forage quality of alfalfa when grown at ambient CO₂ and its perennity for next cutting regrowth cycle when grown under elevated CO₂. Nevertheless, this hypothesis needs to be checked under natural conditions in field.