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     

Waterlogging effect on xylem sap glutamine of nodulated soybean

Waterlogging of soybean plants (Glycine max L.) led to impaired symbiotic N₂ fixation and a marked decline in glutamine (Gln) concentration in xylem bleeding sap. Xylem Gln concentration increased during the growth cycle of the plant and was correlated with nodule formation. Treatments known to impair N₂ fixation, such as exposing the root system to pure N₂ gas or a mixture of Ar and O₂ (80:20; v/v), led to specific declines in xylem sap Gln. The decrease in Gln observed during waterlogging was also seen on transfer of nodulated plants to aerated hydroponics, where the decline was highly correlated with ureide content in the xylem sap. Upon flooding the nodulated root system, the specific decline in xylem sap Gln could be detected within 10 min and reached a minimum within 60 min, indicating that waterlogging has an immediate effect on N₂ fixation. It is concluded that xylem Gln arises directly from N₂-fixation and is a useful indicator of N₂ fixation activity of symbiotic soybean plants.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Changes in the Composition of Xylem Sap during Development of the Spadix of Skunk Cabbage (Symplocarpus foetidus)

The spadix of skunk cabbage, Symplocarpus foetidus, is thermogenic and maintains an internal temperature of around 20 °C even when the ambient air temperature drops below freezing. This homeothermic heat production is observed only during the stigma stage, and thereafter ceases at the male stage when pollen is shed. To clarify the regulatory mechanism by which the stigma stage-specific heat production occurs in the spadix, sugars, organic acids, and amino acids in xylem sap were analyzed and compared with those of post-thermogenic plants. Interestingly, no significant difference was observed in the total volume of xylem sap per fresh weight of the spadix between thermogenic (31.2±24.7 μl h^?1g^?1) and post-thermogenic (50.5±30.4 μl h^?1g^?1) plants. However, concentrations of sugars (sucrose, glucose, and fructose), organic acids (malate and succinate), and amino acids (Asp, Asn, Glu, Gln, Gly, and Ala) in xylem sap decreased remarkably in post-thermogenic plants. Our results indicate that the composition of the xylem sap differs during the development of the spadix of S. foetidus.     

Primary metabolism in N2-fixing Alnus incana-Frankia symbiotic root nodules studied with 15N and 31P nuclear magnetic resonance spectroscopy

The primary nitrogen metabolism of the N₂-fixing root nodule symbiosis Alnus incana (L.)–Frankia was investigated by ³¹P and ¹⁵N nuclear magnetic resonance (NMR) spectroscopy. Perfusion of root nodules in a pulse–chase approach with ¹⁵N- or ¹⁴N-labeled NH₄⁺ revealed the presence of the amino acids alanine (Ala), -amino butyric acid, glutamine (Gln), glutamic acid (Glu), citrulline (Cit) and arginine (Arg). Labeling kinetics of the Gln amide-N and -amino acids suggested that the glutamine synthetase (GS; EC 6.3.1.2)–glutamate synthase (GOGAT; EC 1.4.1.13) pathway was active. Inhibition of the GS-catalyzed reaction by methionine sulphoximine abolished incorporation of ¹⁵N. Cit was labeled in all three N positions but most rapidly in the position, consistent with carbamoyl phosphate as the precursor to which Gln could be the amino donor catalyzed by carbamoyl phosphate synthase (CPS; EC 6.3.5.5). Ala biosynthesis occurred consistent with a flux of N in the sequence Gln–Glu–Ala. ³¹P NMR spectroscopy in vivo and of extracts revealed several metabolites and was used in connection with the ¹⁵N pulse–chase experiment to assess general metabolic status. Stable concentrations of ATP and UDP-glucose during extended perfusions showed that the overall root nodule metabolism appeared undisturbed throughout the experiments. The metabolic pathways suggested by the NMR results were confirmed by high activities of the enzymes GS, NADH-GOGAT and ornithine carbamoyltransferase (OCT; EC 2.1.3.3). We conclude that the primary pathway of NH₄⁺ assimilation in A. incana root nodules occurs through the GS–GOGAT pathway. Biosynthesis of Cit through GS–CPS–OCT is important and is a link between the first amino acid Gln and this final transport and storage form of nitrogen.Abbreviations AlaDH l-Alanine dehydrogenase - Cit Citrulline - CPS Carbamoyl phosphate synthase - GABA -Amino butyric acid - GOGAT Glutamate synthase - GS Glutamine synthetase - MDH Malate dehydrogenase - MSO Methionine sulphoximine - NMR Nuclear magnetic resonance - OCT Ornithine carbamoyltransferase - PEPC Phosphoenolpyruvate decarboxylase - UDPGlc Uridine 5-diphosphoglucose     

Xylem to phloem transfer of solutes in fruiting shoots of legumes,studied by a phloem bleeding technique

Summary Comparisons were made of the levels of various solutes in xylem (tracheal) sap and fruit tip phloem sap of Lupinus albus (L.) and Spartium junceum (L.). Sucrose was present at high concentration (up to 220 mg ml^-1) in phloem but was absent from xylem whereas nitrate was detected in xylem (up to 0.14 mg ml^-1) but not in phloem. Total amino acids reached 0.5–2.5 mg ml^-1 (in xylem) versus 16–40 mg ml^-1 in phloem. Phloem: xylem concentration ratios for mineral nutrients (K, Na, Mg, Ca, Fe, Zn, Mn, Cu) spanned the range 0.7 to 20, the ratios generally reflecting an element's phloem mobility and its availability to the xylem from the roots.The accessibility of nitrate to xylem and phloem was studied in Lupinus. Increasing the nitrate supply to roots from 100 to 1000 mg NO₃–Nl^-1 increased nitrate spill over into xylem, but nitrate always failed to appear in phloem. However, phloem loading of small amounts of nitrate was induced by feeding 750 or 1000 mg NO₃–Nl^-1 directly to cut shoots via the transpiration stream. Transfer of reduced nitrogen to phloem was demonstrated by feeding ¹⁵NO₃ to shoots and recovering ¹⁵N-enriched amides and amino acids in phloem sap. Increased nitrate supply to roots led to increased amino acid levels in xylem and phloem but did not alter markedly the balance between individual amino acids.The fate of xylem-fed ¹⁴C-labelled asparagine, glutamine and aspartic acid and of photosynthetically fed ¹⁴CO₂ was studied in Spartium, with reference to phloem transport to seeds. Substantial fractions of the ¹⁴C of all sources appeared in non-amino compounds. [¹⁴C]asparagine passed largely in unchanged form to the phloem whereas the ¹⁴C from aspartic acid or glutamine appeared in phloem attached to other amino acids (e.g. asparagine and glutamic acid). Serine, asparagine and glutamine were the main amino compounds labelled in phloem sap after feeding ¹⁴CO₂. The wide distribution of ¹⁴C amongst free and bound amino acids of seeds suggested that extensive metabolism of phloem-borne solutes occurred in the fruits.     

Effect of NO3 on N2 fixation and nitrogenous solutes of xylem in two nodulated West African geocarpic legumes,Kersting's bean (Macrotyloma geocarpum L.) and Bambara groundnut (Vigna subterranea L.)

Nodulation, nitrogen (N₂) fixation and xylem sap composition were examined in sand cultured plants of Bambara groundnut (Vigna subterranea L.) and Kersting's bean (Macrotyloma geocarpum L.) inoculated with Bradyrhizobium strain CB756 and supplied via the roots for a 4 week period from the third week onwards with different levels of (¹⁵N)-nitrate (0–15 mM). The separate contributions of nitrate and N₂ to plant nitrogen were measured by isotope dilution. Increasing levels of nitrate inhibited nodule growth (measured as dry matter or nodule N) of both species parallel with decreased dependence on symbiotically-fixed N. Specific nodule activity (N₂ fixed g nodule dry⁻¹ d⁻¹ of nodules) was reduced progressively with time in V. subterranea at higher (5 or 15 mM) levels of NO₃, but this was not so for M. geocarpum. Root xylem bleeding sap of both species showed ureides (allantoin and allantoic acid) as predominant (>90%) solutes of nitrogen when plants were relying solely on atmospheric N. Levels of ureide and glutamine decreased and those of asparagine and nitrate in xylem increased with increasing level of applied nitrate. Relative levels of xylem ureide-N were positively correlated (R²=0.842 for M. geocarpum and 0.556 for V. subterranea), and the ratio of asparagine to glutamine in xylem exudate negatively correlated (R²=0.955 for M. geocarpum and 0.736 for V. subterranea) with plant reliance on nitrogen fixation. The data indicate that xylem sap analyses might be useful for indirect field assays of nitrogen fixation by the species and that Kersting's bean might offer some potential as a symbiosis in which N₂ fixation is relatively tolerant of soil N.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Cycling of amino-nitrogen between shoots and roots in wheat seedlings

Summary One part of a split root system of wheat seedlings received full nutrient solution with¹⁵N-nitrate, the other received an identical solution with unlabelled nitrate. Appearance of labelled amino compounds was measured in the xylem sap exuding from roots not supplied directly with¹⁵N-nitrate after removal of the¹⁵N-nitrate-fed roots. This material indicates cycling of nitrogen from the shoots and through the roots. About 60 per cent of the nitrogen in the xylem appears to be cycling in this way.     

Effect of Defoliation or Excision of Growing Axillary Shoots on the Composition of Labeled Products of Photosynthesis in the Leaves and Xylem Sap of Kidney Bean

The content of ¹⁴C in the products of photosynthesis of the source leaf and xylem sap was investigated in kidney bean (Phaseolus vulgaris L.) plants during the stage of mass tillering. ¹⁴C partition was measured a day after two-minute photoassimilation of ¹⁴CO₂ by an individual mature leaf located in the middle part of the shoot. The source-sink relations were disturbed by the excision of all mature leaves (except the source leaf) or all growing axillary shoots. The leaves or growing axillary shoots were excised 15 min after leaf feeding with ¹⁴C₂. A day later, in plants with excised growing axillary shoots, the content of ¹⁴C in the source leaf was by 18% higher and in those with removed leaves by 15% lower than in control plants. The next day after the excision of growing axillary shoots, radioactivity of the xylem sap increased; after defoliation, both the volume of the xylem sap and its specific radioactivity decreased. In the xylem sap of defoliated plants, the proportion of ¹⁴C in malate decreased more than six times, whereas the proportion of ¹⁴C in amino acids somewhat increased (1.5 times). In two days, the volume of the xylem sap exuded by treated plants became the same as in control plants and its radioactivity decreased almost by an order of magnitude but essentially did not differ in the both types of treatment. It is concluded that the processes occurring in the roots are governed by photosynthesis but its regulatory effect is limited by a photoperiod and largely depends on changes in the ratio between biosynthesis of amino acids in the roots and leaves.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 518–521.Original Russian Text Copyright © 2005 by Chikov, Bakirova, Batasheva, Sergeeva.     

Perfusion of onion root xylem vessels: a method and some evidence of control of the pH of the xylem sap

We describe a method for perfusing the xylem in the stele of excised onion roots with solutions of known composition under a pressure gradient. Tracer studies using [¹⁴C] polyethylene glycol 4000 and the fluorescent dye, Tinopal CBSX, indicated that perfusing solutions passed exclusively through the xylem vessels. The conductance of the xylem was small over the apical 100 mm of the root axis but increased markedly between 100 and 200 mm. Unbuffered perfusion solutions supplied in the range pH 3.7–7.8 emerged after passage through the xylem adjusted to pH 5.2–6.0, indicating the presence of mechanisms for absorbing or releasing protons. This adjustment continued over many hours with net proton fluxes apparently determined by the disparity between the pH of the perfusion solution and the usual xylem sap pH of about 5.5. Mild acidification of the xylem sap by buffered perfusion solutions increased the release of ⁸⁶Rb (K⁺) and ³⁵SO₄ ^2- from the stelar tissue into the xylem stream. The ion-transporting properties of onion roots seemed little changed by excision from the bulbs, or by removal of the apical zones of the root axis. The pH of sap produced by root pressure resembles that found in the outflow solutions of perfused root segments.     

Measurement of xylem sap amino acid concentrations in conjunction with whole tree transpiration estimates spring N remobilization by cherry (Prunus avium L.) trees

Prunus avium trees were grown in sand culture for one vegetative season with contrasting N supplies, in order to precondition their N storage capacities. During the spring of the second year a constant amount of ¹⁵N was supplied to all the trees, and the recovery of unlabelled N in the new biomass production was used as a direct measure of N remobilization. Destructive harvests were taken during spring to determine the pattern of N remobilization and uptake. Measurements of both xylem sap amino acid profiles and whole tree transpiration rates were taken, to determine whether specific amino acids are translocated as a consequence of N remobilization and if remobilization can be quantified by calculating the flux of these amino acids in the xylem. Whereas remobilization started immediately after bud burst, N derived from uptake by root appeared in the leaves only 3 weeks later. The tree internal N status affected both the amount of N remobilization and its dynamics. The concentration of xylem sap amino acids peaked shortly after bud burst, concurrently with the period of fastest remobilization. Few amino acids and amides (Gln, Asn and Asp) were responsible for most of N translocated through the xylem; however, their relative concentration varied over spring, demonstrating that the transport of remobilized N occurred mainly with Gln whereas transport of N taken up from roots occurred mainly with Asn. Coupling measurements of amino acid N in the xylem sap with transpiration values was well correlated with the recovery of unlabelled N in the new biomass production. These results are discussed in relation to the possibility of measuring the spring remobilization of N in field‐grown trees by calculating the flux of N translocation in the xylem.     

Distribution of NO3− reduction between roots and shoots of peachtree seedlings as affected by NO3− uptake rate

The distribution of NO₃^? reduction between roots and shoots was studied in hydro-ponically-grown peach-tree seedlings (Prunus persica L.) during recovery from N starvation. Uptake, translocation and reduction of NO₃^?, together with transport through xylem and phloem of the newly reduced N were estimated, using ¹⁵N labellings, in intact plants supplied for 90 h with 0.5 mM NH₄⁺ and 0.5, 1.5 or 10 mM NO₃^?. Xylem transport of NO₃^? was further investigated by xylem sap analysis in a similar experiment. The roots were the main site of NO₃^? reduction at all 3 levels of NO₃^? nutrition. However, the contribution of the shoots to the whole plant NO₃^? reduction increased with increasing external NO₃^? availability. This contribution was estimated to be 20, 23 and 42% of the total assimilation at 0.5, 1.5 and 10 mM NO₃^?, respectively. Both ¹⁵N results and xylem sap analysis confirmed that this trend was due to an enhancement of NO₃^? translocation from roots to shoots. It is proposed that the lack of NO₃^? export to the shoots at low NO₃^? uptake rate resulted from a competition between NO₃^? reduction in the root epidermis/cortex and NO₃^? diffusion to the stele. On the other hand, net xylem transport of newly reduced N was very efficient since ca 70% of the amino acids synthesized in the roots were translocated to the shoots, regardless of the level of NO₃^? nutrition. This net xylem transport by far exceeded the net downward phloem transport of the reduced N assimilated in shoots. As a consequence, the reduced N resulting from NO₃^? assimilation, principally occurring in the roots, was mainly incorporated in the shoots.     

Form of aluminium for uptake and translocation in buckwheat (Fagopyrum esculentum Moench)

 The forms of Al for uptake by the roots and translocation from the root to the shoot were investigated in a buckwheat (Fagopyrum esculentum Moench, cv. Jianxi) that accumulates Al in its leaves. The Al concentration in the xylem sap was 15-fold higher in the plants exposed to AlCl₃ than in those exposed to an Al-oxalate (1:3) complex, suggesting that the roots take up Al in the ionic form. The Al concentration in the xylem sap was 4-fold higher than that in the external solution after a 1-h exposure to AlCl₃ solution and 10-fold higher after a 2-h exposure. The Al concentration in the xylem sap increased with increasing Al concentration in the external solution. The Al uptake was not affected by a respiratory inhibitor, hydroxylamine, but significantly inhibited by the addition of La. These results suggest that Al uptake by the root is a passive process, and La³⁺ competes for the binding sites for Al³⁺ on the plasma membrane. The form of Al in the xylem sap was identified by ²⁷Al-nuclear magnetic resonance analysis. The chemical shift of ²⁷Al in the xylem sap was around 10.9 ppm, which is consistent with that of the Al-citrate complex. Furthermore, the dominant organic acid in the xylem sap was citric acid, indicating that Al was translocated in the form of Al-citrate complex. Because Al is present as Al-oxalate (1:3) in the root, the present data show that ligand exchange from oxalate to citrate occurs before Al is released to xylem. Received: 10 December 1999 / Accepted: 3 February 2000     

Copper uptake and translocation in chicory (Cichorium intybus L. cv Grasslands Puna) and tomato (Lycopersicon esculentum Mill. cv Rondy) plants grown in NFT system. II. The role of nicotianamine and histidine in xylem sap copper transport

The effect of rooting media Cu concentration (0.05–20 mg Cu L^-1) on amino acid concentrations and copper speciation in the xylem sap of chicory and tomato plants was measured using 6 week old plants grown in a nutrient film technique system (NFT). Irrespective of the Cu concentration in the nutrient solutions, more than 99.68% and 99.74% of total Cu in tomato and chicory xylem sap was in a bound form. When exposed to high Cu concentrations in the rooting media, amino acid concentrations in the sap increased. Relative to other amino acids, the concentrations of glutamine (Gln), histidine (His), asparagine (Asn), valine (Val), nicotianamine (NA) and proline (Pro) in tomato xylem saps, and His, γ-aminobutyric acid (Gaba), glutamic acid (Glu), leucine (Leu), NA and phenylalanine (Phe) in chicory xylem saps showed the greatest increases. The data indicate that induced synthesis of some free amino acids as a specific and proportional response to Cu treatment. For a single complexation amino acid, the solution Cu²⁺concentration vs pH titration curve for NA at 0.06–0.07 mM was most similar, closely followed by His at 0.5–0.6 mM, to the solution Cu²⁺concentration behaviour in both tomato and chicory xylem sap. It is concluded that increased Cu concentrations in the rooting media induced selective synthesis of certain amino acid which include NA, His, Asn and Gln which have high stability constants with Cu. NA and His have the highest binding constants for Cu and the concentrations of NA and His in chicory and tomato xylem saps can account for all the bound Cu carried in the sap. This revised version was published online in June 2006 with corrections to the Cover Date.     

Analysis of essential leucine residue for catalytic activity of novel thermostable chitosanase by site-directed mutagenesis

Bacterial chitosanases share weak amino acid sequence similarities at certain regions of each enzyme. These regions have been assumed to be important for catalytic activities of the enzyme. To verify this assumption, the functional importance of the conserved region in a novel thermostable chitosanase (TCH-2) from Bacillus coagulans CK108 was investigated. Each of the conserved amino acid residues (Leu64, Glu80, Glu94, Asp98, and Gly108) was changed to aspartate and glutamine or asparagine and glutamate by site-directed mutagenesis, respectively. Kinetic parameters for colloidal chitosan hydrolysis were determined with wild-type and 10 mutant chitosanases. The Leu64 Arg and Leu64 Gln mutations were essentially inactive and kinetic parameters such as V _max and k _cat were approximately 1/10⁷ of those of the wild-type enzyme. The Asp98 Asn mutation did not affect the K _m value significantly, but decreased k _cat to 15% of that of wild-type chitosanase. On the other hand, the Asp98 srarr; Glu mutation affected neither K _m nor k _cat. The observation that approximately 15% of activity remained after the substitution of Asp98 by Asn indicated that the carboxyl side chain of Asp98 is not absolutely required for catalytic activity. These results indicate that the Leu64 residue is directly involved in the catalytic activity of TCH-2.     

Uptake of Amino Acids and Their Metabolic Conversion into the Compatible Solute Proline Confers Osmoprotection to Bacillus subtilis

The data presented here reveal a new facet of the physiological adjustment processes through which Bacillus subtilis can derive osmostress protection. We found that the import of proteogenic (Glu, Gln, Asp, Asn, and Arg) and of nonproteogenic (Orn and Cit) amino acids and their metabolic conversion into proline enhances growth under otherwise osmotically unfavorable conditions. Osmoprotection by amino acids depends on the functioning of the ProJ-ProA-ProH enzymes, but different entry points into this biosynthetic route are used by different amino acids to finally yield the compatible solute proline. Glu, Gln, Asp, and Asn are used to replenish the cellular pool of glutamate, the precursor for proline production, whereas Arg, Orn, and Cit are converted into γ-glutamic semialdehyde/Δ¹-pyrroline-5-carboxylate, an intermediate in proline biosynthesis. The import of Glu, Gln, Asp, Asn, Arg, Orn, and Cit did not lead to a further increase in the size of the proline pool that is already present in osmotically stressed cells. Hence, our data suggest that osmoprotection of B. subtilis by this group of amino acids rests on the savings in biosynthetic building blocks and energy that would otherwise have to be devoted either to the synthesis of the proline precursor glutamate or of proline itself. Since glutamate is the direct biosynthetic precursor for proline, we studied its uptake and found that GltT, an Na⁺-coupled symporter, is the main uptake system for both glutamate and aspartate in B. subtilis. Collectively, our data show how effectively B. subtilis can exploit environmental resources to derive osmotic-stress protection through physiological means.     

A proposed role for copper ions in cell wall loosening

The measurement of N₂ fixation by legumes is necessary for gaining an understanding of their contributions to the N economies of agricultural and forestry systems and for their management in those systems. We report research to determine whether N₂ fixation of four of the commonly-grown ureide-producing legumes, soybean (Glycine max), cowpea (Vigna unguiculata), mungbean (V. radiata) and black gram (V. mungo), could be quantified from a single sampling and N-solute analysis of xylem sap. Data were derived from a previously-published experiment involving six genotypes of soybean at five field sites and from a second, irrigated experiment in which two genotypes of soybean, and one each of cowpea, mungbean and black gram were assessed in low- and high-nitrate soils for nodulation, yields of shoot and grain dry matter and N, and N₂ fixation using xylem solute (ureide) and ¹⁵N methods. Regression analysis of the published soybean data set indicated that the early pod-fill (R3.5 and R4) samplings for xylem sap gave estimates of percentage of plant N derived from N₂ fixation (%Ndfa) which agreed well with %Ndfa for the entire growing season obtained from ¹⁵N analysis of the shoots at R6-7. There was a marginal benefit in combining the R3.5 and R4 samplings and using the average of the two, with regression coefficients (r ²) increasing from 0.86 (R3.5 or R4 alone) to 0.92 (average of R3.5+R4). There was no additional benefit in combining R3, R3.5 and R4. In the second experiment, agreement between ¹⁵N-determined %Ndfa and either measured (R4 sampling) or calculated ureide-determined %Ndfa (R3.5 sampling) was also good (r ² of 0.73 (R4) and 0.79 (R3.5)). We conclude that seasonal %Ndfa can be accurately estimated using the xylem solute (ureide) method from a single sampling of xylem sap during early pod-fill (R3.5) and that this simplification of the protocol of the technique may encourage expanded use.     

Pathways of Nitrogen Metabolism in Nodules of Alfalfa (Medicago sativa L.)

Exposure of intact alfalfa nodules to ¹⁵N₂ showed that in bacteroids the greatest flow of ¹⁵N was to NH₃. Label was also detected in glutamic acid, aspartic acid, and asparagine (Glu, Asp and Asn), but at far lower levels. In the host plant cytosols, more ¹⁵N was incorporated into Asn than into other compounds. Detached nodules were also used to study the metabolic pathway of N assimilation after exposure to ¹⁵N₂ or vacuum infiltration with (¹⁵NH₄)₂SO₄ in the presence or absence of different inhibitors of nitrogen assimilation: methionine sulfoximine (MSO), azaserine (AZA), or amino-oxyacetate (AOA). Treatment with MSO, an inhibitor of glutamine synthetase (GS), inhibited the flow of the label to glutamine (Gln)-amide, resulting in subsequently decreased label in Asnamide. Aza, which inhibits the formation of Glu from Gln by glutamate synthase (GOGAT), enhanced the labeling of the amide groups of both Gln and Asn, while that of Asn-amino decreased. When AOA was used to block the transamination reaction very little label was found in Asp and Asn-amino. The results are consistent with the role of GS/GOGAT in the cytosol for the assimilation of NH₃ produced by N₂ fixation in the bacteroids of alfalfa nodules. Asn, a major nitrogen transport compound in alfalfa, is mainly synthesized by a Gln-dependent amidation of Asp, according to feeding experiments using the ¹⁵N-labeled amide group of glutamine. Data from ¹⁵NH₄⁺ feeding support some direct amidation of Asp to form Asn.     

Electrophysiological factors in the influence of nitrate and ammonium ions on calcium uptake and translocation in tomato plants

Abstract Tomato plants (Lycopersicon esculentum Mill. cv. San Marzano), grown in dilute nutrient solutions containing (in meq ˙ 1^-1) 0.5 NaNO₃, 0.5 NH₄NO₃ or 0.25 (NH₄)₂ SO₄ as the nitrogen source, were detopped for collection of xylem sap and measurement of trans-root electrical potentials. The plant parts and the xylem exudate were subsequently analysed for mineral content. The commonly observed effects of NH₄⁺ were noted, including reduction of calcium concentration in the xylem sap, and of calcium content in stems and leaves, compared with NO₃^-fed plants. This effect was attributed principally to the less negative trans-root electrical potential measured in NH₄⁺-fed plants, and the resultant reduction of inward driving force on passively moving divalent cations.