Products of Dark CO(2) Fixation in Pea Root Nodules Support Bacteroid Metabolism

Products of the nodule cytosol in vivo dark [¹⁴C]CO₂ fixation were detected in the plant cytosol as well as in the bacteroids of pea (Pisum sativum L. cv “Bodil”) nodules. The distribution of the metabolites of the dark CO₂ fixation products was compared in effective (fix⁺) nodules infected by a wild-type Rhizobium leguminosarum (MNF 300), and ineffective (fix⁻) nodules of the R. leguminosarum mutant MNF 3080. The latter has a defect in the dicarboxylic acid transport system of the bacterial membrane. The ¹⁴C incorporation from [¹⁴C]CO₂ was about threefold greater in the wild-type nodules than in the mutant nodules. Similarly, in wild-type nodules the in vitro phosphoenolpyruvate carboxylase activity was substantially greater than that of the mutant. Almost 90% of the ¹⁴C label in the cytosol was found in organic acids in both symbioses. Malate comprised about half of the total cytosol organic acid content on a molar basis, and more than 70% of the cytosol radioactivity in the organic acid fraction was detected in malate in both symbioses. Most of the remaining ¹⁴C was contained in the amino acid fraction of the cytosol in both symbioses. More than 70% of the ¹⁴C label found in the amino acids of the cytosol was incorporated in aspartate, which on a molar basis comprised only about 1% of the total amino acid pool in the cytosol. The extensive ¹⁴C labeling of malate and aspartate from nodule dark [¹⁴C]CO₂ fixation is consistent with the role of phosphoenolpyruvate carboxlase in nodule dark CO₂ fixation. Bacteroids from the effective wild-type symbiosis accumulated sevenfold more ¹⁴C than did the dicarboxylic acid transport defective bacteroids. The bacteroids of the effective MNF 300 symbiosis contained the largest proportion of the incorporated ¹⁴C in the organic acids, whereas ineffective MNF 3080 bacteroids mainly contained ¹⁴C in the amino acid fraction. In both symbioses a larger proportion of the bacteroid ¹⁴C label was detected in malate and aspartate than their corresponding proportions of the organic acids and amino acids on a molar basis. The proportion of ¹⁴C label in succinate, 2-oxogultarate, citrate, and fumarate in the bacteroids of the wild type greatly exceeded that of the dicarboxylate uptake mutant. The results indicate a central role for nodule cytosol dark CO₂ fixation in the supply of the bacteroids with dicarboxylic acids.     

Labeling of Carbon Pools in Bradyrhizobium japonicum and Rhizobium leguminosarum bv viciae Bacteroids following Incubation of Intact Nodules with CO(2)

The aim of the work reported here was to ascertain that the patterns of labeling seen in isolated bacteroids also occurred in bacteroids in intact nodules and to observe early metabolic events following exposure of intact nodules to ¹⁴CO₂. Intact nodules of soybean (Glycine max L. Merr. cv Ripley) inoculated with Bradyrhizobium japonicum USDA 110 and pea (Pisum sativum L. cv Progress 9) inoculated with Rhizobium leguminosarum bv viciae isolate 128C53 were detached and immediately fed ¹⁴CO₂ for 1 to 6 min. Bacteroids were purified from these nodules in 5 to 7 min after the feeding period. In the cytosol from both soybean and pea nodules, malate had the highest radioactivity, followed by citrate and aspartate. In peas, asparagine labeling equaled that of aspartate. In B. japonicum bacteroids, malate was the most rapidly labeled compound, and the rate of glutamate labeling was 67% of the rate of malate labeling. Aspartate and alanine were the next most rapidly labeled compounds. R. leguminosarum bacteroids had very low amounts of ¹⁴C and, after a 1-min feeding, malate contained 90% of the radioactivity in the organic acid fraction. Only a trace of activity was found in aspartate, whereas the rate of glutamate and alanine labeling approached that of malate after 6 min of feeding. Under the conditions studied, malate was the major form of labeled carbon supplied to both types of bacteroids. These results with intact nodules confirm our earlier results with isolated bacteroids, which showed that a significant proportion of provided labeled substrate, such as malate, is diverted to glutamate. This supports the conclusion that microaerobic conditions in nodules influence carbon metabolism in bacteroids.     

Labelling of lupine nodule metabolites with 14CO2 assimilated from the leaves

On feeding ¹⁴CO₂ to the shoots of lupine (25 mCi per plant) 30 min was the minimal time needed to determine the incorporation of label into bacteroid compounds. The predominant incorporation, exhibited in all root, nodule and bacteroid samples after 30 min exposure, was into sucrose (45–90% of the corresponding fraction radioactivity) of the neutral fraction; into malate (30–40%) of the acid fraction; into aspartic acid and asparagine (60–80% in sum) of the basic fraction. The composition of carbon compounds containing the greatest amount of ¹⁴C in the cytosol of nodules and in bacteroids was similar. Their radioactivity after 30 min exposure was for bacteroids (nCi per g of bacteroid fr. wt): sucrose 5.73, glucose 1.00, malate 0.15, succinate 0.11; for the nodule cytosol (nCi per g of nodule fr. wt): sucrose 200.00, glucose 8.40, malate 9.34, succinate 8.50. Thus it was demonstrated that in lupine, sucrose is the main photoassimilate entering not only into nodules but also into bacteroids. The biosynthesis of aspartic acid and asparagine occurs during nitrogen fixation in bacteroids.     

Some Enzymes of Hydrogen Peroxide Metabolism in Leaves and Root Nodules of Medicago sativa

Leaves and nodules (bacteroids and cytosol) of alfalfa (Medicago sativa L. cv Aragon) plants inoculated with Rhizobium meliloti strain 102F51 have been analyzed for the presence of the enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (EC 1.11.1.6), and peroxidase (EC 1.11.1.7). All three fractions investigated (leaves, bacteroids, and nodular cytosol) show Cu,Zn-SOD activity. Besides, the bacteroids and cytosol of nodules possess CN⁻-insensitive SOD activities. Studies of SOD inactivation with H₂O₂ indicate that, very likely, a Mn-SOD is present in the bacteroids, and suggest that the cytosol contain both Mn-SOD and Fe-SOD. Bacteroids show high catalase activity but lack peroxidase. By contrast, the nodule cytosol exhibits an elevated peroxidase activity as compared with the foliar tissue; this activity was completely inhibited by 50 to 100 micromolar KCN. The significantly lower contents of H₂O₂ and malondialdehyde (a product of lipid peroxidation) in nodules with respect to those in leaves reveal that the above-mentioned bacteroid and cytosol enzymes act in an efficient and combined manner to preserve integrity of nodule cell membranes and to keep leghemoglobin active.     

Sugar metabolism and partitioning in cytosol and bacteroid fractions of chickpea nodules

The contents of free sugars in nodules of chickpea (Cicer arietinum) were maximum around flowering. In stem and root tissues, the relative incorporation of ¹⁴C from [¹⁴C]-labelled sucrose or glucose into extracted sucrose was over 70 %. In the former tissue, the relative incorporation of ¹⁴C from glutamate into sucrose was about 50 % at 50 d after sowing (DAS) but the same decreased to about 25 % at 80 DAS. However, from glutamate, 63–68 % of ¹⁴C from extracted sugars of root tissue appeared in invert sugars. Feeding via stem [¹⁴C]-glutamate to intact nodules led to intense labelling of sucrose and invert sugars in nodule cytosol. Upon injecting labelled sugars or glutamate into isolated nodules, maximum ¹⁴C appeared in glucose of this nodule fraction. In bacteroids, incorporation of ¹⁴C from glutamate was much higher in amino acids. In the cytosol of younger (50 DAS) nodules, sucrose was cleaved largely by soluble alkaline invertase (EC 3.2.1.26). However, sucrose cleavage in this fraction of older (80 DAS) nodules was catalysed by this enzyme as well as sucrose synthase (reversal, EC 2.4.1.13) and such nodules also contained higher activity of nitrogenase. The bacteroid fraction, which contained 10–17 % of nodule sugars, lacked the activities of sucrose-cleaving enzymes. The activities of ATP-dependent phosphofructokinase (EC 2.7.1.11), glyceraldehyde-3-phosphate dehydrogenase (EC 1.1.1.12), NADP⁺-dependent isocitrate dehydrogenase (EC 1.1.1.41) and malate dehydrogenase (EC 1.1.1.37) were higher in cytosol than bacteroids. However, the reverse was true for glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44). The results suggest that in chickpea nodules sugar metabolism occurs largely via the glycolytic pathway in cytosol and the pentose phosphate pathway in bacteroids and there is some transport of glutamate from cytosol to bacteroids.     

Enzymes of sucrose,maltose, and α,α-trehalose catabolism in soybean root nodules

Crude, Sephadex-filtered extracts of soybean (Glycine max (L.) Merr.) root nodules contained invertase (E.C. 3.2.1.26) activity with pH optima at 5.4 and 7.8, ,-trehalase (E.C. 3.2.1.28) activity with pH optima at 3.8 and 6.6, and maltase (E.C. 3.2.1.20) activity with a broad pH optimum between 4.5 and 5.0. Bacteroids and cytosol were separated using Percoll density gradients. Cellulase and pectinase were employed to separate protoplasts from the infected region from the nodule cortex, which remained intract. Assays of disaccharidases from these nodule fractions indicated the following localization of enzymes: (1) Bacteroids lack invertase activity (pH 5.4 and 7.8). (2) Much, if not most, of the invertase activity may be localized in the nodule cortex; this is especially likely for acid invertase. However, there was substantial invertase activity in cytosol from the infected region. (3) Most of the maltase activity (pH 5.0) and trehalase activity (pH 3.8 and 6.6) were localized in the cytosol. It is likely that most of these disaccharidase activities are in the cytosol of the infected region, in contrast to invertase. (4) Bacteroids contain maltase (pH 5.0) and trehalase (pH 3.8 and 6.6), but the amount of these enzyme activities was less than 15% of total activity in nodules. Bacteroids and nodule cortex were capable of in-vivo hydrolysis of [¹⁴C]trehalose and [¹⁴C]maltose. These disaccharides were also hydrolyzed by soybean roots and hypocotyls. Therefore, while ,-trehalose in soybean nodules is probably synthesized by the bacteroids, the capability for utilization of trehalose was not restricted to the bacteroids.Approved for publication as Journal Article 74–81 of the Ohio Agricultural Research and Development Center     

The Effect of Potassium on the Fixation of Molecular Nitrogen by Root Nodules of Vicia faba

The effect of potassium supply of Vicia faba on the fixation of molecular nitrogen by root nodules was studied by using ¹⁵N-labeled molecular nitrogen. Plants well supplied with potassium showed higher contents of ¹⁵N in the soluble amino fraction and in the protein fraction of various plant organs as compared with plants of a lower potassium status. This effect was evident particularly in the root nodules. Assimilation experiments, carried out with ¹⁴CO₂, revealed that the content of radioactivity in the sugars and amino acids of the root nodules was increased by the potassium supply of the host plants. In particular, the content of ¹⁴C amino acids in the root nodules was influenced beneficially by potassium, which means that potassium favored the provision of reduced nitrogen (NH₃). It is postulated that the better carbohydrate supply of nodules, by plants well supplied with potassium, results in a higher carbohydrate turnover in the nodules and thus the provision of ATP and reducing electrons required by the nitrogenase is enhanced.     

Uptake and Metabolism of Carbohydrates by Bradyrhizobium japonicum Bacteroids

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with ¹⁴C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O₂ (2% in the gas phase). Uptake and conversion of ¹⁴C to CO₂ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO₂, and fructose was respired at a rate at least double that of glucose. Sucrose and glucose were converted to CO₂ at a very low but measurable rate (<0.1 nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO₂ at a rate 30 times greater than the conversion of carbon Number 6 to CO₂, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.     

Properties of the hydrogenase system in Rhizobium japonicum bacteroids

The hydrogenase system which catalyzes the oxyhydrogen reaction in soybean nodules produced by strains of $\text{Rhizobium japonicum}$ is located in the bacteroids. The hydrogenase complex in intact bacteroids has an apparent K_m for H₂ of 2.8 μM and an apparent K_m for O₂ of 1.3 μM. The addition of hydrogen to bacteroids increases oxygen uptake but decreases respiratory CO₂ production, indicating a conservation of endogenous substrates. After correction for the effect of hydrogen on endogenous respiration a ratio of 1.9 ± 0.1 for H₂ to O₂ uptake was determined. Bacteroids from greenhouse or field-grown soybeans that evolved hydrogen showed no measurable oxyhydrogen reaction activity whereas consistent activity was demonstrated by bacteroids from soybean nodules that evolved little or no H₂.     

Effects of Salt Stress on Amino Acid, Organic Acid, and Carbohydrate Composition of Roots, Bacteroids, and Cytosol of Alfalfa (Medicago sativa L.)

Ethanol-soluble organic acid, carbohydrate, and amino acid constituents of alfalfa (Medicago sativa) roots and nodules (cytosol and bacteroids) have been identified by gas-liquid chromatography and high performance liquid chromatography. Among organic acids, citrate was the predominant compound in roots and cytosol, with malonate present in the highest concentration in bacteroids. These two organic acids together with malate and succinate accounted for more than 85% of the organic acid pool in nodules and for 97% in roots. The major carbohydrates in roots, nodule cytosol, and bacteroids were (descending order of concentration): sucrose, pinitol, glucose, and ononitol. Maltose and trehalose appeared to be present in very low concentrations. Asparagine, glutamate, alanine, γ-aminobutyrate, and proline were the major amino acids in cytosol and bacteroids. In addition to these solutes, serine and glutamine were well represented in roots. When alfalfa plants were subjected to 0.15 m sodium chloride stress for 2 weeks, total organic acid concentration in nodules and roots were depressed by more than 40%, whereas lactate concentration increased by 11, 27, and 94% in cytosol, roots, and bacteroids, respectively. In bacteroids, lactate became the most abundant organic acid and might contribute partly to the osmotic adjustment. On the other hand, salt stress induced a large increase in the amino acid and carbohydrate pools. Within the amino acids, proline showed the largest increase, 11.3-, 12.8-, and 8.0-fold in roots, cytosol, and bacteroids, respectively. Its accumulation reflected an osmoregulatory mechanism not only in roots but also in nodule tissue. In parallel, asparagine concentration was greatly enhanced; this amide remained the major nitrogen solute and, in bacteroids, played a significant role in osmoregulation. On the contrary, the salt treatment had a very limited effect on the concentration of other amino acids. Among carbohydrates, pinitol concentration was increased significantly, especially in cytosol and bacteroids (5.4- and 3.4-fold, respectively), in which this cyclitol accounted for more than 35% of the total carbohydrate pool; pinitol might contribute to the tolerance to salt stress. However, trehalose concentration remained low in both nodules and roots; its role in osmoregulation appeared unlikely in alfalfa.     

Fissazione di C14O2 in Colture di Tessuti Vegetali « in Vitro »

Abstract

C¹⁴O₂ fixation in plant tissues « in vitro ». — In the present work it has been examinated the autotrophic and heterotrophic CO₂ fixation of explants of « Helianthus tuberosus » « in vitro » and the photosyntetic efficiency of leaves produced from buds of « in vitro » explants of « Cichorium intybus » compared with that of mature leaves from normal plants of the same species. From our results it is evident that « in vitro » explants of « Helianthus tuberosus », grown, in the light, are able to autotrophically incorporate C¹⁴O₂; the distribution of the radioactivity into the various fractions shows a large influence of the light on the neutral fraction containing sugars (50% of the total radioactivity). In the chlorophyllous explants the dark CO₂ fixation is obviously of heterotrophic type: 97% of the total radioactivity is incorporated in amine acids (43%) and the organic acids (53%); on the other hand in the dark grown explants the radioactivity is differently distributed between amino acids (59%) and organic acids (39%). Mature leaves from normal plants and leaves produced from buds of « in vitro » explants of « Cichorium intybus » incorporate the same quantity of C¹⁴O₂ when expressed per mg of chlorophyll; the different distribution of the radioactivity in the neutral and acid fractions could be explained in terms of a different utilization pathway of the photosynthates in the two tissues.     

A comparative developmental pattern of enzymes of carbon metabolism and pentose phosphate pathway in mungbean and lentil nodules

The activities of enzymes of pentose phosphate pathway (PPP) viz. glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and carbon metabolism viz. phosphoenol pyruvate carboxylase, NADP- isocitrate dehydrogenase and NADP-malic enzyme were measured in the plant and bacteroid fractions of mungbean (ureide exporter) and lentil (amide exporter) nodules along with the developing roots for comparison. The enzymes of pentose phosphate pathway in legume cytosol had higher activities at a stage of maximum nitrogenase activity and higher sucrose metabolism. However, bacteroids had only limited capacity for this pathway. The specific activities of these enzymes were greater in ureide than in amide exporter. CO₂ fixation via higher activity of phosphoenolpyruvate carboxylase in the plant part of the nodules in lentil might have been due to the greater synthesis of four carbon amino acids for amide export. The peak of NADP-isocitrate dehydrogenase in both legumes coincided with the pentose phosphate pathway enzymes at the time of high rates of sucrose metabolism and nitrogen fixation. Higher activities of NADP-malic enzyme were obtained in mungbean than in the lentil nodules. These findings are consistent with the role of these enzymes in providing reductant (NADPH) and substrates for energy yielding metabolism of bacteroids and carbon skeletons for ammonia assimilation.     

Transport and Partitioning of CO(2) Fixed by Root Nodules of Ureide and Amide Producing Legumes

Nodulated and denodulated roots of adzuki bean (Vigna angularis), soybean (Glycine max), and alfalfa (Medicago sativa) were exposed to ¹⁴CO₂ to investigate the contribution of nodule CO₂ fixation to assimilation and transport of fixed nitrogen. The distribution of radioactivity in xylem sap and partitioning of carbon fixed by nodules to the whole plant were measured. Radioactivity in the xylem sap of nodulated soybean and adzuki bean was located primarily (70 to 87%) in the acid fraction while the basic (amino acid) fraction contained 10 to 22%. In contrast, radioactivity in the xylem sap of nodulated alfalfa was primarily in amino acids with about 20% in organic acids. Total ureide concentration was 8.1, 4.7, and 0.0 micromoles per milliliter xylem sap for soybean, adzuki bean, and alfalfa, respectively. While the major nitrogen transport products in soybeans and adzuki beans are ureides, this class of metabolites contained less than 20% of the total radioactivity. When nodules of plants were removed, radioactivity in xylem sap decreased by 90% or more. Pulse-chase experiments indicated that CO₂ fixed by nodules was rapidly transported to shoots and incorporated into acid stable constituents. The data are consistent with a role for nodule CO₂ fixation providing carbon for the assimilation and transport of fixed nitrogen in amide-based legumes. In contrast, CO₂ fixation by nodules of ureide transporting legumes appears to contribute little to assimilation and transport of fixed nitrogen.     

Metabolism of [13C]-labelled photosynthate in plant cytosol and bacteroids of root nodules of Glycine max

Well-nodulated soybean ( Glycine max L. Merr. cv. Akisengoku) plants were allowed to assimilate ¹³CO₂. Plant cytosol and bacteroid fractions were isolated from nodules, and the kinetics of [¹³C]-labelling of soluble carbohydrates, organic acids and amino acids were investigated.
The concentrations of all metabolites, with the exception of trehalose and 3-hydroxy-butyrate, were 10- to 1000-fold higher in plant cell cytosol than in bacteroids. The major portion of trehalose was found in bacteroids and 3-hydroxybutyrate only in bacteroids. Sucrose was most highly labelled with ¹³C in nodules, and the levels and time-course of labelling of sucrose were in good agreement with those of respired CO₂ from the nodules. The levels and time-courses of labelling of sucrose were closely similar in cytosol and bacteroids. Glucose was less labelled than sucrose and the level of labelling was consistently higher in cytosol than in bacteroids. The levels of [¹³C]-labelling of organic acids and amino acids in nodules were lower than those of sucrose and of respired CO₂. Tricarboxylic acid cycle intermediates, particularly succinate, were considerably less labelled in bacteroids than in the cytosol. All amino acids detected were also much more rapidly labelled in the cytosol. The results are discussed in relation to the utilization and possible compartmentation of carbon substrates in nodule tissues.     

Partitioning of C-photosynthate, and long distance translocation of amino acids in preflowering and flowering, nodulated and nonnodulated soybeans

The influence of stage of development (preflowering versus flowering) in nodulated and nonnodulated soybeans (Glycine max [L.] Merr. cv. Wells) on partitioning of ¹⁴C into assimilates following exposure of a soybean leaf to ¹⁴CO₂ by both steady-state and pulse-labeling techniques was studied. Blades on the second fully expanded leaf from the stem apex were exposed to ¹⁴CO₂. Radioactive assimilates were extracted from source leaf blades, petioles, and stems (both the path up and path down from source leaf), were separated into neutral (sugars), basic (amino acids), and acidic (organic acids, sugar phosphates) fractions by ion exchange chromatography. The basic fraction was further resolved using thin layer chromatography and the percentage of radioactivity recovered in each amino acid was determined.     

Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study

The effects of dark-induced stress on the evolution of the soluble metabolites present in senescent soybean (Glycine max L.) nodules were analysed in vitro using ¹³C- and ³¹P-NMR spectroscopy. Sucrose and trehalose were the predominant soluble storage carbons. During dark-induced stress, a decline in sugars and some key glycolytic metabolites was observed. Whereas 84% of the sucrose disappeared, only one-half of the trehalose was utilised. This decline coincides with the depletion of Gln, Asn, Ala and with an accumulation of ureides, which reflect a huge reduction of the N₂ fixation. Concomitantly, phosphodiesters and compounds like P-choline, a good marker of membrane phospholipids hydrolysis and cell autophagy, accumulated in the nodules. An autophagic process was confirmed by the decrease in cell fatty acid content. In addition, a slight increase in unsaturated fatty acids (oleic and linoleic acids) was observed, probably as a response to peroxidation reactions. Electron microscopy analysis revealed that, despite membranes dismantling, most of the bacteroids seem to be structurally intact. Taken together, our results show that the carbohydrate starvation induced in soybean by dark stress triggers a profound metabolic and structural rearrangement in the infected cells of soybean nodule which is representative of symbiotic cessation.     

Effects of Oxygen Concentration and Leghemoglobin on Organic Acid Degradation by Isolated Soybean Nodule Bacteriods

Bacteroids retaining high acetylene reduction activity (nitrogenase activity) were prepared anaerobically from soybean nodules. Addition of succinate (or of both leghemoglobin and succinate) to the acetylene reduction assay system greatly increased the activity of the isolated bacteroids.

When various organic acids were incubated with the bacteroids at 2% oxygen concentration, an optimum condition for bacteroid acetylene reduction, the organic acid degradation by bacteroids was very slow, and both lactate and acetate were accumulated in the incubation system, suggesting the operation of fermentative pathway in bacteroids under such low oxygen conditions.

With 20% oxygen, the added organic acids were degraded rapidly by bacteroids without addition of leghemoglobin to the incubation system.

With leghemoglobin in the incubation system, the organic acid degradation by bacteroids was accelerated extensively even at 2% oxygen, and the formation of lactate and acetate were negligible. No significant difference in the organic acid degradation rate was observed between the 2% and 20% oxygen concentrations when the leghemoglobin was present in the incubation system. Addition of acetylene to the assay system slightly inhibited the organic acid degradation.

This data suggests that bacteroids are unable to oxidize organic acid in low oxygen concentration and that the leghemoglobin allows the rapid organic acid dagradation by bacteroids even in such low oxygen concentrations.     

Partitioning of 14C-labeled photosynthate to allelochemicals and primary metabolites in source and sink leaves of aspen: evidence for secondary metabolite turnover

Theories on allelochemical concentrations in plants are often based upon the relative carbon costs and benefits of multiple metabolic fractions. Tests of these theories often rely on measuring metabolite concentrations, but frequently overlook priorities in carbon partitioning. We conducted a pulse-labeling experiment to follow the partitioning of ¹⁴CO₂-labeled photosynthate into ten metabolic pools representing growth and maintenance (amino acids, organic acids, lipids plus pigments, protein, residue), defense (phenolic glycosides, methanol:water and acetone-soluble tannins/phenolics), and transport and storage (sugars and starch) in source and importing sink leaves of quaking aspen (Populus tremuloides). The peak period of ¹⁴C incorporation into sink leaves occurred at 24 h. Within 48 h of labeling, the specific radioactivity (dpm/mg dry leaf weight) of phenolic glycosides declined by over one-third in source and sink leaves. In addition, the specific radioactivity in the tannin/phenolic fraction decreased by 53% and 28% in source and sink leaves, respectively. On a percent recovery basis, sink leaves partitioned 1.7 times as much labeled photosynthate into phenolic glycosides as source leaves at peak ¹⁴C incorporation. In contrast, source leaves partitioned 1.8 times as much ¹⁴C-labeled photosynthate into tannins/phenolics as importing sink leaves. At the end of the 7-day chase period, sink leaves retained 18%, 52%, and 30% of imported ¹⁴C photosynthate, and labeled source leaves retained 15%, 66%, and 19% of in situ photosynthate in metabolic fractions representing transport and storage, growth and maintenance, and defense, respectively. Analyses of the phenolic fractions showed that total phenolics were twice as great and condensed tannins were 1.7 times greater in sink than in source leaves. The concentration of total phenolics and condensed tannins did not change in source and sink leaves during the 7-day chase period. Received: 31 July 1998 / Accepted: 8 February 1999     

Nonphotosynthetic CO(2) Fixation by Alfalfa (Medicago sativa L.) Roots and Nodules

The dependence of alfalfa (Medicago sativa L.) root and nodule nonphotosynthetic CO₂ fixation on the supply of currently produced photosynthate and nodule nitrogenase activity was examined at various times after phloem-girdling and exposure of nodules to Ar:O₂. Phloemgirdling was effected 20 hours and exposure to Ar:O₂ was effected 2 to 3 hours before initiation of experiments. Nodule and root CO₂ fixation rates of phloem-girdled plants were reduced to 38 and 50%, respectively, of those of control plants. Exposure to Ar:O₂ decreased nodule CO₂ fixation rates to 45%, respiration rates to 55%, and nitrogenase activities to 51% of those of the controls. The products of nodule CO₂ fixation were exported through the xylem to the shoot mainly as amino acids within 30 to 60 minutes after exposure to ¹⁴CO₂. In contrast to nodules, roots exported very little radioactivity, and most of the ¹⁴C was exported as organic acids. The nonphotosynthetic CO₂ fixation rate of roots and nodules averaged 26% of the gross respiration rate, i.e. the sum of net respiration and nonphotosynthetic CO₂ assimilation. Nodules fixed CO₂ at a rate 5.6 times that of roots, but since nodules comprised a small portion of root system mass, roots accounted for 76% of the nodulated root system CO₂ fixation. The results of this study showed that exposure of nodules to Ar:O₂ reduced nodule-specific respiration and nitrogenase activity by similar amounts, and that phloem-girdling significantly reduced nodule CO₂ fixation, nitrogenase activity, nodule-specific respiration, and transport of ¹⁴C photoassimilate to nodules. These results indicate that nodule CO₂ fixation in alfalfa is associated with N assimilation.     

Allantoin and Allantoic Acid in the Nitrogen Economy of the Cowpea (Vigna unguiculata [L.] Walp.)

The ureides, allantoin and allantoic acid, represented major fractions of the soluble nitrogen pool of nodulated plants of cowpea (Vigna unguiculata [L.] Walp. cv. Caloona) throughout vegetative and reproductive growth. Stem and petioles were the principal sites of ureide accumulation, especially in early fruiting.

Labeling studies using ¹⁴CO₂ and ¹⁵N₂ and incubation periods of 25 to 245 minutes indicated that synthesis of allantoin and allantoic acid in root nodules involved currently delivered photosynthate and recently fixed N, and that the ureides were exported from nodule to shoot via the xylem. From 60 to 80% of xylem-borne N consisted of ureides; the remainder was glutamine, asparagine, and amino acids. Allantoin predominated in the soluble N fraction of nodules and fruits, allantoin and allantoic acid were present in approximately equal proportions in xylem exudate, stems, and petioles.

Extracts of the plant tissue fraction of nitrogen-fixing cowpea nodules contained glutamate synthase (EC 2.6.1.53) and glutamine synthetase (EC 6.3.1.2), but little activity of glutamate dehydrogenase (EC 1.4.1.3). High levels of uricase (EC 1.7.3.3) and allantoinase (EC 3.5.2.5) were also detected. Allantoinase but little uricase was found in extracts of leaflets, pods, and seeds.

Balance sheets were constructed for production, storage, and utilization of ureide N during growth. Virtually all (average 92%) of the ureides exported from roots was metabolized on entering the shoot, the compounds being presumably used as N sources for protein synthesis.