A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean

Application of allopurinol (AP; 1H-pyrazolo-[3,5- d ]pyrimidine-4-o1) to intact nodulated roots of ureide-forming legumes causes rapid inhibition of NAD:xanthine dehydrogenase (XDH: EC 1.2.1.37), cessation of ureide synthesis and, subsequently, severe nitrogen deficiency (Atkins et al. 1988. Plant Physiology 88: 1229–1234). Nitrogen deficiency is a result of inhibited nitrogenase (EC 1.7.99.2) activity. Using an open gas exchange system to measure H₂ and CO₂ evolution, short term effects of AP application were examined in a Hup ⁻ soybean symbiosis [ Glycine max (L.) Merr. cv. Harosoy: USDA 16]. The onset of inhibition of nitrogenase was detected after ca 2 h exposure of the roots to AP. At the same time xanthine began to accumulate and ureide levels declined in nodules as a result of inhibition of XDH. The decline in H₂ evolution following AP application was not due to altered electron allocation between N₂ and H⁺ by nitrogenease but was coincident with increased gaseous diffusive resistance of nodules and a decline in intracellular oxygen concentration. A possible scheme for the intermediary metabolism of soybean nodules which might account for a direct connection between nitrogenase activity and ureide synthesis is proposed. The suggested mechanism envisages coupling production of reducing power by cytosolic enzymes of purine oxidation to synthesis of dicarboxylic acid substrates (malate and succinate) required for bacteroid respiration.     

Spittlebug indicators of nitrogen-fixing plants

Abstract.

• 1 Spittlebugs (Homoptera: Cercopoidea) are preferentially associated with nitrogen-fixing angiosperms. Generally, the Aphrophoridae associate with legumes, the Clastopteridae with actinorhizal plants, and the Cercopidae with associative nitrogen-fixing grasses. This is the first reported insect—host association specific to nitrogen-fixing plants.
• 2 It is probable that spittlebugs are attracted to nitrogen-fixing plants because these hosts provide a relatively rich, reliable source of organic nitrogen compounds for xylem-sucking insects.
• 3 Many spittlebugs live on plants that do not fix nitrogen, and ecological, mechanical or chemical factors may bar some spittlebugs from otherwise suitable nitrogen-fixing plants.
• 4 Among nitrogen-fixing hosts, spittlebugs appear to prefer plants that transport fixed nitrogen as amino acids and amides to plants that transport fixed nitrogen as ureides.

     

Allantoinase and asparaginase activities in maturing fruits of nodulated and non -nodulated soybeans

Immature fruits of soybean ( Glycine max L. Merr. cv. Santa Rosa) were found to contain high ureide/amino acid ratios for plants dependent on atmospheric nitrogen (nodulated), but low ratios for plants cultivated on NO⁻₃ (non-nodulated). The pod tissue was responsible for almost all this difference, which reflects the N metabolism of these plants (nodulated:urcide-based; NO⁻₃ dependent: asparagine based). The capacity of fruit tissues to utilize ureides and asparagine via allantoinase (EC 3.5.2.5) and asparaginase (EC 3.5.1.1) was investigated during fruit development. Both enzymes were present in crude desalted extracts of all parts of the fruit analysed (pod, cotyledon and seed coat). Asparaginase was detected in pod tissue only at early stages and with very low activities, whereas high activities of allantoinase (up to 20 [imol pod⁻¹ h⁻¹) were present after this organ reached full expansion. The cotyledons contained most of the allantoinase and asparaginase activities of the seed, the highest activities being recorded during the period of rapid protein accumulation. There was little difference in the activity patterns for nodulated and NO⁻₃-grown plants, despite the large difference in nitrogen nutrition of the fruits.     

Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non‐leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1^OX) under several N regimes. Under normal field conditions, panicles from OsUPS1^OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N‐starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1^OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low‐N soil supplemented with different N‐concentrations, OsUPS1^OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole‐body‐overexpressing plants driven by the GOS2 promoter (OsUPS1^GOS2) as well as silencing plants (OsUPS1^RNAi). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1^GOS2 while in OsUPS1^RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.     

Allantoinase from shoot tissues of soybeans

Allantoinase, catalysing the hydrolysis of allantoin to allantoic acid, was isolated from leaves and fruits of soybeans. The enzyme was only partially     

Increased C availability at elevated carbon dioxide concentration improves N assimilation in a legume

Plant growth is typically stimulated at elevated carbon dioxide concentration ([CO2]), but a sustained and maximal stimulation of growth requires acquisition of additional N in proportion to the additional C fixed at elevated [CO2]. We hypothesized that legumes would be able to avoid N limitation at elevated [CO2]. Soybean was grown without N fertilizer from germination to final senescence at elevated [CO2] over two growing seasons under fully open-air conditions, providing a model legume system. Measurements of photosynthesis and foliar carbohydrate content showed that plants growing at elevated [CO2] had a c. 25% increase in the daily integral of photosynthesis and c. 58% increase in foliar carbohydrate content, suggesting that plants at elevated [CO2] had a surplus of photosynthate. Soybeans had a low leaf N content at the beginning of the season, which was a further c. 17% lower at elevated [CO2]. In the middle of the season, ureide, total amino acid and N content increased markedly, and the effect of elevated [CO2] on leaf N content disappeared. Analysis of individual amino acid levels supported the conclusion that plants at elevated [CO2] overcame an early-season N limitation. These soybean plants showed a c. 16% increase in dry mass at final harvest and showed no significant effect of elevated [CO2] on leaf N, protein or total amino acid content in the latter part of the season. One possible explanation for these findings is that N fixation had increased, and that these plants had acclimated to the increased N demand at elevated [CO2].     

Plant and insect characteristics in response to increasing density of Homalodisca coagulata on three host species: a quantification of assimilate extraction

Three experiments were performed to assess the effect of the number of the leafhopper Homalodisca coagulata (Say) (Homoptera: Auchenorrhyncha: Cicadellidae) on the plant characteristics of Lagerstroemia indica L. (Lythraceae), Prunus salicina Lindl. (Rosaceae) and Glycine max (L.) Merrill (Leguminoseae) and on subsequent consumption rates and fecundity. Leafhoppers were placed in sleeve cages (L. indica or P. salicina) or on caged plants (G. max) for 2–3 weeks at densities of 0–16 per cage. Consumption rates and fecundity were not influenced by leafhopper treatments. Shoot growth and shoot xylem tension were examined on L. indica and G. max, and were not influenced by treatment. For G. max, plant height increased with leafhopper days, however plant dry weight was unaffected. The concentrations of primary organic compounds in xylem fluid were evaluated at the termination of experiments on L. indica (amino acids and organic acids) and G. max (amino acids, ureides, organic acids, and sugars). For L. indica, there was no significant density effect on the concentration of total amino acids or total organic acids. For the smaller legume G. max, concentrations of five primary organic compounds in xylem fluid were moderately reduced (between P = 0.05 and 0.1) with leafhopper days. Consumption rates were inversely related (between P = 0.05 and 0.1) to the concentrations of three organic compounds in xylem fluid. The total quantity of each chemical compound, organic carbon, organic nitrogen, and energy extracted by H. coagulata feeding on G. max was estimated by incorporating consumption rates, leafhopper days, diet composition, and the heat of combustion of each compound quantified. The quantity of water and nutrients (and particularly organic N) depleted by leafhopper feeding at high densities was substantial. Individual H. coagulata extracted 3.9 cm³ water, 57 µmol organic carbon, 21 µmol organic nitrogen, and 2.7 J of chemical bond energy during a day of feeding. Moderate to large sized plants (P. salicina and L. indica) did not exhibit effects of leafhopper feeding, however high amounts of feeding on G. max increased plant height and moderately reduced the concentrations of several compounds in xylem fluid.     

Evidence that carbon dioxide enrichment alleviates ureide-induced decline of nodule nitrogenase activity

The hypothesis that elevated [CO(2)] alleviates ureide inhibition of N(2)-fixation was tested. Short-term responses of the acetylene reduction assay (ARA), ureide accumulation and total non-structural carbohydrate (TNC) levels were measured following addition of ureide to the nutrient solution of hydroponically grown soybean. The plants were exposed to ambient (360 micromol mol(-1)) or elevated (700 micromol mol(-1)) [CO(2)]. Addition of 5 and 10 mM ureide to the nutrient solution inhibited N(2)-fixation activity under both ambient and elevated [CO(2)] conditions. However, the percentage inhibition following ureide treatment was significantly greater under ambient [CO(2)] as compared with that under elevated [CO(2)]. Under ambient [CO(2)] conditions, ARA was less than that under elevated [CO(2)] 1 d after ureide treatment. Under ambient [CO(2)], the application of ureide resulted in a significant accumulation of ureide in all plant tissues, with the highest concentration increases in the leaves. However, application of exogenous ureide to plants subjected to elevated [CO(2)] did not result in increased ureide concentration in any tissues. TNC concentrations were consistently higher under elevated [CO(2)] compared with those under ambient [CO(2)]. For both [CO(2)] treatments, the application of ureide induced a significant decrease of TNC concentrations in the leaves and nodules. For both leaves and nodules, a negative correlation was observed between TNC and ureide levels. Results indicate that product(s) of ureide catabolism rather than tissue ureide concentration itself are critical in the regulation of N(2)-fixation.     

Effects of applied nitrogen upon aspects of nitrogen metabolism and transport in developing nodulated winged bean plants

Nodulated winged bean [Psophocarpus tetragonolobus (L.) DC., cv. UPS 122] were grown under constant environmental conditions and supplied with mineral nutrient solution in which nitrogen was absent or was present as nitrate (12 mg N week^-1 plant^-1). Nitrate treatment dramatically promoted plant growth, increased fruit weight 1.6 fold, was necessary for tuberisation and enhanced nodulation. The in vitro accumulation of ¹⁴C into asparagine and aspartate components of excised nodules supplied with exogenous ¹⁴CO₂ and [¹⁴C]-D-glucose was greater for nitrate-treated plants, whilst accumulation into ureides was reduced by nitrate treatment. Levels of amino acids in xylem sap were greater for plants supplied with a complete nutrient solution, than those grown without applied nitrate, particularly for asparagine, glutamine and proline. Xylem ureide levels were greater for plants grown in the absence of supplementary nitrate. Nitrogen accumulated in leaf, stem and petiole, and root nodule tissues for utilisation during fruit development; peak nitrogen levels and time of anthesis were retarded for plants grown without applied nitrate. The shoot ureide content increased during fruiting, coincident with decreases in the total nitrogen content, indicating that ureide pools are not utilised during the early reproductive phase. However ureide reserves, particularly allantoin, were utilised during the later stages of pod fill. Enzyme activity which metabolised asparagine was found throughout the plant and was identified as K⁺-dependent asparaginase (EC 3.5.1.1) and an aminotransferase. Apart from temporal differences in developmental profiles of enzyme activity, the activity of these enzymes and of allantoinase (EC 3.5.2.5) in developing tissues were similar for both treatments. The main differences were greater asparaginase and asparagine:pyruvate aminotransferase activities in root tissues and fruit of nitrate-supplied plants; allantoinase activity in the primary roots of plants grown without nitrate decreased during development, whilst activity in developing tubers (nitrate-supplied plants) increased.