NMR analysis of plant nitrogen metabolism

The analysis of primary and secondary nitrogen metabolism in plants by nuclear magnetic resonance (NMR) spectroscopy is comprehensively reviewed. NMR is a versatile analytical tool, and the combined use of ¹H, ²H, ¹³C, ¹⁴N and ¹⁵N NMR allows detailed investigation of the acquisition, assimilation and metabolism of nitrogen. The analysis of tissue extracts can be complemented by the in vivo NMR analysis of functioning tissues and cell suspensions, and by the application of solid state NMR techniques. Moreover stable isotope labelling with ²H-, ¹³C- and ¹⁵N-labelled precursors provides direct insight into specific pathways, with the option of both time-course and steady state analysis increasing the potential value of the approach. The scope of the NMR method, and its contribution to studies of plant nitrogen metabolism, are illustrated with a wide range of examples. These include studies of the GS/GOGAT pathway of ammonium assimilation, investigations of the metabolism of glutamate, glycine and other amino acids, and applications to tropane alkaloid metabolism. The continuing development of the NMR technique, together with potential applications in the emerging fields of metabolomics and metabolic flux analysis, leads to the conclusion that NMR will play an increasingly valuable role in the analysis of plant nitrogen metabolism.     

Isolation,Purification, and Characterization of Nitrate Reductase from a Salt-Tolerant <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> Yeast Strain Grown in the Presence of Tungsten

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.     

Responses of Vigna radiata to Foliar Application of 28-Homobrassinolide and Kinetin

Effects of 28-homobrassinolide (HBR) and kinetin (KIN) on photosynthesis, nitrogen metabolism, and the seed yield were studied. The leaves of 25-d-old plants of Vigna radiata (L.) Wilczek were sprayed with 0.01, 1.0 or 100 M aqueous solution of KIN, or 0.0001, 0.01 or 1.0 M that of HBR. KIN and especially HBR increased the activities of nitrate reductase and carbonic anhydrase, chlorophyll and total protein contents and net photosynthetic rate in the leaves, and pod number and seed yield, at harvest.     

A comparison of the effects of carbon dioxide concentration and temperature on respiration, translocation and nitrate reduction in darkened soybean leaves

BACKGROUND AND AIMS: Respiration of autotrophs is an important component of their carbon balance as well as the global carbon dioxide budget. How autotrophic respiration may respond to increasing carbon dioxide concentrations, [CO(2)], in the atmosphere remains uncertain. The existence of short-term responses of respiration rates of plant leaves to [CO(2)] is controversial. Short-term responses of respiration to temperature are not disputed. This work compared responses of dark respiration and two processes dependent on the energy and reductant supplied by dark respiration, translocation and nitrate reduction, to changes in [CO(2)] and temperature. METHODS: Mature soybean leaves were exposed for a single 8-h dark period to one of five combinations of air temperature and [CO(2)], and rates of respiration, translocation and nitrate reduction were determined for each treatment. KEY RESULTS: Low temperature and elevated [CO(2)] reduced rates of respiration, translocation and nitrate reduction, while increased temperature and low [CO(2)] increased rates of all three processes. A given change in the rate of respiration was accompanied by the same change in the rate of translocation or nitrate reduction, regardless of whether the altered respiration was caused by a change in temperature or by a change in [CO(2)]. CONCLUSIONS: These results make it highly unlikely that the observed responses of respiration rate to [CO(2)] were artefacts due to errors in the measurement of carbon dioxide exchange rates in this case, and indicate that elevated [CO(2)] at night can affect translocation and nitrate reduction through its effect on respiration.     

Anion uptake in maize roots: Interactions between chlorate and nitrate

Effects of nitrate, chloride and chlorate ions upon nitrate and chlorate uptake by roots of maize ( Zea mays L., cv. B73) seedlings were examined. Net nitrate uptake, ³⁶ClO₃⁻ influx and ³⁶Cl⁻ influx (the latter two in a background of 0.5 m M KNO₃) displayed similar pH profiles with optima at pH 5.5 and below. External, non-labeled chloride had little effect on the accumulation of ³⁶ClO₃⁻ (both in 5 h and 20 min uptake assays), while nitrate and chlorate had almost identical, marked inhibitory effects. Nitrate pretreatment caused an apparent induction of both ³⁶ClO₃⁻ and ¹⁵NO₃⁻ uptake activities. After 5 h of treatment in nitrate, the uptake activities of chloride- and chlorate-pretreated plants increased to that of nitrate-pretreated plants. During 6 h exposure to chlorate, ³⁶ClO₃⁻ uptake activity of nitrate-pretreated plants decreased to that of chlorate- and chloride-pretreated plants. The results support the existence of a shared nitrate/chlorate transport system in maize roots which is not inhibited by external chloride, and which is induced by nitrate, but not by chlorate or chloride. The suggestion is made that selection of chlorate-resistant mutants of maize can identify nitrate uptake as well as nitrate reductase mutants.     

Influence of nitrogen and sulphur form on manganese acquisition by barley (<Emphasis Type="Italic">shape Hordeum vulgare</Emphasis>)

The influence of various nitrogen (N) and sulphur (S) forms on the uptake of manganese (Mn) in young spring barley (Hordeum vulgare L cv Golf) plants was examined in both a hydroponic system and in a soil-based system. The soil was a typical Danish Mn-deficient soil viz. a sandy loam soil developed on old marine sediments. Plants growing in solution culture with NO₃^– as the only N source had a higher Mn uptake than plants receiving mixtures of NO₃^– and NH₄⁺. These findings were opposite to the results obtained in the soil-based experiments, where plants fertilized with NO₃^– as the only N source accumulated much less Mn than plants fertilized with NH₄⁺. Combining the results of these experiments confirmed that NH₄⁺ acted as a powerful antagonist to Mn²⁺ during uptake but that this antagonistic effect was more than compensated for by the influence of NH₄⁺ in reducing plant-unavailable Mn(IV) to plant-available Mn(II) in the soil. Furthermore the soil experiments showed that fertilizers containing sulphur in the form of reduced S (thiosulphate) had a strong mobilizing effect on Mn, and enabled the plants to accumulate large amounts of Mn in the biomass compared with oxidized S (sulphate). Thus, fertilization with thiosulphate may be very effective in alleviating Mn-deficiency in soils developed on old marine sediments where Mn availability is limiting plant growth.     

Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase

The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.     

Soil nitrogen dynamics in switchgrass seeded to a marginal cropland in South Dakota

The potential ecological impacts of switchgrass (Panicum virgatum L.), as a biofuel feedstock, have been assessed under different environmental conditions. However, limited information is available in understanding the integrated analysis of nitrogen (N) dynamics including soil nitrate (), nitrous oxide (N₂O) emissions, and leaching under switchgrass land management. The specific objective was to explore N dynamics for 2009 through 2015 in switchgrass seeded to a marginally yielding cropland based on treatments of N fertilization rate (N rate; low, 0; medium, 56; high, 112 kg N ha^?1) and landscape position (shoulder, backslope, and footslope). Our findings indicated that N rate impacted soil (0–5 cm depth) and surface N₂O fluxes but did not impact leaching during the observed years. Medium N (56 kg N ha^?1) was the optimal rate for increasing biomass yield with reduced environmental problems. Landscape position impacted the N dynamics. At the footslope position, soil , soil leaching, and N₂O fluxes were higher than the other landscape positions. Soil N₂O fluxes and leaching had downward trends over the observed years. Growing switchgrass on marginally yielding croplands can store soil N, reduce N losses via leaching, and mitigate N₂O emissions from soils to the atmosphere over the years. Switchgrass seeded on marginally yielding croplands can be beneficial in reducing N losses and can be grown as a sustainable bioenergy crop on these marginal lands.