Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

Genetic tools for selective labeling of proteins with α-15N-amino acids

Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any ¹⁵N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations ¹⁵N-amino acid -¹⁵N-amino acid - Cam^R chloramphenicol-resistant - DPA diaminopimelic acid - Hfr high-frequency recombinant - LB Luria broth - Kan^R kanamycin resistant - P1 bacteriophage P1 - pfu plaque-forming units - Str^R streptomycin-resistant - Tet^R tetracycline-resistant     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

15β-hydroxysteroids (Part IV). Steroids of the human perinatal period: The synthesis of 3α,15β,17α-trihydroxy-5α-pregnan-20-one and its A/B-ring configurational isomers

In recent years several 15β-hydroxysteroids have emerged pathognomonic of adrenal disorders in human neonates of which 3α,15β,17α-trihydroxy-5β-pregnan-20-one (2) was the first to be identified in the urine of newborn infants affected with congenital adrenal hyperplasia. In this investigation we report the synthesis of the three remaining 3ξ,5ξ-isomers, namely 3α,15β,17α-trihydroxy-5α-pregnan-20-one (3), 3β,15β,17α-trihydroxy-5α-pregnan-20-one (7) and 3β,15β,17α-trihydroxy-5β-pregnan-20-one (8) for their definitive identification in pathological conditions in human neonates. 3β,15β-Diacetoxy-17α-hydroxy-5-pregnen-20-one (11), a product of chemical synthesis was converted to the isomeric 3 and 7, while conversion of 15β,17α-dihydroxy-4-pregnen-3,20-dione (4), a product of microbiological transformation, resulted in the preparation of 8. In brief, selective acetate hydrolysis of 11 gave 15β-acetoxy-3β,17α-dihydroxy-5-pregnen-20-one (12) which on catalytic hydrogenation gave 15β-acetoxy-3β,17α-dihydroxy-5α-pregnan-20-one (13) a common intermediate for the synthesis of the 3β(and α),5α-isomers. Hydrolysis of the 15β-acetate gave 7, whereas oxidation with pyridinium chlorochromate gave 15β-acetoxy-17α-hydroxy-5α-pregnan-3,20-dione (14) which on reduction with -Selectride and hydrolysis of the 15β-acetate gave 3. Finally, hydrogenation of 4 gave 15β,17α-dihydroxy-5β-pregnan-3,20-dione (10) which on reduction with -Selectride gave 8.     

Molecular-marker-facilitated investigation on the ability to stimulate N2 fixation in the rhizosphere by irrigated rice plants

An F₂ population, consisting of 231 individuals derived from a cross between rice cultivars with a similar growing duration, Palawan and IR42, was utilized to investigate the genetic nature of rice varietal ability to stimulate N₂ fixation in the rice rhizosphere. To assess rhizospheric N₂ fixation, an isotope-enriched ¹⁵N dilution technique was employed, using ¹⁵N-stabilized soil in pots. IR42, an indica variety, had 23% higher N derived from fixation (Ndfa) than Palawan, a javanica genotype. Normal segregation of atom% ¹⁵N excess was obtained in the F₂ population, with an average of 0.218 with 8% of plants below IR42 (0.188) and 10% of plants above Palawan (0.248). One-hundred-and-four RFLP markers mapped on 12 chromosomes were tested for linkage to the putative QTLs. Significant (P<0.01) associations between markers and segregation of atom% ¹⁵N excess were observed for seven marker loci located on chromosomes 1, 3, 6 and 11. Four QTLs defined by the detected marker loci were identified by interval-mapping analysis. Additive gene action was found to be predominant, but for at least one locus, dominance and partial dominance effects were observed. Significant (P<0.01) epistatic effects were also identified. Individual marker loci detected between 8 and 16% of the total phenotypic variation. All four putative QTLs showed recessive gene action, and no phenotypic effects associated with heterozygosity of marker loci were observed. The results of this study suggest that rice genetic factors can be identified which affect levels of atom% ¹⁵N excess in the soil by interacting with diazotrophs in the rice rhizosphere.     

Nitrate fluxes in soybean seedling roots and their response to amino acids: an approach using 15N

This study was undertaken to determine which of the two NO₃^? fluxes (influx or efflux) across plasma membranes of root cells is the target of those amino acids which have been shown to inhibit net NO₃^? uptake (Muller & Touraine 1992, Journal of Experimental Botany 43 , 617–623). Parallel experiments were performed to mea-sure either the time course of ¹⁵NO₃^? release from roots of soybean seedlings previously labelled with this isotope into non-labelled solution, or the time course of ¹⁵N accumulation from labelled ¹⁵NO₃^? solution in non-labelled seedlings. Focusing on the fate of ¹⁵NO₃^? in the cytoplasmic compartment, a model is developed to describe the time courses of the accumulation and release of tracer across the plasma membranes of root cells. Both time courses can be described by the sum of an exponential plus a linear term. In our material, the linear part of the accumulation time course is obscured by the NO₃^? fluxes exiting the cytoplasm, and the curve thus appears to be quasilinear over several minutes. However, we show that the use of the net tracer accumulation rate during this time period as an estimate of NO₃^? influx does not provide accurate estimates of influx and efflux. By contrast, ¹⁵NO₃^? efflux analysis permits calculation of the unidirectional fluxes across plasma membranes of root cells and the kinetic parameters of the cytoplasmic NO₃^? pool. Under our experimental conditions, efflux accounted for 30 to 50% of influx, and the cytoplasmic NO₃^? content was found to be in the 70–400nmol g^?1 fw range. Using this methodology, the effect of amino acid accumulation on unidirectional fluxes of nitrate was then examined. Pretreatments of the seedlings with an amino acid which has been shown to inhibit net NO₃^? uptake led to concomitant decreases in net accumulation rates of ¹⁵NO₃^? and of reduced ¹⁵N in roots and total ¹⁵N in cotyledons. NO₃^? influx was markedly inhibited by these treatments, while NO₃^? efflux remained essentially unaffected, or even decreased. It is concluded that the target of the regulation of NO₃^? uptake by phloemtranslocated amino acids is the influx system.     

15N-ammonium and 15N-nitrate uptake of a 15-year-old Picea abies plantation

Throughfall nitrogen of a 15-year-old Picea abies (L.) Karst. (Norway spruce) stand in the Fichtelgebirge, Germany, was labeled with either ¹⁵N-ammonium or ¹⁵N-nitrate and uptake of these two tracers was followed during two successive growing seasons (1991 and 1992). ¹⁵N-labeling (62 mg ¹⁵N m^-2 under conditions of 1.5 g N m^-2 atmospheric nitrogen deposition) did not increase N concentrations in plant tissues. The ¹⁵N recovery within the entire stand (including soils) was 94%±6% of the applied ¹⁵N-ammonium tracer and 100%±6% of the applied ¹⁵N-nitrate tracer during the 1st year of investigation. This decreased to 80%±24% and 83%±20%, respectively, during the 2nd year. After 11 days, the ¹⁵N tracer was detectable in 1-year-old spruce needles and leaves of understory species. After 1 month, tracer was detectable in needle litter fall. At the end of the first growing season, more than 50% of the ¹⁵N taken up by spruce was assimilated in needles, and more than 20% in twigs. The relative distribution of recovered tracer of both ¹⁵N-ammonium and ¹⁵N-nitrate was similar within the different foliage age classes (recent to 11-year-old) and other compartments of the trees. ¹⁵N enrichment generally decreased with increasing tissue age. Roots accounted for up to 20% of the recovered ¹⁵N in spruce; no enrichment could be detected in stem wood. Although ¹⁵N-ammonium and ¹⁵N-nitrate were applied in the same molar quantities (¹⁵NH ₄ ⁺ : ¹⁵NO ₃ ^- =1:1), the tracers were diluted differently in the inorganic soil N pools (¹⁵NH ₄ ⁺ /NH ₄ ⁺ : ¹⁵NO ₃ ^- /NO ₃ ^- =1:9). Therefore the measured ¹⁵N amounts retained by the vegetation do not represent the actual fluxes of ammonium and nitrate in the soil solution. Use of the molar ammonium-to-nitrate ratio of 9:1 in the soil water extract to estimate ¹⁵N uptake from inorganic N pools resulted in a 2–4 times higher ammonium than nitrate uptake by P. abies.     

Nitrogen fixation in Faidherbia albida,Acacia raddiana,Acacia senegal and Acacia seyal estimated using the 15N isotope dilution technique

A pot experiment was conducted in a greenhouse using the ¹⁵N isotope dilution method and two reference plants, Parkia biglobosa and Tamarindus indica to estimate nitrogen fixed in four Acacia species: A raddiana, A. senegal, A. seyal and Faidherbia albida (synonym Acacia albida). For the reference plants, the ¹⁵N enrichments in leaves, stems and roots were similar. With the fixing plants, leaves and stems had similar ¹⁵N enrichments; they were higher than the ¹⁵N enrichment of roots. The amounts of nitrogen fixed at 5 months after planting were similar using either reference plant. Estimates of the percentage of N derived from fixation (%Ndfa) for the above ground parts, in contrast to %Ndfa in roots, were similar to those for the whole plant. However, none of the individual plant parts estimated accurately total N fixed in the whole plant, and excluding the roots resulted in at least 30% underestimation of the amounts of N fixed. Between species, differences in N₂ fixation were observed, both for %Ndfa and total N fixed. For %Ndfa, the best were A. seyal (average, 63%) and A. raddiana (average, 62%), being at least twice the %Ndfa in A. senegal and F. albida. Because of its very high N content, A. seyal was clearly the best in total N fixed, fixing 1.62 g N plant^–1 compared to an average of 0.48 g N plant^–1 for the other Acacia species. Our results show the wide variability existing between Acacia species in terms of both %Ndfa and total N fixed: A. seyal was classified as having a high N₂ fixing potential (NFP) while the other Acacia species had a low NFP.