Georgia Shearer

Fertilizer nitrogen: contribution to nitrate in surface water in a corn belt watershed.

Measurements of nitrate concentration and relative enrichment in nitrogen-15 were made on samples of the surface waters of a typical Illinois corn belt watershed and the effluent of the subterranean tiles that drain the cropped land in the region. From these measurements, we estimate that at the time of peak nitrate concentration in the spring of 1970 a minimum of 55 to 60 percent of the nitrogen found as nitrate in the surface waters of this watershed originated from fertilizer nitrogen

Estimates of N2-fixation from variation in the natural abundance of 15N in Sonoran desert ecosystems

SummaryThe 15N abundance of tissues of five Prosopis specimens at our primary study site (a Prosopis woodland at Harpers Well in the Sonoran desert of Southern California) was determined over two growing seasons 1980 and 1981. The 15N abundance of soil and of tissues of presumed non-N2-fixing (control) plants was also measured. Prosopis tissues were significantly lower in 15N than either soil N or corresponding tissues of presumed non-N2-fixing plants which derive their N entirely from soil. Soil N was also significantly higher in 15N than atmospheric N2. We conclude that it is feasible to use variations in the natural abundance of 15N as an index of N2-fixation in this kind of ecosystem, and that N2-fixation is of considerable importance to Prosopis growing at this site.We also determined the 15N abundance of leaf tissue of presumed N2-fixing and control plants growing at the same site at six additional sites (five in the Sonoran desert of southern California and one in Baja California, Mexico near the town of Catavina). Four of these additional sites were dominated by Prosopis and two were mixed communities. There were statistically significant differences between the 15N abundances of the pooled legume population and control plants at all sites, although not every legume specimen exhibited this difference. From 15N abundance data we estimated the fractional contribution of biologically fixed N to the N economy of desert legumes. We concluded that N2-fixation is very important to Prosopis at six of seven sites in the Sonoran Desert. At the site where Prosopis did not appear to be fixing N2, N2-fixation was important only for legumes of the sub-family Papilionoideae, Lupinus, Dalea, Astragalus and Lotus.

Seasonal dynamics of nitrogen cycling for a Prosopis woodland in the Sonoran Desert@@@Dinamica estacional del ciclo de nitrógeno de un bosque de Prosopis en el desierto Sonorense

Prosopis woodlands in the Sonoran Desert have levels of above-ground biomass and productivity much higher than those predicted for desert plant communities with such low levels of precipitation. A stand ofP. glandulosa near the Salton Sea, California, has 13,000 kg ha−1 aboveground biomass and a productivity of 3700 kg ha−1 yr−1. Such a high level of productivity is possible because Prosopis is decoupled from the normal limiting factors of water and nitrogen availability. Soil nitrogen contents for the upper 60 cm of soil beneath Prosopis canopies have 1020 g m−2 total nitrogen, 25 per cent of which is in the form of nitrate. Such accumulations of nitrogen may be the result of active symbiotic nitrogen fixation. Early estimates suggest that about 25–30 kg N ha−1 yr−1 is fixed in these stands. Since Prosopis covers only 34% of the ground surface and its water resources are not limiting, much higher levels of nitrogen fixation and productivity may be possible in managed stands at greater densities.ResumenLos bosques de Prosopis en el desierto Sonorense tienen niveles de producción de biomasa (parte aérea) y productividad mucho mayores que las predecibles para comunidades de plantas de desierto con muy bajos niveles de precipitación. Los bosques freatofiticos deP. glandulosa cerca del Mar de Salton, California, producen 13 000 kg ha−1 de biomasa aérea con una productividad 3700 kg ha−1 año−1. Tan alto nivel de productividad es posible porque Prosopis no es afectado por los factores que limitan la aprovechabilidad de agua y nitrógeno.Los primeros 60 cm del perfil del suelo bajo el dosel de Prosopis contienen 1020 g m−2 de nitrógeno total, el 25% existe en la forma de nitrato. Tales acumulaciones de nitrógeno pueden ser el resultado de la fijación simbiótica activa. Los primeros valores estimados sugieren que son fijados entre 25–30 kg N ha−1 año−1 en estos bosques. Puesto que Prosopis cubre solamente 34% de esta área y sus recursos de agua no son limitantes, puede ser posible la obtención de mayores niveles de fijación de nitrógeno y productividad de los cultivos si se manejan con mayores densidades.

Associating protein activities with their genes: rapid identification of a gene encoding a methylglyoxal reductase in the yeast Saccharomyces cerevisiae

Methylglyoxal is associated with a broad spectrum of biological effects, including cytostatic and cytotoxic activities. It is detoxified by the glyoxylase system or by its reduction to lactaldehyde by methylglyoxal reductase. We show that methylglyoxal reductase (NADPH‐dependent) is encoded by GRE2 (YOL151w). We associated this activity with its gene by partially purifying the enzyme and identifying by MALDI–TOF the proteins in candidate bands on SDS–PAGE gels whose relative intensities correlated with specific activity through three purification steps. The candidate proteins were then purified using a glutathione‐S‐transferase tag that was fused to them, and tested for methylglyoxal reductase activity. The advantage of this approach is that only modest protein purification is required. Our approach should be useful for identifying many of the genes that encode the metabolic pathway enzymes that have not been associated with a gene (about 275 in S. cerevisiae, by our estimate). Copyright

SEPARATING THE EFFLUX AND INFLUX COMPONENTS OF NET NITRATE UPTAKE BY SYNECHOCOCCUS R2 UNDER STEADY-STATE CONDITIONS

N isotopic discrimination was used to determine the ratio of influx to efflux in experiments in which NO3 reduction was decreased (and also net NO- 3 uptake, since under steady-state conditions, net NO- 3 uptake is equal to the rate of reduction) by tungstate. Exponential growth rate constants for the cyanobacterium Synechococcus sp. R2 grown in media containing 0.18 μM-molybdate decreased linearly (0.040 to 0.013 h-1) with increasing tungstate concentration (0 to 0.2 mM). Values of the overall, observed N isotope effect (βobs, the ratio of the rate of reaction of 14N- to 15N-bearing molecules, normalized to adjust for unequal concentrations) varied inversely and were linear with exponential growth rate. At high tungstate concentration, when the exponential growth rate approached zero (as a result of negligible NO- 3 reduction), βobs approached 1.0197. In the absence of tungstate, βobs was equal to 1.0037. From this value, we calculated the ratio of efflux to influx to be about 0.19. That is, in the absence of tungstate, more than 80% of the NO- 3 entering the cell underwent reduction with less than 20% released to the medium from the cell. This approach can be used to distinguish changes in influx from changes in metabolic steps in response to factors which affect net uptake rate.

Quantitative estimation of channeling from early glycolytic intermediates to CO2 in intact Escherichia coli

A pathway intermediate is said to be ‘channeled’ when an intermediate just made in a pathway has a higher probability of being a substrate for the next pathway enzyme compared with a molecule of the same species from the aqueous cytoplasm. Channeling is an important phenomenon because it might play a significant role in the regulation of metabolism. Whereas the usual mechanism proposed for channeling is the (often) transient interaction of sequential pathway enzymes, many of the supporting data come from results with pure enzymes and dilute cell extracts. Even when isotope dilution techniques have utilized whole‐cell systems, most often only a qualitative assessment of channeling has been reported. Here we develop a method for making a quantitative calculation of the fraction channeled in glycolysis from in vivo isotope dilution experiments. We show that fructose‐1,6‐bisphosphate, in whole cells of Escherichia coli, was strongly channeled all the way to CO2, whereas fructose‐6‐phosphate was not. Because the signature of channeling is lost if any downstream intermediate prior to CO2 equilibrates with molecules in the aqueous cytosol, it was not possible to evaluate whether glucose‐6‐phosphate was channeled in its transformation to fructose‐6‐phosphate. The data also suggest that, in addition to pathway enzymes being associated with one another, some are free in the aqueous cytosol. How sensitive the degree of channeling is to growth or experimental conditions remains to be determined.

Nitrogen fixation efficiency, natural15N abundance, and morphology of mesquite (Prosopis glandulosa) root nodules

SummaryThe relative nitrogen fixation efficiencies (RE 1-[H2 evolved÷C2H2 reduced]·100) of four mesquite (Prosopis glandulosa var.torreyana) rhizobia (Strains WR 1001, WR 1002, L5, L9) and a cowpea rhizobia (Strain 176A32) on mesquite were evaluated in a glasshouse experiment. Plant yield, shoot N accumulation, and the natural15N abundance (δ15N) of nodule tissue were determined. Strain WR 1002 failed to nodulate mesquite and strain L5 produced ineffective nodules. Among the three effective strains (WR 1001, L9, 176A32) the cowpea strain (176A32) and strain L9 had significantly higher RE than strain WR 1001. Differences in RE, however, were not accompanied by significantly higher plant yield and shoot N accumulation. The difference in15N abundance between foliar tissue and nodules (nodules minus leaves) was 0.47 δ15N for the ineffective L5 nodules, while for the effective WR 1001, L9, and 176A32 nodules, respectively, this difference was 8.35, 7.81, and 8.35 δ15N. This indicates a similar relationship between N2-fixing effectiveness and natural15N enrichment of nodules that was previously observed in soybeans (Glycine max, L. Merr.). Strains WR 1001 and L9 produced elongate, indeterminate nodules typical for mesquite. The ineffective L5 nodules had few infected cells and an abundance of cortical amyloplasts. Mesquite nodules produced by the cowpea strain were spherical and were somewhat more similar in internal morphology to determinate nodules typical of cowpea than indeterminate nodules normally associated with mesquite.

Natural 15N abundance as a method of estimating the contribution of biologically fixed nitrogen to N2-fixing systems: Potential for non-legumes

The15N abundance of plants usually closely reflects the15N abundance of their major immediate N source(s); plant-available soil N in the case of non-N2-fixing plants and atmospheric N2 in the case of N2 fixing plants. The15N abundance values of these sources are usually sufficiently different from each other that a significant and systematic difference in the15N abundance between the two kinds of plants can be detected. This difference provides the basis for the natural15N abundance method of estimating the relative contribution of atmospheric N2 to N2-fixing plants growing in natural and agricultural settings. The natural15N abundance method has certain advantages over more conventional methods, particularly in natural ecosystems, since disturbance of the system is not required and the measurements may be made on samples dried in the field. This method has been tested mainly with legumes in agricultural settings. The tests have demonstrated the validity of this method of arriving at semi-quantitative estimates of biological N2-fixation in these settings. More limited tests and applications have been made for legumes in natural ecosystems. An understanding of the limits and utility of this method in these systems is beginning to emerge. Examples of systematic measurements of differences in15N abundance between non-legume N2-fixing systems and neighbouring non-fixing systems are more unusual. In principle, application of the method to estimate N2-fixation by nodulated non-legumes, using the natural15N abundance method, is as feasible as estimating N2-fixation by legumes. Most of the studies involving N2-fixing non-legumes are with this type of system (e.g., Ceanothus, Chamabatia, Eleagnus, Alnus, Myrica, and so forth). Resuls of these studies are described. Applicability for associative N2-fixation is an empirical question, the answer to which probably depends upon the degree to which fixed N goes predominantly to the plant rather than to the soil N pool. The natural15N abundance method is probably not well suited to assessing the contribution of N2-fixation by free-living microorganisms in their natural habitat, particularly soil microorganisms.

RNA synthesis in tobacco leaves infected with tobacco mosaic virus

Abstract The ribonucleic acid (RNA) synthesis of tobacco leaf tissue was inhibited by about 90% when the tissue was infiltrated with actinomycin D (AMD) at 50 μg/ml for 2 hours before labeling with uracil-14C without affecting pool size. Smaller doses given for longer periods, or higher doses for shorter periods, were less effective. Long exposures, even to small doses of the drug, reduced the uptake of uracil-14C by both healthy and tobacco mosaic virus (TMV)-infected tissue. Application of the optimal conditions of treatment for the suppression of cellular RNA synthesis to infected tissue enhanced the synthesis of TMV-RNA. Moreover, infected tissue exhibited an excess AMD-resistant RNA synthesis not accounted for by the RNA incorporated into virus particles. This RNA sedimented mainly in the position occupied by the light ribosomal RNA in sucrose density gradients.

The use of soils lightly enriched in15N to screen for N2-fixing activity

SummaryThis short communication proposes the use of soils whose organic N has been lightly enriched in15N to screen plants for N2-fixing activity. The major advantage of this approach is that it provides a reliable, integrated estimate of N2-fixation up to any point in the life cycle of the plant, while using very few plants. The feasibility of the method, from the points of view of cost and availability of suitable soils, is discussed.