Nitrous oxide emissions from agricultural fields: Assessment, measurement and mitigation

In this paper we discuss three topics concerning N₂O emissions from agricultural systems. First, we present an appraisal of N₂O emissions from agricultural soils (Assessment). Secondly, we discuss some recent efforts to improve N₂O flux estimates in agricultural fields (Measurement), and finally, we relate recent studies which use nitrification inhibitors to decrease N₂O emissions from N-fertilized fields (Mitigation).To assess the global emission of N₂O from agricultural soils, the total flux should represent N₂O from all possible sources; native soil N, N from recent atmospheric deposition, past years fertilization, N from crop residues, N₂O from subsurface aquifers below the study area, and current N fertilization. Of these N sources only synthetic fertilizer and animal manures and the area of fields cropped with legumes have sufficient global data to estimate their input for N₂O production. The assessment of direct and indirect N₂O emissions we present was made by multiplying the amount of fertilizer N applied to agricultural lands by 2% and the area of land cropped to legumes by 4 kg N₂O-N ha^-1. No regard to method of N application, type of N, crop, climate or soil was given in these calculations, because the data are not available to include these variables in large scale assessments. Improved assessments should include these variables and should be used to drive process models for field, area, region and global scales.Several N₂O flux measurement techniques have been used in recent field studies which utilize small and ultralarge chambers and micrometeorological along with new analytical techniques to measure N₂O fluxes. These studies reveal that it is not the measurement technique that is providing much of the uncertainty in N₂O flux values found in the literature but rather the diverse combinations of physical and biological factors which control gas fluxes. A careful comparison of published literature narrows the range of observed fluxes as noted in the section on assessment. An array of careful field studies which compare a series of crops, fertilizer sources, and management techniques in controlled parallel experiments throughout the calendar year are needed to improve flux estimates and decrease uncertainty in prediction capability.There are a variety of management techniques which should conserve N and decrease the amount of N application needed to grow crops and to limit N₂O emissions. Using nitrification inhibitors is an option for decreasing fertilizer N use and additionally directly mitigating N₂O emissions. Case studies are presented which demonstrate the potential for using nitrification inhibitors to limit N₂O emissions from agricultural soils. Inhibitors may be selected for climatic conditions and type of cropping system as well as the type of nitrogen (solid mineral N, mineral N in solution, or organic waste materials) and applied with the fertilizers.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Conservation of the alternative oxidase and enhancement of cyanide-resistant respiration in suspension-cultured pear fruit cells by inhibitors of macromolecular synthesis

The contribution of the alternative pathway to the respiration of suspension-cultured pear ( Pyrus communis cv. Passa Crasanne) cells was enhanced, often severalfold, within 2 to 4 days following the addition of cycloheximide, actinomycin D, or 2-(4-methyl-2,6-dinitroanalino)- N -methyl propionamide (D-MDMP). Concomitant inhibition of cellular protein synthesis by cycloheximide and actinomycin D was transient and incomplete. However, inhibition by D-MDMP was virtually complete (>97%) and persisted over several days. [³⁵S]-labelling and polyacrylamide gel separation indicated that cycloheximide precluded the appearance of discernable new proteins in mitochondria. Probes with monoclonal antibodies revealed a conservation of alternative oxidase protein levels in the mitochondria of inhibitor-treated cells. The data, appraised within the complexities of cell-culture dynamics, lead to the conclusion that the observed increases in capacity for cyanide-resistant respiration are the consequence, likely indirect, of inhibited protein synthesis with resultant retention and activation of constitutive alternative oxidase.     

Fibre length and gibberellins A1 and A20 are decreased in Eucalyptus globules by acylcyclohexanedione injected into the stem

Trinexapacethyl (TriEt), an acylcyclohexanedionetype inhibitor of gibberellin (GA) biosynthesis, was applied to 3-year-old Eucalyptus globules saplings by localised injection near the base of each stem. The objective was to alter cambial region GA levels and to study the effects on secondary xylem fibre development. Seven weeks later wood samples, with bark and cambial region intact, were removed 10 and 30 cm above the point of injection. Fusiform cambial cell dimensions were compared with those of fibre-tracheids in the most recently formed 100 um of secondary xylem. Increasing TriEt applications from 5 to 5 000 mg active ingredient significantly reduced average fibre length, and to a lesser extent average fusiform cambial cell length. Also reduced was the number of cells in the cambial zone and the number of differentiating fibres with primary walls. However, no trends were evident for changes in fibre diameter, the proportion of vessel elements or the ratio of cambial ray cells to fusiform cambial cells. Two gibberellins (GA₁ and GA₂₀), indole-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in cambial region tissues by gas chromatographymass spectrometry using stable isotope labelled internal standards. Increasing TriEt application reduced both GA₁ and GA₂₀ levels. Effects on IAA and ABA were not significant, although their levels tended to be lower at the highest TriEt application rate. The elongation of secondary xylem fibres was positively correlated with higher levels of endogenous GA₁ (r_s= 0.74, P < 0.01) and GA₂₀ (r_s= 0.72, P < 0.01). These results support a causal role for GA₁ in cambial cell division. They are also consistent with the hypothesis that the elongation of differentiating secondary xylem fibres in woody an–giosperms is dependent on GA₁ levels in the cambial region.     

Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

Physiological and growth responses of two African species, Acacia karroo and Themeda triandra, to combined increases in CO2 and UV-B radiation

The interactive effects of increased carbon dioxide (CO₂) concentration and ultraviolet-B (UV-B, 280–320 nm) radiation on Acacia karroo Hayne, a C₃ tree, and Themeda triandra Forsk., a C₄ grass, were investigated. We tested the hypothesis that A. karroo would show greater CO₂-induced growth stimulation than T. triandra, which would partially explain current encroachment of A. karroo into C₄ grasslands, but that increased UV-B could mitigate this advantage. Seedlings were grown in open-top chambers in a greenhouse in ambient (360 μmol mol^-1) and elevated (650 μmol mol^-1) CO₂, combined with ambient (1.56 to 8.66 kJ m^-2 day^-1) or increased (2.22 to 11.93 kJ m^-2 day^-1) biologically effective (weighted) UV-B irradiances. After 30 weeks, elevated CO₂ had no effect on biomass of A. karroo, despite increased net CO₂ assimilation rates. Interaction between UV-B and CO₂ on stomatal conductance was found, with conductances decreasing only where elevated CO₂ and UV-B were supplied separately. Increases in water use efficiencies, foliar starch concentrations, root nodule numbers and total nodule mass were measured in elevated CO₂. Elevated UV-B caused only an increase in foliar carbon concentrations. In T. triandra, net CO₂ assimilation rates were unaffected in elevated CO₂, but stomatal conductances and foliar nitrogen concentrations decreased, and water use efficiencies increased. Biomass of all vegetative fractions, particularly leaf sheaths, was increased in elevated CO₂. and was accompanied by increased leaf blade lengths and individual leaf and leaf sheath masses. However, tiller numbers were reduced in elevated CO₂. Significantly moderating effects of elevated UV-B were apparent only in individual masses of leaf blades and sheaths, and in total sheath and shoot biomass. The direct CO₂-induced growth responses of the species therefore do not support the hypothesis of CO₂-driven woody encroachment of C₄ grasslands. Rather, differential changes in resource use efficiency between grass and woody species, or morphological responses of grass species, could alter the competitive balance. Increased UV-B radiation is unlikely to substantially alter the CO₂ response of these species.     

The influence of plant density on the responses of Sinapis alba to CO2 and windspeed

Plants in nature live in populations of variable density, a characteristic which may influence individual plant responses to the environment. We investigated how the responses of Sinapis alba plants to different wind speeds and CO₂ concentrations could be modified by plant density. In our wind-density experiment the expectation that mechanical and physiological effects of wind will be ameliorated by growing in high density, as a result of positive plant interactions, was realised. Although individual plants were smaller at higher densities, the effect of increasing windspeed was much less than at lower plant densities. A similar reduced sensitivity of individual plant growth under high densities was also observed under CO₂ enrichment. When measured as a population or stand response, there was no effect of density on the CO₂ responses, with all stands showing very similar increases in total biomass with CO₂ enrichment. In the wind speed experiment, total biomass per stand increased significantly with density, although there was no effect of density on the wind speed response. Specific leaf area decreased with increasing wind speed and this response was significantly affected by the density at which the plants grew.     

Co-purification,co-imniunoprecipitation,and coordinate expression of acetyl-coenzyme A carboxylase activity,biotin carboxylase,and biotin carboxyl carrier protein of higher plants

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a regulatory enzyme of fatty acid synthesis, and in some higher-plant plastids is a multi-subunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxyl transferase (CT). We recently described a Nicotiana tabacum L. (tobacco) cDNA with a deduced amino acid sequence similar to that of prokaryotic BC. We here provide further biochemical and immunological evidence that this higher-plant polypeptide is an authentic BC component of ACCase. The BC protein co-purified with ACCase activity and with BCCP during gel permeation chromatography of Pisum sativum L. (pea) chloroplast proteins. Antibodies to the Ricinus communis L. (castor) BC co-precipitated ACCase activity and BCCP. During castor seed development, ACCase activity and the levels of BC and BCCP increased and subsequently decreased in parallel, indicating their coordinate regulation. The BC protein comprised about 0.8% of the soluble protein in developing castor seed, and less than 0.05% of the protein in young leaf or root. Polypeptides cross-reacting with antibodies to castor BC were detected in several dicotyledons and in the monocotyledons Hemerocallis fulva L. (day lily), Iris L., and Allium cepa L. (onion), but not in the Gramineae species Hordeum vulgare L. (barley) and Panicum virgatum L. (switchgrass). The castor endosperm and pea chloroplast ACCases were not significantly inhibited by long-chain acyl-acyl carrier protein, free fatty acids or acyl carrier protein. The BC polypeptide was detected throughout Brassica napus L. (rapeseed) embryo development, in contrast to the multi-functional ACCase isoenzyme which was only detected early in development. These results firmly establish the identity of the BC polypeptide in plants and provide insight into the structure, regulation and roles of higherplant ACCases.Abbreviations ACCase acetyl-CoA carboxylase - ACP acyl carrier protein - BC biotin carboxylase - BCCP biotin carboxyl carrier protein - CT carboxyl transferase - MF multi-functional - MS multi-subunit We thank our colleagues Nicki Engeseth and Vicki Eccleston for advice on fatty acid analysis and Sarah Hunter for providing the developing Iris seed. This work was supported in part by grant MCB 9406466 from NSF. Acknowledgement is also made to the Michigan Agriculture Experiment Station for its support of this research.     

ADP-ribosylation of actin from the green algaChara corallina byClostridium botulinum C2 toxin andClostridium perfringens iota toxin

Summary The ability of two bacterial toxins to modify a plant actin by covalent ADP-ribosylation was tested in the green algaChara corallina. Using [³²P]NAD, bothClostridium botulinum C2 toxin andClostridium perfringens iota toxin labelled a protein of M_r 42 kDa which comigrated with actin and was immunoprecipitated by a monoclonal anti-actin antibody. ADP-ribosylation ofChara actin was more efficient with iota toxin than with C2 toxin. The actin bundles in perfusedChara cells were not affected by toxin-containing media competent for ADP-ribosylation. The data indicate that monomeric plant actin is substrate for ADP-ribosylation by the bacterial toxins.Abbreviations ADP adenosine-diphosphate - EGTA ethyleneglycol-bis-(-aminoethyl)N,N,N,N-tetraacetic acid - NAD nicotinamide dinucleotide - pCA -log [Ca²⁺] - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement