Exchange of nitrogen dioxide (NO 2) between plants and the atmosphere under laboratory and field conditions

Nitrogen is an essential nutrient. It is for human, animal and plants a constituent element of proteins and nucleic acids. Although the majority of the Earth’s atmosphere consists of elemental nitrogen (N2, 78 %) only a few microorganisms can use it directly. To be useful for higher plants and animals elemental nitrogen must be converted to a reactive oxidized form. This conversion happens within the nitrogen cycle by free-living microorganisms, symbiotic living Rhizobium bacteria or by lightning. Humans are able to synthesize reactive nitrogen through the Haber-Bosch process since the beginning of the 20th century. As a result food security of the world population could be improved noticeably. On the other side the increased nitrogen input results in acidification and eutrophication of ecosystems and in loss of biodiversity. Negative health effects arose for humans such as fine particulate matter and summer smog. Furthermore, reactive nitrogen plays a decisive role at atmospheric chemistry and global cycles of pollutants and nutritive substances.rnNitrogen monoxide (NO) and nitrogen dioxide (NO2) belong to the reactive trace gases and are grouped under the generic term NOx. They are important components of atmospheric oxidative processes and influence the lifetime of various less reactive greenhouse gases. NO and NO2 are generated amongst others at combustion process by oxidation of atmospheric nitrogen as well as by biological processes within soil. In atmosphere NO is converted very quickly into NO2. NO2 is than oxidized to nitrate (NO3-) and to nitric acid (HNO3), which bounds to aerosol particles. The bounded nitrate is finally washed out from atmosphere by dry and wet deposition. Catalytic reactions of NOx are an important part of atmospheric chemistry forming or decomposing tropospheric ozone (O3). In atmosphere NO, NO2 and O3 are in photosta¬tionary equilibrium, therefore it is referred as NO-NO2-O3 triad. At regions with elevated NO concentrations reactions with air pollutions can form NO2, altering equilibrium of ozone formation.rnThe essential nutrient nitrogen is taken up by plants mainly by dissolved NO3- entering the roots. Atmospheric nitrogen is oxidized to NO3- within soil via bacteria by nitrogen fixation or ammonium formation and nitrification. Additionally atmospheric NO2 uptake occurs directly by stomata. Inside the apoplast NO2 is disproportionated to nitrate and nitrite (NO2-), which can enter the plant metabolic processes. The enzymes nitrate and nitrite reductase convert nitrate and nitrite to ammonium (NH4+). NO2 gas exchange is controlled by pressure gradients inside the leaves, the stomatal aperture and leaf resistances. Plant stomatal regulation is affected by climate factors like light intensity, temperature and water vapor pressure deficit. rnThis thesis wants to contribute to the comprehension of the effects of vegetation in the atmospheric NO2 cycle and to discuss the NO2 compensation point concentration (mcomp,NO2). Therefore, NO2 exchange between the atmosphere and spruce (Picea abies) on leaf level was detected by a dynamic plant chamber system under labo¬ratory and field conditions. Measurements took place during the EGER project (June-July 2008). Additionally NO2 data collected during the ECHO project (July 2003) on oak (Quercus robur) were analyzed. The used measuring system allowed simultaneously determina¬tion of NO, NO2, O3, CO2 and H2O exchange rates. Calculations of NO, NO2 and O3 fluxes based on generally small differences (∆mi) measured between inlet and outlet of the chamber. Consequently a high accuracy and specificity of the analyzer is necessary. To achieve these requirements a highly specific NO/NO2 analyzer was used and the whole measurement system was optimized to an enduring measurement precision.rnData analysis resulted in a significant mcomp,NO2 only if statistical significance of ∆mi was detected. Consequently, significance of ∆mi was used as a data quality criterion. Photo-chemical reactions of the NO-NO2-O3 triad in the dynamic plant chamber’s volume must be considered for the determination of NO, NO2, O3 exchange rates, other¬wise deposition velocity (vdep,NO2) and mcomp,NO2 will be overestimated. No significant mcomp,NO2 for spruce could be determined under laboratory conditions, but under field conditions mcomp,NO2 could be identified between 0.17 and 0.65 ppb and vdep,NO2 between 0.07 and 0.42 mm s-1. Analyzing field data of oak, no NO2 compensation point concentration could be determined, vdep,NO2 ranged between 0.6 and 2.71 mm s-1. There is increasing indication that forests are mainly a sink for NO2 and potential NO2 emissions are low. Only when assuming high NO soil emissions, more NO2 can be formed by reaction with O3 than plants are able to take up. Under these circumstance forests can be a source for NO2.

Impact of metal and metal oxide engineered nanoparticles in soil and plant systems

Nanotechnology promises huge benefits for society and capital invested in this new technology is steadily increasing, therefore there is a growing number of nanotechnology products on the market and inevitably engineered nanomaterials will be released in the atmosphere with potential risks to humans and environment. This study set out to extend the comprehension of the impact of metal (Ag, Co, Ni) and metal oxide (CeO2, Fe3O4, SnO2, TiO2) nanoparticles (NPs) on one of the most important environmental compartments potentially contaminated by NPs, the soil system, through the use of chemical and biological tools. For this purpose experiments were carried out to simulate realistic environmental conditions of wet and dry deposition of NPs, considering ecologically relevant endpoints. In detail, this thesis involved the study of three model systems and the evaluation of related issues: (i) NPs and bare soil, to assess the influence of NPs on the functions of soil microbial communities; (ii) NPs and plants, to evaluate the chronic toxicity and accumulation of NPs in edible tissues; (iii) NPs and invertebrates, to verify the effects of NPs on earthworms and the damaging of their functionality. The study highlighted that NP toxicity is generally influenced by NP core elements and the impact of NPs on organisms is specie-specific; moreover experiments conducted in media closer to real conditions showed a decrease in toxicity with respect to in vitro test or hydroponic tests. However, only a multidisciplinary approach, involving physical, chemical and biological skills, together with the use of advanced techniques, such as X-ray absorption fine structure spectroscopy, could pave the way to draw the right conclusions and accomplish a deeper comprehension of the effects of NPs on soil and soil inhabitants.

Water, sediment and soil physicochemical interactions in freshwater, brackish and saline systems

The physicochemical interactions between water, sediment and soil deeply influence the formation and development of the ecosystem. In this research, different freshwater, brackish and saline subaqueous environments of Northern Italy were chosen as study area to investigate the physicochemical processes which occur at the interface between water and sediments, as well as the effects of soil submergence on ecosystem development. In the freshwater system of the Reno river basin, the main purpose was to define the heavy metals hazard in water and sediments of natural and artificial water courses. Heavy metals partitioning and speciation allowed to assess the environmental risk linked to the critical action of dredging canal sediments, for the maintenance of the hydraulic safety of plain lands. In addition, some bioremediation techniques were experimented for protecting sediments from heavy metals contamination, and for giving an answer to the problem of sediments management. In the brackish system of S. Vitale park, the development of hydromorphic and subaqueous soils was investigated. The study of soil profiles highlighted the presence of a soil continuum among pedons subjected to different saturation degrees. This investigation allowed to the identification of both morphological and physicochemical indicators, which characterize the formation of subaqueous soils and describe the soil hydromorphism in transitional soil systems. In the saline system of Grado lagoon, an ecosystem approach was used to define the role of water oscillation in soil characterization and plants colonization. This study highlighted the close relationship and the mutual influence of soil submergence and aeration, tide oscillation and vegetation cover, on the soil development. In view of climate change, this study contribute to understand and suppose how soil and landscape could evolve. However, a complete evaluation of hydromorphic soil functionality will be achieved only involving physiological and biochemical expertise in these kind of studies.

Three-dimensional electron microscopy of myriapod hemocyanin, planorbid snail acetylcholine-binding protein and cyanobacterial Vipp1

Three-dimensional electron microscopy (3-D EM) provides a framework for the analysis of large protein quaternary structures. The advantage over the generally higher resolving meth- od of X-ray crystallography is the embedding of the proteins in their physiological environ- ment. However, results of the two methods can be combined to obtain superior structural information. In this work, three different protein types – (i) Myriapod hemocyanin, (ii) vesi- cle-inducing protein in plastids 1 (Vipp1) and (iii) acetylcholine-binding protein (AChBP) – were structurally analyzed by 2-D and 3-D EM and, where possible, functionally interpreted.rnMyriapod hemocyanins have been previously shown to be 6x6-meric assemblies that, in case of Scutigera coleoptrata hemocyanin (ScoHc), show two 3x6-mer planes whith a stag- gering angle of approximately 60°. Here, previously observed structural differences between oxy- and deoxy-ScoHc could be substantiated. A 4° rotation between hexamers of two dif- ferent 3x6-mer planes was measured, which originates at the most central inter-hexamer in- terface. Further information about allosteric behaviour in myriapod hemocyanin was gained by analyzing Polydesmus angustus hemocyanin (PanHc), which shows a stable 3x6-mer and divergent histidine patterns in the inter-hexamer interfaces when compared to ScoHc. Both findings would conclusively explain the very different oxygen binding properties of chilopod and diplopod hemocyanin.rnVipp1 is a protein found in cyanobacteria and higher plants which is essential for thyla- koid membrane function and forms highly variable ring-shaped structures. In the course of this study, the first 3-D analysis of Vipp1 was conducted and yielded reconstructions of six differently sized Vipp1 rings from negatively stained images at resolutions between 20 to 30 Å. Furthermore, mutational analyses identified specific N-terminal amino acids that are essential for ring formation. On the basis of these analyses and previously published results, a hypothetical model of the Vipp1 tertiary and quaternary structure was generated.rnAChBP is a water-soluble protein in the hemolymph of mollusks. It is a structural and functional homologue of the ligand-binding domain of nicotinic acetylcholine receptors. For the freshwater snail Biomphalaria glabrata, we previously described two types of AChBP (BgAChBP1 and BgAChBP2). In this work, a 6 Å 3-D reconstruction of native BgAChBP is presented, which shows a dodecahedral assembly that is unprecedented for an AChBP. Single particle analysis of recombinantely expressed BgAChBP types led to preliminary results show- ing a dodecahedral assembly of BgAChBP1 and a dipentameric assembly of BgAChBP2. This indicates divergent biological functions of the two types.

Distribution of metals in several vineyards of North Italy: from soil to wine

Nowadays we live in densely populated regions and this leads to many environmental issues. Among all pollutants that human activities originate, metals are relevant because they can be potentially toxic for most of living beings. We studied the fate of Cd, Cr, Cu, Fe, Mn, Ni, Pb and Zn in a vineyard environment analysing samples of plant, wine and soil. Sites were chosen considering the type of wine produced, the type of cultivation (both organic and conventional agriculture) and the geographic location. We took vineyards that cultivate the same grape variety, the Trebbiano). We investigated 5 vineyards located in the Ravenna district (Italy): two on the Lamone Valley slopes, one in the area of river-bank deposits near Ravenna city, then a farm near Lugo and one near Bagnacavallo in interfluve regions. We carried out a very detailed characterization of soils in the sites, including the analysis of: pH, electric conductivity, texture, total carbonate and extimated content of dolomite, active carbonate, iron from ammonium oxalate, Iron Deficiency Chlorosis Index (IDCI), total nitrogen and organic carbon, available phosphorous, available potassium and Cation Exchange Capacity (CEC). Then we made the analysis of the bulk chemical composition and a DTPA extraction to determine the available fraction of elements in soils. All the sites have proper ground to cultivate, with already a good amount of nutrients, such as not needing strong fertilisations, but a vineyard on hills suffers from iron deficiency chlorosis due to the high level of active carbonate. We found some soils with much silica and little calcium oxide that confirm the marly sandstone substratum, while other soils have more calcium oxide and more aluminium oxide that confirm the argillaceous marlstone substratum. We found some critical situations, such as high concentrations of Chromium, especially in the farm near Lugo, and we noticed differences between organic vineyards and conventional ones: the conventional ones have a higher enrichment in soils of some metals (Copper and Zinc). Each metal accumulates differently in every single part of grapevines. We found differences between hill plants and lowland ones: behaviors of plants in metal accumulations seems to have patterns. Metals are more abundant in barks, then in leaves or sometimes in roots. Plants seem trying to remove excesses of metal storing them in bark. Two wines have excess of acetic acid and one conventional farm produces wine with content of Zinc over the Italian law limit. We already found evidence of high values relating them with uncontaminated environments, but more investigations are suggested to link those values to their anthropogenic supplies.

Summer drought reduces total and litter-derived soil CO2 effluxes in temperate grassland - clues from a 13C litter addition experiment

Current climate change models predict significant changes in rainfall patterns across Europe. To explore the effect of drought on soil CO2 efflux (FSoil) and on the contribution of litter to FSoil we used rain shelters to simulate a summer drought (May to July 2007) in an intensively managed grassland in Switzerland by reducing annual precipitation by around 30% similar to the hot and dry year 2003 in Central Europe. We added 13C-depleted as well as unlabelled grass/clover litter to quantify the litter-derived CO2 efflux (FLitter). Soil CO2 efflux and the 13C/12C isotope ratio (δ13C) of the respired CO2 after litter addition were measured during the growing season 2007. Drought significantly decreased FSoil in our litter addition experiment by 59% and FLitter by 81% during the drought period itself (May to July), indicating that drought had a stronger effect on the CO2 release from litter than on the belowground-derived CO2 efflux (FBG, i.e. soil organic matter (SOM) and root respiration). Despite large bursts in respired CO2 induced by the rewetting after prolonged drought, drought also reduced FSoil and FLitter during the entire 13C measurement period (April to October) by 26% and 37%, respectively. Overall, our findings show that drought decreased FSoil and altered its seasonality and its sources. Thus, the C balance of temperate grassland soils respond sensitively to changes in precipitation, a factor that needs to be considered in regional models predicting the impact of climate change on ecosystems C balance.

Geographic information system-based decision support for soil conservation planning in Tajikistan

Soil erosion on sloping agricultural land poses a serious problem for the environment, as well as for production. In areas with highly erodible soils, such as those in loess zones, application of soil and water conservation measures is crucial to sustain agricultural yields and to prevent or reduce land degradation. The present study, carried out in Faizabad, Tajikistan, was designed to evaluate the potential of local conservation measures on cropland using a spatial modelling approach to provide decision-making support for the planning of spatially explicit sustainable land use. A sampling design to support comparative analysis between well-conserved units and other field units was established in order to estimate factors that determine water erosion, according to the Revised Universal Soil Loss Equation (RUSLE). Such factor-based approaches allow ready application using a geographic information system (GIS) and facilitate straightforward scenario modelling in areas with limited data resources. The study showed first that assessment of erosion and conservation in an area with inhomogeneous vegetation cover requires the integration of plot-based cover. Plot-based vegetation cover can be effectively derived from high-resolution satellite imagery, providing a useful basis for plot-wise conservation planning. Furthermore, thorough field assessments showed that 25.7% of current total cropland is covered by conservation measures (terracing, agroforestry and perennial herbaceous fodder). Assessment of the effectiveness of these local measures, combined with the RUSLE calculations, revealed that current average soil loss could be reduced through low-cost measures such as contouring (by 11%), fodder plants (by 16%), and drainage ditches (by 53%). More expensive measures such as terracing and agroforestry can reduce erosion by as much as 63% (for agroforestry) and 93% (for agroforestry combined with terracing). Indeed, scenario runs for different levels of tolerable erosion rates showed that more cost-intensive and technologically advanced measures would lead to greater reduction of soil loss. However, given economic conditions in Tajikistan, it seems advisable to support the spread of low-cost and labourextensive measures.

Purification, cDNA structure and biological significance of a single insulin-like growth factor-binding domain protein (SIBD-1) identified in the hemocytes of the spider Cupiennius salei

Cupiennius salei single insulin-like growth factor-binding domain protein (SIBD-1), which exhibits an IGFBP N-terminal domain-like profile, was identified in the hemocytes of the spider C. salei. SIBD-1 was purified by RP-HPLC and the sequence determined by a combination of Edman degradation and 5'-3'- RACE PCR. The peptide (8676.08 Da) is composed of 78 amino acids, contains six intrachain disulphide bridges and carries a modified Thr residue at position 2. SIBD-1 mRNA expression was detected by quantitative real-time PCR mainly in hemocytes, but also in the subesophageal nerve mass and muscle. After infection, the SIBD-1 content in the hemocytes decreases and, simultaneously, the temporal SIBD-1 expression seems to be down-regulated. Two further peptides, SIBD-2 and IGFBP-rP1, also exhibiting IGFBP N-terminal domain variants with unknown functions, were identified on cDNA level in spider hemocytes and venom glands. We conclude that SIBD-1 may play an important role in the immune system of spiders.

No adverse effect of genetically modified antifungal wheat on decomposition dynamics and the soil fauna community - a field study

The cultivation of genetically modified (GM) plants has raised several environmental concerns. One of these concerns regards non-target soil fauna organisms, which play an important role in the decomposition of organic matter and hence are largely exposed to GM plant residues. Soil fauna may be directly affected by transgene products or indirectly by pleiotropic effects such as a modified plant metabolism. Thus, ecosystem services and functioning might be affected negatively. In a litterbag experiment in the field we analysed the decomposition process and the soil fauna community involved. Therefore, we used four experimental GM wheat varieties, two with a race-specific antifungal resistance against powdery mildew (Pm3b) and two with an unspecific antifungal resistance based on the expression of chitinase and glucanase. We compared them with two non-GM isolines and six conventional cereal varieties. To elucidate the mechanisms that cause differences in plant decomposition, structural plant components (i.e. C:N ratio, lignin, cellulose, hemicellulose) were examined and soil properties, temperature and precipitation were monitored. The most frequent taxa extracted from decaying plant material were mites (Cryptostigmata, Gamasina and Uropodina), springtails (Isotomidae), annelids (Enchytraeidae) and Diptera (Cecidomyiidae larvae). Despite a single significant transgenic/month interaction for Cecidomyiidae larvae, which is probably random, we detected no impact of the GM wheat on the soil fauna community. However, soil fauna differences among conventional cereal varieties were more pronounced than between GM and non-GM wheat. While leaf residue decomposition in GM and non-GM wheat was similar, differences among conventional cereals were evident. Furthermore, sampling date and location were found to greatly influence soil fauna community and decomposition processes. The results give no indication of ecologically relevant adverse effects of antifungal GM wheat on the composition and the activity of the soil fauna community.

Lactococcus lactis HemW (HemN) is a haem-binding protein with a putative role in haem trafficking

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 μM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

Study Of The Binding Of Substrate By The Phe557Val Mutant Soybean Lipoxygenase-1

Lipoxygenases are a class of enzymes which consist of non-heme iron dioxygenases that are produced by fungi, plants, and mammals and catalyze the oxygenation of polyunsaturated fatty acid substrates to unsaturated fatty acid hydroperoxide products. The unsaturated fatty acid hydroperoxide products are stereo- and regiospecific. One such lipoxygenase, soybean lipoxygenase-1 (SBLO-1), catalyzes the conversion of linoleate to 13-hydroperoxy-9(Z),11(E)-octadecadienoate (13-HPOD) and a small amount of 9-hydroperoxy-10(E),12(Z)-octadecadienoate (9-HPOD). Although the structure of SBLO-1 is known and it is the most widely studied lipoxygenase, how it binds to substrate is still poorly understood. Two competing binding hypotheses that have been used to understand and explain the binding are the head first binding model and the tail first binding model. The head first binding model predicts linoleate binds with its polar carboxylate group in the binding pocket and the methyl terminus at the surface of the binding pocket. The tail first binding model predicts that linoleate binds with its methyl terminus end in the binding pocket and the polar carboxylate group at the surface of the binding pocket. Both binding models have been used in the explanation of previous work. In previous work the replacement of phenylalanine with valine has been performed to produce the phe557val mutant SBLO-1. The mutant SBLO-1 was then used in the enzymatic oxygenation of linoleate. With this mutant, the amount of 9-HPOD that is formed increases. This result has been interpreted using the head-first binding model in which the smaller valine residue allows linoleate to bind with the polar carboxylate group of linoleate interacting with arginine-707. The work presented in this thesis confirms the regiochemical results of the previous work and further tests the head-first binding model. If head-first binding occurs, the 9-HPOD is expected to have primarily S configuration. Utilizing chiral-phase HPLC, it was found that the 9-HPOD produced by the phe557val mutant SBLO-1 is primarily S, consistent with head-first binding. The head-first binding model was also tested using linoleyl dimethylamine (LDMA), which has been shown to be a good substrate for SBLO-1 at pH 7.0, where LDMA is thought to be positively charged. This model predicts that less of the 9-peroxide should be produced with this substrate. Through the use of gas chromatography/mass spectrometry, it was found that the conversion of LDMA by the phe557val mutant SBLO-1 resulted in the formation of a 46:54 mixture of the 13-peroxide:9-peroxide. The higher amount of 9-peroxide is the opposite of what is expected for the currently proposed model suggesting that the proposed model may not be entirely correct. The results thus far have been consistent with reverse binding but not with the proposed interaction of the polar end of the substrate with arginine-707.