Anthropogenic control over wintertime oxidation of atmospheric pollutants.

During winter in the mid-latitudes, photochemical oxidation is significantly slower than in summer and the main radical oxidants driving formation of secondary pollutants, such as fine particulate matter and ozone, remain uncertain, owing to a lack of observations in this season. Using airborne observations, we quantify the contribution of various oxidants on a regional basis during winter, enabling improved chemical descriptions of wintertime air pollution transformations. We show that 25-60% of NOx is converted to N2O5 via multiphase reactions between gas-phase nitrogen oxide reservoirs and aerosol particles, with ~93% reacting in the marine boundary layer to form >2.5 ppbv ClNO2. This results in >70% of the oxidizing capacity of polluted air during winter being controlled, not by typical photochemical reactions, but from these multiphase reactions and emissions of volatile organic compounds, such as HCHO, highlighting the control local anthropogenic emissions have on the oxidizing capacity of the polluted wintertime atmosphere.

Laboratory studies on secondary organic aerosol formation from crude oil vapors.

Airborne measurements of aerosol composition and gas phase compounds over the Deepwater Horizon (DWH) oil spill in the Gulf of Mexico in June 2010 indicated the presence of high concentrations of secondary organic aerosol (SOA) formed from organic compounds of intermediate volatility. In this work, we investigated SOA formation from South Louisiana crude oil vapors reacting with OH in a Potential Aerosol Mass flow reactor. We use the dependence of evaporation time on the saturation concentration (C*) of the SOA precursors to separate the contribution of species of different C* to total SOA formation. This study shows consistent results with those at the DWH oil spill: (1) organic compounds of intermediate volatility with C* = 10(5)-10(6) µg m(-3) contribute the large majority of SOA mass formed, and have much larger SOA yields (0.37 for C* = 10(5) and 0.21 for C* = 10(6) µg m(-3)) than more volatile compounds with C*≥10(7) µg m(-3), (2) the mass spectral signature of SOA formed from oxidation of the less volatile compounds in the reactor shows good agreement with that of SOA formed at DWH oil spill. These results also support the use of flow reactors simulating atmospheric SOA formation and aging.

Investigations of the role of the main light-harvesting chlorophyll-protein complex in thylakoid membranes. Reconstitution of depleted membranes from intermittent-light-grown plants with the isolated complex.

The functions of the light-harvesting complex of photosystem II (LHC-II) have been studied using thylakoids from intermittent-light-grown (IML) plants, which are deficient in this complex. These chloroplasts have no grana stacks and only limited lamellar appression in situ. In vitro the thylakoids showed limited but significant Mg2+-induced membrane appression and a clear segregation of membrane particles into such regions. This observation, together with the immunological detection of small quantities of LHC-II apoproteins, suggests that the molecular mechanism of appression may be similar to the more extensive thylakoid stacking seen in normal chloroplasts and involve LHC-II polypeptides directly. To study LHC-II function directly, a sonication-freeze-thaw procedure was developed for controlled insertion of purified LHC-II into IML membranes. Incorporation was demonstrated by density gradient centrifugation, antibody agglutination tests, and freeze-fracture electron microscopy. The reconstituted membranes, unlike the parent IML membranes, exhibited both extensive membrane appression and increased room temperature fluorescence in the presence of cations, and a decreased photosystem I activity at low light intensity. These membranes thus mimic normal chloroplasts in this regard, suggesting that the incorporated LHC-II interacts with photosystem II centers in IML membranes and exerts a direct role in the regulation of excitation energy distribution between the two photosystems.

Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules.

Nitrogen-fixing bacteroids in legume root nodules are surrounded by the plant-derived peribacteroid membrane, which controls nutrient transfer between the symbionts. A nodule complementary DNA (GmSAT1) encoding an ammonium transporter has been isolated from soybean. GmSAT1 is preferentially transcribed in nodules and immunoblotting indicates that GmSAT1 is located on the peribacteroid membrane. [14C]methylammonium uptake and patch-clamp analysis of yeast expressing GmSAT1 demonstrated that it shares properties with a soybean peribacteroid membrane NH4^{+ channel described elsewhere. GmSAT1 is likely to be involved in the transfer of fixed nitrogen from the bacteroid to the host.}

Reflection across plant cell boundaries in confocal laser scanning microscopy.

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.

Aqueous-phase mechanism for secondary organic aerosol formation from isoprene: application to the Southeast United States and co-benefit of SO2 emission controls.

Isoprene emitted by vegetation is an important precursor of secondary organic aerosol (SOA), but the mechanism and yields are uncertain. Aerosol is prevailingly aqueous under the humid conditions typical of isoprene-emitting regions. Here we develop an aqueous-phase mechanism for isoprene SOA formation coupled to a detailed gas-phase isoprene oxidation scheme. The mechanism is based on aerosol reactive uptake coefficients (γ) for water-soluble isoprene oxidation products, including sensitivity to aerosol acidity and nucleophile concentrations. We apply this mechanism to simulation of aircraft (SEAC4RS) and ground-based (SOAS) observations over the Southeast US in summer 2013 using the GEOS-Chem chemical transport model. Emissions of nitrogen oxides (NOx ≡ NO + NO2) over the Southeast US are such that the peroxy radicals produced from isoprene oxidation (ISOPO2) react significantly with both NO (high-NOx pathway) and HO2 (low-NOx pathway), leading to different suites of isoprene SOA precursors. We find a mean SOA mass yield of 3.3 % from isoprene oxidation, consistent with the observed relationship of total fine organic aerosol (OA) and formaldehyde (a product of isoprene oxidation). Isoprene SOA production is mainly contributed by two immediate gas-phase precursors, isoprene epoxydiols (IEPOX, 58% of isoprene SOA) from the low-NOx pathway and glyoxal (28%) from both low- and high-NOx pathways. This speciation is consistent with observations of IEPOX SOA from SOAS and SEAC4RS. Observations show a strong relationship between IEPOX SOA and sulfate aerosol that we explain as due to the effect of sulfate on aerosol acidity and volume. Isoprene SOA concentrations increase as NOx emissions decrease (favoring the low-NOx pathway for isoprene oxidation), but decrease more strongly as SO2 emissions decrease (due to the effect of sulfate on aerosol acidity and volume). The US EPA projects 2013-2025 decreases in anthropogenic emissions of 34% for NOx (leading to 7% increase in isoprene SOA) and 48% for SO2 (35% decrease in isoprene SOA). Reducing SO2 emissions decreases sulfate and isoprene SOA by a similar magnitude, representing a factor of 2 co-benefit for PM2.5 from SO2 emission controls.

Organic nitrate chemistry and its implications for nitrogen budgets in an isoprene- and monoterpene-rich atmosphere: constraints from aircraft (SEAC4RS) and ground-based (SOAS) observations in the Southeast US.

Formation of organic nitrates (RONO2) during oxidation of biogenic volatile organic compounds (BVOCs: isoprene, monoterpenes) is a significant loss pathway for atmospheric nitrogen oxide radicals (NOx), but the chemistry of RONO2 formation and degradation remains uncertain. Here we implement a new BVOC oxidation mechanism (including updated isoprene chemistry, new monoterpene chemistry, and particle uptake of RONO2) in the GEOS-Chem global chemical transport model with ∼25 × 25 km2 resolution over North America. We evaluate the model using aircraft (SEAC4RS) and ground-based (SOAS) observations of NOx, BVOCs, and RONO2 from the Southeast US in summer 2013. The updated simulation successfully reproduces the concentrations of individual gas- and particle-phase RONO2 species measured during the campaigns. Gas-phase isoprene nitrates account for 25-50% of observed RONO2 in surface air, and we find that another 10% is contributed by gas-phase monoterpene nitrates. Observations in the free troposphere show an important contribution from long-lived nitrates derived from anthropogenic VOCs. During both campaigns, at least 10% of observed boundary layer RONO2 were in the particle phase. We find that aerosol uptake followed by hydrolysis to HNO3 accounts for 60% of simulated gas-phase RONO2 loss in the boundary layer. Other losses are 20% by photolysis to recycle NOx and 15% by dry deposition. RONO2 production accounts for 20% of the net regional NOx sink in the Southeast US in summer, limited by the spatial segregation between BVOC and NOx emissions. This segregation implies that RONO2 production will remain a minor sink for NOx in the Southeast US in the future even as NOx emissions continue to decline.

Evidence for a link between translocation and processing during protein import into soybean mitochondria.

The effect of metal chelators on protein import was investigated using isolated soybean mitochondria and soybean precursor proteins. Adding 1,10-phenanthroline, a metal chelator that can cross both mitochondrial membranes abolished import of both the alternative oxidase, and the F(A)d subunit of the ATP synthase, a matrix located protein. Other metal chelators such as EDTA, 1,7-phenanthroline and 4,7-phenanthroline, which cannot cross the mitochondrial membranes, had no effect on import. When processing, a known metal-dependent step inside mitochondria, was inhibited using a mutagenesis approach (changing a -2 arginine to a -2 glycine in the pre-piece of the precursor), so was import. Thus it would appear that in soybean, at least, translocation of proteins across the mitochondrial membrane, as well as processing, relies on a metal dependent step. Taken together, the data suggest the two processes may be directly connected in these mitochondria.

The effect of calcium on the respiratory responses of corn mitochondria.

Tightly coupled respiring corn mitochondria (Zea mays L.) respond to calcium addition with a transitory respiratory increase, proton extrusion, and Ca2+ binding. The extent of response is dependent upon the level of endogenous phosphate, and a large sustained respiratory increase can be obtained with addition of phosphate. However, calcium does not act as a permeant cation in that it will not penetrate with acetate. It appears that the transitory respiratory increase must be linked to the uptake of a calcium phosphate complex, but there is no evidence that transport of the complex serves to produce an electrophoretic calcium uniport. It is believed that calcium phosphate transport in corn is a constitutive property, and not produced by membrane damage.

Identification and Characterization of an Inducible NAD(P)H Dehydrogenase from Red Beetroot Mitochondria.

Exogenous NADH oxidation of mitochondria isolated from red beetroots (Beta vulgaris L.) increased dramatically upon slicing and aging the tissue. Anion-exchange chromatography of soluble fractions derived by sonication from fresh and aged beetroot mitochondria yielded three NADH dehydrogenase activity peaks. The third peak from aged beetroot mitochondria was separated into two activities by blue-affinity chromatography. One of these (the unbound peak) readily oxidized dihydrolipoamide, whereas the other (the bound peak) did not. The latter was an NAD(P)H dehydrogenase with high quinone and ferricyanide reductase activity and was absent from fresh beet mitochondria. Further affinity chromatography of the NAD(P)H dehydrogenase indicated enrichment of a 58-kD polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We propose that this 58-kD protein is the inducible, external NADH dehydrogenase.

Iron Uptake by Symbiosomes from Soybean Root Nodules.

To identify possible iron sources for bacteroids in planta, soybean (Glycine max L. Merr.) symbiosomes (consisting of the bacteroid-containing peribacteroid space enclosed by the peribacteroid membrane [PBM]) and bacteroids were assayed for the ability to transport iron supplied as various ferric [Fe(III)]-chelates. Iron presented as a number of Fe(III)-chelates was transported at much higher rates across the PBM than across the bacteroid membranes, suggesting the presence of an iron storage pool in the peribacteroid space. Pulse-chase experiments confirmed the presence of such an iron storage pool. Because the PBM is derived from the plant plasma membrane, we reasoned that it may possess a ferric-chelate reductase activity similar to that present in plant plasma membrane. We detected ferric-chelate reductase activity associated with the PBM and suggest that reduction of Fe(III) to ferrous [Fe(II)] plays a role in the movement of iron into soybean symbiosomes.

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria.

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.

Regulation of Alternative Oxidase Activity by Pyruvate in Soybean Mitochondria.

The regulation of alternative oxidase activity by the effector pyruvate was investigated in soybean (Glycine max L.) mitochondria using developmental changes in roots and cotyledons to vary the respiratory capacity of the mitochondria. Rates of cyanide-insensitive oxygen uptake by soybean root mitochondria declined with seedling age. Immunologically detectable protein levels increased slightly with age, and mitochondria from younger, more active roots had less of the protein in the reduced form. Addition of pyruvate stimulated cyanide-insensitive respiration in root mitochondria, up to the same rate, regardless of seedling age. This stimulation was reversed rapidly upon removal of pyruvate, either by pelleting mitochondria (with succinate as substrate) or by adding lactate dehydrogenase with NADH as substrate. In mitochondria from cotyledons of the same seedlings, cyanide-insensitive NADH oxidation was less dependent on added pyruvate, partly due to intramitochondrial generation of pyruvate from endogenous substrates. Cyanide-insensitive oxygen uptake with succinate as substrate was greater than that with NADH, in both root and cotyledon mitochondria, but this difference became much less when an increase in external pH was used to inhibit intramitochondrial pyruvate production via malic enzyme. Malic enzyme activity in root mitochondria declined with seedling age. The results indicate that the activity of the alternative oxidase in soybean mitochondria is very dependent on the presence of pyruvate: differences in the generation of intramitochondrial pyruvate can explain differences in alternative oxidase activity between tissues and substrates, and some of the changes that occur during seedling development.

Alternative Oxidase Activity in Tobacco Leaf Mitochondria (Dependence on Tricarboxylic Acid Cycle-Mediated Redox Regulation and Pyruvate Activation).

Transgenic Nicotiana tabacum (cv Petit Havana SR1) containing high levels of mitochondrial alternative oxidase (AOX) protein due to the introduction of a sense transgene(s) of Aox1, the nuclear gene encoding AOX, were used to investigate mechanisms regulating AOX activity. After purification of leaf mitochondria, a large proportion of the AOX protein was present as the oxidized (covalently associated and less active) dimer. High AOX activity in these mitochondria was dependent on both reduction of the protein by DTT (to the noncovalently associated and more active dimer) and its subsequent activation by certain [alpha]-keto acids, particularly pyruvate. Reduction of AOX to its more active form could also be mediated by intramitochondrial reducing power generated by the oxidation of certain tricarboxylic acid cycle substrates, most notably isocitrate and malate. Our evidence suggests that NADPH may be specifically required for AOX reduction. All of the above regulatory mechanisms applied to AOX in wild-type mitochondria as well. Transgenic leaves lacking AOX due to the introduction of an Aox1 antisense transgene or multiple sense transgenes were used to investigate the potential physiological significance of the AOX-regulatory mechanisms. Under conditions in which respiratory carbon metabolism is restricted by the capacity of mitochondrial electron transport, feed-forward activation of AOX by mitochondrial reducing power and pyruvate may act to prevent redirection of carbon metabolism, such as to fermentative pathways.

Nitric oxide inhibits the cytochrome oxidase but not the alternative oxidase of plant mitochondria.

Oxygen consumption via the cytochrome pathway in isolated soybean (Glycine max [L.] Merr.) cotyledon mitochondria was inhibited by nitric oxide (NO) while respiration via the cyanide-insensitive alternative oxidase was not significantly affected. Inhibition of cytochrome pathway activity was rapidly reversible upon depletion of the added NO. NO production was also detected in solutions of NaNO2 plus ascorbate and the extent of cytochrome pathway inhibition was dependent on the NO2- concentration. Little inhibition of alternative pathway respiration was observed under similar conditions. The alternative oxidase may play a role in nitric oxide tolerance in higher plants and in organisms such as trypanosomes which contain a plant-like alternative oxidase.

How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.

Organic acid activation of the alternative oxidase of plant mitochondria.

Alternative oxidase activity (oxygen uptake in the presence of KCN, antimycin or myxothiazol) in mitochondria isolated from the roots of soybean seedlings was very slow, even with succinate as substrate. This activity was stimulated substantially (100-400%) by the addition of pyruvate, with half maximal stimulation occurring at 0.1 mM pyruvate. Mitochondria from soybean shoots displayed high alternative oxidase activity with succinate and malate as substrates but lower activity with exogenous NADH; addition of pyruvate stimulated the activity with NADH up to that seen with succinate. This stimulation of cyanide-insensitive NADH oxidation was seen also with mitochondria from other species. Hydroxypyruvate and oxoglutarate could substitute for pyruvate, although higher concentrations were required to achieve maximum stimulation. Pyruvate stimulation of cyanide-insensitive oxygen uptake was observed with exogenous quinols as substrates, with sub-mitochondrial particles, and in the presence of the pyruvate transport inhibitor, cyanohydroxycinnamic acid, but was not observed with detergent-solubilised mitochondria. It is suggested that pyruvate acts allosterically on the alternative oxidase to stimulate its activity. The implications of these findings for respiration in vivo are discussed.

An alternative oxidase monoclonal antibody recognises a highly conserved sequence among alternative oxidase subunits.

The alternative oxidase is found in the inner mitochondrial membranes of plants and some fungi and protists. A monoclonal antibody raised against the alternative oxidase from the aroid lily Sauromatum guttatum has been used extensively to detect the enzyme in these organisms. Using an immunoblotting strategy, the antibody binding site has been localised to the sequence RADEAHHRDVNH within the soybean alternative oxidase 2 protein. Examination of sequence variants showed that A2 and residues C-terminal to H7 are required for recognition by the monoclonal antibody raised against the alternative oxidase. The recognition sequence is highly conserved among all alternative oxidase proteins and is absolutely conserved in 12 of 14 higher plant sequences, suggesting that this antibody will continue to be extremely useful in studying the expression and synthesis of the alternative oxidase.

Proteomic identification of divalent metal cation binding proteins in plant mitochondria.

Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analysed by mobility shifts in the presence of divalent cations during two-dimensional diagonal sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Tandem mass spectrometry and searches of the predicted Arabidopsis protein dataset were used in an attempt to identify 34 of the proteins which shifted. This analysis identified a total of 23 distinct protein spots as the products of at least 11 different Arabidopsis genes. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identified. These included: succinyl CoA ligase beta subunit, Mn-superoxide dismutase (SOD), an Fe-S centred component of complex I and the REISKE iron-sulphur protein of the b/c(1) complex. A further set of four proteins of known function but without known divalent binding properties were also identified: the Vb subunit of cytochrome c oxidase, a subunit of ATP synthase (orfB), the acyl carrier protein, and the translocase of the outer membrane (TOM20). Three other proteins, of unknown function, were also found to shift in the presence of divalent cations. This approach has broad application for the identification of sub-proteomes based on the metal interaction of polypeptides.

Protein phosphorylation stimulates the rate of malate uptake across the peribacteroid membrane of soybean nodules.

Incubation of intact isolated symbiosomes with [gamma-32P]ATP, followed by isolation of the peribacteroid membrane and polypeptide analysis, showed that a single major polypeptide at 26 kDa was labelled. Antibodies raised against nodulin 26 reacted with a similar sized polypeptide. Incubation of the symbiosomes with alkaline phosphatase removed the label from this polypeptide. Pre-incubation with ATP stimulated malate accumulation by isolated symbiosomes, but only slightly (10-30%). Pre-treatment of symbiosomes with alkaline phosphatase inhibited malate uptake substantially and this inhibition was completely relieved by addition of ATP. The ATP stimulation of malate uptake was not affected by ATPase inhibitors. It is suggested that the rate of malate uptake across the peribacteroid membrane is controlled by phosphorylation of nodulin 26.