Cadmium-induced subcellular accumulation of O2·− and H2O2 in pea leaves

Cadmium is a toxic metal that produces disturbances in plant antioxidant defences giving rise to oxidative stress. The effect of this metal on H₂O₂ and O₂^·? production was studied in leaves from pea plants growth for 2 weeks with 50 µm Cd, by histochemistry with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT), respectively. The subcellular localization of these reactive oxygen species (ROS) was studied by cytochemistry with CeCl₃ and Mn/DAB staining for H₂O₂ and O₂^·?, respectively, followed by electron microscopy observation. In leaves from pea plants grown with 50 µm CdCl₂ a rise of six times in the H₂O₂ content took place in comparison with control plants, and the accumulation of H₂O₂ was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells. In mesophyll cells a small accumulation of H₂O₂ was observed in mitochondria and peroxisomes. Experiments with inhibitors suggested that the main source of H₂O₂ could be a NADPH oxidase. The subcellular localization of O₂^·? production was demonstrated in the tonoplast of bundle sheath cells, and plasma membrane from mesophyll cells. The Cd‐induced production of the ROS, H₂O₂ and O₂^·?, could be attributed to the phytotoxic effect of Cd, but lower levels of ROS could function as signal molecules in the induction of defence genes against Cd toxicity. Treatment of leaves from Cd‐grown plants with different effectors and inhibitors showed that ROS production was regulated by different processes involving protein phosphatases, Ca²⁺ channels, and cGMP.     

O2 regulation and O2‐limitation of nitrogenase activity in root nodules of pea and lupin

The gas exchange characteristics of intact attached nodulated roots of pea (Pisum sativum cv. Finale X) and lupin (Lupinus albus cv. Ultra) were studied under a number of environmental conditions to determine whether or not the nodules regulate resistance to oxygen diffusion. Nitrogenase activity (H₂ evolution) in both species was inhibited by an increase in rhizosphere pO₂ from 20% to 30%, but recovered within 30 min without a significant increase in nodulated root respiration (CO₂ evolution). These data suggest that the nodules possess a variable barrier to O₂ diffusion. Also, nitrogenase activity in both species declined when the roots were either exposed to an atmosphere of Ar:O₂ or when the shoots of the plants were excised. These declines could be reversed by elevating rhizosphere pO₂, indicating that the inhibition of nitrogenase activity resulted from an increase in gas diffusion resistance and consequent O₂-limitation of nitrogenase-linked respiration. These results indicate that nodules of pea and lupin regulate their internal O₂ concentration in a manner similar to nodules of soybean, despite the distinct morphological and biochemical differences that exist between the nodules of the 3 species. Experiments in which total nitrogenase activity (TNA = H₂ production in Ar:O₂) in pea and lupin nodules was monitored while rhizosphere pO₂ was increased gradually to 100%, showed that the resistance of the nodules to O₂ diffusion maintains nitrogenase activity at about 80% of its potential activity (PNA) under normal atmospheric conditions. The O₂-limitation coefficient of nitrogenase (OLC_N= TNA/PNA) declined significantly with prolonged exposure to Ar:O₂ or with shoot excision. Together, these results indicate a significant degree of O₂-limitation of nitrogenase activity in pea and lupin nodules, and that yields may be increased by realizing full potential activity.     

Influence of soil O2 and CO2 on root respiration for Agave deserti

Respiration measured as CO₂ efflux was determined at various soil O₂ and CO₂ concentrations for individual, attached roots of a succulent perennial from the Sonoran Desert, Agave deserti Engelm. The respiration rate increased with increasing O₂ concentration up to about 16% O₂ for established roots and 5% O₂ for rain roots (fine branch roots on established roots induced by wetting of the soil) and then remained fairly constant up to 21% O₂. When O₂ was decreased from 21 to 0%, the respiration rates were similar to those obtained with increasing O₂ concentration. The CO₂ concentration in the root zone, which for the shallow-rooted A. deserti in the field was about 1 000 μl l^-1, did not affect root respiration at concentrations up to 2 000 μl l^-1, but higher concentrations reduced it, respiration being abolished at 20 000 μl l^-1 (2%) CO₂ for both established and rain roots. Upon lowering CO₂ to 1 000 μl l^-1 after exposure to concentrations up to 10000 μl l^-1 CO₂, inhibition of respiration was reversible. Uptake of the vital stain neutral red by root cortical cells was reduced to zero, indicating cell death, in about 4 h at 2% CO₂, substantiating the detrimental effects of high soil CO₂ concentrations on roots of A. deserti . This CO₂ response may explain why roots of desert succulents tend to occur in porous, well-aerated soils.     

Utilization of O2 in the metabolic optimization of C4 photosynthesis

The combined effects of O₂ on net rates of photosynthesis, photosystem II activity, steady‐state pool size of key metabolites of photosynthetic metabolism in the C₄ pathway, C₃ pathway and C₂ photorespiratory cycle and on growth were evaluated in the C₄ species Amaranthus edulis and the C₃ species Flaveria pringlei. Increasing O₂ reduced net CO₂ assimilation in F. pringlei due to an increased flux of C through the photorespiratory pathway. However, in A. edulis increasing O₂ up to 5–10% stimulated photosynthesis. Analysis of the pool size of key metabolites in A. edulis suggests that while there is some O₂ dependent photorespiration, O₂ is required for maximizing C₄ cycle activity to concentrate CO₂ in bundle sheath cells. Therefore, the response of net photosynthesis to O₂ in C₄ plants may result from the balance of these two opposing effects. Under 21 versus 5% O₂, growth of A. edulis was stimulated about 30% whereas that of F. pringlei was inhibited about 40%.     

Aerenchyma formation and radial O2 loss along adventitious roots of wheat with only the apical root portion exposed to O2 deficiency

This study investigated aerenchyma formation and function in adventitious roots of wheat (Triticum aestivum L.) when only a part of the root system was exposed to O₂ deficiency. Two experimental systems were used: (1) plants in soil waterlogged at 200 mm below the surface; or (2) a nutrient solution system with only the apical region of a single root exposed to deoxygenated stagnant agar solution with the remainder of the root system in aerated nutrient solution. Porosity increased two‐ to three‐fold along the entire length of the adventitious roots that grew into the water‐saturated zone 200 mm below the soil surface, and also increased in roots that grew in the aerobic soil above the water‐saturated zone. Likewise, adventitious roots with only the tips growing into deoxygenated stagnant agar solution developed aerenchyma along the entire main axis. Measurements of radial O₂ loss (ROL), taken using root‐sleeving O₂ electrodes, showed this aerenchyma was functional in conducting O_2. The ROL measured near tips of intact roots in deoxygenated stagnant agar solution, while the basal part of the root remained in aerated solution, was sustained when the atmosphere around the shoot was replaced by N₂. This illustrates the importance of O₂ diffusion into the basal regions of roots within an aerobic zone, and the subsequent longitudinal movement of O₂ within the aerenchyma, to supply O₂ to the tip growing in an O₂ deficient zone.     

Biological oxidation of hydrogen in soils flushed with a mixture of H2, CO2, O2 and N2

Abstract A stainless steel cylinder filled with soil was flushed upstream with a H₂/CO₂/air mixture. The consequence was a strong enrichment of the aerobic, autotrophic hydrogen-oxidising microflora, which reached densities enabling them to oxidize 84.5 ml H₂· dm⁻²· h⁻¹ in the first 25-cm layer. H₂ concentration profiles, hydrogen uptake activity and cell numbers correlated well with each other. Most of the organisms isolated were dinitrogen fixers. Thus, soils containing hydrogen-oxidising bacteria may act as a biological shield between H₂-rich environments and air, and may be utilized as biofilters, e.g., in the waste-processing industry.     

Decomposition of soybean grown under elevated concentrations of CO2 and O3

A critical global climate change issue is how increasing concentrations of atmospheric CO₂ and ground‐level O₃ will affect agricultural productivity. This includes effects on decomposition of residues left in the field and availability of mineral nutrients to subsequent crops. To address questions about decomposition processes, a 2‐year experiment was conducted to determine the chemistry and decomposition rate of aboveground residues of soybean (Glycine max (L.) Merr.) grown under reciprocal combinations of low and high concentrations of CO₂ and O₃ in open‐top field chambers. The CO₂ treatments were ambient (370 μmol mol^?1) and elevated (714 μmol mol^?1) levels (daytime 12 h averages). Ozone treatments were charcoal‐filtered air (21 nmol mol^?1) and nonfiltered air plus 1.5 times ambient O₃ (74 nmol mol^?1) 12 h day^?1. Elevated CO₂ increased aboveground postharvest residue production by 28–56% while elevated O₃ suppressed it by 15–46%. In combination, inhibitory effects of added O₃ on biomass production were largely negated by elevated CO₂. Plant residue chemistry was generally unaffected by elevated CO₂, except for an increase in leaf residue lignin concentration. Leaf residues from the elevated O₃ treatments had lower concentrations of nonstructural carbohydrates, but higher N, fiber, and lignin levels. Chemical composition of petiole, stem, and pod husk residues was only marginally affected by the elevated gas treatments. Treatment effects on plant biomass production, however, influenced the content of chemical constituents on an areal basis. Elevated CO₂ increased the mass per square meter of nonstructural carbohydrates, phenolics, N, cellulose, and lignin by 24–46%. Elevated O₃ decreased the mass per square meter of these constituents by 30–48%, while elevated CO₂ largely ameliorated the added O₃ effect. Carbon mineralization rates of component residues from the elevated gas treatments were not significantly different from the control. However, N immobilization increased in soils containing petiole and stem residues from the elevated CO₂, O₃, and combined gas treatments. Mass loss of decomposing leaf residue from the added O₃ and combined gas treatments was 48% less than the control treatment after 20 weeks, while differences in decomposition of petiole, stem, and husk residues among treatments were minor. Decreased decomposition of leaf residues was correlated with lower starch and higher lignin levels. However, leaf residues only comprised about 20% of the total residue biomass assayed so treatment effects on mass loss of total aboveground residues were relatively small. The primary influence of elevated atmospheric CO₂ and O₃ concentrations on decomposition processes is apt to arise from effects on residue mass input, which is increased by elevated CO₂ and suppressed by O₃.     

The effects of reduced and elevated CO2 and O2 on the seaweed Lomentaria articulata

We grew a non-bicarbonate using red seaweed, Lomentaria articulata (Huds.) Lyngb., in media aerated with four O₂ concentrations between 10 and 200% of current ambient [O₂] and four CO₂ concentrations between 67 and 500% of current ambient [CO₂], in a factorial design, to determine the effects of gas composition on growth and physiology. The relative growth rate of L. articulata increased with increasing [CO₂] up to 200% of current ambient [CO₂] but was unaffected by [O₂]. The relative growth enhancement, on a carbon basis, was 52% with a doubling of [CO₂] but fell to 23% under 5× ambient [CO₂]. Plants collected in winter responded more extremely to [CO₂] than did plants collected in the summer, although the overall pattern was the same. Discrimination between stable carbon isotopes (Δ¹³C) increased with increasing [CO₂] as would be expected for diffusive CO₂ acquisition. Tissue C and N were inversely related to [CO₂]. Growth in terms of biomass appeared to be limited by conversion of photosynthate to new biomass rather than simply by diffusion of CO₂, suggesting that non-bicarbonate-using macroalgae, such as L. articulata, may not be directly analogous to C3 higher plants in terms of their responses to changing gas composition.     

Interannual climatic variation mediates elevated CO2 and O3 effects on forest growth

We analyzed growth data from model aspen (Populus tremuloides Michx.) forest ecosystems grown in elevated atmospheric carbon dioxide ([CO₂]; 518 μL L^?1) and ozone concentrations ([O₃]; 1.5 × background of 30–40 nL L^?1 during daylight hours) for 7 years using free‐air CO₂ enrichment technology to determine how interannual variability in present‐day climate might affect growth responses to either gas. We also tested whether growth effects of those gasses were sustained over time. Elevated [CO₂] increased tree heights, diameters, and main stem volumes by 11%, 16%, and 20%, respectively, whereas elevated ozone [O₃] decreased them by 11%, 8%, and 29%, respectively. Responses similar to these were found for stand volume and basal area. There were no growth responses to the combination of elevated [CO₂+O₃]. The elevated [CO₂] growth stimulation was found to be decreasing, but relative growth rates varied considerably from year to year. Neither the variation in annual relative growth rates nor the apparent decline in CO₂ growth response could be explained in terms of nitrogen or water limitations. Instead, growth responses to elevated [CO₂] and [O₃] interacted strongly with present‐day interannual variability in climatic conditions. The amount of photosynthetically active radiation and temperature during specific times of the year coinciding with growth phenology explained 20–63% of the annual variation in growth response to elevated [CO₂] and [O₃]. Years with higher photosynthetic photon flux (PPF) during the month of July resulted in more positive growth responses to elevated [CO₂] and more negative growth responses to elevated [O₃]. Mean daily temperatures during the month of October affected growth in a similar fashion the following year. These results indicate that a several‐year trend of increasingly cloudy summers and cool autumns were responsible for the decrease in CO₂ growth response.     

Influence of O2 availability on NO and N2O release by nitrification and denitrification in soils

The availability of O₂ is believed to be one of the main factors regulating nitrification and denitrification and the release of NO and N₂O. The availability of O₂ in soil is controlled by the O₂ partial pressure in the gas phase and by the moisture content in the soil. Therefore, we investigated the influence of O₂ partial pressures and soil moisture contents on the NO and N₂O release in a sandy and a loamy silt and differentiated between nitrification and denitrification by selective inhibition of nitrification with 10 Pa acetylene. At 60% whc (maximum water holding capacity) NO and N₂O release by denitrification increased with decreasing O₂ partial pressure and reached a maximum under anoxic conditions. Under anoxic conditions NO and N₂O were only released by denitrification. NO and N₂O release by nitrification also increased with decreasing O₂ partial pressure, but reached a maximum at 0.1–0.5% O₂ and then decreased again. Nitrification was the main source of NO and N₂O at O₂ partial pressures higher than 0.1–0.5% O₂. At lower O₂ partial pressures denitrification was the main source of NO and N₂O. With decreasing O₂ partial pressure N₂O release increased more than NO release, indicating that the N₂O release was more sensitive against O₂ than the NO release. At ambient O₂ partial pressure (20.5% O₂) NO and N₂O release by denitrification increased with increasing soil moisture content. The maximum NO and N₂O release was observed at soil moisture contents of 65–80% whc and 100% whc, respectively. NO and N₂O release by nitrification also increased with increasing soil moisture content with a maximum at 45–55% whc and 90% whc, respectively. Nitrification was the main source of NO and N₂O at soil moisture contents lower than 90% whc and 80% whc, respectively. Higher soil moisture contents favoured NO and N₂O release by denitrification. Soil texture had also an effect on the release of NO and N₂O. The coarse-textured sandy silt released more NO than N₂O compared with the fine-textured loamy silt. At high soil moisture contents (80–100% whc) the fine-textured soil showed a higher N₂O release by denitrification than the coarse-textured soil. We assume that the fine-textured soil became anoxic at a lower soil moisture content than the coarse-textured soil. In conclusion, the effects of O₂ partial pressure, soil moisture and soil texture were consistent with the theory that denitrification increasingly contributes to the release of NO and in particular N₂O when conditions for soil microorganisms become increasingly anoxic.     

Aphid individual performance may not predict population responses to elevated CO2 or O3

Changes in atmospheric composition affect plant quality and herbivore performance. We used the Aspen Free Air CO₂ Enrichment (FACE) facility to investigate the impacts of elevated carbon dioxide (CO₂) and ozone (O₃) on the performance of the aphid Cepegillettea betulaefoliae Granovsky feeding on paper birch (Betula papyrifera Marsh.). In Year 1, we simultaneously measured individual performance and population growth rates, and in Year 2 we surveyed natural aphid, predator and parasitoid populations throughout the growing season. Aphid growth and development (relative growth rate (RGR), development time, adult weight, embryo number and the birth weight of newborn nymphs) were unaffected by CO₂ and O₃. Aphid fecundity decreased on trees grown at elevated CO₂, O₃ and CO₂+O₃. Neither nymphal performance nor adult size were reliable indicators of future fecundity at elevated CO₂ and/or O₃. Aphid populations protected from natural enemies were unaffected by elevated CO₂, but increased significantly at elevated O₃. Individual fecundity in elevated CO₂ and O₃ atmospheres did not predict population growth rates, probably because of changes in the strength of intraspecific competition or the ability of the aphids to induce nutrient sinks. Natural aphid, predator and parasitoids populations (Year 2) showed few significant responses to CO₂ and O₃, although CO₂ and O₃ did affect the timing of aphid and natural enemy peak abundance. Elevated CO₂ and O₃ affected aphid and natural enemy populations independently: no CO₂× O₃ interactions were observed. We conclude that: (1) aphid individual performance did not predict population responses to CO₂ and O₃ and (2) elevated CO₂ and O₃ atmospheres are unlikely to affect C. betulaefoliae populations in the presence of natural enemy communities.