Contribution of Larvae Developing on Corn and Dry Beans to the Adult Population of Western Bean Cutworm in Michigan.

The western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is historically a pest of both corn (Zea mays L. (Poales: Poaceae)) and dry beans (Phaseolus sp. L. (Fabales: Fabaceae)) in the western Great Plains. However, it has recently undergone an eastward range expansion establishing itself across the Corn Belt in 25 states and 4 Canadian provinces. To mitigate the effects of infestation in Michigan, foliar insecticides are used in dry beans, whereas management of the pest in corn relies more heavily on the use of Bt-expressing hybrids. In this study stable carbon isotope analysis was used to determine what crop adult moths developed on as larvae with analysis showing that very few of the adult moths developed on dry beans. These results suggest that beans and corn are not suitable as co-refuges and that mainly adults which developed on corn are contributing to the next generation of western bean cutworm in Michigan.

Stomatal Development and Conductance of a Tropical Forage Legume Are Regulated by Elevated [CO2] Under Moderate Warming.

The opening and closing of stomata are controlled by the integration of environmental and endogenous signals. Here, we show the effects of combining elevated atmospheric carbon dioxide concentration (eCO 2; 600 µmol mol-1) and warming (+2°C) on stomatal properties and their consequence to plant function in a Stylosanthes capitata Vogel (C3) tropical pasture. The eCO 2 treatment alone reduced stomatal density, stomatal index, and stomatal conductance (gs ), resulting in reduced transpiration, increased leaf temperature, and leading to maintenance of soil moisture during the growing season. Increased CO2 concentration inside leaves stimulated photosynthesis, starch content levels, water use efficiency, and PSII photochemistry. Under warming, plants developed leaves with smaller stomata on both leaf surfaces; however, we did not see effects of warming on stomatal conductance, transpiration, or leaf water status. Warming alone enhanced PSII photochemistry and photosynthesis, and likely starch exports from chloroplasts. Under the combination of warming and eCO 2, leaf temperature was higher than that of leaves from the warming or eCO 2 treatments. Thus, warming counterbalanced the effects of CO2 on transpiration and soil water content but not on stomatal functioning, which was independent of temperature treatment. Under warming, and in combination with eCO 2, leaves also produced more carotenoids and a more efficient heat and fluorescence dissipation. Our combined results suggest that control on stomatal opening under eCO 2 was not changed by a warmer environment; however, their combination significantly improved whole-plant functioning.

Patch use in the arctic ground squirrel: effects of micro-topography and shrub encroachment in the Arctic Circle.

We investigated the roles of vegetation structure, micro-topographic relief, and predator activity patterns (time of day) on the perception of predatory risk of arctic ground squirrels (Urocitellus parryii), an abundant pan-Arctic omnivore, in Arctic Circle tundra on the North Slope of Alaska, where tundra vegetation structure has been predicted to change in response to climate. We quantified foraging intensity by measuring the giving-up densities (GUDs) of the arctic ground squirrels in experimental foraging patches along a heath-graminoid-shrub moist tundra gradient. We hypothesized that foraging intensity of arctic ground squirrels would be greatest and GUDs lowest, where low-stature vegetation or raised micro-topography improves sightlines for predator detection. Furthermore, GUDs should vary with time of day and reflect 24-h cycles of varying predation risk. Foraging intensity varied temporally, being highest in the afternoon and lowest overnight. During the morning, foraging intensity was inversely correlated with the normalized difference vegetation index (NDVI), a proxy for vegetation productivity and cover. Foraging was additionally measured within landscapes of fear, confirming that vegetative and topographic obstructions of sightlines reduces foraging intensity and increases GUDs. We conclude that arctic ground squirrels may affect Arctic Circle vegetation of tundra ecosystems, but these effects will vary spatially and temporally.

Does elevated atmospheric CO2affect soil carbon burial and soil weathering in a forest ecosystem?

Most experimental studies measuring the effects of climate change on terrestrial C cycling have focused on processes that occur at relatively short time scales (up to a few years). However, climate-soil C interactions are influenced over much longer time scales by bioturbation and soil weathering affecting soil fertility, ecosystem productivity, and C storage. Elevated CO2can increase belowground C inputs and stimulate soil biota, potentially affecting bioturbation, and can decrease soil pH which could accelerate soil weathering rates. To determine whether we could resolve any changes in bioturbation or C storage, we investigated soil profiles collected from ambient and elevated-CO2plots at the Free-Air Carbon-Dioxide Enrichment (FACE) forest site at Oak Ridge National Laboratory after 11 years of 13C-depleted CO2 release. Profiles of organic carbon concentration, δ13C values, and activities of 137Cs, 210Pb, and 226Ra were measured to ∼30 cm depth in replicated soil cores to evaluate the effects of elevated CO2 on these parameters. Bioturbation models based on fitting advection-diffusion equations to 137Cs and 210Pb profiles showed that ambient and elevated-CO2 plots had indistinguishable ranges of apparent biodiffusion constants, advection rates, and soil mixing times, although apparent biodiffusion constants and advection rates were larger for 137Cs than for 210Pb as is generally observed in soils. Temporal changes in profiles of δ13C values of soil organic carbon (SOC) suggest that addition of new SOC at depth was occurring at a faster rate than that implied by the net advection term of the bioturbation model. Ratios of (210Pb/226Ra) may indicate apparent soil mixing cells that are consistent with biological mechanisms, possibly earthworms and root proliferation, driving C addition and the mixing of soil between ∼4 cm and ∼18 cm depth. Burial of SOC by soil mixing processes could substantially increase the net long-term storage of soil C and should be incorporated in soil-atmosphere interaction models.

Experimental Air Warming of a Stylosanthes capitata, Vogel Dominated Tropical Pasture Affects Soil Respiration and Nitrogen Dynamics.

Warming due to global climate change is predicted to reach 2°C in tropical latitudes. There is an alarming paucity of information regarding the effects of air temperature on tropical agroecosystems, including foraging pastures. Here, we investigated the effects of a 2°C increase in air temperature over ambient for 30 days on an established tropical pasture (Ribeirão Preto, São Paulo, Brazil) dominated by the legume Stylosanthes capitata Vogel, using a T-FACE (temperature free-air controlled enhancement) system. We tested the effects of air warming on soil properties [carbon (C), nitrogen (N), and their stable isotopic levels (δ13C and δ15N), as well as soil respiration and soil enzymatic activity] and aboveground characteristics (foliar C, N, δ13C, δ15N, leaf area index, and aboveground biomass) under field conditions. Results show that experimental air warming moderately increased soil respiration rates compared to ambient temperature. Soil respiration was positively correlated with soil temperature and moisture during mid-day (when soil respiration was at its highest) but not at dusk. Foliar δ13C were not different between control and elevated temperature treatments, indicating that plants grown in warmed plots did not show the obvious signs of water stress often seen in warming experiments. The 15N isotopic composition of leaves from plants grown at elevated temperature was lower than in ambient plants, suggesting perhaps a higher proportion of N-fixation contributing to tissue N in warmed plants when compared to ambient ones. Soil microbial enzymatic activity decreased in response to the air warming treatment, suggesting a slower decomposition of organic matter under elevated air temperature conditions. Decreased soil enzyme capacity and increases in soil respiration and plant biomass in plots exposed to high temperature suggest that increased root activity may have caused the increase seen in soil respiration in this tropical pasture. This response along with rapid changes in soil and plant 15N may differ from what has been shown in temperate grasslands.

Winter precipitation and snow accumulation drive the methane sink or source strength of Arctic tussock tundra.

Arctic winter precipitation is projected to increase with global warming, but some areas will experience decreases in snow accumulation. Although Arctic CH4 emissions may represent a significant climate forcing feedback, long-term impacts of changes in snow accumulation on CH4 fluxes remain uncertain. We measured ecosystem CH4 fluxes and soil CH4 and CO2 concentrations and (13) C composition to investigate the metabolic pathways and transport mechanisms driving moist acidic tundra CH4 flux over the growing season (Jun-Aug) after 18 years of experimental snow depth increases and decreases. Deeper snow increased soil wetness and warming, reducing soil %O2 levels and increasing thaw depth. Soil moisture, through changes in soil %O2 saturation, determined predominance of methanotrophy or methanogenesis, with soil temperature regulating the ecosystem CH4 sink or source strength. Reduced snow (RS) increased the fraction of oxidized CH4 (Fox) by 75-120% compared to Ambient, switching the system from a small source to a net CH4 sink (21 ± 2 and -31 ± 1 mg CH4  m(-2)  season(-1) at Ambient and RS). Deeper snow reduced Fox by 35-40% and 90-100% in medium- (MS) and high- (HS) snow additions relative to Ambient, contributing to increasing the CH4 source strength of moist acidic tundra (464 ± 15 and 3561 ± 97 mg CH4  m(-2)  season(-1) at MS and HS). Decreases in Fox with deeper snow were partly due to increases in plant-mediated CH4 transport associated with the expansion of tall graminoids. Deeper snow enhanced CH4 production within newly thawed soils, responding mainly to soil warming rather than to increases in acetate fermentation expected from thaw-induced increases in SOC availability. Our results suggest that increased winter precipitation will increase the CH4 source strength of Arctic tundra, but the resulting positive feedback on climate change will depend on the balance between areas with more or less snow accumulation than they are currently facing.

The world's biomes and primary production as a triple tragedy of the commons foraging game played among plants.

Plants appear to produce an excess of leaves, stems and roots beyond what would provide the most efficient harvest of available resources. One way to understand this overproduction of tissues is that excess tissue production provides a competitive advantage. Game theoretic models predict overproduction of all tissues compared with non-game theoretic models because they explicitly account for this indirect competitive benefit. Here, we present a simple game theoretic model of plants simultaneously competing to harvest carbon and nitrogen. In the model, a plant's fitness is influenced by its own leaf, stem and root production, and the tissue production of others, which produces a triple tragedy of the commons. Our model predicts (i) absolute net primary production when compared with two independent global datasets; (ii) the allocation relationships to leaf, stem and root tissues in one dataset; (iii) the global distribution of biome types and the plant functional types found within each biome; and (iv) ecosystem responses to nitrogen or carbon fertilization. Our game theoretic approach removes the need to define allocation or vegetation type a priori but instead lets these emerge from the model as evolutionarily stable strategies. We believe this to be the simplest possible model that can describe plant production.

Responses of temperate forest productivity to insect and pathogen disturbances.

Pest and pathogen disturbances are ubiquitous across forest ecosystems, impacting their species composition, structure, and function. Whereas severe abiotic disturbances (e.g., clear-cutting and fire) largely reset successional trajectories, pest and pathogen disturbances cause diffuse mortality, driving forests into nonanalogous system states. Biotic perturbations that disrupt forest carbon dynamics either reduce or enhance net primary production (NPP) and carbon storage, depending on pathogen type. Relative to defoliators, wood borers and invasive pests have the largest negative impact on NPP and the longest recovery time. Forest diversity is an important contributing factor to productivity: NPP is neutral, marginally enhanced, or reduced in high-diversity stands in which a small portion of the canopy is affected (temperate deciduous or mixed forests) but very negative in low-diversity stands in which a large portion of the canopy is affected (western US forests). Pests and pathogens reduce forest structural and functional redundancy, affecting their resilience to future climate change or new outbreaks. Therefore, pests and pathogens can be considered biotic forcing agents capable of causing consequences of similar magnitude to climate forcing factors.

Mechanistic insights on the responses of plant and ecosystem gas exchange to global environmental change: lessons from Biosphere 2.

Scaling up leaf processes to canopy/ecosystem level fluxes is critical for examining feedbacks between vegetation and climate. Collectively, studies from Biosphere 2 Laboratory have provided important insight of leaf-to-ecosystem investigations of multiple environmental parameters that were not before possible in enclosed or field studies. B2L has been a testing lab for the applicability of new technologies such as spectral approaches to detect spatial and temporal changes in photosynthesis within canopies, or for the development of cavity ring-down isotope applications for ecosystem evapotranspiration. Short and long term changes in atmospheric CO2, drought or temperature allowed for intensive investigation of the interactions between photosynthesis and leaf, soil and ecosystem respiration. Experiments conducted in the rainforest biome have provided some of the most comprehensive dataset to date on the effects of climate change variables on tropical ecosystems. Results from these studies have been later corroborated in natural rainforest ecosystems and have improved the predictive capabilities of models that now show increased resilience of tropics to climate change. Studies of temperature and CO2 effects on ecosystem respiration and its leaf and soil components have helped reconsider the use of simple first-order kinetics for characterizing respiration in models. The B2L also provided opportunities to quantify the rhizosphere priming effect, or establish the relationships between net primary productivity, atmospheric CO2 and isoprene emissions.

Synthesis and modeling perspectives of rhizosphere priming.

The rhizosphere priming effect (RPE) is a mechanism by which plants interact with soil functions. The large impact of the RPE on soil organic matter decomposition rates (from 50% reduction to 380% increase) warrants similar attention to that being paid to climatic controls on ecosystem functions. Furthermore, global increases in atmospheric CO2 concentration and surface temperature can significantly alter the RPE. Our analysis using a game theoretic model suggests that the RPE may have resulted from an evolutionarily stable mutualistic association between plants and rhizosphere microbes. Through model simulations based on microbial physiology, we demonstrate that a shift in microbial metabolic response to different substrate inputs from plants is a plausible mechanism leading to positive or negative RPEs. In a case study of the Duke Free-Air CO2 Enrichment experiment, performance of the PhotoCent model was significantly improved by including an RPE-induced 40% increase in soil organic matter decomposition rate for the elevated CO2 treatment--demonstrating the value of incorporating the RPE into future ecosystem models. Overall, the RPE is emerging as a crucial mechanism in terrestrial ecosystems, which awaits substantial research and model development.

Ecosystem-level controls on root-rhizosphere respiration.

Recent advances in the partitioning of autotrophic from heterotrophic respiration processes in soils in conjunction with new high temporal resolution soil respiration data sets offer insights into biotic and environmental controls of respiration. Besides temperature, many emerging controlling factors have not yet been incorporated into ecosystem-scale models. We synthesize recent research that has partitioned soil respiration into its process components to evaluate effects of nitrogen, temperature and photosynthesis on autotrophic flux from soils at the ecosystem level. Despite the widely used temperature dependence of root respiration, gross primary productivity (GPP) can explain most patterns of ecosystem root respiration (and to some extent heterotrophic respiration) at within-season time-scales. Specifically, heterotrophi crespiration is influenced by a seasonally variable supply of recent photosynthetic products in the rhizosphere. The contribution of stored root carbon (C) to root respiratory fluxes also varied seasonally, partially decoupling the proportion of photosynthetic C driving root respiration. In order to reflect recent insights, new hierarchical models, which incorporate root respiration as a primary function of GPP and which respond to environmental variables by modifying Callocation belowground, are needed for better prediction of future ecosystem C sequestration.

Stored carbon partly fuels fine-root respiration but is not used for production of new fine roots.

The relative use of new photosynthate compared to stored carbon (C) for the production and maintenance of fine roots, and the rate of C turnover in heterogeneous fine-root populations, are poorly understood. We followed the relaxation of a (13)C tracer in fine roots in a Liquidambar styraciflua plantation at the conclusion of a free-air CO(2) enrichment experiment. Goals included quantifying the relative fractions of new photosynthate vs stored C used in root growth and root respiration, as well as the turnover rate of fine-root C fixed during [CO(2)] fumigation. New fine-root growth was largely from recent photosynthate, while nearly one-quarter of respired C was from a storage pool. Changes in the isotopic composition of the fine-root population over two full growing seasons indicated heterogeneous C pools; < 10% of root C had a residence time < 3 months, while a majority of root C had a residence time > 2 yr. Compared to a one-pool model, a two-pool model for C turnover in fine roots (with 5 and 0.37 yr(-1) turnover times) doubles the fine-root contribution to forest NPP (9-13%) and supports the 50% root-to-soil transfer rate often used in models.

Plasticity in bundle sheath extensions of heterobaric leaves.

PREMISE OF THE STUDY: Leaf venation is linked to physiological performance, playing a critical role in ecosystem function. Despite the importance of leaf venation, associated bundle sheath extensions (BSEs) remain largely unstudied. Here, we quantify plasticity in the spacing of BSEs over irradiance and precipitation gradients. Because physiological function(s) of BSEs remain uncertain, we additionally explored a link between BSEs and water use efficiency (WUE). METHODS: We sampled leaves of heterobaric trees along intracrown irradiance gradients in natural environments and growth chambers and correlated BSE spacing to incident irradiance. Additionally, we sampled leaves along a precipitation gradient and correlated BSE spacing to precipitation and bulk δ(13)C, a proxy for intrinsic WUE. BSE spacing was quantified using a novel semiautomatic method on fresh leaf tissue. KEY RESULTS: With increased irradiance or decreased precipitation, Liquidambar styraciflua decreased BSE spacing, while Acer saccharum showed little variation in BSE spacing. Two additional species, Quercus robur and Platanus occidentalis, decreased BSE spacing with increased irradiance in growth chambers. BSE spacing correlated with bulk δ(13)C, a proxy for WUE in L. styraciflua, Q. robur, and P. occidentalis leaves but not in leaves of A. saccharum. CONCLUSIONS: We demonstrated that BSE spacing is plastic with respect to irradiance or precipitation and independent from veins, indicating BSE involvement in leaf adaptation to a microenvironment. Plasticity in BSE spacing was correlated with WUE only in some species, not supporting a function in water relations. We discuss a possible link between BSE plasticity and life history, particularly canopy position.

Plastic and adaptive responses of plant respiration to changes in atmospheric CO(2) concentration.

The concentration of atmospheric CO2 has increased from below 200 microl l(-1) during last glacial maximum in the late Pleistocene to near 280 microl l(-1) at the beginning of the Holocene and has continuously increased since the onset of the industrial revolution. Most responses of plants to increasing atmospheric CO2 levels result in increases in photosynthesis, water use efficiency and biomass. Less known is the role that respiration may play during adaptive responses of plants to changes in atmospheric CO2. Although plant respiration does not increase proportionally with CO2-enhanced photosynthesis or growth rates, a reduction in respiratory costs in plants grown at subambient CO2 can aid in maintaining a positive plant C-balance (i.e. enhancing the photosynthesis-to-respiration ratio). The understanding of plant respiration is further complicated by the presence of the alternative pathway that consumes photosynthate without producing chemical energy [adenosine triphosphate (ATP)] as effectively as respiration through the normal cytochrome pathway. Here, we present the respiratory responses of Arabidopsis thaliana plants selected at Pleistocene (200 microl l(-1)), current Holocene (370 microl l(-1)), and elevated (700 microl l(-1)) concentrations of CO2 and grown at current CO2 levels. We found that respiration rates were lower in Pleistocene-adapted plants when compared with Holocene ones, and that a substantial reduction in respiration was because of reduced activity of the alternative pathway. In a survey of the literature, we found that changes in respiration across plant growth forms and CO2 levels can be explained in part by differences in the respiratory energy demand for maintenance of biomass. This trend was substantiated in the Arabidopsis experiment in which Pleistocene-adapted plants exhibited decreases in respiration without concurrent reductions in tissue N content. Interestingly, N-based respiration rates of plants adapted to elevated CO2 also decreased. As a result, ATP yields per unit of N increased in Pleistocene-adapted plants compared with current CO2 adapted ones. Our results suggest that mitochondrial energy coupling and alternative pathway-mediated responses of respiration to changes in atmospheric CO2 may enhance survival of plants at low CO2 levels to help overcome a low carbon balance. Therefore, increases in the basal activity of the alternative pathway are not necessarily associated to metabolic plant stress in all cases.

The effect of carbon dioxide enrichment on apparent stem respiration from Pinus taeda L. is confounded by high levels of soil carbon dioxide.

Respiration supports growth and maintenance processes and returns a substantial portion of the CO(2) fixed by photosynthesis to the atmosphere each year. Investigating stem respiration using CO(2) flux measurements is complicated by uncertainty surrounding the source of CO(2) diffusing from tree stems. Over 2 years we measured the stem efflux from 24 trees exposed to ambient or elevated CO(2). The rate of stem CO(2) efflux increased with annual tree diameter increment and the estimated uptake of dissolved CO(2) from the soil. To determine the source of CO(2) diffusing from tree stems, we used the fumigation gas at the Duke Forest Atmosphere Carbon Transfer and Storage-1 elevated-CO(2) experiment as a (13)C tracer and measured the presence of soil CO(2) in stem efflux on a subset of these trees. The isotopic composition of soil CO(2) explained a considerable portion of the variation in the composition of CO(2) in stem efflux. We also found that direct measurements of the isotopic composition of phloem-respired CO(2), unlike the CO(2) found in stem efflux, was less variable and distinct from the isotopic composition of soil CO(2). Tree growth rates and soil CO(2) concentrations found at the site together explained 56% of the variance in stem CO(2) efflux among trees. These results suggest that the uptake of CO(2) dissolved in soil water and transported through the vascular system can potentially confound efforts to interpret stem efflux measurements in trees exposed to elevated CO(2) and that previous studies may have overestimated the effects of elevated CO(2) on autotrophic respiration in tree stems.

Changes in respiratory mitochondrial machinery and cytochrome and alternative pathway activities in response to energy demand underlie the acclimation of respiration to elevated CO2 in the invasive Opuntia ficus-indica.

Studies on long-term effects of plants grown at elevated CO(2) are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO(2), the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO(2) concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO(2) during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO(2) also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO(2), the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO(2). Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO(2), the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO(2) suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO(2). However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO(2), total mitochondrial ATP production was decreased by plant growth at elevated CO(2) when compared to ambient-grown plants. Because plant growth at elevated CO(2) increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O(2) consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO(2) results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested.

Plant respiration and elevated atmospheric CO2 concentration: cellular responses and global significance.

BACKGROUND: Elevated levels of atmospheric [CO2] are likely to enhance photosynthesis and plant growth, which, in turn, should result in increased specific and whole-plant respiration rates. However, a large body of literature has shown that specific respiration rates of plant tissues are often reduced when plants are exposed to, or grown at, high [CO2] due to direct effects on enzymes and indirect effects derived from changes in the plant's chemical composition. SCOPE: Although measurement artefacts may have affected some of the previously reported effects of CO2 on respiration rates, the direction and magnitude for the effects of elevated [CO2] on plant respiration may largely depend on the vertical scale (from enzymes to ecosystems) at which measurements are taken. In this review, the effects of elevated [CO2] from cells to ecosystems are presented within the context of the enzymatic and physiological controls of plant respiration, the role(s) of non-phosphorylating pathways, and possible effects associated with plant size. CONCLUSIONS: Contrary to what was previously thought, specific respiration rates are generally not reduced when plants are grown at elevated [CO2]. However, whole ecosystem studies show that canopy respiration does not increase proportionally to increases in biomass in response to elevated [CO2], although a larger proportion of respiration takes place in the root system. Fundamental information is still lacking on how respiration and the processes supported by it are physiologically controlled, thereby preventing sound interpretations of what seem to be species-specific responses of respiration to elevated [CO2]. Therefore the role of plant respiration in augmenting the sink capacity of terrestrial ecosystems is still uncertain.

Impacts of fine root turnover on forest NPP and soil C sequestration potential.

Estimates of forest net primary production (NPP) demand accurate estimates of root production and turnover. We assessed root turnover with the use of an isotope tracer in two forest free-air carbon dioxide enrichment experiments. Growth at elevated carbon dioxide did not accelerate root turnover in either the pine or the hardwood forest. Turnover of fine root carbon varied from 1.2 to 9 years, depending on root diameter and dominant tree species. These long turnover times suggest that root production and turnover in forests have been overestimated and that sequestration of anthropogenic atmospheric carbon in forest soils may be lower than currently estimated.

Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis.

The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP. Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other alpha-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of alpha-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.

Role of sugars and organic acids in regulating the concentration and activity of the alternative oxidase in Poa annua roots.

Detached roots of Poa annua were used to study alternative oxidase protein expression upon the addition of sucrose, glucose, fructose, inositol, mannitol, citrate or malate, at a concentration of 1 or 10 mM for 24 h. After 24 h the capacity of cytochrome c oxidase was decreased equally in all treatments. Only citrate induced the expression of the alternative oxidase, especially at a concentration of 1 mM (15-fold). The activity of the alternative pathway (measured with the (18)O-fractionation technique) was not affected by the addition of sucrose for 24 h as compared with time zero. However, after the addition of citrate or mannitol the activity of the alternative pathway decreased to almost zero. The discrepancy between the large increase in alternative oxidase protein concentration when citrate was applied and the concomitant decrease in alternative pathway activity is discussed.