Arbuscular mycorrhizal fungi increase organic carbon decomposition under elevated CO2.

The extent to which terrestrial ecosystems can sequester carbon to mitigate climate change is a matter of debate. The stimulation of arbuscular mycorrhizal fungi (AMF) by elevated atmospheric carbon dioxide (CO(2)) has been assumed to be a major mechanism facilitating soil carbon sequestration by increasing carbon inputs to soil and by protecting organic carbon from decomposition via aggregation. We present evidence from four independent microcosm and field experiments demonstrating that CO(2) enhancement of AMF results in considerable soil carbon losses. Our findings challenge the assumption that AMF protect against degradation of organic carbon in soil and raise questions about the current prediction of terrestrial ecosystem carbon balance under future climate-change scenarios.

Dissecting Arabidopsis Gß signal transduction on the protein surface.

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gß-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gß-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits.

Field assessment of a snap bean ozone bioindicator system under elevated ozone and carbon dioxide in a free air system.

Ozone-sensitive (S156) and -tolerant (R123 and R331) genotypes of snap bean (Phaseolus vulgaris L.) were tested as a plant bioindicator system for detecting O(3) effects at current and projected future levels of tropospheric O(3) and atmospheric CO(2) under field conditions. Plants were treated with ambient air, 1.4× ambient O(3) and 550 ppm CO(2) separately and in combination using Free Air Concentration Enrichment technology. Under ambient O(3) concentrations pod yields were not significantly different among genotypes. Elevated O(3) reduced pod yield for S156 (63%) but did not significantly affect yields for R123 and R331. Elevated CO(2) at 550 ppm alone did not have a significant impact on yield for any genotype. Amelioration of the O(3) effect occurred in the O(3) + CO(2) treatment. Ratios of sensitive to tolerant genotype pod yields were identified as a useful measurement for assessing O(3) impacts with potential applications in diverse settings including agricultural fields.

Re-evaluating the role of ascorbic acid and phenolic glycosides in ozone scavenging in the leaf apoplast of Arabidopsis thaliana L.

Phenolic glycosides are effective reactive oxygen scavengers and peroxidase substrates, suggesting that compounds in addition to ascorbate may have functional importance in defence responses against ozone (O(3)), especially in the leaf apoplast. The apoplastic concentrations of ascorbic acid (AA) and phenolic glycosides in Arabidopsis thaliana L. Col-0 wild-type plants were determined following exposure to a range of O(3) concentrations (5, 125 or 175 nL L(-1)) in controlled environment chambers. AA in leaf apoplast extracts was almost entirely oxidized in all treatments, suggesting that O(3) scavenging by direct reactions with reduced AA was very limited. In regard to phenolics, O(3) stimulated transcription of numerous phenylpropanoid pathway genes and increased the apoplastic concentration of sinapoyl malate. However, modelling of O(3) scavenging in the apoplast indicated that sinapoyl malate concentrations were too low to be effective protectants. Furthermore, null mutants for sinapoyl esters (fah1-7), kaempferol glycosides (tt4-1) and the double mutant (tt4-1/fah1-7) were equally sensitive to chronic O(3) as Ler-0 wild-type plants. These results indicate that current understanding of O(3) defence schemes deserves reassessment as mechanisms other than direct scavenging of O(3) by extracellular AA and antioxidant activity of some phenolics may predominate in some plant species.

Digestive utilization of ozone-exposed forage by rabbits (Oryctolagus cuniculus).

A mixture of common Southern Piedmont (USA) grassland species (Lolium arundinacea, Paspalum dilatatum, Cynodon dactylon and Trifolium repens) was exposed to O(3) [ambient (non-filtered; NF) and twice-ambient (2X) concentrations] and fed to individually caged New Zealand white rabbits (Oryctolagus cuniculus) in a digestibility experiment. Forages and feed refusals were analyzed for concentrations of total cell wall constituents, lignin, crude protein, and soluble and hydrolyzable phenolic fractions. Neutral detergent fiber and acid detergent fiber digestibility by rabbits were significantly lower for 2X than NF forage. Decreased digestibility could not be attributed to lignin concentrations, but was associated with increased concentrations of acid-hydrolyzable and saponifiable phenolics. Exposure of forage to elevated O(3) resulted in decreased digestible dry matter intake by rabbits. Elevated O(3) concentrations could be expected to have a negative impact on forage quality, resulting in decreased nutrient utilization by mammalian herbivores in Southern Piedmont grasslands under projected future climate scenarios.

Influence of atmospheric vapour pressure deficit on ozone responses of snap bean (Phaseolus vulgaris L.) genotypes.

Environmental conditions influence plant responses to ozone (O(3)), but few studies have evaluated individual factors directly. In this study, the effect of O(3) at high and low atmospheric vapour pressure deficit (VPD) was evaluated in two genotypes of snap bean (Phaseolus vulgaris L.) (R123 and S156) used as O(3) bioindicator plants. Plants were grown in outdoor controlled-environment chambers in charcoal-filtered air containing 0 or 60 nl l(-1) O(3) (12 h average) at two VPDs (1.26 and 1.96 kPa) and sampled for biomass, leaf area, daily water loss, and seed yield. VPD clearly influenced O(3) effects. At low VPD, O(3) reduced biomass, leaf area, and seed yield substantially in both genotypes, while at high VPD, O(3) had no significant effect on these components. In clean air, high VPD reduced biomass and yield by similar fractions in both genotypes compared with low VPD. Data suggest that a stomatal response to VPD per se may be lacking in both genotypes and it is hypothesized that the high VPD resulted in unsustainable transpiration and water deficits that resulted in reduced growth and yield. High VPD- and water-stress-induced stomatal responses may have reduced the O(3) flux into the leaves, which contributed to a higher yield compared to the low VPD treatment in both genotypes. At low VPD, transpiration increased in the O(3) treatment relative to the clean air treatment, suggesting that whole-plant conductance was increased by O(3) exposure. Ozone-related biomass reductions at low VPD were proportionally higher in S156 than in R123, indicating that differential O(3) sensitivity of these bioindicator plants remained evident when environmental conditions were conducive for O(3) effects. Assessments of potential O(3) impacts on vegetation should incorporate interacting factors such as VPD.

Ambient ozone effects on gas exchange and total non-structural carbohydrate levels in cutleaf coneflower (Rudbeckia laciniata L.) growing in Great Smoky Mountains National Park.

Ozone-sensitive and -tolerant individuals of cutleaf coneflower (Rudbeckia laciniata L.) were compared for their gas exchange characteristics and total non-structural carbohydrates at Purchase Knob, a high elevation site in Great Smoky Mountains National Park, USA. Photosynthesis and stomatal conductance decreased with increased foliar stipple. Sensitive plants had lower photosynthetic rates for all leaves, except the very youngest and oldest when compared to tolerant plants. Stomatal conductance decreased with increasing leaf age, but no ozone-sensitivity differences were found. Lower leaves had less starch than upper ones, while leaves on sensitive plants had less than those on tolerant plants. These results show that ambient levels of ozone in Great Smoky Mountains National Park can adversely affect gas exchange, water use efficiency and leaf starch content in sensitive coneflower plants. Persistence of sensitive genotypes in the Park may be due to physiological recovery in low ozone years.

Genetic effects on total phenolics, condensed tannins and non-structural carbohydrates in loblolly pine (Pinus taeda L.) needles.

Carbon allocation to soluble phenolics (total phenolics, proanthocyanidins (PA)) and total non-structural carbohydrates (TNC; starch and soluble sugars) in needles of widely planted, highly productive loblolly pine (Pinus taeda L.) genotypes could impact stand resistance to herbivory, and biogeochemical cycling in the southeastern USA. However, genetic and growth-related effects on loblolly pine needle chemistry are not well characterized. Therefore, we investigated genetic and growth-related effects on foliar concentrations of total phenolics, PA and TNC in two different field studies. The first study contained nine different genotypes representing a range of genetic homogeneity, growing in a 2-year-old plantation on the coastal plain of North Carolina (NC), USA. The second study contained eight clones with different growth potentials planted in a 9-year-old clonal trial replicated at two sites (Georgia (GA) and South Carolina (SC), USA). In the first study (NC), we found no genetic effects on total phenolics, PA and TNC, and there was no relationship between genotype size and foliar biochemistry. In the second study, there were no differences in height growth between sites, but the SC site showed greater diameter (diameter at breast height (DBH)) and volume, most likely due to greater tree mortality (lower stocking) which reduced competition for resources and increased growth of remaining trees. We found a significant site × clone effect for total phenolics with lower productivity clones showing 27-30% higher total phenolic concentrations at the GA site where DBH and volume were lower. In contrast to the predictions of growth-defense theory, clone volume was positively associated with total phenolic concentrations at the higher volume SC site, and PA concentrations at the lower volume GA site. Overall, we found no evidence of a trade-off between genotype size and defense, and genetic potential for improved growth may include increased allocation to some secondary metabolites. These results imply that deployment of more productive loblolly pine genotypes will not reduce stand resistance to herbivory, but increased production of total phenolics and PA associated with higher genotype growth potential could reduce litter decomposition rates and therefore, nutrient availability.

Soil microbial responses to elevated CO2 and O3 in a nitrogen-aggrading agroecosystem.

Climate change factors such as elevated atmospheric carbon dioxide (CO2) and ozone (O3) can exert significant impacts on soil microbes and the ecosystem level processes they mediate. However, the underlying mechanisms by which soil microbes respond to these environmental changes remain poorly understood. The prevailing hypothesis, which states that CO2- or O3-induced changes in carbon (C) availability dominate microbial responses, is primarily based on results from nitrogen (N)-limiting forests and grasslands. It remains largely unexplored how soil microbes respond to elevated CO2 and O3 in N-rich or N-aggrading systems, which severely hinders our ability to predict the long-term soil C dynamics in agroecosystems. Using a long-term field study conducted in a no-till wheat-soybean rotation system with open-top chambers, we showed that elevated CO2 but not O3 had a potent influence on soil microbes. Elevated CO2(1.5×ambient) significantly increased, while O3 (1.4×ambient) reduced, aboveground (and presumably belowground) plant residue C and N inputs to soil. However, only elevated CO2 significantly affected soil microbial biomass, activities (namely heterotrophic respiration) and community composition. The enhancement of microbial biomass and activities by elevated CO2 largely occurred in the third and fourth years of the experiment and coincided with increased soil N availability, likely due to CO2-stimulation of symbiotic N2 fixation in soybean. Fungal biomass and the fungi∶bacteria ratio decreased under both ambient and elevated CO2 by the third year and also coincided with increased soil N availability; but they were significantly higher under elevated than ambient CO2. These results suggest that more attention should be directed towards assessing the impact of N availability on microbial activities and decomposition in projections of soil organic C balance in N-rich systems under future CO2 scenarios.

Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2].

While exposure of C3 plants to elevated [CO2] would be expected to reduce production of reactive oxygen species (ROS) in leaves because of reduced photorespiratory metabolism, results obtained in the present study suggest that exposure of plants to elevated [CO2] can result in increased oxidative stress. First, in Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated [CO2]. In soybean, increased carbonyl content was often associated with loss of leaf chlorophyll and reduced enhancement of leaf photosynthetic rate (Pn) by elevated [CO2]. Second, two-dimensional (2-DE) difference gel electrophoresis (DIGE) analysis of proteins extracted from leaves of soybean plants grown at elevated [CO2] or [O3] revealed that both treatments altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. The 2-DE analysis of leaf proteins was facilitated by a novel and simple procedure to remove ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from soluble soybean leaf extracts. Collectively, these findings add a new dimension to our understanding of global change biology and raise the possibility that oxidative signals can be an unexpected component of plant response to elevated [CO2].

Optimizing the statistical estimation of the parameters of the Farquhar-von Caemmerer-Berry model of photosynthesis.

The model of Farquhar, von Caemmerer and Berry is the standard in relating photosynthetic carbon assimilation and concentration of intercellular CO(2). The techniques used in collecting the data from which its parameters are estimated have been the object of extensive optimization, but the statistical aspects of estimation have not received the same attention. The model segments assimilation into three regions, each modeled by a distinct function. Three parameters of the model, namely the maximum rate of Rubisco carboxylation (V(c max)), the rate of electron transport (J), and nonphotorespiratory CO(2) evolution (R(d)), are customarily estimated from gas exchange data through separate fitting of the component functions corresponding to the first two segments. This disjunct approach is problematic in requiring preliminary arbitrary subsetting of data into sets believed to correspond to each region. It is shown how multiple segments can be estimated simultaneously, using the entire data set, without predetermination of transitions by the investigator. Investigation of the number of parameters that can be estimated in the two-segment model suggests that, under some conditions, it is possible to estimate four or even five parameters, but that only V(c max), J, and R(d), have good statistical properties. Practical difficulties and their solutions are reviewed, and software programs are provided.

Leaf extracellular ascorbate in relation to O(3) tolerance of two soybean cultivars.

Soybean [Glycine max (L.) Merr.] cultivars Essex and Forrest that exhibit differences in ozone (O(3)) sensitivity were used in greenhouse experiments to investigate the role of leaf extracellular antioxidants in O(3) injury responses. Charcoal-filtered air and elevated O(3) conditions were used to assess genetic, leaf age, and O(3) effects. In both cultivars, the extracellular ascorbate pool consisted of 80-98% dehydroascorbic acid, the oxidized form of ascorbic acid (AA) that is not an antioxidant. For all combinations of genotype and O(3) treatments, extracellular AA levels were low (1-30nmolg(-1) FW) and represented 3-30% of the total antioxidant capacity. Total extracellular antioxidant capacity was twofold greater in Essex compared with Forrest, consistent with greater O(3) tolerance of Essex. The results suggest that extracellular antioxidant metabolites in addition to ascorbate contribute to detoxification of O(3) in soybean leaves and possibly affect plant sensitivity to O(3) injury.

The role of ozone flux and antioxidants in the suppression of ozone injury by elevated CO2 in soybean.

The projected rise in atmospheric CO2 concentration is expected to increase growth and yield of many agricultural crops. The magnitude of this stimulus will partly depend on interactions with other components of the atmosphere such as tropospheric O3. Elevated CO2 concentrations often lessen the deleterious effects of O3, but the mechanisms responsible for this response have received little direct examination. Previous studies have indicated that protection against O3 injury by elevated CO2 can be attributed to reduced O3 uptake, while other studies suggest that CO2 effects on anti-oxidant metabolism might also be involved. The aim of this experiment was to test further the roles of O3 flux and antioxidant metabolism in the suppression of O3 injury by elevated CO2. In a two-year experiment, soybean [Glycine max (L.) Merr.] was exposed from emergence to maturity to charcoal-filtered air or charcoal-filtered air plus a range of O3 concentrations in combination with ambient or approximately twice-ambient CO2 concentrations in open-top field chambers. Experimental manipulation of O3 concentrations and estimates of plant O3 uptake indicated that equivalent O3 fluxes that suppressed net photosynthesis, growth, and yield at ambient concentrations of CO2 were generally much less detrimental to plants treated concurrently with elevated CO2. These responses appeared unrelated to treatment effects on superoxide dismutase, glutathione reductase, and peroxidase activities and glutathione concentration. Total ascorbic acid concentration increased by 28-72% in lower canopy leaves in response to elevated CO2 and O3 but not in upper canopy leaves. Increasing concentrations of atmospheric CO2 will likely ameliorate O3 damage to many crops due to reduced O3 uptake, increased carbon assimilation, and possibly as yet undetermined additional factors. The results of this study further suggest that elevated CO2 may increase the threshold O3 flux for biomass and yield loss in soybean.

Influence of ozone on ribonuclease activity in wheat (Triticum aestivum) leaves.

Ribonucleases (RNases) degrade RNA and exert a major influence on gene expression during development and in response to biotic and abiotic stresses. RNase activity typically increases in response to pathogen attack, wounding and phosphate (P(i)) deficiency. Activity also increases during senescence and other programmed cell death processes. The air pollutant ozone (O(3)) often induces injury and accelerated senescence in many plants, but the biochemical mechanisms involved in these responses remain unclear. The objective of this study was to determine whether RNase activity and isozyme expression was stimulated in wheat (Triticum aestivum L.) flag leaves following treatment with O(3). Plants were treated in open-top chambers with charcoal-filtered air (27 nmol O(3) mol(-1)) (control) or non-filtered air plus O(3) (90 nmol O(3) mol(-1)) (O(3)) from seedling to reproductive stage. After exposure for 56 days, RNase activity was 2.1 times higher in flag leaf tissues from an O(3)-sensitive cultivar in the O(3) treatment compared with the control, which generally coincided with foliar injury and lower soluble protein concentration, but not soluble leaf [P(i)]. Soluble [P(i)] in leaf tissue extracts from the O(3) and control treatments was not significantly different. RNase activity gels indicated the presence of three major RNases and two nucleases, and their expression was enhanced by the O(3) treatment. Isozymes stimulated in the O(3) treatment were also stimulated in naturally senescent flag leaf tissues from plants in the control. However, soluble [P(i)] in extracts from naturally senescent flag leaves was 50% lower than that found in green flag leaves in the control treatment. Thus, senescence-like pathological responses induced by O(3) were accompanied by increased RNase and nuclease activities that also were observed in naturally senescent leaves. However, [P(i)] in the leaf tissue samples suggested that O(3)-induced injury and accelerated senescence was atypical of normal senescence processes in that P(i) export was not observed in O(3)-treated plants.

Differential responses of G-protein Arabidopsis thaliana mutants to ozone.

• Ground-level ozone (O3 ) curtails agricultural production in many regions worldwide. However, the etiology of O3 toxicity remains unclear. Activated oxygen species appear to inflict biochemical lesions and propagate defense responses that compound plant injury. Because some plant defense responses involve membrane-delimited GTPases (G proteins), we evaluated the O3 sensitivity of Arabidopsis mutants altered in the heterotrimeric G-protein pathway. • Eight genotypes were treated with a range of O3 concentrations (0, 100, 175 and 250 nmol mol-1 ) for 13 d in controlled environment chambers. • After treatment with O3 , the epinasty typically observed for wild type leaves did not occur in mutant plants lacking the alpha subunit of the G-protein complex (gpa1). O3 -induced suppression of leaf chlorophyll levels and leaf mass per unit leaf area were less for gpa1 mutants and were not due to differences in O3 flux. • There was a positive correlation between the lack of a G-protein alpha subunit and decreased O3 sensitivity. Our results suggest that a heterotrimeric G-protein is critically involved in the expression of O3 effects in plants.

Mineral nutrition, resin flow and phloem phytochemistry in loblolly pine.

Southern pine beetles and associated pathogenic fungi represent the largest biotic threat to pine forests in the southeastern USA. The two primary defensive mechanisms of the tree to the beetle-fungal complex are the primary oleoresin flow and the concentrations of preformed and induced secondary compounds. We compared oleoresin flow and concentrations of phloem nutrients, soluble sugars, starch, total phenolics and proanthocyanidins in Pinus taeda L. trees in fertilized and control plots in the Sandhills region of North Carolina. Four blocks of 10 trees per treatment were sampled on five dates from May to November 1995. Phloem nitrogen and potassium concentrations were elevated in trees on fertilized plots, whereas phloem calcium concentrations were decreased. Fertilization significantly enhanced (10-20%) concentrations of phloem phenolics and proanthocyanidins. In contrast, phloem soluble sugars and starch concentrations were up to 30% lower in fertilized trees than in control trees. Increased phenolic concentrations and lower nonstructural carbohydrates should correlate with reduced tissue palatability and decreased pathogen susceptibility in fertilized trees; however, resin flows were significantly lower (30-100%) in fertilized trees compared with control trees, which may facilitate pine bark beetle establishment. Furthermore, fertilization-induced increases in phloem nitrogen concentration may be more important than tissue carbohydrate or phenolic content in determining tissue palatability.