2008 Annual Report 1a.Objectives (from AD-416) Quantify the combined effects of elevated carbon dioxide and ozone concentrations on soil carbon dynamics in a soybean-wheat field crop system; quantify the effects of temperature on the physiology, biomass production and seed yield of selected crop plants under conditions of elevated carbon dioxide and ozone; and identify soybean germplasm with enhanced growth and yield potential under elevated levels of carbon dioxide and ozone, and determine the underlying mechanisms and associated genes that control plant responses to these gases. Application of results from this research in models and decision support systems will ultimately support management strategies that optimize sequestration of carbon in agricultural soils to offset carbon dioxide emissions. Research results should also aid in the development of crops that tolerate ozone and utilize carbon dioxide more effectively. 1b.Approach (from AD-416) Research will be conducted using field and greenhouse chambers for control of ozone and carbon dioxide concentrations and temperature. Effects on yield, development, and quality for specific crops (e.g. soybean, snap bean, wheat and selected forages) and on changes in soil carbon will be tested. To evaluate the interactive effects of ozone, carbon dioxide, and temperature on plants, studies will include measurements of leaf conductance, photosynthesis, chlorophyll fluorescence, carbon metabolism, and biochemical systems that detoxify reactive oxygen intermediates formed from ozone. To evaluate the effects on agricultural soils, studies of plant response to ozone and carbon dioxide will be accompanied by measurements of soil microbial biomass, microbial community structure, and microbial activity, soil carbon distribution among carbon pools, soil carbon turnover using delta 13C mass spectrometry, and nitrogen mineralization. Genetic variability of response to ozone and carbon dioxide will be assessed through a combination of screening and pedigree analysis. Development and testing of Arabidopsis mutants will be conducted to identify genes associated with plant response to ozone. 3.Progress Report Year 3 of a soybean-wheat no-till experiment to investigate long-term effects of elevated ozone (O3) and CO2 on plant-soil interactions and soil carbon dynamics was completed. Elevated CO2 increased aboveground biomass and seed yield of both crops; added O3 had no significant effect on these parameters. Wheat grain protein was decreased 7% at elevated CO2. The soybean cultivar used past two years appeared to be relatively tolerant to O3. Results to date suggest that rising atmospheric CO2 will have significant effects on residue production, litter decomposition, soil respiration and possibly soil carbon sequestration and soil organic matter content while O3 effects on soil carbon as mediated by lower residue inputs may be slow to appear or are marginally important. In combination, however, elevated O3 curtails elevated CO2 effects on biomass and residue production. The first year of O3 x temperature interaction study using O3-sensitive and O3 -tolerant soybean lines was completed. Preliminary analysis indicates that O3 sensitive line showed a substantial decrease in seed yield at higher temperatures while O3 sensitivity at lower O3 levels was increased. The O3-tolerant line had lower yields in clean air than the sensitive line but showed no significant effects of either O3 or temperature. No temperature treatments were significantly different at high O3 levels. Antioxidant metabolism plays a role in mitigating plant response to O3 stress. Research is often directed toward soluble antioxidant compounds such as vitamin C while much less is known about the antioxidant capacity of insoluble components. A method was developed to measure the antioxidant capacity of insoluble leaf cell wall material. Leaf cell walls were shown to contain significant antioxidant capacity that may contribute to O3 detoxification. Genetic mechanisms involved in the role of G-proteins in regulating O3 injury in plants were investigated using Arabidopsis wild type and G-protein null mutants. Messenger ribonucleic acid was isolated from leaf tissue following the initial 3 hours of exposure to 150 parts per billion O3 and again at the end of two 6-hour exposure periods conducted over two days. Gene expression was analyzed to determine O3 responses in general and specifically the role of G-proteins in O3-induced changes in transcript abundance. Analysis of a very large microarray data set is in progress. Preliminary results show that O3 induced expression of genes associated with redox control, transcription factors, detoxification, ethylene biosynthesis, pathogen responses, and unknown function. Notably, transcripts for the beta subunit of the G-protein complex were elevated by O3. Progress is related to the National Program (NP) 204 Global Change, Component III Agricultural Ecosystem Impacts, Goal 2A to measure and predict plant responses to multiple interactions of abiotic and biotic stresses with rising carbon dioxide and NP 203 Air Quality, Component IV Ozone Impacts, Goal 2B to determine environmental influences on crop response to ozone and Goal 4A identify specific aspects of physiology and metabolism that distinguish ozone-sensitive and ozone-tolerant plants. 4.Accomplishments 1. An improved method for analyzing photosynthetic gas exchange data. A rapid and detailed statistical method was developed for analyzing A/Ci gas exchange curves without arbitrary sub-setting of data. The common model describing photosynthesis is divided into 3 segments, each limited by a specific physiological process. These processes may be estimated from gas exchange data over a range of external CO2 concentrations. Parameter estimates were usually conducted by separately fitting the three component functions of the model. This disjunctive approach requires arbitrary sub-setting of the data believed to correspond to each separate region. We showed how the multiple segments can be estimated simultaneously using the entire data set without any arbitrary predetermination of the transitions. National Program 204 Global Change, Component III Agricultural Ecosystem Impacts, Problem Statement – Cropping Systems. 2. Container-grown plants exhibit similar yield enhancement under elevated carbon dioxide as plants grown in the ground. Previous comparisons of yield enhancement ratio (yield of treated plants divided by yield of control plants) resulting from elevated CO2 was similar whether plants were grown in pots or in the ground. However, planting densities varied substantially in these studies so direct yield comparisons were difficult. We therefore grew soybean plants in open-top chambers either in large pots or in the ground but at equal planting densities. It was shown that the relative enhancement ratios were similar but the yield per plant in pots was still less than the ground-grown plants regardless of the CO2 level. The only observed environmental difference was higher daytime root zone temperatures for plants in pots but the robust enhancement ratios suggest that neither above- or below-ground resource limitations would account for the yield discrepancies. National Program 204 Global Change, Component III Agricultural Ecosystem Impacts, Problem Statement – Cropping Systems. 3. Plant competition for resources may limit yield under elevated carbon dioxide. High levels of ozone have long been known to decrease crop yields and conversely that elevated CO2 levels tend to increase yields. However, the yield increase due to elevated CO2 (CO2 enhancement ratio) in clean air is highly variable suggesting other potential resource limitations. A two year 3-way interaction study (ozone x CO2 x planting density) with rice revealed that intra-plant competition for resources such as light, space, nutrients and water also plays a part in determining the magnitude of the CO2 enhancement ratio. We showed that in high ozone, fewer of the available resources (53%) were fixed into biomass than in clean air (70%), while in elevated CO2 that proportion reached 85% regardless of the ozone level. The greatest impact of this work may be that projections of increased productivity due to elevated CO2 may require larger infusions of fertilizer and water in order to be realized. National Program 204 Global Change, Component III Agricultural Ecosystem Impacts, Problem Statement – Cropping Systems. 4. Antioxidant compounds in soybean are associated with ozone tolerance. Development of ozone tolerant plants is one approach to alleviate the adverse effects air pollution on agricultural crops. Progress toward improvement of ozone tolerance requires knowledge of the critical points in plant metabolism that can be manipulated to provide protection against ozone stress without sacrificing yield or other desirable characteristics. Soybean genotypes exhibiting differential ozone sensitivity were compared to determine whether ozone tolerance was related to the concentration of ascorbic acid (vitamin C) in the fluid surrounding leaf cells, a cellular location where antioxidants have the potential to neutralize ozone before cellular injury can occur. Antioxidant compounds other than ascorbic acid were elevated in the extracellular fluid of the tolerant genotype. Identification of these compounds and their reactions with ozone is a critical topic for future research into the development of ozone tolerant crops. National Program 203 Air Quality, Component IV Ozone Impacts, Problem Statement – Mechanisms of Ozone Response. 5. Increased oxidative stress in leaves of Arabidopsis and soybean in response to elevated CO2 and ozone. In-depth understanding of crop responses to rising levels of atmospheric CO2 is critical for adapting and predicting future crop responses to global climate change. While exposure of C3 plants to elevated CO2 would be expected to reduce production of reactive oxygen species in leaves because of reduced photorespiratory metabolism, new results suggest that exposure of plants to elevated CO2 can result in increased oxidative stress. In Arabidopsis and soybean, leaf protein carbonylation, a marker of oxidative stress, was often increased when plants were exposed to elevated CO2 or ozone. Two-dimensional difference gel electrophoresis analysis of soybean leaf proteins revealed that elevated CO2 or ozone altered the abundance of a similar subset of proteins, consistent with the idea that both conditions may involve an oxidative stress. 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. National Program 204 Global Change, Component III Agricultural Ecosystem Impacts, Problem Statement – Cropping Systems. 5.Significant Activities that Support Special Target Populations None 6.Technology Transfer Number of New Germplasm Releases 1 Number of Other Technology Transfer 2 Review Publications Dubois, J.B., Fiscus, E.L., Booker, F.L., Flowers, M., Reid, C.D. 2007. Resolving bias and inaccuracy in estimation of the parameters of the Farquhar-von Caemmerer-Berry model of photosynthesis. New Phytologist. 176: 402-414. Fiscus, E.L., Booker, F.L., Dubois, J.B., Rufty, T., Burton, J.W., Pursley, W.A. 2007. Co2 enhancement effects in container- versus ground-grown soybeans at equal planting densities. Crop Science. 47:2486-2494. Flowers, M.D., Fiscus, E.L., Burkey, K.O., Booker, F.L., Dubois, J.B. 2007. Photosynthesis, chlorophyll fluorescence and yield of snap bean (phaseolus vulgaris l.) genotypes differing in sensitivity ozones. Environmental and Experimental Botany. 61:190-198. Reid, C.D., Fiscus, E.L. 2008. Ozone and density affect the response of biomass and seed yield to elevated CO2 in rice.. Global Change Biology. 14:60-76. Sinclair, T.R., Fiscus, E.L., Rufty, T., Wherley, B., Durham, M.W. 2007. Humidity critical for grass growth in warming climates. Planta. 227:273-276. Cheng, F., Burkey, K.O., Robinson, J., Booker, F.L. 2007. Leaf extracellular ascorbte in relation to 03 tolerance in two soybean cultivars. Environmental Pollution. 150:355-362 Krupa, S., Booker, F.L., Bowersox, V., Grantz, D., Lehmann, C. 2008. Uncertainties in the current knowledge of some atmospheric trace gases associated with US agriculture. Journal of Air and Waste Management Association. Vol 58, pp. 986-993. Qiu, Q., Huber, J., Booker, F.L., Jain, V.L., Leakey, A., Fiscus, E.L., Yau, P., Ort, D.R., Huber, S.C. 2008. Increased protein carbonylation in leaves of Arabidopsis and soybean in response to elevated [CO2]. Photosynthesis Research. Vol 97, pp. 155-166.