Brief Summary of Minutes of Annual Meeting

The 2011 meeting of the NE-1030 Multi-State Project Technical Committee was held at Schoodic Environmental Center in Acadia National Park, Winter Harbor, ME July 27-29. The meeting was called to order at 8:10 am 27 July 2011 by chairman Art Chappelka. The meeting agenda was discussed followed by introductions by those attending the meeting in person and remotely via conference telephone and GoToMeeting web connection for presentations. Station reports were then presented. Kent Burkey (NC) reported on soybean introductions previously shown to exhibit differential ozone tolerance were subjected to season long ozone exposures in open-top-chambers. Using these data certain soybean varieties were selected for crossing. A genetic cross has been made between Fiskeby III and Mandarin Ottawa and a set of 240 random inbred lines developed for future studies to map ozone tolerance genes in soybean. Teodora Best (PA) reported on data used to develop genomic resources for the analysis of traits related to ozone response in black cherry. There were two objectives 1) Develop a sequence database for expressed genes in black cherry using RNA isolated from the ozone-treated tissues of ozone-tolerant/-sensitive trees. Two cDNA libraries derived from Prunus serotina tissues were sequenced using Roche 454 technology, 2) Construct a genetic linkage map for two full-sib black cherry populations segregating for ozone sensitivity, which will serve as the reference map for the species. David Grantz (CA) described recent research focusing on diurnal trends in ozone sensitivity of vegetation. While the diurnal nature of ozone sensitivity is confirmed, the mechanism remains to be elucidated. These data indicate that parameterization of models of ozone injury to vegetation will require measures of inherent defense capability, for which time of day may be a key determinant. Ozone impacts on plants with C4 photosynthetic systems is also being investigated. Application of methyl jasmonate was shown to have an additive, but no protective effect, on ozone tolerance of Pima cotton. Continued studies of genotoxicity of ozone in Pima cotton, using the Comet assay (alkaline single cell electrophoresis) demonstrated that fine roots do not exhibit chromosomal damage. Art Chappelka (AL) reported that clover biomass and nutritive quality were affected by ozone, however, grasses growing the same community were not. Rabbits fed elevated ozone forages were found to have lower DM, ADF, and NDF digestibility. Forages treated with elevated ozone increased bound phenolic composition which were correlated with lower DM, ADF, and NDF ingestibility. Digestibility depression in forages contributed to lower ingestion and utilization of energy. Dennis Decoteau (PA) presented information regarding a teaching module for demonstrating ozone effects on plants that has been developed at The Air Quality Learning and Demonstration Center at Penn State. The ozone module has been tested and is already being used in high school classrooms in Pennsylvania. The module is available for download by educators and the general public at various public websites. Research was continued in 2010 on Long Island, NY, (McGrath, NY) at Cornells research center (LIHREC) in Riverhead to further an investigation of the impact of ambient ozone on plant productivity using the snap bean system developed by project cooperators. Ozone injury occurred in all plantings. Quaking aspen, cutleaf coneflower and spreading dogbane were set up in plots in national parks and monitored during 2006-10 (Kohut, guest). Plots with elevated soil moisture were selected. Different symptoms of ozone injury were observed on cutleaf coneflower in the Rocky Mt and Smoky Mt national parks. After the presentations a general discussion occurred regarding the station reports. Reduced funding for ozone research and challenges experienced obtaining funding were also discussed. Concern was voiced and discussed about the future of the study of plant response to ozone as a whole generation of scientists is being lost now due to lack of funding with no new scientists being trained, while this air pollutant continues to be an increasing problem. Ray Knighton gave a presentation via conference call. General budgetary constraints and funding challenges at the federal level were described. Lack of funding for travel was the reason Ray was only able to attend the meeting remotely, as was also the case for most committee members who did not attend in person. The Agriculture and Food Research Initiative (AFRI) was described, including opportunities in this program for research pertaining to air pollution. Mechanisms of plant response to stresses is a priority under Biology of Agriculture Plants. A challenge with the APRI program is the need for large, multi-institutional grants that integrate research with extension and/or education. Climate Change Program is a Challenge Area that should be an appropriate fit for a proposal from our group. A field trip to Acadia National Park was taken by the members on July 28. Sites used in previous ozone bioinidicator studies were examined and a presentation was made by park personnel regarding their research in the park. Business Meeting (27 July and 29 July) The future of this Multistate Ozone Effects Project was discussed. Our proposal was approved to renew the project as a Coordinating Committee. The project expires on 30 September 2012. The final report probably is due 6 months after this date; this needs to be confirmed. Preparing the report was discussed. Fitz Booker, David Grantz and Art Chappelka agreed to take leadership on preparing the final report. Grant proposal topics and potential funding sources were discussed. There was a lengthy discussion of collaborations that could work with the range of expertise among the committee members. Some topics included: 1. examining the effect modern variety development has had on ozone sensitivity by comparing heirloom versus modern lines of vegetable, 2. building on interesting results from initial work undertaken by committee members on ozone and herbivore interactions, 3. relationship to ozone sensitivity and defense mechanisms of the timing and seasonality of high ozone episodes, and 4. the relationship between ozone episodes and drought stress comparing natural and agricultural ecosystems. Bend, OR in May  June 2012 was selected as the next meeting site and time period, which Nancy Grulke agreed to host. Meeting adjourned at 10:30 am on 29 July 2011. Minutes prepared by Margaret McGrath, Cornell University.

Objective 1. Describe the spatial - temporal characteristics of the adverse effects of current ambient O3 levels on crop productivity, including the development of numerical models to establish cause-effect relationships that apportion the ozone contribution. (IL) Ozone impacts on soybean quality in IL was reported by Ainsworth. An experiment involving 10 soybean cultivars was performed to evaluate agronomic and seed composition changes caused by exposure to elevated ozone concentrations. The 10 cultivars selected have all contributed significantly to North American soybean breeding efforts. Field observations were made to evaluate appropriate response variables and estimate their correlation with yield loss due to elevated ozone. Evaluated responses included foliar damage, leaf chlorophyll content, photosynthetic rates, plant height, stem diameter, leaf size, time to maturity, seed weight, seed oil/protein content, and yield. Highly significant relationships were observed between all of these responses and ozone concentration, while the strongest correlations with yield loss due to ozone were with physiological responses such as plant height, leaf size, and foliar damage. Although little effect on seed oil and protein content was observed, seed from plants grown in elevated ozone showed an altered fatty acid profile, resulting in seed with higher levels of undesirable polyunsaturated fatty acids. (NY) Research on ozone-sensitive and ozone-resistant snap bean lines was continued in 2010 (McGrath, NY) using field-grown plants. The lines, sensitive S156 and resistant (tolerant) R331, were developed at the USDA-ARS Air Quality Research Unit in Raleigh, NC, to be used to investigate the impact of ambient ozone (O3) on plant productivity. Exposure to ozone caused acute foliar injury in all three plantings. The visible symptom was bronzing. The sensitive line was more severely affected than the tolerant one. The O3-sensitive snap bean line S156 yielded numerically less than the tolerant line R331 when grown under ambient O3 conditions on Long Island in 2010. However, yield was not always significantly less. (NJ) Using these same lines Zilinskas (NJ) analyzed 2009 growing season data. A statistically significant decrease in total pod fresh weight of marketable snapbeans was observed in S156 relative to R331 in three of the five harvest dates, where the fresh weight of marketable pods of the ozone-sensitive cultivar was between 37% and 73% less than that of the ozone-tolerant cultivar. A final harvest of snapbeans was conducted at 84 days after planting. The number of seeds and the dry weight of seeds and pods from the two cultivars were significantly different, with yield reductions in the sensitive relative to the tolerant cultivar of 26%, 47% and 50%, respectively. There was no significant difference in the pod number of the sensitive cultivar relative to the tolerant cultivar. (CA) Ozone impacts on plants with C4 photosynthetic systems. Novel feedstocks for biofuel production will require evaluation of environmental responses, including to tropospheric ozone. In potential production areas in southeastern California, ozone exceedances persist due to in-migration of urban plumes. We have showed previously that growth of a range of Saccharum clones was differentially responsive to ozone. This was reinforced with data on photosynthetic responses, including carbon assimilation responses to photon flux density and to intercellular carbon dioxide. This further confirms the conclusion that C4 crops are similar to C3 crops in exhibiting a range of ozone sensitivity. It is not warranted to assume that C4 crops will exhibit the high levels of ozone tolerance observed in early studies. (NC) Open-top chamber studies were initiated to test the feasibility of identifying ozone-tolerant soybean cultivars based on pedigree analysis. Two ozone-tolerant soybean ancestors, two cultivars genetically related to the tolerant ancestors, and two ozone-sensitive ancestors are being compared using season long exposures to four different ozone concentrations ranging from sub-ambient to twice current ambient levels. Ozone responses are being evaluated in terms of foliar injury, seed yield, and antioxidant metabolism. Objective 2. Assess the effects of O3 on structure, function and inter-species competition in managed and native plant populations, including alterations in their nutrient quality. (PA) To evaluate tree of heaven as a bioindicator for ozone pollution, seed from six locations across the country were exposed to ozone using continuously stirred tank reactor (CSTR) chambers on the campus of Penn State University, PA. During fumigation, seven of the 16 CSTR chambers were programmed to 75 ppb ozone concentration (the low ozone treatment), seven were programmed to 120 ppb (the high ozone treatment), and two chambers were programmed to 0 ppb ozone (the control treatment). During fumigation of these tree of heaven seed sources, it was observed that the most susceptible of these seed locations was Corvallis, Oregon and the least susceptible was Far Rockaway, New York. (MA) Weekly foliar sprays of EDU reduced ozone injury symptoms on leaves of Hibiscus syriacus L. Minerva growing at the Millerose Park Research Station outside Turin, Italy. Leaves located on the western side of the hedge (exposed to more light) exhibited @3X more ozone injury than leaves on the eastern side of the hedge. When transferred to open-top chambers seedlings exposed to NF air exhibited ozone symptoms, but those in CF air did not. This validated symptoms from Mellerose Park and demonstrated that ozone-sensitivity is a heritable trait in this species. (AL) Two separate studies to quantify effects on community structure and function were conducted and reported on: 1): Southern Piedmont plant communities exposed to different ozone concentrations in combination with altered rainfall predictions over a growing season: 2): Grassland forage species grown under different ozone concentrations and fed to rabbits in feeding trial to estimate digestibility and energy differences between treatments. Regarding experiment 1, Sensitive species (clover) biomass and nutritive quality adversely affected by ozone Individual grasses: fescue and Bermuda. Grasses as a growth type insensitive, tended to have greater biomass and nutritive quality. Precipitation had minimal effects due to water treatment blocks and also high rainfall year. The conclusions regarding experiment 2 are as follows; 2X forages determined to have lower DM, ADF, and NDF digestibility. 2X forages increased bound phenolic composition which was correlated with lower DM, ADF, and NDF ingestibility. Implications for lower nutritive quality in pastures exposed to ozone. Digestibility depression in 2X forages contributed to lower ingestion and utilization of energy. Objective 3. Examine the joint effects of O3 with other growth regulating factors (e.g., CO2, temperature) that are expected to vary with ongoing climate change on crop growth and productivity. (NC) Climate change factors such as rising atmospheric CO2 and ozone can exert significant impacts on crop growth, but it remains largely unexplored how the soil microbes in agricultural systems respond to these factors. This severely hinders our ability to predict soil carbon sequestration potential. Using a long-term field study conducted in a no-till wheat-soybean rotation system with open-top chambers, ARS researchers in Raleigh, NC showed that elevated CO2 stimulated plant biomass production and ozone lowered it, but only elevated CO2 significantly affected soil microbial biomass, respiration and community composition. Enhancement of microbial biomass and activities by elevated CO2 coincided with increased soil nitrogen availability, likely due to stimulation of soybean nitrogen-fixation under elevated CO2. These results highlight the need to consider the interactive effects of carbon and nitrogen availability on microbial activities when projecting soil carbon balance under future CO2 scenarios. The addition of nitrogen to agricultural systems through fertilizers or legume crops may stimulate microbial decomposition processes and limit carbon sequestration potential. Our results also suggest that projected ozone concentrations under future climate scenarios may reduce plant productivity but have limited impact on soil microbial processes. (NC) A prototype Air Enrichment System (AES) was designed and built that provides a clean-air (charcoal-filtered) environment along with elevated temperature, ozone and CO2 treatment capabilities (NC). The AES uses passive solar heating and electrical resistance heaters to elevate the air temperature 2.2 ± 0.4 °C and adds moisture to maintain relative humidity. Two prototype units and ambient plots were planted with soybean in June 2011. Harvest and environmental data are being summarized for use in a crop growth computer model. (NC) ARS researchers developed the "water stress field", a form of precipitation exclusion technology, for the multiple purposes of studying the physiology of water stress, comparing genetic lines of soybean and snap bean for desirable water stress characteristics and to examine the interactions between soil moisture levels and the toxic effects of ambient atmospheric ozone. The 2008 and 2009 data were used to develop a model for soybean predicting leaf conductance from measurements of soil volumetric water content in the profile down to 40 cm. Coupled with ambient ozone measurements a flux based risk assessment model, based on soil moisture is also being developed. (NC) Five wheat varieties with different levels of susceptibility/resistance to the wheat stripe rust pathogen, Puccinia striiformis, were vernalized and grown under combinations of elevated carbon dioxide, ozone, and atmospheric vapor pressure deficit. Ozone significantly damaged the growth and seed production of all five varieties. Carbon dioxide promoted growth and lessened the effects of ozone. Vapor pressure deficits were too low to allow for rust development in the leaves. In a second experiment in open-top chambers, 60 varieties of wheat, barley, and oat juvenile plants were shown to vary widely in response to ozone exposure, with oat and barley varieties exhibiting greater leaf discoloration than wheat. Objective 4. Examine the physiological and molecular basis of O3 toxicity and tolerance in plants. (PA) A new project expanded the analysis of the genetic response of hardwood tree species to ozone. Messenger RNA was isolated from leaf tissues of control and ozone-treated Red Oak seedlings. The mRNA preps were sequenced by the 454-Roche technology. A database of over 1 Million sequence reads was created; the reads were assembled into transcript contigs which were annotated by BLAST. The transcript contigs fell into a very similar array of functional categories as observed with black cherry. They observed that expression of chloroplast stress-response genes were up-regulated after 30 days, while stress-response genes in the mitochondria were down-regulated. Also, both heat shock proteins and ubiquitins were up-regulated. HAG1 was up-regulated in the nucleus as well, which functions in unfolding chromatin, for the binding of transcription factors. (PA) The Carlson Lab focused their research on characterizing and understanding the genetic basis of variation in sensitivity to ozone stress in forest trees. They developed a community resource for comparative and functional genomics in Prunus serotina which provides tools for black cherry tree improvement and for sustainable forest management. They observed by comparing black cherry data from the EST sequencing with data from Arabidopsis obtained by microarray analysis of ozone treatments, that in black cherry fewer genes were both up-regulated and down-regulated than in Arabidopsis. (IL) Variation in ozone tolerance in five agriculturally important legume species was reported by Ainsworth. The O3 response of Glycine max, Pisum sativum, Phaseolus vulgaris, Cicer arietinum and Medicago sativa - all commercially relevant crops, important for global food security was compared in a growth chamber experment. When grown in elevated chronic O3, in situ measurements of stomatal conductance and photosynthesis revealed a broad range of O3 sensitivities. The most sensitive species, P. vulgaris and M. sativa, each had decreases in stomatal conductance of 55%, while the most tolerant species, P. sativum, was not significantly different from ambient-grown plants. Whole plant characteristics such as leaf longevity, total plant leaf area and biomass, as well as biochemical markers of antioxidant capabilities such as apoplastic ascorbate, were consistent with the O3 sensitivities determined from gas exchange analysis. The results reveal a large degree of variation in tolerance among legume species, much more so than within soybean alone. (PA, NC) A collaborative project (C. Wiese) in collaboration with Dr. Kent Burkey and Dr. Fitz Booker in the Plant Science Unit at USDA-ARS in Raleigh, NC, is to determine the role of the apoplast in plant defense responses to oxidative stress. The specific objectives of this study are to investigate whether there are differences in the apoplastic constituents of two genotypes each of soybean and snap bean which differ in their O3 tolerance, and whether exposure to O3 affects the quantity and types of apoplastic constituents in these genotypes. The tolerant R123 and sensitive S156 snap bean genotypes were exposed to charcoal-filtered (CF) air or elevated O3. Leaf intercellular wash fluid (IWF) was analyzed for ascorbic acid and also by reversed-phase HPLC for other apoplastic constituents of interest. While there were initial differences in apoplastic ascorbate between the two genotypes, these differences were no longer apparent after several days of O3 exposure. Exposure to O3 resulted in an increase in total ascorbic acid quantities in both genotypes. Phaselic acid (caffeoylmalate) was identified by HPLC-Mass Spectrometry as a major constituent of the snap bean apoplast. The quantities of phaselic acid were significantly higher in the O3-treated samples than the CF samples of both snap bean cultivars at both time-points in the exposure. On the first day of the exposure, phaselic acid quantities were also significantly higher in the tolerant R123 genotype than in the sensitive S156. Therefore, it is possible that phaselic acid, due to the large quantities found in the apoplast and the increases in phaselic acid in O3-treated plants, may play a role in determining plant sensitivity to O3. Further investigations will address the role of phaselic acid in the apoplast, its interactions with other apoplast constituents and its role in potential detoxification reactions occurring in the apoplast. (CA, NC) A collaboration of the University of California at Riverside (R. Heath) University of California Kearney Agricultural Center (D. Grantz), and USDA/ARS (K. Burkey) has developed the first demonstration that defense capacity of leaves against ozone varies diurnally. This year we have developed a more straightforward means of analyzing the diurnal trends in ozone sensitivity of Pima cotton, cv. S-6, grown in a greenhouse. Preliminary data were presented at last years meeting. The experiment was repeated with constant and with variable irriadiance. The additional effect of light stress was evaluated and found to not be a factor in the diurnal sensitivity. The new analysis uses slopes of responses to ozone flux to define a sensitivity parameter. These parameters are better behaved than the extrapolated damage parameter presented previously. These sensitivity parameters may prove useful in support of efforts to model extensive regional impacts of ozone on vegetation. Total antioxidant capacity, ascorbic acid and dehydroascorbic acid content were determined on non-exposed leaves at different times of day, but did not explain a substantial part of the diurnal variability. Regression relationships between sensitivity to O3 injury and various measures of antioxidant status were not significant. While the diurnal nature of ozone sensitivity is confirmed, the mechanism remains to be elucidated. These data indicate that parameterization of models of O3 injury to vegetation will require measures of inherent defense capability, for which time of day may be a key determinant. (NC) Final steps were completed in the development of a soybean population to map stress tolerance genes for drought, iron deficiency chlorosis, ozone, salt, and toxic soil aluminum. The mapping population consists of 240 random inbred lines developed from a cross between Fiskeby III and Mandarin Ottawa plant introductions. Seed increases were completed and initial screening of the population for ozone, drought and iron deficiency chlorosis begun. DNA for use in SNP marker assays was extracted from leaf tissue for approximately 90% of the random inbred lines. Objective 5. Develop educational tools and conduct advanced training for K-12 public school teachers, college level instructors, and outreach educators regarding the effects of ambient O3 pollution on plants. (CA) Grantz in California continues to provide greenhouse exposure chambers that are used to demonstrate ozone symptoms to visiting school and industry groups and international visitors from commodity outreach programs. (CA) Grantz continues to address school groups and industry organizations, and worked with individual commodity groups to explain the importance of air quality improvement. Active involvement continues with efforts to reduce the carbon footprint of energy production. (NC) Participated in the UNECE/ICP Vegetation ambient ozone biomonitoring project (snap bean project). Sensitive and tolerant snap bean lines were grown at our field site and monitored for visible injury, stomatal conductance, photosynthesis and yield. Results of the experiment along with meteorological data and hourly ozone measurements were forwarded to project organizers for incorporation into their multi-location study and flux modeling investigations. (NC) Seeds of ozone-sensitive and ozone-tolerant snap bean were provided to UNECE/ICP Vegetation for use in their multi-location biomonitoring and flux-modeling projects. (NC) Ozone-sensitive and ozone-tolerant snap bean lines were used in laboratory exercises in two classes at NC State University (Environmental Technology 202 and Crop Physiology 714) to teach students about the impacts of ozone on plants. Students measured photosynthesis, stomatal conductance, chlorophyll fluorescence, biomass and leaf area of both genotypes following treatment with clean air or 75 ppb ozone for 20 days. Data were compiled and students presented the results in classroom discussions. (NC) Maintenance of a web educational presence. Booker of USDA in NC, the Web Master for the NE-1030 Project, developed and maintained a project web page at (http://www.ncsu.edu/project/usda-ne-1013/index.htm). This web page is frequently updated with current news items, project annual report and news of the project such as minutes of the 20010 annual meeting. (NC).

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