Brief Summary of Minutes of Annual Meeting

The meeting was convened at the Oregon State University Foundation Center in Portland, Oregon by local host, Dr. Nancy Grulke of the U.S. Forest Service, Western Wildland Environmental Threat Assessment Center. 10 July 2012 08:00 Meeting was called to order by Chair Art Chappelka. Minutes were taken by Acting Secretary David Grantz. Welcome: Dr. Grulke provided an overview of the facility, local area, and the field trip (below) to be led by Dr. Linda Geiser of the U.S. Forest Service. Policy discussion: Dr. Grulke made a case for the NE-1030 group to compose a letter urging the Obama administration to move strongly forward on a Secondary Standard for Welfare Effects on Vegetation. She also raised the possibility of a subcommittee of our group going to a meeting at USFS Headquarters in Washington DC to push for relevant research to support a Secondary Standard. The issue was discussed, but no conclusion made. It will be discussed again at future meetings. The group again grappled with the concept of identifying a suite of protocols for O3 exposure experiments that would be most informative to the next Integrated Science Assessment, and that would be most likely to be cited in support of a Secondary Standard. There was some support for a recommendation to use a flux- rather than concentration-based metric of exposure. There was considerable support for experiments to be aimed at mechanistic conclusions that could eventually be used in process models. Station Reports North Carolina: Dr. Burkey described a series of soybean experiments in open top chambers (OTCs) with 12 hr mean O3 of approximately 25, 60, 90, and 120 ppb. The relationship between foliar injury and yield was reported for several soybean plant introductions that are being used in studies to identify ozone tolerance mechanisms and map ozone tolerance genes. Fiskeby III was confirmed as the most tolerant genotype with the least foliar injury and no yield loss across the full range of O3 treatment, and Mandarin Ottawa was confirmed as a sensitive genotype for comparison. Another genotype, Fiskeby 840-7-3, was found to exhibit an intermediate response, suggesting variation on ozone tolerance within the Fiskeby germplasm. The Booker lab in Raleigh adapted histochemical techniques using diaminobenzidine staining for peroxide and nitroblue tetrazolium staining for superoxide and showed that both of these reactive-oxygen species are involved in the injury response of the ozone sensitive Mandarin Ottawa soybean genotype. A comparison of Fiskeby III and Mandarin Ottawa suggested the differences in sensitivity to ozone are not related to ascorbate and glutathione pools, peroxidase enzymes, glutathione reductase, or superoxide dismutase. The potential role of polyamines is being investigated in collaboration with Dr. Minocha at the University of New Hampshire.A collaboration with Dr. Schlueter at UNC-Charlotte is underway to examine gene expression in soybean following ozone exposure. Expression is being analyzed in the tolerant Fiskeby III and sensitive Mandarin Ottawa genotypes following 1.5 hour and 4 hour exposures to 25 or 75 ppb O3. Preliminary assessment of the data suggests there are distinct differences in gene expression between the two genotypes. A soybean population consisting of 240 soybean RILS (random inbred lines) derived from a genetic cross between Fiskeby III and Mandarin Ottawa has been developed and is currently being screened in CSTRs in order to map genes of ozone tolerance. Pennsylvania: Dr. Decoteau reported that work on O3 impacts is currently being conducted by himself, Don Davis, John Carlson, Teo Best, and Lauren Seiler. He reported on use of the Tree of Heaven plant (Ailanthus altisima) as a potential bioindicator species. New Hampshire: Dr. Minocha described analysis of antioxidant metabolites he has been conducting using soybean genetic material provided by Burkey. He has focused on the polyamines, including putrescine, spermidine and spermine. These respond to diverse stresses, providing some protection, including drought, NH3 and O3 and others, but are not effective as osmolytes They appear to stabilize membranes. He observed that low levels of constitutive polyamines are predictive in forests of susceptibility to stress. Large induction of polyamines (generally by 3- to 5-fold) was found to be a component of resistance to O3. Interestingly, he found that induction of <3-fold was not protective, but also that induction of >10-fold was also not protective. This remains to be explained. These results were supported by the soybean work, which found that both low constitutive polyamines and a low level of induction by stress were characteristic of the O3 sensitive lines. Illinois: Ms. Betzelberger described studies at the SoyFACE site in Illinois where seven soybean genotypes were subjected to 9 hour exposures of a square wave O3 treatment at eight concentrations up to 200 ppb. Gas exchange experiments were conducted using a large number of Li-Cor6400 systems in parallel to rapidly sample treatments at similar time of day. All genotypes had similar responses so the data were pooled. Photosynthesis (A), stomatal conductance (gs), maximum Rubisco activity (Vc,max), and maximum electron transport (Jmax) all declined with increasing O3 exposure, suggesting that all of these processes are targets for improving plant response to O3 stress. California: Dr. Grantz reported on the current status of the U.S. Air Force project on perchlorate, work that is being undertaken in collaboration with NE-1030 members McGrath and Burkey. This work is to help determine the cause of widespread environmental contamination by perchlorate, an iodide mimic that interferes with human thyroid metabolism. Grantz noted that very high O3 can convert Cl- into ClO4-, but that yields are very low. The current research has established that leaves accumulate perchlorate, and that species differ widely in accumulation of perchlorate present in the rhizosphere (in this case from fertilizer used to grow the plants). However, there was no consistent relationship between ozone exposure of plants in CSTR chambers and perchlorate concentration in the leaves. Because the range of O3 spanned a wide ambient concentration gradient, these data appear to demonstrate conclusively that ambient O3 is not a contributing factor to perchlorate accumulation in plants. 11 JULY 2012 Field Trip: 08:45 Trip departed the Portland Federal Building, proceeding by van along a transect of putative air pollution impacts from Portland to the Wind River Crane site. The subject was lichens and the changes in species composition along this aridity/air pollution gradient. Evening Session: With all attendees participating, the business meeting was convened. Election for officers was held--Burkey was elected Chair for a two year term and Grantz was elected Secretary for a two year term for the newly approved NECC1013 project. The venue for the first NECC1013 meeting was discussed. Consensus achieved to pursue both Charleston SC and Washington DC as possibilities for next year, as well as University of New Hampshire. During discussions on July 12, Dr. Minocha suggested that June-July would be the best months to hold the meeting at University of New Hampshire. Dr. Knighton indicated his willingness to host the group at the Waterfront Center in Washington DC next year. The Washington venue could facilitate meetings at some other agencies (e.g. USFS) and possibly with Congressional staff. 12 JULY 2012 Administrative Reports Ray Knighton: There is a new Director of NIFA, a former Dean and trained as an entomologist. One consequence is that AFRI is returning to its former pattern of small grants ($750k over 5 years), though under the same topic headings, and with a possible renewed emphasis on production agriculture. Targeted funding of Specialty Crop research is being phased out. A possible new area of focus is water. Dr Knighton suggested that NE-1030 consider application as a group or as individuals to the Air Quality program, whose RFAs are due out in October 2012. This program was last offered in 2010. He also suggested that the revised Climate Change program, which had formerly focused on mitigation, would now be focused on adaptationi.e. genetic and management techniques to retain productivity in the face of changing biotic and abiotic stresses. Margaret Smith: Dr. Smith discussed the groups recent transition from a Research Group to a Coordinating Committee. We retain a research focus until 30 September 2012 and can request research funds from the Experiment Station Directors. Once we become a Coordinating Committee on 1 October 2012 we are no longer eligible for Hatch funds except to cover travel to these meetings. These funds must also be requested from the Experiment Station Directors. An annual report will still be expected, but it should be lighter on research, and emphasize the groups role in sharing results and approaches. We have no further commitment to do research as a group. The annual report should focus on the objectives outlined in the Coordinating Committee proposal. Station Reports Oregon: Dr. Grulke reported work undertaken with D. Grantz and E. Paoletti on stomatal dynamics. This work was reported on at the Air Pollution Workshop held in Lithuania. This work aims to provide more accurate stomatal responses to short and long term O3 exposure for mechanistic process models. Grulke noted that a Dutch model shows that stomata open in response to O3, while all other models, including those used most widely, assume only stomatal closure. The work with the HOC specialized gas exchange system will directly address this issue. The system has been established in California in the Grantz laboratory, an ongoing collaboration of this group. Alabama: Dr. Chappelka reported on work with Dr. Russ Muntifering. This work was reported on in preliminary form at last years meeting, involving the effect of O3 fumigation of forage on nutritional impacts on grazing rabbits fed the clipped material. Future work will involve contrasting grazing animals, perhaps voles, and potentially placed directly in the exposure chambers. Additional work underway at Auburn will examine the interaction of drought, O3 and fungal pathogens. A new area of research reported by Dr. Chappelka involved a ground based LIDAR system which has the capability of determining non-destructively O3 effects on canopy characteristics. A potential collaboration with the biofuel production product in California was discussed. 15:00 Meeting was adjourned by Chair, Art Chappelka

Accomplishments 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. Snap bean ozone bio-indicator system Predicting ambient O3 impacts on crops for a specific location and growing season is difficult because plant response is dependent on multiple factors including those unique to the local environment. Direct measurement of effects is impractical for most situations due to the lack of a clean air control necessary for quantifying impacts. Bio-indicator plants provide one approach to circumvent some of these challenges. Ozone-sensitive and tolerant genotypes of snap bean, random inbred lines developed by the USDA-ARS group in Raleigh, were tested as bio-indicators to evaluate ambient O3 effects on crops. Two tolerant (R123, R331) and one sensitive (S156) genotypes were utilized by multiple NE-1030 locations. After initial protocol development, R331 and S156 were compared in irrigated ambient air field plots at locations in NY, NJ, NC, and PA during 2007-2010 growing seasons. Marketable green pod and mature pod yields were measured along with seasonal profiles of ambient O3 and weather conditions at each location in each year. Yields of the R331 and S156 genotypes across locations reflected the expected differential O3 response. A portion of the NY data was published in the Journal of Integrated Plant Biology as a collaborative publication by several NE-1030 members (see Booker et al., 2009). A significant relationship was found between the R331/S156 yield ratio and the AOT40 O3 metric. Analysis of the larger dataset combining results from all locations is underway using numerical models that incorporate yield data with meteorological and O3 data to establish a relationship between ambient O3 exposures and crop responses. A field trial at the SoyFACE site in IL showed that the snap bean bio-indicator system has potential to detect O3 effects in non-irrigated plots. Ratios of sensitive to tolerant genotype pod yields were identified as a useful measurement for assessing O3 impacts. The results suggest that this snap bean system could be used to quantify O3 effects in specific locations with potential applications in diverse environments including agricultural fields. Modeling ozone response of forest species NE-1030 members are modeling O3 impacts on red spruce-mixed conifer ecosystems in the Northeast using a combined physiological-phenological-atmospheric model to identify when red spruce are most physiological at risk to chronic O3 exposures. Three main (field) components were phenological monitoring, characterization of seasonal gas exchange, and foliar injury surveys. Participants from Europe worked to advance models that assess risk to trees and forests based on measurement of stomatal whole-tree O3 uptake and the effective O3 dose. Modeling effective dose requires protocols to describe the responsiveness of biological processes per unit of O3 uptake. Current methodologies to establish the spatio-temporal scaling of the first components have been demonstrated using a combination of sapflow on individual trees or branches, and eddy covariance at the level of entire stands. The eddy covariance measurements also allow an estimate of the non-stomatal deposition of O3. Modeling ozone impacts on crops A multiple linear regression model combined soybean crop yield data from a 5-year period in the Midwest of the United States with measurements of ambient O3 during the same period to estimate present day yield losses on the order of 10%. Yield loss trends based on both conventional ground-based instrumentation and satellite-derived tropospheric O3 measurements were statistically significant and were consistent with results obtained from open-top chambers and an open-air experimental facility (SoyFACE) in central Illinois. Extrapolation of these findings supports previous studies that estimate the global economic loss to the farming community of more than $10 billion annually. Ozone impacts on biofuel feedstocks Relatives of sugarcane (Saccharum spp.) represent potential biofuel crops. Genotypes of the Saccharum complex being considered as sources of biofuel, both through easily fermented sugars from commercial sugarcane clones, but also for lingo-cellulosic feedstocks from high fiber energy canes, were screened for O3 sensitivity. A locally grown clone of sugarcane, favored by farmers of Southeast Asian descent, was the most sensitive. A commercial sugarcane clone from Texas was reduced in biomass production by 30%, The southeast Asian clone was reduced by about 55%, while two clones with high percentage of the wild relative, Saccharum spontaneum, were not significantly affected. The most sensitive clone was inhibited in dry matter production by 38% at 12 hour mean O3 of 59 ppm, and by 75% at 117 ppm. This is substantial sensitivity to ozone, relative even to sensitive crops such as Pima cotton. C4 crops are similar to C3 crops in exhibiting a range of O3 sensitivity. It is not warranted to assume that C4 crops will exhibit the high levels of O3 tolerance observed in early studies. Diurnal changes Inherent plant defense capacity against O3 stress was shown to vary diurnally, and the mechanism explored using Pima cotton, cv. S-6, grown in a greenhouse. Injury was determined from digital photo analysis of necrosis, chlorophyll content and summed abaxial and adaxial stomatal conductance 6-7 days after exposure. Leaves were most sensitive near 3:00 p.m. in repeated experiments. Antioxidant levels of foliar ascorbic acid and of total foliar antioxidant capacity exhibited a moderate peak near midday, but leaf injury was also greatest at this time. Regression relationships between sensitivity to O3 injury and various measures of antioxidant status were not significant. While the diurnal nature of O3 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. Perchlorate in the environment Perchlorate in the environment is toxic to human health and is now detected across large areas, particularly in arid regions. Although potential sources include rocket fuel, fireworks, highway flares, and Chilean nitrate fertilizer, perchlorate is now showing up in areas that cannot be attributed to these sources. It has been suggested that plants growing in O3 polluted atmospheres may be a source of de novo synthesis of perchlorate. Ten species of plants were grown in Greenhouse CSTRs and exposed to O3. The average for all ten species measured, suggested that there was no O3 effect on foliar perchlorate levels of daily fumigation over a range of 0 to 120 ppb, 12 hour mean. There were substantial differences in perchlorate accumulation among plants, the source being the fertilizer used to provide nutrients for plant growth. Sugarcane consistently accumulated perchlorate to less than 100 µg/g, whereas broccoli and cotton accumulated between 350-675 µg/g, and spinach accumulated over 675 µg/g, representing differences in uptake or exclusion from the rhizosphere. There is no robust relationship between foliar perchlorate and O3 exposure of a magnitude that could contribute meaningfully to the widespread environmental contamination by perchlorate observed. 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. Forage quality The effects of tropospheric O3 and various precipitation regimes on a semi-natural grassland characteristic of the Piedmont region of the US (mixture of tall fescue, common bermudagrass, dallisgrass and white clover were assessed in open-top chambers (modified with rain-exclusion caps) located at the Auburn University Atmospheric Deposition Site. A multifactor design with two ozone treatments [nonfiltered (NF, ambient) and 2X × NF] and 3 water regimes (30-yr average, +20% and -20%) were replicated 2 times. Ozone exposures and rain treatments were initiated June 1, 2009. Primary growth and re-growth forage were harvested monthly during the growing season. In addition, a point-sampling technique was used to determine species abundance and diversity. Forage samples were analyzed for concentrations of cell-wall constituents and crude protein. Biomass of O3-sensitive clover was adversely affected by O3 and nutritive quality decreased as reflected in elevated neutral detergent fiber, acid detergent fiber, and lignin in cell wall material. Grasses as a growth type were generally insensitive to O3, and tended to have greater biomass and nutritive quality. Precipitation had minimal effects due to water treatment block effects and high rainfall. These same species were exposed to ambient (non-filtered; NF) and twice-ambient (2X) O3 concentrations and fed to individually caged New Zealand white rabbits 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 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 O3 resulted in decreased digestible dry matter intake by rabbits. These findings suggest that ozone air pollution can have a negative impact on forge quality, resulting in decreased nutrient utilization by mammalian herbivores. Native bio-indicator species The Tree of Heaven plant (Ailanthus altisima) was identified as a native bioindicator species with sensitivity to ambient O3 similar to staghorn sumac, black cherry, common milkweed, and dogbane. Evaluation of Ailanthus seed sources from six locations across the country suggested significant genetic variation in foliar injury in response to O3 exposure with plants originating from Corvallis, Oregon significantly more susceptible to O3 pollution than the other locations. Two varieties of cutleaf coneflower (Rudbeckia laciniata), one from Great Smoky Mountains National Park (GRSM, var. digitata) and one from Rocky Mountain National Park (RMNP, var. ampla), were compared in ambient air plots and exposure chambers with elevated O3 treatments. In ambient air plots, foliar injury development on plants from GRSM greater than those from RMNP, suggesting a much reduced sensitivity in the RMNP plants. Both varieties showed similar responses during chamber experiments, but there was some indication that injury was more severe on the GRSM plants than those from RMNP. Maximum stomatal conductance was about a third higher in the GRSM plants than RMNP, and some of the differential foliar responses may be attributed to lower uptake by RMNP plants compared to GRSM plants. Forests Jeffrey pine stands in the western Sierra Nevada are subject to nitrogen deposition and O3 impacts, along with gradients of aridity. Slow release urea was applied to mature Jeffrey pine in perennially moist and dry microsites in the southern Sierra Nevada to simulate N deposition and canopy health was assessed over a 10-year period. Under moderately high O3 exposure, the proportion of poor health trees increased with N deposition but the proportion of healthy trees was reduced in moist microsites. In dry microsites, N amendment improved the health of the healthiest trees except in years of extreme drought year, suggesting N deposition increased tree susceptibility to extreme drought. Simulated N deposition also modified herbivory and mortality. In moist microsites, N amendment increased both needle scale and mortality. In dry microsites, N amendment decreased both scale and mortality. For a limited number of sites where O3 data were available, ozone concentrations in the 42-58 ppb range had little effect on almost all attributes assessed, including needle herbivory, bark beetle, and tree mortality after 3 years of assessment. Drought dominated the canopy response and confounds response to O3. Weeds Horseweed is an increasingly important weed in CA partly because it has developed resistance to the herbicide glyphosate. It is newly invasive, though it is a native species to North America. It was shown that O3 allows glyphosate-sensitive genotypes to escape the impact of the herbicide, potentially accelerating the fixation of alleles for glyphosate resistance in O3 impacted air basins. Yellow nutsedge is a noxious weed that is difficult to control in many warm agricultural systems. Biomass productivity of nutsedge is sensitive to O3. Nutsedge became more competitive with respect to Pima cotton with increased O3 exposure. In contrast, tomato was initially less competitive with nutsedge at moderate O3 but recovered its competitive ability at further increased O3, as nutsedge began to exhibit substantial growth inhibition. These studies indicate that the effect of O3 on crop weed interaction will be determined by the relative sensitivities of specific crops with respect to yellow nutsedge. 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. Forest species Genome-level responses to O3 were compared in two hybrid poplar clones that are known to vary in sensitivity to O3. Ozone-sensitive (NE 388) and O3-tolerant (NE 245) clones were exposed to damaging levels of O3, with half of the plants also being subjected to gypsy moth caterpillars, Lymantria dispar, a common defoliator of Populus spp. Microarray hybridizations were conducted with the recently developed whole genome microarray for Populus from Nimblegen. Results showed that 73% of the genes that are typically regulated in poplar by herbivory under controlled conditions were not regulated by herbivory when the plants were exposed to O3, while only 15% of the herbivore-regulated genes were activated independent of O3. The results provide insights into plant adaptations to biotic and abiotic stressors (i.e. O3 and herbivores) by identifying genes and gene regulation networks that are activated in multiply stressed plants. Knowledge of genome-level interactions and will be useful in developing strategies that might provide tolerance to both abiotic and biotic stresses in plants. Crops Agricultural soils are thought to be a C sink in a changing global climate because rising CO2 often enhances plant-derived C inputs belowground. To investigate this concept, a long-term no-till soybean-wheat study was conducted to examine the effects of elevated CO2 and O3 on plant-soil interactions, including effects on soil respiration, root length, litter decomposition and soil C. Elevated CO2 stimulated plant biomass production and O3 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. Projected O3 concentrations under future climate scenarios may reduce plant productivity but have limited impact on soil microbial processes. A long-term field study on the effects of ambient O3 levels on the growth and production of two wine grape varieties revealed that the grape cultivar Charbourcin is sensitive to O3 levels typically experienced during the summer months in Pennsylvania. Ozone injury to Charbourcin grape foliage varied from year to year and appeared to be influenced by weather conditions with less injury in dry years. The Vidal variety of grape, which is considered tolerant to O3 injury, typically exhibited little to no foliar injury to ambient O3 levels. Further evaluations are needed to examine the influence of O3 levels and its variability during the season on fruit quality. Atmospheric vapor pressure deficit is a critical factor in plant response Differential atmospheric vapor pressure deficit (vpd) experiments showed that vpd affects both yield potential and O3 response in the S156 and R123 bioindicator snap beans. Ozone-induced reductions in snap bean growth and yield under low vpd (high humidity) did not occur under high vpd (low humidity), although overall yield potential was also limited by high vpd conditions. These results suggest that efforts to model climate change impacts on vegetation must consider interacting environmental factors and that vpd is a critical factor to consider when predicting the effects of O3 air pollution. Objective 4. Examine the physiological and molecular basis of O3 toxicity and tolerance in plants. Stomatal conductance A novel gas exchange system was designed and used to directly measure foliar O3 uptake and elucidate stomatal kinetics in response to environmental challenges (changes in VPD, light) with and without biologically relevant ozone concentrations. Increased stomatal conductance in response to short term high O3 exposure was observed in Pinus ponderosa, Quercus kelloggii, Q. douglasii, Phaseolus vulgaris, and Fagus sylvatica. Decreased conductance in response to short term high O3 exposure was found for Gossypium hirsutum, Saccharum officinarum, Malus pumila, and Pinus taeda. Many of these latter species have been highly selected for high production or yield. In general, O3 exposure reduced the rate of stomatal response and attenuated closing responses in woody species which in the long term would increase average stomatal conductance and thus O3 flux, as well as increase water use and degrade plant water relations in ecosystems with limited rainfall. Isoprene The biogenically-produced volatile hydrocarbon, isoprene, is proposed to protect some plants from O3 injury by scavenging O3 in the leaf boundary layer and apoplast, although reaction products may be toxic and overall efficacy of the proposed mechanism is uncertain. Isoprene biosynthesis in transgenic Arabidopsis had no influence on visible injury, decreased rosette diameter and lower biomass accumulation caused by O3 exposure. Velvet bean (Mucuna pruriens) lines that displayed varying extents of foliar visible injury symptoms following acute O3 exposures were found to emit isoprene at similar rates when grown in clean air. Treatment of plants with an antibiotic (fosmidomycin), which suppressed isoprene emission, was ineffective in altering plant responses to O3. These results raise significant questions about the proposed role of isoprene in modifying O3 injury in isoprene-emitting plants. Elevated temperature increased isoprene emission but there was no interaction between isoprene emission rates and O3 effects on net photosynthesis, biomass production, peroxidase activity and ascorbate levels. Increased temperature increased stomatal conductance and O3 effects on plants, suggesting that ozone x temperature interactions deserve further study. Genetics For black cherry, a wide range of O3-sensitivity is known, with some genotypes being so sensitive as to serve as O3 indicator plants. Molecular genomic analyses were conducted on previously identified O3 tolerant and ozone sensitive families of black cherry to characterize and understand the physiological and molecular basis of O3 toxicity and tolerance in trees. An EST database for black cherry was generated. Two cDNA libraries from RNA isolated from O3-treated leaves of O3-sensitive and O3-tolerant seedlings. Functionally, 13% of the expressed DNA sequences of the sensitive family are from genes known to be involved in response to biotic and/or abiotic stresses. About 1% of genes in black cherry are involved in signal transduction. Within these gene sequences we have indentified unique microsatellite DNAs that we are presently using to construct genetic linkage maps for black cherry, with which we plan to map the loci for O3 tolerance. The full-sib black cherry families that we have selected for mapping will be maintained as reference populations for future research and a community resource for comparative and functional genomics in Prunus serotina. Ten soybean cultivars that have contributed significantly to North American soybean breeding efforts were evaluated for agronomic and seed composition changes caused by exposure to elevated O3 concentrations at the SoyFACE research site. On average, soybean yields are reduced by ~38 kg ha-1 per ppb of O3 over ambient concentrations. Evaluated O3 responses included foliar damage, leaf chlorophyll content, photosynthetic capacity, 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 O3 concentration, while the strongest correlations with yield loss due to O3 were with physiological responses such as plant height, leaf size, and foliar damage. Water use efficiency of the soybean also decreased with increasing O3 concentration, suggesting that selection for more water use efficient lines may be a strategy to dealing with O3 pollution. Although little effect on seed oil and protein content was observed, seed from plants grown in elevated O3 showed an altered fatty acid profile, resulting in seed with higher levels of undesirable polyunsaturated fatty acids. Thirty soybean ancestors representing 92% of the genetic base of North American soybean were screened for O3-induced foliar injury and the results combined with pedigree analysis techniques to predict O3 resistance of 247 publically-released soybean cultivars. Ancestors with the greatest O3 resistance were not major contributors to current US cultivars. Predicted injury scores suggested that cultivars from the Midwest may be more sensitive to O3-induced foliar injury, on average, than Southern cultivars. Two of the ancestors, Fiskeby III (O3-tolerant) and Mandarin Ottawa (O3-sensitive), were used as parents to develop a population consisting of 240 random inbred lines for mapping O3 tolerance genes. In collaborative work outside the context of NE-1030, Fiskeby III was also identified as a source of tolerance genes for drought, iron deficiency chlorosis, salt stress, and toxic soil aluminum. Thus, the mapping population will provide a unique opportunity to map and compare genes for a wide range of abiotic stresses. Open-top chambers were employed to test the potential for identifying O3-tolerant soybean cultivars on the basis of pedigree analysis. Two O3-tolerant soybean ancestors (Fiskeby III and Fiskeby 840-7-3), two modern cultivars genetically related to these tolerant ancestors (Maple Ridge and Maple Amber), and an O3-sensitive ancestor (Mandarin Ottawa) were compared using season long exposures to four different O3 concentrations ranging from sub-ambient to twice current ambient levels. Seed yield of Fiskeby III was not reduced under any O3 treatment employed, Fiskeby 840-7-3 yield was reduced at high O3 concentrations, and Mandarin Ottawa yields declined as severity of the O3 treatment increased. Cultivars derived from the O3-tolerant parents did not consistently exhibit the parental O3 response, suggesting that pedigree analysis must be combined with direct screening of germplasm to effectively evaluate the O3 tolerance. Genetic silencing of G-protein genes in the model plant, Arabidopsis thaliana, had had little effect on many processes commonly associated with plant response to O3 stress. This suggests that the G-protein signaling pathway is not a significant target for altering O3 tolerance. Future research to enhance O3 tolerance of crops should be directed toward other aspects of metabolism. Metabolism Leaf infiltration techniques were employed to identify phenolic compounds in the leaf apoplast that could mediate plant defense responses to O3 stress. In Arabidopsis, sinapoyl malate was identified by HPLC-mass spectrometry was a major apoplast component that increased upon O3 exposure, but the concentrations were too low to be effective protectants. In snap bean, phaselic acid (caffeoyl malate) was identified by as the major phenolic constituent of the leaf apoplast. Phaselic acid concentrations were higher in the O3-tolerant R123 genotype than in O3-sensitive S156 genotype during the early phases of O3 exposure, so it is possible that phaselic acid may play a role in determining snap bean sensitivity to O3. Methyl jasmonate Methyl jasmonate is a key signaling metabolite, synthesized from the membrane constituent, linolenic acid. It functions with other signaling compounds, including salicylic acid and ethylene, in controlling programmed cell death in response to pathogens and abiotic stress such as acute O3, and possibly in mediating plant responses to chronic O3. While methyl jasmonate provided protection in tobacco and Arabidopsis against O3 exposure, in Pima cotton it did not. Growth and allocation of Pima cotton responded to a concentration gradient of methyl jasmonate in a manner similar to responses to increasing O3 exposure. A low concentration of methyl jasmonate had no impact on growth or allocation, and did not alter the response to O3. A higher application rate reduced growth and allocation to roots but did not interact with the O3 response, resulting in parallel O3 response curves. Thus there was no protection against chronic O3 damage by methyl jasmonate in Pima cotton. Leaf anatomy Physical properties of cells (cuticular cell wall and mesophyll cell wall thickness), and leaves (thickness, number of layers of palisade mesophyll cells, cell packing or density, exposed internal cell surface area, the tortuosity of the diffusional pathway, and stomatal densities and sizes) may play a role in determining O3 sensitivity in plants. Leaf anatomical properties were investigated for O3-sensitive and O3-insensitive individuals of the O3 bioindicator plant cutleaf coneflower (Rudbeckia laciniata var. digitata) in Great Smoky Mountains National Park. In the two cases where plant sensitivity was statistically significant (adaxial epidermal wall/cuticle thickness and spongy mesophyll cell area) it was greater in the sensitive plants, which is contrary to what models would predict for increasing sensitivity. Therefore, we conclude that, leaf anatomical differences do not appear to contribute to the observed sensitivity differences in cutleaf coneflower. 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. The NE-1030 project web page (http://www.ncsu.edu/project/usda-ne-1013/index.htm) was maintained and updated with current news items, project annual report and minutes of annual meetings. Ozone-sensitive and O3-tolerant snap bean lines were used in laboratory exercises in two classes at North Carolina State University (Environmental Technology 202 and Crop Physiology 714) to teach students about the impacts of O3 on plants and the impact of genetic diversity. Students measured photosynthesis, stomatal conductance, chlorophyll fluorescence, biomass and leaf area of both genotypes following treatment with clean air or 75 ppb O3. Data were compiled and students presented the results in classroom discussions. Middle school students at Rosman High School, Rosman, NC were provided seeds of O3-sensitive and O3-resistant snap bean genotypes and guidance for a 9th grade science fair project on the effects of light and stomatal conductance on plant responses to O3. Cooperative Extension presentations and greenhouse exposure chamber demonstrations were provided to farm groups, environmental groups, industry organizations, and middle school career days, to show impacts of O3 on plants in the San Joaquin Valley of California and explain the importance of air quality improvement. These efforts are beginning to interact with groups seeking to reduce the carbon footprint of energy production, a specific initiative at this time in California. Information on ambient O3 and its impacts was presented annually during a class on vegetable diseases conducted annually during the Master Gardener Training Program in Suffolk County, NY. Penn State maintains the Air Quality Learning and Demonstration center, which continues to provide hands on learning experiences for classes at Penn State and the general public around Centre County in Pennsylvania. This center provided the basis for developing a teaching module An Environmental Education Technique For Demonstrating Ozone Pollution Effects On Vegetation that can be implemented into high school level curricula to educate individuals about ground level O3 pollution and its effects on vegetation. After being tested the module was uploaded onto a website for the public to access.

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