Brief Summary of Minutes of Annual Meeting

The meeting was held in the Mission Inn in Riverside California. The meeting was called to order by Chairman David Grantz at 8:15 on May 5, 2009. The meeting proceeded as a series of technical reports from each of the participating Experiment Stations or other collaborating institutions. These are captured in the accomplishments section of this report. A field trip to the greenhouse exposure experiments of Dr. Pam Padgett, at the University of California, and to the kinetic gas exchange experiments of Dr. Nancy Grulke at the US Forest Service, was incorporated as part of the meeting. The location of the next meeting was discussed. The possibility of holding it in association with the Air Pollution Workshop, scheduled to be held in Puerto Rico, was discussed. The Chair is to poll the membership regarding the desirability of this. The meeting was adjourned at 5:00 on 6 May 2009. An optional post meeting field trip to the Salton Sea Ecological area was also provided for interested participants.

Accomplishments during the period 1 October 2008 through 30 September 2009 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. Risk assessment of trees and forests needs to be related to a measure of stomatal whole-tree O3 uptake and to the effective O3 dose. This second requirement will necessitate development of measurements and modelling protocols to describe the responsiveness of biological process per unit of O3 uptake. Current methodologies to establish the spatio-temporal scaling of the first components have been demonstrated during this reporting period, 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 O3 flux. Progress towards assessment of O3 sensitivity remain in progress. (Germany) Relatives of sugarcane (Saccharum spp.) are highly diverse. Interest in sustainable biofuel production has led to a reexamination of wild relatives of commercial sugarcane as potential purpose-grown energy crops in California. The most likely locations are the inland valleys of central and southern California, subject to high temperatures, aridity and highly productive irrigated agriculture. Using GIS techniques we have demonstrated that the most likely microhabitats for cultivation of Saccharum species are also subject to elevated ambient O3. This information has initiated a series of studies to screen genotypes for tolerance to O3, and to evaluate the genus for typical C4 characteristics, including tolerance to O3. (CA) Red spruce-mixed conifer ecosystems in the Northeast are of high ecological importance. A modeling approach (DO3SE) was undertaken in the forested ecosystem located in the GMNF Lye Brook Class I Wilderness Area and surrounding airshed. The primary objective was to quantify ozone uptake using a combined physiological-phenological-atmospheric model, and identify when red spruce are most physiological at risk to chronic ozone exposures. High-elevation physiological sampling sites were expanded in 2009 due to decommissioning of Vermonts only CASTNET site, located within the project area. Three main (field) components were phenological monitoring, characterization of seasonal gas exchange, and foliar injury surveys. Highest average exposures tended to occur close to the period of flushing of current-year needles. Physiological age may be protective against ozone injury, as needles have not yet entered the phase of seasonal maximum stomatal conductance (gmax). Because the 2009 ozone season was exceptionally wet, the project will require one additional season to capture representative physiological responses to air pollution events.(MA) Summertime ozone persistence at remote higher elevations is characterized by well-documented level diel exposures, with elevated nocturnal exposures of several consecutive hours common. Significant ozone reductions have been claimed (nationally) during this reporting period of documented climate warming. Up to 18 years of archival ozone data were available for analysis of long-term trends at 4 distinct higher elevation sites in New England: Mt. Washington, NH, Vermont CASTNET, Mt. Greylock, MA, Pack Monadnock, NH. A fifth site, Whiteface Mt. Air quality at surveyed high elevation sites shows no trend (neither improvement nor deterioration) at southern and northern New England locations, with highest levels in spring. A substantial proportion of exposure in the highest concentration ranges (e80 ppb) occurs nocturnally at these higher elevations, often exceeding valley locations. Average night/day ratios of AOT40 and SUM60 exposure indices range from 0.52 - 0.77. 2008 was an unusual year at one site, indicating that nighttime ozone exposures can exceed daytime exposures in southern New England. 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. Jeffrey pine stands in the western Sierra Nevada are subject to nitrogen deposition and O3 impacts, along with gradients of aridity. During this reporting period we applied slow release urea to mature Jeffrey pine in perennially mesic and xeric microsites in the southern Sierra Nevada. This represents the most recent stage of annual applications that have been done for 10 years. Canopy health was assessed using the following attributes: needle and branch elongation growth, branchlet diameter, needle retention, and the level of chlorotic mottle observed. Under moderately high O3 exposure, the proportion of poor health trees increased with N deposition but the proportion of healthy trees was reduced in mesic microsites. In xeric microsites, N amendment improved the health of the healthiest trees (e.g., an increase in canopy growth was observed). However, in an extreme drought year, increased leaf area was reduced the most in trees that were fertilized in xeric microsites. Simulated N deposition increased tree susceptibility to extreme drought. Simulated N deposition also modified herbivory and mortality in Jeffrey pine. In mesic microsites, N amendment increased both needle scale and mortality (decadal rate: 9%). In xeric microsites, N amendment decreased both scale and mortality (decreased decadal mortality rate from 23 to 9%). There are few O3 concentration data in remote eastern Sierra Nevada sites. However, for those sites that we do have data for, concentrations average ca. 42 (NE of Mammoth Lakes) to 58 (north of Lake Tahoe). Canopy response to this O3 exposure level, after 3 years of assessment is undetectable for almost all attributes assessed, including needle herbivory, bark beetle, and tree mortality (see above paragraph). Drought dominates the canopy response and confounds response to O3. Sites along a 900 km transect of the eastern Sierra Nevada and Transverse range were chosen to represent the range of the species as well as provide gradients in evapotranspiration, precipitation, and therefore the level of drought stress experienced by individual trees. We used this system to define a mechanistic link between tree drought stress and risk of mortality from pine bark beetles in Jeffrey pine. Tree-tree competition and stand density further drive differences in tree drought stress within sites. The objective of this 3 year study (now in the 3rd year) is to correlate tree drought stress with bole protein and carbohydrate content, resin production and chemistry, and terpene emissions. Further, we want to identify a quantitative threshold to beetle resistance (high resin production) and beetle outbreak (high terpene emissions, low resin flow, increase in bole palatability). Jeffrey pine susceptibility to bark beetle under drought stress is relevant to understanding O3 exposure because both drought stress and O3 increase oxidation of zeazanthin, which initiates the processes by which jasmonate and resin production is upregulated. To date, our research team has documented a relationship between increased tree drought stress and increased resin production at the annual time scale. The entomologists are working on qualifying terpene emissions from trees in stands of differing densities and levels of drought stress. The biochemists are working on quantifying the degree of jasmonate upregulation in the same stands. (USFS) Yellow nutsedge (Cyperus esculentus) is a noxious and difficult to control weed in many warm agricultural systems, particularly with irrigation. We have previously reported that biomass productivity of nutsedge is sensitive to O3. We found in these previous studies that 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. In these studies there were indications that the stress of O3 on nutsedge enhanced allocation of current biomass to reproductive structures, belowground. In the current reporting period we tested this hypothesis explicitly. While biomass was again reduced, the reductions were similar above and below ground, and slightly greater below ground. There was no enhancement of allocation to tubers. 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. The competitiveness of nutsedge will increase in many cases, but the abundance of propagules will not be increased directly by the oxidant stress of ambient O3. Nutsedge and sugarcane are C4 species. As such they were not expected to be sensitive to O3. We tested the impact of O3 on the photosynthetic systems of nutsedge and a commercial clone of sugarcane. In both cases, midday levels of carbon assimilation declined with increasing exposure to O3. In both cases, intercellular CO2 concentrations increased, suggesting direct inhibition of mesophyll photosynthesis rather than induced stomatal closure. In the case of sugarcane a simple model was developed that linked SPAD (indirect chlorophyll measurements) to carbon assimilation at each leaf insertion level. As SPAD is a rapid measurement compared with gas exchange, it was possible to predict whole plant carbon assimilation from a vertical series of SPAD measurements and leaf dimensions up the stalk. Preliminary studies indicate that wild relatives of sugarcane, which are known to be more stress hardy than commercial clones, may also exhibit greater tolerance of O3. (CA) A major objective of the NE-1030 Multistate Project is to describe the spatial-temporal characteristics of the adverse effects of current ambient ozone levels on crop productivity, including the development of numerical models to establish cause-effect relationships. A refined protocol was developed by Dr. K. Burkey (USDAARS, NC) and Dr. M. McGrath (Cornell, Long Island Field Station) and strictly followed by our NJ research team, as well as by several other members of the multi-state project in various parts of the US where ambient ozone levels and meteorological conditions vary. We monitored the effects of ambient ozone on the productivity of two snapbean cultivars R331 (ozone-tolerant) and S156 (ozone-sensitive) over the 2008 growing season. We planted the two snapbean cultivars in East Brunswick, NJ, on June 13, 2008, according to the field design and conditions agreed upon by the four field stations in the US that are collaborating on this project. Throughout the growing season, ambient ozone levels and meteorological data were recorded at each site. At each field station, in order for the data to be more compatible for the statistical model to handle data from various locations, we made multiple harvests of marketable pods at 49, 56, 66, 73 and 81 days after planting. In the 2008 season, peak pod number and fresh weight (of both cultivars) occurred at the mid (third) harvesting date. A statistically significant decrease in total pod fresh weight of marketable snapbeans was observed in S156 relative to R331 in two of the five harvest dates, where the fresh weight of marketable pods of the ozone-sensitive cultivar was less than 50% of that of the ozone-tolerant cultivar. A final harvest of snapbeans was conducted at 84 days after planting. At this harvest date in the 2008 growing season, a significant proportion of the pods were immature (without seeds) or green, and this was more pronounced in the ozone-tolerant cultivar. Nonetheless, to be consistent with the investigators cooperating on this project, all stations collected pods at this date. The number of seeds and dry weights of seeds and pods from the two cultivars were significantly different, with yield reductions in the sensitive relative to the tolerant cultivar of 44%, 56% and 48%, respectively. Although there was a trend toward reduction in the pod number of the sensitive cultivar when compared to the tolerant cultivar, the decrease was not significant at a p-value of 0.05 or less. The meteorological and ozone data, coupled with the crop yield data, will be analyzed for the several states where this field experiment has been conducted and incorporated into a numerical model by Dr. S. Krupa (MN) to establish a relationship between ambient ozone exposures and crop responses. The continuation of this study will strengthen our understanding of the impact of ambient ozone on plants and crop productivity. (NJ) The poplar project tries to answer to the question Do ozone (O3) concentrations relevant to Pennsylvania forests alter induced responses of poplars to insect herbivory? We treated hybrid poplar OGY (P. deltoides x nigra) with ozone (80 ppb) and gypsy moth (Lymantria dispar L.) herbivory in environmentally controlled chambers. RNA was isolated and subjected to an EST custom microarray with probes for ~6500 unique genes. The preliminary results showed that ozone treatments supressed Transcriptome-Level Dynamic Responses of Poplar Leaves to Insect Herbivory. These past weeks we repeated the experiment using NE-388 (sensitive) and NE-245(tolerant) hybrid poplar clones. No results to report. (PA) 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. This study examined the effects of elevated carbon dioxide and ozone on plant-soil interactions, including effects on soil respiration, root length, litter decomposition and soil C, in a no-till soybean-wheat system. The experiment was started in 2005 and is ongoing. Elevated carbon dioxide increased soil respiration by 26%, due in part to increased microbial respiration. Added ozone suppressed soil respiration during the latter part of the soybean growing season. Total root length in the elevated carbon dioxide treatments was not different from the control in September while root length was 45% lower with added ozone, likely due to suppressed growth and early senescence. Litter inputs were higher with elevated carbon dioxide and lower with added ozone. Residue decomposition / input ratios indicated that decomposition was similar among treatments although C input to the soil was altered. Decreased 13C and higher %C in the coarse 0-5 cm deep soil fraction indicated that elevated carbon dioxide increased soil organic matter even though soil respiration was higher. Ozone effects were not apparent. (NC). A new study was initiated in 2009 to determine the effects of tropospheric ozone and various climate change (precipitation) on a semi-natural grassland characteristic of the Piedmont region of the US (mixture of tall fescue, common bermudagrass, dallisgrass and white clover). Twelve, large (4.8 m ht. × 4.5 m dia.) OTCs (modified with rain-exclusion caps) located at the Auburn University Atmospheric Deposition (AtDep) Site are used in this study. A multifactor design with two ozone treatments [nonfiltered (NF, ambient) and 1.5 × NF] and 3 water regimes (30-yr average, +20% and -20%) is replicated 2 times. Ozone exposures and rain treatments were initiated June 1, 2009. Primary growth and regrowth will be harvested monthly during the growing season. Various field and laboratory methodologies will be used to test the specific hypotheses. Results will provide critical information on structure and functioning of managed grassland ecosystems using projected climate scenarios of elevated ozone and differing amounts of rainfall, with emphasis on interspecific relationships among the various processes examined. Integration of various measures of diversity and productivity and underlying physiological and biochemical responses will enable a more complete characterization and modeling of potential impacts of future climate change scenarios on these plant communities. Non-fumigated forage from our site was harvested on April 21 and May 12, 2008, after which they were exposed to either ambient, non-filtered air (NF) or twice-ambient O3 air (2 × NF) air and harvested on June 9 and July 2. Forages are being processed for nutritive quality analysis and fabrication into 50-g cubes that will be fed to New Zealand White rabbits in nutrient-utilization/diet-selection experiments beginning in late 2009. In collaboration with scientists at the Swiss Federal Research Station for Agroecology and Agriculture, we have completed the fifth year of a seven-year experiment in the Swiss Alps in which we are investigating productivity and forage quality of semi-natural herbaceous vegetation exposed to three concentrations of ground-level ozone (ambient, ambient + 20 ppb O3 and ambient + 40 ppb O3) and five levels of atmospherically deposited nitrogen (0, 5, 10, 15, 20 and 25 kg N/ha). Forage samples are awaiting processing for nutritive quality analysis. (Auburn University) The biogenically-produced volatile hydrocarbon, isoprene, may be influential in protecting some plants from ozone injury. Isoprene emission is correlated with tolerance to high temperature and oxidative stress. Isoprene can also scavenge ozone in the leaf boundary layer and apoplast, although reaction products may be toxic and overall efficacy of the proposed mechanism is uncertain. A series of experiments conducted in Raleigh, NC investigated potential interactions between biogenically-synthesized isoprene, temperature and ozone using an isoprene-emitting legume species, velvet bean (Mucuna pruriens). Preliminary screening experiments indicated that isoprene emission was not correlated with ozone sensitivity. Velvet bean lines that displayed varying extents of foliar visible injury symptoms following acute ozone 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 ozone. Elevated temperature increased isoprene emission but there was no interaction between isoprene emission rates and ozone effects on net photosynthesis, biomass production, peroxidase activity and ascorbate levels. Increased temperature increased stomatal conductance and ozone effects on plant biomass, ascorbate levels and redox status. It was apparent that increased temperature exacerbated ozone injury, suggesting that ozone x temperature interactions deserve further study. These results raise significant questions about the proposed role of isoprene in modifying ozone injury in isoprene-emitting plants. (Fitz Booker, Ed Fiscus, USDA-ARS, Raleigh, NC). To develop robust models of landscape scale O3 impacts it is critical to related single organ and single organism data to extensive canopy data. During this reporting period, a team of gas exchange ecophysiologists tested for comparability between eddy correlation estimates of stand flux of O3 and CO2, with canopy level O3 uptake based on a near-leaf surface, chamberless gas sampling system, with O3 uptake calculated from both canopy transpiration measurements and leaf-level gas exchange measurements. Following a proof-of-concept project in a cultivated orange grove, we will measure fluxes in an oak woodland as well as in a pine forest. These data will demonstrate certain key gas exchange technologies, and provide infrastructure for landscape scale assessments of O3 damage across important California ecosystems. (USFS) Objective 4. Examine the physiological and molecular basis of O3 toxicity and tolerance in plants. The specific root respiration (per unit root weight) is enhanced in Pima cotton by exposure of the shoot to O3. Similarly, we have shown that phloem loading is inhibited by O3 in Pima cotton. Using alkaline single cell electrophoresis of root tip cells, we have sought to document genotoxicity of O3 exposure of the shoot to the developing roots. Although we have previously reported increasing trends in damage to fine roots with increasing O3, it has been difficult despite much replication to establish convincing statistical significance with this tissue. Preliminary experiments with older roots suggest similar trends, and statistical significance. Further experiments will be required to determine if the older roots which have longer experience with O3-impacted shoot tissue, may exhibit such genotoxicity. Similarly, preliminary experiments with young leaf tissue exhibit increasing trends, but do not reach the level of statistical significance. The question of genotoxicity of O3 exposure remains important, but unresolved as of this reporting period. 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 (40 micrograms per plant, twice weekly) had no impact on growth or allocation, and did not affect the response to O3. A higher application rate (160 micrograms per plant, twice weekly) 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. (CA) Completed a study of ozone effects on leaf peroxidase isozymes in Arabidopsis. Native gels revealed induction of a major cationic isozyme following a 2-day exposure to moderate levels of ozone. The ozone-responsive cationic isozyme was induced in Col-O wild-type plants as well as a number of mutants. This enzyme has the potential to serve as a marker for ozone stress prior to the appearance of foliar injury. Results from prior year greenhouse screening of soybean ancestors for ozone-induced foliar injury were combined with pedigree analysis techniques to predict ozone resistance of 247 publically-released soybean cultivars. Ancestors with the greatest ozone resistance were not major contributors to current US cultivars. Predicted injury scores suggested that cultivars from the Midwest may be more sensitive to ozone-induced foliar injury, on average, than Southern cultivars. (NC) The biogenically-produced volatile hydrocarbon, isoprene, may be influential in protecting some plants from ozone injury. Isoprene emission is correlated with tolerance to high temperature and oxidative stress. Isoprene can also scavenge ozone in the leaf boundary layer and apoplast, although reaction products may be toxic and overall efficacy of the proposed mechanism is uncertain. A series of experiments conducted in Raleigh, NC investigated potential interactions between biogenically-synthesized isoprene, temperature and ozone using an isoprene-emitting legume species, velvet bean (Mucuna pruriens). Preliminary screening experiments indicated that isoprene emission was not correlated with ozone sensitivity. Velvet bean lines that displayed varying extents of foliar visible injury symptoms following acute ozone 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 ozone. Elevated temperature increased isoprene emission but there was no interaction between isoprene emission rates and ozone effects on net photosynthesis, biomass production, peroxidase activity and ascorbate levels. Increased temperature increased stomatal conductance and ozone effects on plant biomass, ascorbate levels and redox status. It was apparent that increased temperature exacerbated ozone injury, suggesting that ozone x temperature interactions deserve further study. These results raise significant questions about the proposed role of isoprene in modifying ozone injury in isoprene-emitting plants. (Fitz Booker, Ed Fiscus, USDA-ARS, Raleigh, NC). (NC) Expression of Pyrococcus furiosus superoxide reductase in Arabidopsis enhances tolerance to heat and paraquat, but not ozone Uncontrolled production of reactive oxygen species such as superoxide in response to environmental stressors can result in cell death. Superoxide dismutase participates in quenching superoxide in plants but attempts to alter its expression in vivo have been challenging. Another approach to manipulating control of superoxide in vivo was tried in this experiment. A superoxide reductase (SOR) gene from the archaeal hyperthermophile, Pyrococcus furiosus, was expressed in Arabidopsis. SOR is a cytosolic, thermostable enzyme that reduces superoxide to hydrogen peroxide. Although transgenic plants expressing SOR were more tolerant than wild-type plants to heat stress and the herbicide paraquat (which generates superoxide in the chloroplast), plant responses to chronic and acute ozone exposures were not significantly different between wild-type and transgenic lines. This suggests that superoxide may not have a major role in ozone toxicity effects in plants. In a related experiment, plants treated with continuous light for 24 h showed much reduced ozone treatment effects and significantly higher leaf anthocyanin concentrations. This finding suggests that anthocyanins may be effective in protecting plants from ozone injury, provided that ozone uptake was unaffected by the light treatment. Further experiments are underway to test this possibility. (NC) Using a novel gas exchange system that concurrently measures water, O3, and CO2 flux at the leaf level, we investigated the direct effects of O3 exposure on stomatal behavior. The following species had increased stomatal conductance in response to short term high O3 exposure: Pinus ponderosa, Quercus kelloggii, Q. douglasii, Phaseolus vulgaris, Fagus sylvatica. The following species had decreased gs in response to short term high O3 exposure: Gossypium hirsutum, Saccharum officinarum, Malus pumila, and Pinus taeda. Many of these latter species have been highly selected for high production or yield, and so there may be a fundamental differences in control of gas exchange (exception: snapbean). At high CO2, stomata of Quercus ilex was completely unresponsive to moderate or high O3 exposure. The variation in stomatal response at different cuvette humidities and light levels was discussed. The following research needs were identified to improve understanding of stomatal behavior concurrent with O3 exposure: cooperators to work on biochemical aspects of stomatal responses; real-time imaging of stomatal behavior; and cooperators to electronically clean up system. (USFS) In development of models of O3 sensitivity of extensive stands of vegetation, including forests, it is reqired to develop a measure of O3 sensitivity as it changes over time of day and over the season. Stable isotope analysis provides such a mechanistically based, long-term integration of metabolic O3 responsiveness and its temporal variation. This and other proxies may be suitable for developing new risk modelling tools. This area of O3 research remains in a relatively early phase. (Germany) This project was conducted in collaboration with Dr. Kent Burkey, USDA-ARS in Raleigh, NC to further investigate the role of the apoplast and cell wall in the differential sensitivity of snap bean and soybean cultivars to O3; this project focuses on objective 4. Two soybean cultivars, Fiskeby (O3 tolerant) and Mandarin Ottowa (O3 sensitive), and two snap bean cultivars, R123 (O3 tolerant) and S156 (O3 sensitive), were used in these experiments. Prior to beginning the O3 exposures, o-anisic acid, which was included in the apoplast extraction buffer (0.1M KPO4, pH 6.5), was analyzed for use as an internal standard. Apoplast wash fluid was extracted from both snap bean and soybean plants and the recovery of the internal standard measured by HPLC. O-anisic acid was determined to be an appropriate internal standard, showing good recovery quantities in the apoplast wash fluid and eluting as a distinct peak in the chromatogram. Three week old soy bean and snap bean plants were given a pre-treatment and exposed to either 0 or 25 ppb O3 for 5 days. Plants were then exposed to either 0, 25, or 75 ppb O3 for an additional 6 days, after which the apoplastic wash fluid was extracted from the leaves and analyzed by HPLC. Initial results indicate differences in the quantity of several peaks between the Fiskeby (O3 tolerant) and Mandarin Ottowa (O3 sensitive) cultivars of soybean, with greater quantities of the peaks appearing in the Fiskeby cultivar, regardless of treatment. These cultivar differences were not observed as consistently in the snap beans. In the Mandarin Ottowa plants, the chromatograms indicate that several compounds decreased in quantity in the high O3 treatments; this was not observed in the Fiskeby plants. There were no treatment effects observed in the snap beans. (Misericordia University) The black cherry project focuses on the development of genomic resources for the analysis of traits related to ozone response in black cherry. We are generating EST database for black cherry by 454 pyrosequencing of leaf cDNAs from ozone tolerant and sensitive genotypes exposed to varying levels and durations of ozone stress. Half-sib families of black cherry were selected based on heritable differences in O3 sensitivity (Lee et al, 1999 and 2002) - tolerant (M-21), and sensitive (R-14). We are also developing reference populations segregating for ozone sensitivity for QTL mapping, in collaboration with the Pennsylvania Bureau of Forestry. (PA) 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 updated with current news items, project annual report and minutes of the 2008 annual meeting. (NC). A laboratory for NCSU students in the Environmental Technology course, Plants Soils and Natural Systems (ET202), was taught using the ozone-sensitive and resistant snap bean lines to explore effects on photosynthesis, stomatal conductance, biomass production and visible injury due to ozone. The genetic component of differential ozone sensitivity between genotypes was highlighted. (NC). A number of Cooperative Extension presentations were made to farm groups, environmental groups, and middle school career days, related to O3 impacts on plants in the San Joaquin Valley of California. (CA) An Environmental Education Technique For Demonstrating Ozone Pollution Effects On Vegetation was developed as a graduate student thesis. This research focused on the development and testing the effectiveness of a teaching module, used to educate individuals about ground level ozone pollution and its effects on vegetation. This research resulted in the development of a teaching module that can be implemented into high school level curricula to educate students and the public on the effects of ground level ozone on vegetation. The research facility utilized for this project was the Air Quality Learning and Demonstration Center located at the Penn State University Arboretum. The methods used to conduct this research were broken into four phases. Initially, photographs showing plant injury due to ozone were analyzed along with weather and air pollution data collected at the research facility; this data was then used for the development of the teaching module. Next, pre-service teachers about to begin their student teaching were presented with the teaching module. Prior to being presented with the module, these students completed a pre-module quiz, which tested their knowledge on the subject matter. After the module, the students were given a post-module quiz, which was identical to the pre quiz. Both the pre and post-module quiz were analyzed to determine the effectiveness of the teaching module. A paired t-test was used for statistical analysis, which demonstrated that there was an increase between the pre and post quiz means (p=0.000, mean pre-quiz=6.63, mean post-quiz=13.06, n=16). After being tested the module was uploaded onto a website for the public to access. Some of this material is already available on the Air Quality Learning and Demonstration website (http://www.aireffects.psu.edu/learning/index.htm), at the PA Department of Environmental Protection website and on the website of the National Literacy Council (http://www.enviroliteracy.org/article.php/74.html). (PA)

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