NIMSS: View = Report

SAES-422 Multistate Research Activity Accomplishments Report =

Approved =

Project No. and Title:	NE10= 30=20 Characterization and Mechanisms of Plant Responses to Ozone in the = U.S.=20
Period Covered:	10-2008 to 09-2009
Date of Report:	30-May-2009
Annual Meeting Dates:	05-May-2009 to 07-May-2009

Participants

Booker, Fitzgerald (fitz.booker@ars.usda.gov) - USDA-ARS Plant = Science=20 Unit, Raleigh, NC=20
Burkey, Kent (kent.burkey@ars.usda.gov) - USDA-ARS Plant Science = Unit,=20 Raleigh, NC=20
Chappelka, Art (chappah@auburn.edu) - Auburn University, Auburn, = AL=20
Grantz, David (david@uckac.edu) - University of California - = Riverside,=20 Riverside, CA=20
Grulke, Nancy (ngrulke@fs.fed.us) - US Forest Service, Pacific = Southwest=20 Research Station, Riverside, CA=20
Heath, Bob (heath@ucr.edu) - University of California - Riverside, = Riverside, CA=20
Matyssek, Rainer (matyssek@wzw.tum.de) - Technical University of = Munich,=20 Munich, Germany=20
McGrath, Margaret (mtm3@cornell.edu) - Cornell University, = Riverhead, NY=20
Smith, Margaret (mes25@cornell.edu) - Cornell University, Ithaca, = NY=20
Wieser, Gerhard (gerhard.wieser@uibk.ac.at) - Innsbruk, Austria =

Brief Summary of Minutes of Annual Meeting

The = meeting=20 was held in the Mission Inn in Riverside California. The meeting was = called to=20 order by Chairman David Grantz at 8:15 on May 5, 2009. The meeting = proceeded as=20 a series of technical reports from each of the participating Experiment = Stations=20 or other collaborating institutions. These are captured in the = accomplishments=20 section of this report. A field trip to the greenhouse exposure = experiments of=20 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=20 incorporated as part of the meeting. The location of the next meeting = was=20 discussed. The possibility of holding it in association with the Air = Pollution=20 Workshop, scheduled to be held in Puerto Rico, was discussed. The Chair = is to=20 poll the membership regarding the desirability of this. The meeting was=20 adjourned at 5:00 on 6 May 2009. An optional post meeting field trip to = the=20 Salton Sea Ecological area was also provided for interested = participants.=20

Accomplishments

Accomplishments during the = period 1=20 October 2008 through 30 September 2009=20

Objective 1. Describe the spatial - temporal characteristics of the = adverse=20 effects of current ambient O3 levels on crop productivity, including the = development of numerical models to establish cause / effect = relationships that=20 apportion the ozone contribution. Risk assessment of trees and forests = needs to=20 be related to a measure of stomatal whole-tree O3 uptake and to the = effective O3=20 dose. This second requirement will necessitate development of = measurements and=20 modelling protocols to describe the responsiveness of biological process = per=20 unit of O3 uptake. Current methodologies to establish the = spatio-temporal=20 scaling of the first components have been demonstrated during this = reporting=20 period, using a combination of sapflow on individual trees or branches, = and eddy=20 covariance at the level of entire stands. The eddy covariance = measurements also=20 allow an estimate of the non-stomatal O3 flux. Progress towards = assessment of O3=20 sensitivity remain in progress. (Germany)=20

Relatives of sugarcane (Saccharum spp.) are highly diverse. Interest = in=20 sustainable biofuel production has led to a reexamination of wild = relatives of=20 commercial sugarcane as potential purpose-grown energy crops in = California. The=20 most likely locations are the inland valleys of central and southern = California,=20 subject to high temperatures, aridity and highly productive irrigated=20 agriculture. Using GIS techniques we have demonstrated that the most = likely=20 microhabitats for cultivation of Saccharum species are also subject to = elevated=20 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=20 characteristics, including tolerance to O3. (CA)=20

Red spruce-mixed conifer ecosystems in the Northeast are of high = ecological=20 importance. A modeling approach (DO3SE) was undertaken in the forested = ecosystem=20 located in the GMNF Lye Brook Class I Wilderness Area and surrounding = airshed.=20 The primary objective was to quantify ozone uptake using a combined=20 physiological-phenological-atmospheric model, and identify when red = spruce are=20 most physiological at risk to chronic ozone exposures. High-elevation=20 physiological sampling sites were expanded in 2009 due to = decommissioning of=20 Vermont=19s only CASTNET site, located within the project area. Three = main (field)=20 components were phenological monitoring, characterization of seasonal = gas=20 exchange, and foliar injury surveys. Highest average exposures tended to = occur=20 close to the period of flushing of current-year needles. Physiological = age may=20 be protective against ozone injury, as needles have not yet entered the = phase of=20 seasonal maximum stomatal conductance (gmax). Because the 2009 ozone = season was=20 exceptionally wet, the project will require one additional season to = capture=20 representative physiological responses to air pollution events.(MA)=20

Summertime ozone persistence at remote higher elevations is = characterized by=20 well-documented level diel exposures, with elevated nocturnal exposures = of=20 several consecutive hours common. Significant ozone reductions have been = claimed=20 (nationally) during this reporting period of documented climate warming. = Up to=20 18 years of archival ozone data were available for analysis of long-term = trends=20 at 4 distinct higher elevation sites in New England: Mt. Washington, NH, = Vermont=20 CASTNET, Mt. Greylock, MA, Pack Monadnock, NH. A fifth site, Whiteface = Mt. Air=20 quality at surveyed high elevation sites shows no trend (neither = improvement nor=20 deterioration) at southern and northern New England locations, with = highest=20 levels in spring. A substantial proportion of exposure in the highest=20 concentration ranges (e80 ppb) occurs nocturnally at these higher = elevations,=20 often exceeding valley locations. Average night/day ratios of AOT40 and = SUM60=20 exposure indices range from 0.52 - 0.77. 2008 was an unusual year at one = site,=20 indicating that nighttime ozone exposures can exceed daytime exposures = in=20 southern New England.=20

Objective 2. Assess the effects of O3 on structure, function and=20 inter-species competition in managed and native plant populations, = including=20 alterations in their nutrient quality. Jeffrey pine stands in the = western Sierra=20 Nevada are subject to nitrogen deposition and O3 impacts, along with = gradients=20 of aridity. During this reporting period we applied slow release urea to = mature=20 Jeffrey pine in perennially mesic and xeric microsites in the southern = Sierra=20 Nevada. This represents the most recent stage of annual applications = that have=20 been done for 10 years. Canopy health was assessed using the following=20 attributes: needle and branch elongation growth, branchlet diameter, = needle=20 retention, and the level of chlorotic mottle observed. Under moderately = high O3=20 exposure, the proportion of poor health trees increased with N = deposition but=20 the proportion of healthy trees was reduced in mesic microsites. In = xeric=20 microsites, N amendment improved the health of the healthiest trees = (e.g., an=20 increase in canopy growth was observed). However, in an extreme drought = year,=20 increased leaf area was reduced the most in trees that were fertilized = in xeric=20 microsites. Simulated N deposition increased tree susceptibility to = extreme=20 drought. Simulated N deposition also modified herbivory and mortality in = Jeffrey=20 pine. In mesic microsites, N amendment increased both needle scale and = mortality=20 (decadal rate: 9%). In xeric microsites, N amendment decreased both = scale and=20 mortality (decreased decadal mortality rate from 23 to 9%). There are = few O3=20 concentration data in remote eastern Sierra Nevada sites. However, for = those=20 sites that we do have data for, concentrations average ca. 42 (NE of = Mammoth=20 Lakes) to 58 (north of Lake Tahoe). Canopy response to this O3 exposure = level,=20 after 3 years of assessment is undetectable for almost all attributes = assessed,=20 including needle herbivory, bark beetle, and tree mortality (see above=20 paragraph). Drought dominates the canopy response and confounds response = to O3.=20 Sites along a 900 km transect of the eastern Sierra Nevada and = Transverse range=20 were chosen to represent the range of the species as well as provide = gradients=20 in evapotranspiration, precipitation, and therefore the level of drought = stress=20 experienced by individual trees. We used this system to define a = mechanistic=20 link between tree drought stress and risk of mortality from pine bark = beetles in=20 Jeffrey pine. Tree-tree competition and stand density further drive = differences=20 in tree drought stress within sites. The objective of this 3 year study = (now in=20 the 3rd year) is to correlate tree drought stress with bole protein and=20 carbohydrate content, resin production and chemistry, and terpene = emissions.=20 Further, we want to identify a quantitative threshold to beetle = resistance (high=20 resin production) and beetle outbreak (high terpene emissions, low resin = flow,=20 increase in bole palatability). Jeffrey pine susceptibility to bark = beetle under=20 drought stress is relevant to understanding O3 exposure because both = drought=20 stress and O3 increase oxidation of zeazanthin, which initiates the = processes by=20 which jasmonate and resin production is upregulated. To date, our = research team=20 has documented a relationship between increased tree drought stress and=20 increased resin production at the annual time scale. The entomologists = are=20 working on qualifying terpene emissions from trees in stands of = differing=20 densities and levels of drought stress. The biochemists are working on=20 quantifying the degree of jasmonate upregulation in the same stands. = (USFS)=20

Yellow nutsedge (Cyperus esculentus) is a noxious and difficult to = control=20 weed in many warm agricultural systems, particularly with irrigation. We = have=20 previously reported that biomass productivity of nutsedge is sensitive = to O3. We=20 found in these previous studies that nutsedge became more competitive = with=20 respect to Pima cotton with increased O3 exposure. In contrast, tomato = was=20 initially less competitive with nutsedge at moderate O3 but recovered = its=20 competitive ability at further increased O3, as nutsedge began to = exhibit=20 substantial growth inhibition. In these studies there were indications = that the=20 stress of O3 on nutsedge enhanced allocation of current biomass to = reproductive=20 structures, belowground. In the current reporting period we tested this=20 hypothesis explicitly. While biomass was again reduced, the reductions = were=20 similar above and below ground, and slightly greater below ground. There = was no=20 enhancement of allocation to tubers. These studies indicate that the = effect of=20 O3 on crop weed interaction will be determined by the relative = sensitivities of=20 specific crops with respect to yellow nutsedge. The competitiveness of = nutsedge=20 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=20 are C4 species. As such they were not expected to be sensitive to O3. We = tested=20 the impact of O3 on the photosynthetic systems of nutsedge and a = commercial=20 clone of sugarcane. In both cases, midday levels of carbon assimilation = declined=20 with increasing exposure to O3. In both cases, intercellular CO2 = concentrations=20 increased, suggesting direct inhibition of mesophyll photosynthesis = rather than=20 induced stomatal closure.=20

In the case of sugarcane a simple model was developed that linked = SPAD=20 (indirect chlorophyll measurements) to carbon assimilation at each leaf=20 insertion level. As SPAD is a rapid measurement compared with gas = exchange, it=20 was possible to predict whole plant carbon assimilation from a vertical = series=20 of SPAD measurements and leaf dimensions up the stalk. Preliminary = studies=20 indicate that wild relatives of sugarcane, which are known to be more = stress=20 hardy than commercial clones, may also exhibit greater tolerance of O3. = (CA)=20

A major objective of the NE-1030 Multistate Project is to describe = the=20 spatial-temporal characteristics of the adverse effects of current = ambient ozone=20 levels on crop productivity, including the development of numerical = models to=20 establish cause-effect relationships. A refined protocol was developed = by Dr. K.=20 Burkey (USDA=13ARS, NC) and Dr. M. McGrath (Cornell, Long Island Field = Station)=20 and strictly followed by our NJ research team, as well as by several = other=20 members of the multi-state project in various parts of the US where = ambient=20 ozone levels and meteorological conditions vary. We monitored the = effects of=20 ambient ozone on the productivity of two snapbean cultivars R331=20 (ozone-tolerant) and S156 (ozone-sensitive) over the 2008 growing = season. We=20 planted the two snapbean cultivars in East Brunswick, NJ, on June 13, = 2008,=20 according to the field design and conditions agreed upon by the four = field=20 stations in the US that are collaborating on this project. Throughout = the=20 growing season, ambient ozone levels and meteorological data were = recorded at=20 each site. At each field station, in order for the data to be more = compatible=20 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=20 cultivars) occurred at the mid (third) harvesting date. A statistically=20 significant decrease in total pod fresh weight of marketable snapbeans = was=20 observed in S156 relative to R331 in two of the five harvest dates, = where the=20 fresh weight of marketable pods of the ozone-sensitive cultivar was less = than=20 50% of that of the ozone-tolerant cultivar. A final harvest of snapbeans = was=20 conducted at 84 days after planting. At this harvest date in the 2008 = growing=20 season, a significant proportion of the pods were immature (without = seeds) or=20 green, and this was more pronounced in the ozone-tolerant cultivar. = Nonetheless,=20 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=20 seeds and pods from the two cultivars were significantly different, with = yield=20 reductions in the sensitive relative to the tolerant cultivar of 44%, = 56% and=20 48%, respectively. Although there was a trend toward reduction in the = pod number=20 of the sensitive cultivar when compared to the tolerant cultivar, the = decrease=20 was not significant at a p-value of 0.05 or less. The meteorological and = ozone=20 data, coupled with the crop yield data, will be analyzed for the several = states=20 where this field experiment has been conducted and incorporated into a = numerical=20 model by Dr. S. Krupa (MN) to establish a relationship between ambient = ozone=20 exposures and crop responses. The continuation of this study will = strengthen our=20 understanding of the impact of ambient ozone on plants and crop = productivity.=20 (NJ)=20

The poplar project tries to answer to the question =1CDo ozone (O3)=20 concentrations relevant to Pennsylvania forests alter induced responses = of=20 poplars to insect herbivory?=1D We treated hybrid poplar OGY (P. = deltoides x=20 nigra) with ozone (80 ppb) and gypsy moth (Lymantria dispar L.) = herbivory in=20 environmentally controlled chambers. RNA was isolated and subjected to = an EST=20 custom microarray with probes for ~6500 unique genes. The preliminary = results=20 showed that ozone treatments supressed Transcriptome-Level Dynamic = Responses of=20 Poplar Leaves to Insect Herbivory. These past weeks we repeated the = experiment=20 using NE-388 (sensitive) and NE-245(tolerant) hybrid poplar clones. No = results=20 to report. (PA)=20

Objective 3. Examine the joint effects of O3 with other growth = regulating=20 factors (e.g., CO2, temperature) that are expected to vary with ongoing = climate=20 change on crop growth and productivity. This study examined the effects = of=20 elevated carbon dioxide and ozone on plant-soil interactions, including = effects=20 on soil respiration, root length, litter decomposition and soil C, in a = no-till=20 soybean-wheat system. The experiment was started in 2005 and is ongoing. = Elevated carbon dioxide increased soil respiration by 26%, due in part = to=20 increased microbial respiration. Added ozone suppressed soil respiration = during=20 the latter part of the soybean growing season. Total root length in the = elevated=20 carbon dioxide treatments was not different from the control in = September while=20 root length was 45% lower with added ozone, likely due to suppressed = growth and=20 early senescence. Litter inputs were higher with elevated carbon dioxide = and=20 lower with added ozone. Residue decomposition / input ratios indicated = that=20 decomposition was similar among treatments although C input to the soil = was=20 altered. Decreased 13C and higher %C in the coarse 0-5 cm deep soil = fraction=20 indicated that elevated carbon dioxide increased soil organic matter = even though=20 soil respiration was higher. Ozone effects were not apparent. (NC).=20

A new study was initiated in 2009 to determine the effects of = tropospheric=20 ozone and various climate change (precipitation) on a semi-natural = grassland=20 characteristic of the Piedmont region of the US (mixture of tall fescue, = common=20 bermudagrass, dallisgrass and white clover). Twelve, large (4.8 m ht. = =C3=97 4.5 m=20 dia.) OTCs (modified with rain-exclusion caps) located at the Auburn = University=20 Atmospheric Deposition (AtDep) Site are used in this study. A = multifactor design=20 with two ozone treatments [nonfiltered (NF, ambient) and 1.5 =C3=97 NF] = and 3 water=20 regimes (30-yr average, +20% and -20%) is replicated 2 times. Ozone = exposures=20 and rain treatments were initiated June 1, 2009. Primary growth and = regrowth=20 will be harvested monthly during the growing season. Various field and=20 laboratory methodologies will be used to test the specific hypotheses. = Results=20 will provide critical information on structure and functioning of = managed=20 grassland ecosystems using projected climate scenarios of elevated ozone = and=20 differing amounts of rainfall, with emphasis on interspecific = relationships=20 among the various processes examined. Integration of various measures of = diversity and productivity and underlying physiological and biochemical=20 responses will enable a more complete characterization and modeling of = potential=20 impacts of future climate change scenarios on these plant communities.=20 Non-fumigated forage from our site was harvested on April 21 and May 12, = 2008,=20 after which they were exposed to either ambient, non-filtered air (NF) = or=20 twice-ambient O3 air (2 =C3=97 NF) air and harvested on June 9 and July = 2. Forages=20 are being processed for nutritive quality analysis and fabrication into = 50-g=20 cubes that will be fed to New Zealand White rabbits in=20 nutrient-utilization/diet-selection experiments beginning in late 2009. = In=20 collaboration with scientists at the Swiss Federal Research Station for=20 Agroecology and Agriculture, we have completed the fifth year of a = seven-year=20 experiment in the Swiss Alps in which we are investigating productivity = and=20 forage quality of semi-natural herbaceous vegetation exposed to three=20 concentrations of ground-level ozone (ambient, ambient + 20 ppb O3 and = ambient +=20 40 ppb O3) and five levels of atmospherically deposited nitrogen (0, 5, = 10, 15,=20 20 and 25 kg N/ha). Forage samples are awaiting processing for nutritive = quality=20 analysis. (Auburn University)=20

To develop robust models of landscape scale O3 impacts it is critical = to=20 related single organ and single organism data to extensive canopy data. = During=20 this reporting period, a team of gas exchange ecophysiologists tested = for=20 comparability between eddy correlation estimates of stand flux of O3 and = CO2,=20 with canopy level O3 uptake based on a near-leaf surface, chamberless = gas=20 sampling system, with O3 uptake calculated from both canopy = transpiration=20 measurements and leaf-level gas exchange measurements. Following a=20 proof-of-concept project in a cultivated orange grove, we will measure = fluxes in=20 an oak woodland as well as in a pine forest. These data will demonstrate = certain=20 key gas exchange technologies, and provide infrastructure for landscape = scale=20 assessments of O3 damage across important California ecosystems. (USFS)=20

Objective 4. Examine the physiological and molecular basis of O3 = toxicity and=20 tolerance in plants. The specific root respiration (per unit root = weight) is=20 enhanced in Pima cotton by exposure of the shoot to O3. Similarly, we = have shown=20 that phloem loading is inhibited by O3 in Pima cotton. Using alkaline = single=20 cell electrophoresis of root tip cells, we have sought to document = genotoxicity=20 of O3 exposure of the shoot to the developing roots. Although we have = previously=20 reported increasing trends in damage to fine roots with increasing O3, = it has=20 been difficult despite much replication to establish convincing = statistical=20 significance with this tissue. Preliminary experiments with older roots = suggest=20 similar trends, and statistical significance. Further experiments will = be=20 required to determine if the older roots which have longer experience = with=20 O3-impacted shoot tissue, may exhibit such genotoxicity. Similarly, = preliminary=20 experiments with young leaf tissue exhibit increasing trends, but do not = reach=20 the level of statistical significance. The question of genotoxicity of = O3=20 exposure remains important, but unresolved as of this reporting period. = Methyl=20 jasmonate is a key signaling metabolite, synthesized from the membrane=20 constituent, linolenic acid. It functions with other signaling = compounds,=20 including salicylic acid and ethylene, in controlling programmed cell = death in=20 response to pathogens and abiotic stress such as acute O3, and possibly = in=20 mediating plant responses to chronic O3. While Methyl Jasmonate provided = protection in tobacco and Arabidopsis against O3 exposure, in Pima = cotton it did=20 not. Growth and allocation of Pima cotton responded to a concentration = gradient=20 of Methyl Jasmonate in a manner similar to responses to increasing O3 = exposure.=20 A low concentration of Methyl Jasmonate (40 micrograms per plant, twice = weekly)=20 had no impact on growth or allocation, and did not affect the response = to O3. A=20 higher application rate (160 micrograms per plant, twice weekly) reduced = growth=20 and allocation to roots but did not interact with the O3 response, = resulting in=20 parallel O3 response curves. Thus there was no protection against = chronic O3=20 damage by Methyl Jasmonate in Pima cotton. (CA)=20

Completed a study of ozone effects on leaf peroxidase isozymes in=20 Arabidopsis. Native gels revealed induction of a major cationic isozyme=20 following a 2-day exposure to moderate levels of ozone. The = ozone-responsive=20 cationic isozyme was induced in Col-O wild-type plants as well as a = number of=20 mutants. This enzyme has the potential to serve as a marker for ozone = stress=20 prior to the appearance of foliar injury.=20

Results from prior year greenhouse screening of soybean ancestors for = ozone-induced foliar injury were combined with pedigree analysis = techniques to=20 predict ozone resistance of 247 publically-released soybean cultivars. = Ancestors=20 with the greatest ozone resistance were not major contributors to = current US=20 cultivars. Predicted injury scores suggested that cultivars from the = Midwest may=20 be more sensitive to ozone-induced foliar injury, on average, than = Southern=20 cultivars. (NC)=20

The biogenically-produced volatile hydrocarbon, isoprene, may be = influential=20 in protecting some plants from ozone injury. Isoprene emission is = correlated=20 with tolerance to high temperature and oxidative stress. Isoprene can = also=20 scavenge ozone in the leaf boundary layer and apoplast, although = reaction=20 products may be toxic and overall efficacy of the proposed mechanism is=20 uncertain. A series of experiments conducted in Raleigh, NC investigated = potential interactions between biogenically-synthesized isoprene, = temperature=20 and ozone using an isoprene-emitting legume species, velvet bean (Mucuna = pruriens). Preliminary screening experiments indicated that isoprene = emission=20 was not correlated with ozone sensitivity. Velvet bean lines that = displayed=20 varying extents of foliar visible injury symptoms following acute ozone=20 exposures were found to emit isoprene at similar rates when grown in = clean air.=20 Treatment of plants with an antibiotic (fosmidomycin), which suppressed = isoprene=20 emission, was ineffective in altering plant responses to ozone. Elevated = temperature increased isoprene emission but there was no interaction = between=20 isoprene emission rates and ozone effects on net photosynthesis, biomass = production, peroxidase activity and ascorbate levels. Increased = temperature=20 increased stomatal conductance and ozone effects on plant biomass, = ascorbate=20 levels and redox status. It was apparent that increased temperature = exacerbated=20 ozone injury, suggesting that ozone x temperature interactions deserve = further=20 study. These results raise significant questions about the proposed role = of=20 isoprene in modifying ozone injury in isoprene-emitting plants. (Fitz = Booker, Ed=20 Fiscus, USDA-ARS, Raleigh, NC). (NC) Expression of Pyrococcus furiosus=20 superoxide reductase in Arabidopsis enhances tolerance to heat and = paraquat, but=20 not ozone Uncontrolled production of reactive oxygen species such as = superoxide=20 in response to environmental stressors can result in cell death. = Superoxide=20 dismutase participates in quenching superoxide in plants but attempts to = alter=20 its expression in vivo have been challenging. Another approach to = manipulating=20 control of superoxide in vivo was tried in this experiment. A superoxide = reductase (SOR) gene from the archaeal hyperthermophile, Pyrococcus = furiosus,=20 was expressed in Arabidopsis. SOR is a cytosolic, thermostable enzyme = that=20 reduces superoxide to hydrogen peroxide. Although transgenic plants = expressing=20 SOR were more tolerant than wild-type plants to heat stress and the = herbicide=20 paraquat (which generates superoxide in the chloroplast), plant = responses to=20 chronic and acute ozone exposures were not significantly different = between=20 wild-type and transgenic lines. This suggests that superoxide may not = have a=20 major role in ozone toxicity effects in plants. In a related experiment, = plants=20 treated with continuous light for 24 h showed much reduced ozone = treatment=20 effects and significantly higher leaf anthocyanin concentrations. This = finding=20 suggests that anthocyanins may be effective in protecting plants from = ozone=20 injury, provided that ozone uptake was unaffected by the light = treatment.=20 Further experiments are underway to test this possibility. (NC)=20

Using a novel gas exchange system that concurrently measures water, = O3, and=20 CO2 flux at the leaf level, we investigated the direct effects of O3 = exposure on=20 stomatal behavior. The following species had increased stomatal = conductance in=20 response to short term high O3 exposure: Pinus ponderosa, Quercus = kelloggii, Q.=20 douglasii, Phaseolus vulgaris, Fagus sylvatica. The following species = had=20 decreased gs in response to short term high O3 exposure: Gossypium = hirsutum,=20 Saccharum officinarum, Malus pumila, and Pinus taeda. Many of these = latter=20 species have been highly selected for high production or yield, and so = there may=20 be a fundamental differences in control of gas exchange (exception: = snapbean).=20 At high CO2, stomata of Quercus ilex was completely unresponsive to = moderate or=20 high O3 exposure. The variation in stomatal response at different = cuvette=20 humidities and light levels was discussed. The following research needs = were=20 identified to improve understanding of stomatal behavior concurrent with = O3=20 exposure: cooperators to work on biochemical aspects of stomatal = responses;=20 real-time imaging of stomatal behavior; and cooperators to = electronically =18clean=20 up=19 system. (USFS)=20

In development of models of O3 sensitivity of extensive stands of = vegetation,=20 including forests, it is reqired to develop a measure of O3 sensitivity = as it=20 changes over time of day and over the season. Stable isotope analysis = provides=20 such a mechanistically based, long-term integration of metabolic O3=20 responsiveness and its temporal variation. This and other proxies may be = suitable for developing new risk modelling tools. This area of O3 = research=20 remains in a relatively early phase. (Germany)=20

This project was conducted in collaboration with Dr. Kent Burkey, = USDA-ARS in=20 Raleigh, NC to further investigate the role of the apoplast and cell = wall in the=20 differential sensitivity of snap bean and soybean cultivars to O3; this = project=20 focuses on objective 4. Two soybean cultivars, Fiskeby (O3 tolerant) and = Mandarin Ottowa (O3 sensitive), and two snap bean cultivars, R123 (O3 = tolerant)=20 and S156 (O3 sensitive), were used in these experiments. Prior to = beginning the=20 O3 exposures, o-anisic acid, which was included in the apoplast = extraction=20 buffer (0.1M KPO4, pH 6.5), was analyzed for use as an internal = standard.=20 Apoplast wash fluid was extracted from both snap bean and soybean plants = and the=20 recovery of the internal standard measured by HPLC. O-anisic acid was = determined=20 to be an appropriate internal standard, showing good recovery quantities = in the=20 apoplast wash fluid and eluting as a distinct peak in the chromatogram. = Three=20 week old soy bean and snap bean plants were given a =1Cpre-treatment=1D = and exposed=20 to either 0 or 25 ppb O3 for 5 days. Plants were then exposed to either = 0, 25,=20 or 75 ppb O3 for an additional 6 days, after which the apoplastic wash = fluid was=20 extracted from the leaves and analyzed by HPLC. Initial results indicate = differences in the quantity of several peaks between the Fiskeby (O3 = tolerant)=20 and Mandarin Ottowa (O3 sensitive) cultivars of soybean, with greater = quantities=20 of the peaks appearing in the Fiskeby cultivar, regardless of treatment. = These=20 cultivar differences were not observed as consistently in the snap = beans. In the=20 Mandarin Ottowa plants, the chromatograms indicate that several = compounds=20 decreased in quantity in the high O3 treatments; this was not observed = in the=20 Fiskeby plants. There were no treatment effects observed in the snap = beans.=20 (Misericordia University)=20

The black cherry project focuses on the development of genomic = resources for=20 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=20 from ozone tolerant and sensitive genotypes exposed to varying levels = and=20 durations of ozone stress. Half-sib families of black cherry were = selected based=20 on heritable differences in O3 sensitivity (Lee et al, 1999 and 2002) - = tolerant=20 (M-21), and sensitive (R-14). We are also developing reference = populations=20 segregating for ozone sensitivity for QTL mapping, in collaboration with = the=20 Pennsylvania Bureau of Forestry. (PA)=20

Objective 5. Develop educational tools and conduct advanced training = for K-12=20 public school teachers, college level instructors, and outreach = educators=20 regarding the effects of ambient O3 pollution on plants.=20

The NE-1030 project web page=20 (http://www.ncsu.edu/project/usda-ne-1013/index.htm) was updated with = current=20 news items, project annual report and minutes of the 2008 annual = meeting. (NC).=20

A laboratory for NCSU students in the Environmental Technology = course, Plants=20 Soils and Natural Systems (ET202), was taught using the ozone-sensitive = and=20 =13resistant snap bean lines to explore effects on photosynthesis, = stomatal=20 conductance, biomass production and visible injury due to ozone. The = genetic=20 component of differential ozone sensitivity between genotypes was = highlighted.=20 (NC).=20

A number of Cooperative Extension presentations were made to farm = groups,=20 environmental groups, and middle school career days, related to O3 = impacts on=20 plants in the San Joaquin Valley of California. (CA)=20

An Environmental Education Technique For Demonstrating Ozone = Pollution=20 Effects On Vegetation was developed as a graduate student thesis. This = research=20 focused on the development and testing the effectiveness of a teaching = module,=20 used to educate individuals about ground level ozone pollution and its = effects=20 on vegetation. This research resulted in the development of a teaching = module=20 that can be implemented into high school level curricula to educate = students and=20 the public on the effects of ground level ozone on vegetation. The = research=20 facility utilized for this project was the Air Quality Learning and=20 Demonstration Center located at the Penn State University Arboretum. The = methods=20 used to conduct this research were broken into four phases. Initially,=20 photographs showing plant injury due to ozone were analyzed along with = weather=20 and air pollution data collected at the research facility; this data was = then=20 used for the development of the teaching module. Next, pre-service = teachers=20 about to begin their student teaching were presented with the teaching = module.=20 Prior to being presented with the module, these students completed a = pre-module=20 quiz, which tested their knowledge on the subject matter. After the = module, the=20 students were given a post-module quiz, which was identical to the pre = quiz.=20 Both the pre and post-module quiz were analyzed to determine the = effectiveness=20 of the teaching module. A paired t-test was used for statistical = analysis, which=20 demonstrated that there was an increase between the pre and post quiz = means=20 (p=3D0.000, mean pre-quiz=3D6.63, mean post-quiz=3D13.06, n=3D16). After = being tested=20 the module was uploaded onto a website for the public to access. Some of = this=20 material is already available on the Air Quality Learning and = Demonstration=20 website (http://www.aireffects.psu.edu/learning/index.htm), at the PA = Department=20 of Environmental Protection website and on the website of the National = Literacy=20 Council (http://www.enviroliteracy.org/article.php/74.html). (PA)=20

Impacts

This Multi-State Project provided data to state and federal = regulatory=20 bodies and Agricultural Air Quality Task Force as air quality = standards and=20 policies are revised. Wilderness and National Park managers have = utilized=20 Project data to document long term impacts of ozone on unmanaged = vegetation.=20 Across a broad spectrum of stakeholders, tropospheric ozone is = recognized as=20 an element of global change that interacts with other elements, such = as=20 temperature, moisture and nitrogen.
Growers and extension educators in California are recognizing = ozone=20 impacts as part of climate change on the dynamics of important = agricultural=20 weeds, including horseweed and yellow nutsedge. This influences = regulatory=20 acceptance, and may lead to altered vegetation management = protocols.
Integrated measurements and modeling protocols are being developed = to=20 inform the next generation of flux based regulatory standards for = ozone,=20 bringing European and North American researchers together. The = dynamics of=20 ozone sensitivity has been identified as a research need to develop = ozone dose=20 s a unifying factor.
Development of agriculturally relevant plant growth regulators is=20 beginning to take ozone into account. Experimental use of methyl = jasmonate in=20 grapes and citrus in California and Florida has the potential to = interact with=20 the ethylene-salicylic acid-jasmonate signaling system that controls = plant=20 response to acute ozone exposure. Work by this Multi-State project and = others=20 has begun to evaluate the potential for phytoprotection by = manipulation of the=20 elements of these signaling systems.
Public educational facilities are in operation in California and=20 Pennsylvania, and a comprehensive web presence is maintained in North = Carolina=20 to provide information that is relevant locally, nationally and=20 internationally, with respect to ozone air pollution.

Publications

Albertine, JM, WJ Manning. 2009. = Elevated=20 night soil temperatures result in earlier incidence and increased extent = of=20 foliar ozone injury to common bean (Phaseolus vulgaris L.). = Environmental=20 Pollution 157:711-713. Booker, FL, R Muntifering, M McGrath, KO Burkey, = D=20 Decoteau, EL Fiscus, W Manning, S Krupa, A Chappelka, DA Grantz. 2009. = The ozone=20 component of global change: Potential effects on agricultural and = horticultural=20 plant yield, product quality and interactions with invasive species. = Journal of=20 Integrative Plant Biology 51:337-351.=20

Burkey, KO and TE Carter. 2009. Foliar resistance to ozone injury in = the=20 genetic base of U.S. and Canadian soybean and prediction of resistance = in=20 descendent cultivars using coefficient of parentage. Field Crop Research = 111:207-217.=20

Davis, D.D., J.M. Skelly, D.R. Decoteau, L.J. Kline, J.A. Ferdinand, = J.E.=20 Savage, and T. Orendovici-Best. 2008. Susceptibility and Foliar Response = of=20 Broadleaved Species Exposed to Ozone. USDA =13 Forest Service Forest = Health=20 Monitoring Program, 50 pp.=20

Ditchkoff, S.S., J.S. Lewis, J.C. Lin, R.B. Muntifering, and A.H. = Chappelka.=20 2009. Nutritive quality of highbush blackberry (Rubus argutus) exposed = to=20 tropospheric ozone. Rang. Ecol. & Mang. (In press, available online, = DOI:=20 10.2111/08-222.1).=20

Gonzalez-Fernadez, I, D. Bass, R. Muntifering, G. Mills and J. = Barnes. 2008.=20 Impacts of ozone pollution on productivity and forage quality of = grass/clover=20 swards. Atmos. Environ. 42: 8755-8769.=20

Grantz, DA and H-B Vu. 2009. O3 sensitivity in a potential C4 = bioenergy crop:=20 Sugarcane in California. Crop Science 49:643-650.=20

Kline, L.J., D.D. Davis. J.M. Skelly, and D.R. Decoteau 2009. = Variation in=20 Ozone Sensitivity Within Indian Hemp andCommon Milkweed Selections from = the=20 Midwest. Northeastern Naturalist 16:307-313.=20

Leakey, ADB, F Xu, KM Gillespie, JM McGrath, EA Ainsworth and DR Ort. = 2009.=20 Genomic basis for stimulated respiration by plants growing under = elevated carbon=20 dioxide. Proceedings of the National Academy of Sciences 106:3597-3602.=20

Leakey, ADB, EA Ainsworth, CJ Bernacchi, A Rogers, SP Long and DR = Ort. 2009.=20 Elevated CO2 effects on plant carbon, nitrogen, and water relations: six = important lessons from FACE. Journal of Experimental Botany: In press.=20

Liu, L, JS King, CP Giardina, FL Booker. 2009. The influence of = chemistry,=20 production and community composition on leaf litter decomposition under = elevated=20 atmospheric CO2 and tropospheric O3 in a northern hardwood ecosystem. = Ecosystems=20 12:401-416.=20

Liu, L, JS King, FL Booker, CP Giardina, HL Allen, S Hu. 2009. = Enhanced=20 litter input rather than changes in litter chemistry drive soil carbon = and=20 nitrogen cycles under elevated CO2: a microcosm study. Global Change = Biology=20 15:441-453.=20

Lucier, A., Ayres, M., Karnosky, D., Thompson, I., Loehle, C., Percy, = K.,=20 Sohngen, B. 2009. Future environmental impacts and vulnerabilities. pp. = 29-52 In=20 Sepp=C3=A4l=C3=A4, R., Buck A., Katila, P. (Eds.) Adaptation of Forests = and People to=20 Climate Change =13 A Global Assessment Report. International Union of = Forest=20 Research Organizations (IUFRO) World Series Vol. 22, Vienna, Austria. = 224pp=20

Paoletti, E, AM Ferrara, V Calatayud, J Cervero, F Giannetti, MJ = Sanz, WJ=20 Manning. 2009. Deciduous shrubs for ozone bioindication: Hibiscus = syriacus as an=20 example. Environmental Pollution 157:865-870.=20

Paoletti, E, N Contran, WJ Manning, AM Ferrara. 2009. Use of the = antiozonant=20 ethylenediurea (EDU) in Italy: Verification of the effects of ambient = ozone on=20 crop plants and trees and investigation of EDU's mode of action. = Environmental=20 Pollution 157:1453-1460.=20

Papinchak, H.L., E.J. Holcomb, T.O. Best and D.R. Decoteau. 2009.=20 Effectiveness of houseplants in reducing the indoor air pollutant ozone. = HortTechnology 19:286-290.=20

Percy, KE, S Manninen, K-H Haberle, C Heerdt, H Werner, GW Henderson, = R=20 Matyssek. 2009. Effect of 3 years' free-air exposure to elevated ozone = on mature=20 Norway spruce (Picea abies (L.) Karst.) needle epicuticular wax = physicochemical=20 characteristics. Environmental Pollution 157:1657-1665.=20

Percy, K.E., Nosal, M., Heilman, W., Dann, T., Karnosky, D.F. 2009.=20 Standards-based ozone exposure-response functions that predict forest = growth.=20 pp. 269-293 In A. H. Legge (Ed.) Relating Atmospheric Source = Apportionment to=20 Vegetation Effects: Establishing Cause and Effect Relationships. = Elsevier=20 Environmental Science Series Vol. 9, Oxford, UK.=20

Percy, Kevin E. , Sirkku Manninen, Karl-Heinz Haberle, C. Heerdt, H. = Werner,=20 Henderson, G.W., Rainer Matyssek, R. 2009. Effect of 3 years=19 free-air = exposure=20 to elevated ozone on mature Norway spruce (Picea abies (L.) Karst.) = needle=20 epicuticular wax physicochemical characteristics. Environmental = Pollution 157:=20 1657-1665 Rodolfo SE, BA Humberto, MA Violeta, SA Pablo, BL Emma, S = Krupa. 2009.=20 Levels and source apportionment of volatile organic compounds in = southwestern=20 area of Mexico City. Environmental Pollution 157:1038-1044.=20

Suvi Nikula, Sirkku Manninen, Kevin Percy, Maarit Falck, Elina = Oksanen and=20 Toni Holopainen 2009. Elevated O3 induced minor changes in growth and = foliar=20 traits of European and hybrid aspen. Boreal Environment Research 14(A): = 29-47.=20

Szantoi Z, AH Chappelka, RB Muntifering, GL Somers. 2009. Cutleaf = coneflower=20 (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU).=20 Environmental Pollution 157:840-846.=20